Meptazinol: unusual in vivo opioid receptor activity at supraspinal and spinal sites.

Systemic (1-10 mg/kg, s.c.), intracerebroventricular (i.c.v. 20-80 micrograms) and spinal intrathecal (i.t., 5-20 micrograms) administration of meptazinol hydrochloride produced dose-related inhibition of reflex contractions of the urinary bladder, recorded isometrically in urethane-anesthetized rats. The effects of meptazinol were reversed by naloxone administered by the same route. Indeed, this was achieved with intracerebroventricular or intrathecal administration of naloxone (2 micrograms), which also selectively antagonized the mu-receptor ligand [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). However ICI 174,864 (3 micrograms, i.c.v. or i.t.), a delta-opioid receptor antagonist, did not affect the actions of meptazinol given intracerebroventricularly or intrathecally though it consistently abolished the equieffective actions of a selective delta-receptor ligand (2-D-penicillamine, 5-L-penicillamine) enkephalin (DPLPE). Naloxonazine (5 micrograms, i.c.v. or i.t.), an irreversible mu 1-opioid receptor antagonist, produced prolonged antagonism of the effects of DPLPE and meptazinol. The effects of DPLPE partially or completely recovered by 24 hr, indicating that naloxonazine produced prolonged antagonism of delta-opioid receptors. The effects of maptazinol however only recovered after 72 hr, suggesting that antagonism by naloxonazine of this ligand was irreversible and was mediated through a unique opioid receptor interaction. Subthreshold doses of meptazinol (10 micrograms, i.c.v.; 3 micrograms, i.t.) consistently antagonized the effects of morphine given intracerebroventricularly or intrathecally but not the equieffective doses of DPLPE or DAGO. These observations suggest that meptazinol inhibited reflex contractions of the bladder by supraspinal and spinal mu-opioid receptor activation. Furthermore, its agonistic effect and its antagonistic actions were compatible with interactions at a subpopulation of opioid receptors, possibly mu 1-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Naloxonazine and opioid-induced inhibition of reflex urinary bladder contractions.

Spontaneous volume-induced contractions of the urinary bladder were recorded isometrically in urethane-anesthetized rats. Contractions were inhibited by alternate submaximal but equieffective doses of the selective mu and delta-opioid ligands [D-Ala2-Me-Phe4,Gly(ol)5] enkephaline (DAGO) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE), respectively, administered by the intracerebroventricular (i.c.v.) or spinal intrathecal (i.t.) route. Naloxonazine, postulated to be an irreversible mu 1-opioid receptor antagonist, administered by the same route, antagonized the effects of both DAGO and DPDPE. The antagonism of the effect of DAGO was reversed 3-4 hr later but that of DPDPE was more prolonged. Recovery of the effect of DPDPE was observed some 24 hr later. A similar pattern of activity against DAGO and DPDPE given intraventricularly or intrathecally was observed following intravenous injection of naloxonazine (10 mg/kg). Also naloxonazine (i.c.v., i.t. or i.v.) antagonized the effect of morphine given intraventricularly or intrathecally, but antagonism was not observed when morphine was retested 3-4 hr and 24 hr later. Naloxonazine increased the frequency of contraction of the bladder after each route of administration. This effect lasted 1-3 hr and was not seen 24 hr later. Systemic administration of naloxone (10 mg/kg, i.v) also increased the frequency of bladder contraction and attenuated or abolished the effect of DAGO given intraventricularly or intrathecally and the delta-receptor agonist [2-D-penicillamine, 5-L-penicillamine] enkephaline (DPLPE).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the in vivo and in vitro opioid activity of bridged hexahydroaporphine and isoquinoline molecules.

Four novel racemic bridged hexahydroaporphine (1 and 2) and isoquinoline (3 and 4) analogues have been synthesized in an attempt to generate bicyclic derivatives of the morphinan ring system. The opioid activity of these analogues has been assessed through membrane-binding studies, in vitro studies in isolated guinea pig ileum and mouse vas deferens, and in vivo studies utilizing the mouse hot plate technique. The bridged isoquinoline precursor molecules were inactive as antinociceptives. Both the racemic phenolic hexahydroaporphine 1 and its 10-methoxy congener 2 demonstrated dose-dependent, albeit weak, antinociceptive activity when administered icv, but they induced lethal convulsions when given subcutaneously. The antinociception elicited by 1 appeared to show very weak opioid character while that caused by 2 was totally nonopioid.

Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific mu opioid receptor antagonists with greatly reduced binding at somatostatin receptors.

A series of conformationally restricted, cyclic octapeptides containing a conformationally stable tetrapeptide sequence related to somatostatin, -Tyr-D-Trp-Lys-Thr-, as a template, were designed and synthesized with the goal of developing highly potent and selective mu opioid antagonists with minimal or no somatostatin-like activity. Three distinct structures of the peptide became targets of chemical modifications and constraints; the N- and C-terminal amino acids and the cyclic 20-membered ring moiety. Based on the conformational analysis of active and inactive analogues of the parent peptide D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Pen7+ ++-Thr8-NH2, CTP (Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 44, 697-710), we designed analogues to include the tetrahydroisoquinolinecarboxylate (Tic) moiety as the N-terminal amino acid instead of D-Phe, since Tic can exist only as a gauche (-) or a gauche (+) conformer. In this series, the following peptides were synthesized and pharmacologically evaluated: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (TCTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (TCTOP), and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (TCTAP). In rat brain membrane opioid radioligand binding assays, all three peptides displayed high affinity for mu opioid receptors (IC50 = 1.2, 1.4, 1.2 nM, respectively), and exceptional mu vs delta opioid receptor selectivity: 7770, 11,396, and 1060, respectively. TCTOP and TCTAP also possess exceptional mu vs somatostatin receptor selectivity: 14,574 and 28,613, respectively. In the peripheral in vitro GPI bioassay, TCTP, TCTOP, and TCTAP were highly effective antagonists of the potent mu opioid receptor agonist PL017, with pA2 = 8.69 for TCTAP, 8.10 for TCTP, and 7.38 for TCTOP. Our results show that a 10-fold higher affinity and selectivity for mu opioid receptors (in both central and peripheral studies) over delta and somatostatin receptor was gained as a result of the D-Tic1 substitution. These three peptides, TCTP, TCTOP, and TCTAP, are the most potent and selective mu opioid antagonists known. CTP has been shown to possess prolonged biological action, much longer than that of naloxone. This renders these analogues potentially useful ligands for investigating the physiological functions of the mu opioid receptor. Analogues of TCTP in which the 20-membered disulfide ring was contracted by deletion of D-Trp4, and/or Lys5, and/or Thr6 led to compounds with greatly reduced potency at the mu opioid receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Design and synthesis of highly potent and selective cyclic dynorphin A analogues.

We have designed and synthesized several cyclic disulfide-containing peptide analogues of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogues exhibit unexpected selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogues has resulted in the kappa/mu opioid receptor ligands [Cys5,Cys11]Dyn A1-11-NH2 (1) and [Cys5,Cys11,D-Ala8]Dyn A1-11-NH2 (2), which possess high kappa and mu opioid receptor affinities centrally (guinea pig brain, GPB), but only weak activity at peripheral kappa and mu opioid receptors (guinea pig ileum, GPI). On the other hand, [Cys8,Cys13]Dyn A1-13-NH2 and [D-Cys8,D-Cys13]Dyn A1-13-NH2 (5) display high kappa potencies and selectivities at the peripheral (GPI) but not at the central (GPB) kappa opioid receptor. The lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays suggests the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral nervous systems.

Design and synthesis of highly potent and selective cyclic dynorphin A analogs. 2. New analogs.

We have designed and synthesized several cyclic disulfide-containing peptide analogs of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogs exhibit unexpected apparent selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogs has resulted in the kappa/mu opioid receptor ligands [L-Pen5,Cys11]Dyn A1-11-NH2 (4), [Cys5,Cys10]Dyn A1-11-NH2 (5), [Cys5,Cys9]DynA1-11-NH2 (6), and [Cys4,Cys9,Arg10]DynA1-11-NH2(7). All of these analogs possess high kappa and mu opioid receptor affinities for the central receptor (guinea pig brain), but effect only weak potency at peripheral kappa and mu opioid receptors (GPI). In fact cyclic dynorphin A analog 4 shows > 19,000-fold differences between central kappa opioid affinity and potency in the guinea pig ileum (GPI). Additionally analog 4 is not an antagonist in the GPI, suggesting possible receptor differences between these sites. Substitution of Tyr1 by Phe1 in the cyclic 1-11 series gave the analog [Phe1,Cys5,Cys11]Dyn A1-11-NH2 (1) that was surprisingly potent in the guinea pig brain binding assay (IC50 = 15.1 nM) at the kappa receptor, but was inactive in the GPI and mouse vas deferens bioassays. D-Ala2 and Tic4 analogs of 1 had lower affinity at brain kappa receptors and had very weak potencies in the GPI and MVD bioassays. On the other hand, [Cys6,Cys10]DynA1-11-NH2 (8), [Cys8,D-Cys13]DynA1-13-NH2 (9), [D-Cys8,D-Cys12]DynA1-13-NH2 (10), and [D-Pro10,Cys5,Cys13]-Dyn A1-13-NH2 (11) were surprisingly potent in the GPI bioassay, though considerable apparent selectivity for central receptors is still retained. The apparent lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays, particularly with 1 and 4, may suggest the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral systems.

Central delta-opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat.

The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. The conformationally restricted enkephalin analogues [2-D-penicillamine, 5-L-cysteine] enkephalin (DPLCE), [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. Both the novel delta-opioid receptor antagonist ICI 154,129 (200-600 micrograms) [N,N-bisallyl-Tyr-Gly-Gly-Psi-(CH2S)-Phe-Leu-OH) and ICI 174,864 (1-3 micrograms) [N,N-dially-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the mu-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate delta-receptor antagonism. Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 micrograms, i.c.v. or i.t.) but could be abolished by higher (10-15 micrograms) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by mu-opioid receptor. beta-Endorphin (0.2-1.0 micrograms, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of delta-receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic activity. The inhibitory effect of a submaximal dose of DPDPE was also potentiated by beta-endorphin under these circumstances. These observations suggest that supra-spinal and spinal delta-opioid receptors are involved in the opioid-mediated inhibition of reflex bladder contractions in the rat. Moreover beta-endorphin may be important in regulating central delta-opioid receptors.

Induction of protective immunity against herpes simplex virus with DNA encoding the immediate early protein ICP 27.

Using a mouse zosteriform model that mimics human herpes simplex virus (HSV) infection in several aspects, the effectiveness of plasmid DNA encoding the immediate early protein ICP 27 was evaluated as a vaccine. Animals were immunized intramuscularly twice with DNA, then either challenged with virus or killed, and the nature of the immune response induced was measured. After intramuscular injection with plasmid DNA encoding ICP 27 (pc-ICP 27), solid protection was evident in 70-80% of mice and the lesions were delayed in the remaining animals. Immune splenocytes obtained from pc-ICP 27 immune mice showed HSV-specific lymphoproliferation, MHC-class I restricted cytotoxic T-lymphocyte (CTL) activity, and type 1 cytokine production. These animals also exhibited delayed-type hypersensitivity (DTH) reactions. Adoptive transfer studies conducted on syngeneic nude mice revealed that those recipients of immune CD4+ T cells, but not CD8+ T cells, were protected from subsequent HSV-1 (strain 17) challenge. Thus pc-ICP 27 DNA immunization protected the mice principally by CD4+ T cells and it is likely that these cells were Th-1 type because only type 1 cytokines were detectable after in vitro antigen stimulation. Our results indicate the potential value of DNA encoding nonstructural viral proteins as vaccines against HSV.

Neuropeptide Y depresses reflex urinary bladder contractions in rats and modifies central activity of opioid agonists.

The 36 amino acid peptide neuropeptide Y (NPY) has been found distributed in central structures associated with nociception and the actions of opioid analgesics. We therefore studied its central actions on reflex bladder contractions which we have shown to be inhibited by supraspinal and spinal opioid administrations in urethane anesthetized rats. Neuropeptide Y produced a dose related (0.5-2 micrograms per rat) inhibition of bladder contractions following intracerebroventricular (ICV) and spinal intrathecal (IT) administrations. These effects could not be antagonized by naloxone (2 micrograms, ICV or IT) or by ICI 174,864 [N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] (3 micrograms, ICV or IT). NPY (0.5-1 micrograms) reduced the ICV and IT effects of morphine but potentiated the action of the selective delta-receptor ligand [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE). The effect of the mu-selective opioid ligand [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) were unaffected as were the submaximal ICV and IT actions of noradrenaline. It was concluded that NPY-induced inhibition of bladder activity was not due to a direct opioid receptor interaction. However since NPY consistently changed the activity of opioids (morphine and DPLPE), this suggested a possible physiological role in the regulation of opioid receptors, central neural excitability and thereby visceral activity.

Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors.

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.

Centrally administered beta-endorphin produces prolonged changes in delta-opioid ligand activity in vivo.

Spontaneous reflex bladder contractions were recorded isometrically in urethane anesthetized rats. Bladder contractions were depressed by intracerebroventricular injections of the mu-opioid receptor agonist [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and the delta-agonist [2D-penicillamine,5D-penicillamine]enkephalin (DPDPE) respectively. The effect of DPDPE was selectively antagonized by ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib = alpha-aminoisobutyric acid). However following the administration of beta-endorphin the antagonistic action of ICI 174,864 could no longer be observed. In addition ICI 174,864 exhibited agonistic activity following beta-endorphin and the effects of DPDPE were prolonged in a dose related manner by beta-endorphin. These observations suggest that beta-endorphin may produce complex changes in central delta-opioid receptor activity.

Differentiation between rat brain and mouse vas deferens delta opioid receptors.

Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.

Prolonged in vivo antagonism of central mu- and delta-opioid receptor activity by beta-funal trexamine.

beta-Funaltrexamine (beta-FNA) was tested in the spinal cord and supraspinally against inhibition of reflex bladder contractions produced in the anesthetized rat by the opioid-receptor selective agonists [D-Ala2, MePhe4, Gly (ol)5]enkephalin (DAGO, mu-agonist) and [D-Pen2, D-Pen5] enkephalin (DPDPE, delta-agonist). All agents were microinjected either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). beta-FNA (1-8 micrograms) produced long-lasting antagonism of both DAGO and DPDPE. Complete recovery from its effects was only observed some 24-32 h later. Higher doses of beta-FNA (4 and 8 micrograms i.t.) produced short-lived agonistic activity though the selectivity of this was not determined. It was concluded that beta-FNA was a potent, long-lasting antagonist at central opioid receptors in vivo but was unselective for the mu and delta opioid receptor.

Proenkephalin A fragments exhibit spinal and supraspinal opioid activity in vivo.

The inhibition of reflex urinary bladder contractions, recorded isometrically in the urethane-anesthesized rat, was used as an index of central opioid activity. Inhibition of bladder activity has been shown to be mediated both spinally and supraspinally by mu and delta opioid receptors. Using this model a number of neuropeptide fragments of the proenkephalin A molecule (peptide F, peptide E, BAM 22P, BAM 12P, Met5-enkephalin-Arg6,Phe7,Leu8, Met5-enkephalin-Arg6,Phe7, Met-enkephalin, Leu-enkephalin) were tested for in vivo activity. Each of the fragments inhibited reflex bladder contractions when administered by bolus microinjection into a lateral ventricle (i.c.v.) or intrathecally into the spinal subarachnoid space (between L3 and L4 vertebra). The larger molecular weight peptides produced more prolonged inhibition of bladder activity than the smaller molecular weight ones, with the rank order of activity being similar at spinal and supraspinal sites: peptide F greater than or equal to peptide E = BAM 22P greater than BAM 12P greater than Met5-enkephalin-Arg6,Phe7,Leu8 = Met5-enkephalin-Arg6,Phe7 greater than or equal to Met-enkephalin = Leu-enkephalin. The relative receptor selectivity of each fragment was further determined using the opioid antagonists naloxone (mu receptors) and ICI 174,864 (N, N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) (delta receptors). The activity (Ke) of each antagonist against equieffective doses of the highly selective opioid receptor ligands [D-Ala2,Me-Phe4,Gly(ol)5]enkephalin (mu receptors) and [D-Pen2,D-Pen5]enkephalin (delta receptors) was compared with that against the proenkephalin A fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes.

Plasmid DNA encoding proteins represent a convenient novel approach to vaccination. We have investigated this "genetic immunization" approach as a means to protect against herpes simplex virus (HSV) infection using a mouse zosteriform model that mimics several aspects of reactivated HSV infection of humans. After i.m. immunization with plasmid DNA-encoding glycoprotein B (gB), (pc-gB), 80% of BALB/c mice were completely protected and lesions were delayed in the remaining animals. Upon pc-gB vaccination, the animals developed both gB- and HSV-specific IgG Ab response and the isotype examination revealed a predominance of IgG2a. These mice also have low levels (1/16) of HSV-neutralizing Abs. Immune splenocytes obtained from pc-gB-immunized mice, when restimulated in vitro with HSV resulted in production of type 1 cytokines. Evidence for CD(8+)-mediated cytotoxic T lymphocyte response was equivocal. Protection could be adoptively transferred to nude mice recipients by CD4+ T cells from pc-gB-immunized mice but not by CD8+ T cells. Our results demonstrate that genetic immunization is a potent means of inducing protection against HSV and that the mechanism of immunity responsible for clearing virus from cutaneous sites is principally by CD4+ T cells. It is likely that these cells are Th1 cells because type 1 cytokines were the major cytokines detected upon in vitro Ag stimulation.

Characterization of herpes simplex virus type-1 infection and herpetic stromal keratitis development in IFN-gamma knockout mice.

Herpetic stromal keratitis (HSK) has an immune-mediated pathogenesis that involves T cells that have a type 1 cytokine profile. IFN-gamma is suspected to be the type 1 cytokine involved in ocular pathology, and to test this notion more directly the pathogenesis of HSK was compared in mice deficient in the IFN-gamma gene (gamma knockout or gko) and control mice (wild-type littermates or BALB/c mice). The clinical course of HSK in gko mice closely paralleled that in control mice, yet virus persisted in the corneas of gko mice for an extended period of time, severe periocular skin lesions developed, and gko mice were far more susceptible to encephalitis. Delayed-type hypersensitivity to viral Ag was present, though diminished, in knockout mice, and serum herpes simplex virus-specific IgG isotypes indicated a Th2 shift. No differences existed in proliferative responses to in vitro Ag stimulation in gko vs control mice nor in T cell or proinflammatory cytokine mRNA levels in the corneas of infected mice. However, up-regulation of Th2 cytokine mRNA did occur in in vitro Ag-stimulated gko immune splenocytes. Histopathologic lesions were not statistically different between any of the groups of mice analyzed. These observations indicate that although IFN-gamma plays an important role in the clearance of virus from the eye, the pathogenesis of HSK lesions most likely involves additional cytokines, inflammatory mechanisms, or immune responses to nonviral Ags.

Opioid receptor selectivity of beta-endorphin in vitro and in vivo: mu, delta and epsilon receptors.

The relative contributions of mu and delta opioid receptors in the response to Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn - Ala-Ileu-Ileu-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (B-endorphin) were assessed as reductions in B-endorphin potency in the presence of mu and delta receptor selective antagonists in the guinea pig ileum, mouse vas deferens, rat vas deferens and in analgesic and gastrointestinal transit time tests in mice. We used the nonselective antagonist naloxone, the mu antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) and the delta antagonist N,N,diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864) in each test system at concentrations that effectively antagonized the respective mu and delta agonists, Tyr-Pro-N-MePhe-D-Pro-NH2 and Tyr-D-Pen-Gly-Phe-D-Pen. In the guinea pig ileum, the inhibitory effects of 1 microM B-endorphin were blocked by 1 microM CTP and 1 microM naloxone, but not by 1 microM ICI 174,864. In the mouse vas deferens, B-endorphin (0.2 microM) was antagonized by 1 microM CTP, 1 microM ICI 174,864 and by 1 microM naloxone. In contrast, in the rat vas deferens, B-endorphin (0.01-1 microM) produced potent inhibitory actions that were blocked by 1 microM naloxone, but not by 1 microM-CTP or by 1 microM ICI 174,864. The mu agonist, Tyr-Pro-N-MePhe-D-Pro-NH2 (0.1-10 microM), like B-endorphin, also had inhibitory actions in the rat vas deferens, but its effects were blocked by 1 microM CTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologic evaluation of a cyclic somatostatin analog with antagonist activity at mu opioid receptors in vitro.

The cyclic somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2(CTP) was evaluated for agonist and opioid antagonist actions and receptor selectivity in two bioassays: electrically stimulated guinea pig isolated ileum (GPI) and mouse isolated vas deferens (MVD). CTP (100, 300, 1000 nM) produced concentration-dependent antagonism of the mu agonist [Me-Phe3,D-Pro4]morphiceptin (PL017) in both the GPI and MVD. Schild analysis of the interactions between CTP and PL017 indicated competitive antagonism between these peptides (Schild slope GPI -0.97 +/- 0.16, Schild slope MVD -1.4 +/- 0.4), and also suggested that the mu receptors in the two tissues are not different (pA2 GPI 7.1 +/- 0.17, pA2 MVD 6.9 +/- 0.16). The effects of the delta selective agonist [D-Pen2,D-Pen5]enkephalin in the MVD were not antagonized by CTP. Likewise, in the GPI, CTP did not antagonize the kappa agonist (trans-3-4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenea cetamine (U50, 488H). In comparison, naloxone antagonized both PL017 and U50,488H in the GPI, as well as [D-Pen2,D-Pen5]enkephalin and PL017 in the MVD. In the MVD, CTP also exerted weak somatostatin-like actions (35% maximal inhibition) that could not be demonstrated in somatostatin-tolerant tissues. It also showed inhibitory actions at very high concentrations (3000 and 10,000 nM) that were blocked by both naloxone and the delta antagonist N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH (ICI 174,864). ICI 174,864 antagonized [D-Pen2,D-Pen5]enkephalin in the MVD, but did not affect PL017. These results indicate that CTP is a selective mu receptor antagonist in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological activities of cyclic enkephalin pseudopeptides containing thioamides as amide bond replacements.

Analogs of H-Tyr-cyclo(N epsilon-D-Lys-Gly-Phe-Leu) have been prepared which contain thioamides at the 3-4 position (monothio), 3-4 and 5-2 positions (dithio), and 2-3, 3-4, and 5-2 positions (trithio). These compounds have been tested for opioid activity in mu- and delta-receptor selective bio- and binding assays. As the number of sulfurs increased, the biological activities dropped on the guinea pig ileum and fluctuated modestly on the mouse vas deferens assay. Surprisingly, the compounds displayed increasing delta selectivity as the number of sulfurs increased. In the binding assay, the thioamide analogs tended to retain affinity toward the mu receptor. The mono- and dithio-analogs were more mu selective than the parent, while the trithio-analog was more delta selective. These results suggest that the subtle exchange of sulfur for oxygen can have a significant impact on receptor selectivity and affinity, and probably reflect the different conformation/structural requirements for binding vs. the biological transduction event.

Novel peptidic mu opioid antagonists: pharmacologic characterization in vitro and in vivo.

A series of six synthetic octapeptides, structurally related to somatostatin, demonstrate high affinity and selectivity for mu opioid receptors in radioligand binding assays. The compounds, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), D-tetrahydroisoquinoline carboxylic acid (D-Tic)-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (D-Tic-CTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (D-Tic-CTOP) and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (D-Tic-CTAP), were tested in vitro and in vivo for agonist and antagonist potency and selectivity. In vitro bioassays included the guinea pig ileum, mouse vas deferens and rabbit vas deferens. In vivo tests included hotplate antinociception and gastrointestinal transit inhibition, performed in mice. In vitro, all six derivatives were competitive, highly selective mu antagonists (pA2 values from 6.4-7.9). The compounds demonstrated varying degrees of intrinsic agonist activity especially in the mouse vas deferens, the least active being CTAP and D-Tic-CTAP, which showed no mu or kappa agonist actions, and delta activity only at very high (greater than 3 microM) concentrations. In vivo, none of these compounds showed antinociceptive actions when administered i.c.v. in mice. All were competitive mu antagonists in the hotplate antinociception test.(ABSTRACT TRUNCATED AT 250 WORDS)