Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 127 records · Page 7Linked to original sources

PubMed1996

Synthesis and biological properties of gamma-glutamyl-dermorphin, a prodrug.

The possibility of using the gamma-glutamyl-transpeptidase system for transformation of inactive propeptide, gamma-glutamyl-neuropeptides into active neuropeptides has been tested on dermorphin and its gamma-glutamyl analogue. Gamma-glutamyl-dermorphin 2 showed little affinity for opioid receptors. Nonetheless, systemic (intraperitoneal (i.p.), or intravenous (i.v.)) application of this compound induced significant antinociceptive effects, although ten to twenty-fold higher doses were required compared to the parent dermorphin 1. On the other hand, the analogue 2 showed high, antinociceptive activity when injected intrathecally (i.t.). When compared to dermorphin, 2 was one third as potent, but did show a significant prolonged duration of the effect. These results suggest that in the periphery, the peptidase metabolism which results in degradation of bioactivity, is offset by gamma-glutamyl-transpeptidase (GGTP) activity that liberates bioactive peptide 2. On the other hand, in the central nervous system, the activity of gamma-glutamyl-transpeptidase system seems to be more effective than other peptidase systems, resulting in formation of active peptide 2 in a significant amount. These data suggests that gamma-glutamyl analogues of neuropeptides can be considered as potential prodrugs, especially for synthetic analogues which themselves are resistant to peptidase action.

Analgesics, Opioid↗

PubMed1996

Evaluation of melanotan-II, a superpotent cyclic melanotropic peptide in a pilot phase-I clinical study.

A pilot phase I study was conducted with a cyclic heptapeptide analog of alpha-melanocyte stimulating hormone (alpha-MSH). The lactam-bridged molecule, called Melanotan-II (MT-II), has the structure Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10 alpha-MSH4-10-NH2 (MT-II) and has superpotent melanotropic activity in vitro. A single-blind, alternating day (saline or MT-II), placebo-controlled trial was conducted in 3 normal male volunteers at the starting dose of 0.01 mg/kg of MT-II. Subcutaneous injections of MT-II or saline were given daily (Monday-Friday) for 2 consecutive weeks. Two subjects were escalated by 0.005 mg/kg increments to 0.03 mg/kg and one to 0.025 mg/kg. The 0.03 mg/kg dose produced Grade II somnolence and fatigue in one of two subjects (WHO standards). Mild nausea, not requiring antiemetic treatment, was reported at most MT-II dose levels. A stretching and yawning complex appeared to correlate with the onset of spontaneous, penile erections which were intermittently experienced for 1-5 hours after MT-II dosing, depending on the MT-II dose. Two subjects had increased pigmentation in the face, upper body and buttock, as measured by quantitative reflectance and by visual perception 1 week after MT-II dosing ended. These results demonstrate that MT-II has tanning activity in humans given only 5 low doses every other day by subcutaneous injection. The recommended single MT-II dose for future Phase I studies is 0.025 mg/kg/day.

Adult↗

PubMed1996

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

Amino Acid Sequence↗

PubMed1996

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.

Analgesics, Opioid↗

PubMed1996

Passage of a delta-opioid receptor selective enkephalin, [D-penicillamine2,5] enkephalin, across the blood-brain and the blood-cerebrospinal fluid barriers.

[D-Penicillamine2,5] enkephalin (DPDPE) is an enzymatically stable, delta-opioid receptor-selective peptide, which produces analgesia when given intracerebroventricularly. However, because only modest analgesic effects were seen after subcutaneous administration of DPDPE, it has been inferred that it does not cross the blood-brain barrier well. In this present study, a vascular brain perfusion technique in anesthetized rats was used to measure directly whether [3H]DPDPE could cross the blood-brain and/or the blood-CSF barriers. The results indicated that the brain uptake of [3H]DPDPE was significantly greater than that of [14C]sucrose, a vascular marker (p < 0.01), and than that of [3H]DPDPE into the CSF (p < 0.01). Furthermore, HPLC analysis confirmed the integrity of the 3H to DPDPE and demonstrated that intact [3H]DPDPE entered the brain. Although 1 mM leucine-enkephalin failed to inhibit uptake of [3H]DPDPE, unlabeled DPDPE (100 microM) caused a significant inhibition of the brain uptake (p < 0.01) but not the CSF uptake of [3H]DPDPE. These data provide evidence that intact [3H]DPDPE enters the CNS of anesthetized rats by saturable and nonsaturable mechanisms. In addition, the saturable mechanism is likely to be found at the blood-brain barrier, with the blood-CSF barrier playing only a minor role in the brain uptake of this peptide.

Animals↗

PubMed1996

Lipid membrane permeability of modified c[D-Pen2, D-Pen5]enkephalin peptides.

Permeability coefficients of a series of analogues of a potent opioid peptide, c[D-Pen2, D-Pen5]enkephalin, were measured in a model membrane system. The analogues included hydrophobic amino acid substitutions on position 3. Liposomes of a mixed composition consisting of zwitterionic lipids and cholesterol served as the model membranes. The obtained permeability coefficients range between 0.38 x 10(-12) and 2.9 x 10(-12) cm/s. These data were correlated with the hydrophobicity scale of Nozaki and Tanford (J. Biol. Chem. 246, 1971, 2211-2217) (correlation coefficient = 0.9933) and with determinations of lipid order perturbation by differential scanning calorimetry (correlation coefficient = -0.9779). The reasonably good correlation obtained within the family of analogues substituted on position 3 (Gly, Ala, Leu, Phe) indicates that changes in permeabilities are primarily related to increases in the partition coefficient of the peptide. However, Phe residue added on the N-terminal end of the peptide (position 0) does not appear to follow the observed trend, showing stronger lipid perturbation and lower permeability compared to the Phe3 analog. This observation demonstrates that each class of peptide modifications requires a new basis of permeability analysis and predictions.

Calorimetry, Differential Scanning↗

PubMed1996

Melanotropic peptide receptors: membrane markers of human melanoma cells.

The objectives of this research were to determine whether melanotropin receptors are characteristic (constant) membrane markers of human melanoma cells. Methodologies were developed to visualize these receptors by fluorescence microscopy. Multiple copies (10-20) of both [Nle4,D-Phe7]alpha-MSH, a superpotent analog of alpha-melanocyte stimulating hormone (alpha-MSH), and a fluorophore, were conjugated to polyvinyl alcohol (PVA). Incubation in the presence of the multivalent macromolecular conjugate (FITC-PVA-MSH) resulted in binding of human epidermal melanocytes and keratinocytes and human melanoma cells (both melanotic and amelanotic) to the fluorescent conjugate. Binding of the conjugate to the cells exhibited a unique cluster pattern (capping) suggesting a receptor internalization related phenomenon. Most importantly, every cell of every melanoma cell line, melanotic or amelanotic, possessed receptors as visualized by fluorescence microscopy. Since the cells were not synchronized, some binding apparently took place during all phases of the cell cycle. Therefore, receptor expression appears not to be cell-cycle dependent. Specificity of binding of FITC-PVA-MSH was demonstrated by several studies. (i) Binding of the conjugate to melanoma cells could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; [Nle4,D-Phe7]alpha-MSH. (ii) The macromolecular conjugate lacking bound ligand (FITC-PVA) did not bind to the melanoma cells. (iii) Another peptide, a substance-P analog, attached to the substrate (FITC-PVA-SP) failed to bind to the cells. (iv) With the exception of keratinocytes, other cells of nonmelanocyte origin (e.g., fibroblasts, spleen, liver, kidney cells, and mammary cancer cells, lung cancer cells) did not bind to the conjugate. Thus, cell-specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes, keratinocytes, and melanoma cells. In several human melanoma cell lines these receptors appeared to be functional since [Nle4,D-Phe7]alpha-MSH stimulated tyrosinase activity. Fluorescent melanotropin conjugates might prove useful in determining whether all human melanoma (primary and metastatic) tumors possess such receptors. These receptors might then provide targets for melanotropic peptides for the identification, localization, and chemotherapy of melanoma.

Animals↗

PubMed1996

Melanocortin receptors: identification and characterization by melanotropic peptide agonists and antagonists.

Hormones are chemical messengers released from cells to act on and control the activity of other cells. Hormonal ligands initiate their actions by interacting with receptive substances (Langley, 1906) of the target cells. These receptors are proteins that are either integral components of the cell membrane or are localized cytoplasmically within cells. Ligand-receptor interaction results in either the stimulation or inhibition of cellular activity. Since most hormones bind rather specifically to receptors possessed by their target cells, labeling of hormonal ligands can be utilized to identify and localize cells within an animal. In this report we discuss what is presently known about melanocortin receptors (MCRs) as studied by the use of labeled melanotropic peptide ligands.

Amino Acid Sequence↗

PubMed1996

Human epidermal melanocyte and keratinocyte melanocortin receptors: visualization by melanotropic peptide conjugated microspheres (latex beads).

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle4,D-Phe7]alpha-MSH, a superpotent analog of alpha-melanocyte stimulating hormone (alpha-MSH), were conjugated to a macromolecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) microspheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.

Cells, Cultured↗

PubMed1996

Low level cyclic adenosine 3',5'-monophosphate accumulation analysis of [des-His1, des- Phe6, Glu9] glucagon-NH2 identifies glucagon antagonists from weak partial agonists/antagonists.

[des-His1, des-Phe6,Glu9]Glucagon-NH2 is a newly designed glucagon antagonist. This analog has a binding IC50 of 48 nM (compared to glucagon IC50 of 1.5 nM) and demonstrates pure antagonism in an adenylate cyclase assay. Although the number of glucagon antagonists has grown rapidly recently, closer examination suggested that many of these antagonists retained very low, almost imperceptible levels of cAMP accumulation that were sufficient to elicit an in vivo biological response. To investigate more carefully this secondary biological signal, we measured cAMP accumulation in a revised assay using isolated hepatocytes in the presence of the phosphodiesterase (PDE) inhibitor Rolipram. The PDE inhibitors Rolipram and isobutyl-1-methylxanthine (IBMX) increased the sensitivity of the cAMP accumulation assay from approximately 10-fold for the native hormone to 35-fold above basal levels. On the other hand, amrinone, another PDE inhibitor, did not affect the cAMP accumulation caused by glucagon. The use of PDE inhibitors indicated that three glucagon analogs that had previously been reported to have strong antagonist properties in classical adenylate cyclase assays were actually weak partial agonists in this new assay system. [N alpha-Trinitrophenyl-His1, homo-Arg12]glucagon, [des-amino-His1,D-Phe4,Tyr5, Arg12, Lys17,18,Glu21]glucagon, and [des-His1,Glu9]glucagon-NH2 demonstrated 233%, 21%, and 5.5% cAMP accumulation relative to the native hormone in the presence of 25 microM Rolipram. On the other hand, [des-His1,des-Phe6,Glu9]glucagon-NH2, a newly designed glucagon antagonist, did not activate adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, indicating that it was a pure antagonist of glucagon-induced adenylate cyclase activity and also the first one in this class. This compound and others were tested in a glycogen phosphorylase assay. As [des-His1,des- Phe6,Glu9]glucagon-NH2 did not activate phosphorylase activity, it was chosen as our candidate for in vivo testing in streptozotocin-induced diabetic rats. An initial dose of 0.75 mg/kg was found to cause the greatest lowering of blood glucose levels (to 63% of the initial levels in 15 min) when the bolus was followed by continuous infusion of 25 micrograms/kgxmin for 1 h.

1-Methyl-3-isobutylxanthine↗

PubMed1996

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.

Analgesics↗

PubMed1996

Enkephalin analog prodrugs: assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability.

To improve the blood-brain barrier penetration of the delta-opioid receptor peptides [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Pen2, L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2)to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3)to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe0-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe0-DPDPE from 3.9 min to > 450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe0-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.

Analgesics↗

PubMed1996

Enhancement of morphine antinociception by a CCKB antagonist in mice is mediated via opioid delta receptors.

This study investigated the possible involvement of opioid delta receptors in the modulation of morphine antinociceptive potency produced by L365,260 a CCKB antagonist. Intracerebroventricular (i.c.v.), intratheoal (i.th.) or subcutaneous (s.c.) L365,260 alone did not produce any antinociceptive actions in the mouse warm-water tail-nick test. Treatment with L365,260 by any of these routes produced a leftward shift of the corresponding morphine dose-effect curve that was blocked by pretreatment with a receptor selective dose of s.c. naltrindole, an opioid delta receptor antagonist. Pretreatment with i.c.v. antisera to [Leu5]enkephalin also blocked the leftward displacement of the i.c.v. morphine dose-effect curve resulting from L365,260 but did not directly alter the i.c.v. morphine dose-effect curve; antisera to [Met5]enkephalin did not alter the effects of morphine or the modulation of morphine antinociception produced by L365,260. Repeated pretreatment with L365,260 resulted in a progressive decrease in the magnitude of the morphine modulatory action (i.e., L365,260 "tolerance"). In these "L365,260-tolerant" mice, the dose-effect curve for i.c.v. [D-Ala2, Glu4]deltrophin (a selective delta agonist) was displaced to the right by approximately 8.2-fold. The i.c.v. administration of [Leu5]enkephalin produced a leftward displacement of the i.c.v. morphine dose-effect curve that diminished after repeated administration (i.e., [Leu5]enkephalin "tolerance"). In "[Leu5]enkephalin-tolerant" mice, L365,260 failed to produce the leftward shift of the morphine dose-effect curve seen in control animals. That is, two-way antinociceptive cross-tolerance was observed between an opioid delta agonist and a CCKB receptor antagonist. Intracerebroventricular thiorphan, a peptidase inhibitor, did not elicit antinociception directly. Co-administration of thiorphan with L365,260 elicited significant antinociception that was blocked by naltrindole or antisera to [Leu5]enkephalin; antisera to [Met5]enkephalin had no effect. Repeated administration of i.c.v. [D-Ala2, Glu4]deltorphin resulted in a progressively decreasing antinociceptive effect (i.e., [D-Ala2, Glu4]deltorphin "tolerance"). In "[D-Ala2, Glu4]deltorphin-tolerant" mice, the thiorphan/L365,260 antinociceptive effect was inhibited. Collectively, these data suggest that CCK interacts at the CCKB receptor to inhibit tonically the release and/or availability of an endogenous substance acting at opioid delta receptors. The subsequent enhancement of morphine antinociceptive potency may reflect the well-known modulation of morphine antinociception produced by opioid delta receptor agonists. In this case, the latter may be [Leu5]enkephalin or a [Leu5]enkephalin-like substance.

Animals↗

PubMed1996

Blood-to-central nervous system entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives.

Biphalin (Tyr-D-Ala-Gly-Phe-NH)2 is a unique opioid peptide analog that contains two active enkephalin pharmacophores and is more potent than morphine and etorphine in eliciting analgesia after intrathecal administration. After systemic administration, only a small amount was detected in the brain, but analgesia was observed. Because halogenation of enkephalin analogs has been shown to increase the brain uptake after systemic administration, our research group synthesized both p-[Cl-Phe4,4']biphalin and p-[F-Phe4,4']biphalin. The aim of the present study was to characterize and compare the blood-to-central nervous system (CNS) pharmacokinetics and biological stability of biphalin and related halogenated analogs. The initial screening used an in vitro blood-brain barrier model and identified p-[Cl-Phe4,4')biphalin as the enkephalin analog with the best potential for greater CNS entry. The CNS uptake and stability of biphalin and p-[Cl-Phe4,4']biphalin was examined further using an in situ brain perfusion technique coupled to high-performance liquid chromatography analysis. Both biphalin and its chlorohalogenated analog, were found to significantly enter the CNS through both the blood-brain and blood-cerebrospinal fluid barriers. Chlorohalogenation of biphalin was shown to both improve CNS entry, most likely through an enhancement in lipophilicity, and increase biological stability. This study suggests that incorporation of chlorohalogens at the p-Phe4,4' position is a promising structural modification in the development of biphalin as a successful opioid drug for the clinic.

Analgesics↗

PubMed1996

Human delta opioid receptor: functional studies on stably transfected Chinese hamster ovary cells after acute and chronic treatment with the selective nonpeptidic agonist SNC-80.

The SNC-80 series of nonpeptidic agonists for the delta-opioid receptor are being developed as potential analgesic drugs. It is important to understand their acute and chronic effects at human delta-opioid receptors. Thus, we measured the ability of SNC-80 and [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin to inhibit forskolin-stimulated adenylyl cyclase activity in recombinant Chinese hamster ovary cells stably expressing the cloned human delta-opioid receptor. The calculated EC50 values for [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin and SNC-80 were 0.6 +/- 0.1 nM and 6.3 +/- 0.1 nM, respectively. Pretreatment of these cells with SNC-80 (100 nM) for 24 hr produced 1) a time-dependent reduction of delta receptor density, as measured by radioligand binding studies with [3H]naltrindole; 2) a shift in the EC50 value of SNC-80 from 7.7 +/- 4.2 nM to 44.1 +/- 12 nM, as measured by the cyclic AMP assay; 3) a reduction in the maximum inhibition of adenylyl cyclase activity from 86% to 48%; 4) a marked increase in the forskolin stimulation of basal cyclic AMP accumulation by nearly 100% (from 442 pmol/mg of protein to 824 pmol/mg of protein); and 5) a 5-fold increase in forskolin-stimulated cyclic AMP accumulation after addition of naltrindole. These studies showed that SNC-80 produced desensitization and down-regulation of human delta-opioid receptors in recombinant Chinese hamster ovary cells after chronic treatment and that this effect was associated with an increase in adenylyl cyclase activity.

Animals↗

PubMed1996

The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists.

In the present study, we replaced the third extracellular loop of the human delta-opioid receptor with that of the human mu-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the delta-selective peptide agonists cyclo[D-Pen2,4'Cl-Phe4,D-Pen5]enkephalin and deltorphin II and the delta-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the mu-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the mu-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.

Amino Acid Sequence↗

PubMed1996

Three-dimensional molecular models of the hMC1R melanocortin receptor: complexes with melanotropin peptide agonists.

Three-dimensional molecular models of the human melanocortin receptor (hMC1R) have been developed based upon the electron cryo-microscopic structure of bacteriorhodopsin and the electron density footprint of bovine rhodopsin. alpha-Melanocyte-stimulating hormone, Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (alpha-MSH, alpha-melanotropin), and the superpotent, prolonged acting agonists, Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-MSH) and Ac-Nle4-c[Asp5-His6-DPhe7-Arg8-Trp9-Lys10]-NH2 (MTII), have been modeled into the proposed binding sites with specific ligand-receptor interactions identified. The melanotropin sidechain pharmacophores, DPhe7 and Trp9, are proposed to interact with a hydrophobic network of receptor aromatic residues in transmembrane regions 4, 5, 6, and 7. In addition, a hydrophilic network involving the ligand Arg8 and polar receptor residues located in transmembrane regions 2 and 3 were identified. Biological studies on alpha-MSH, NDP-MSH, MTII, and related peptides have been correlated with the proposed hMC1R model in terms of agonism, affinity, and prolongation. Finally, limited MC1R mutagenesis studies comparing alpha-MSH and NDP-MSH are interpreted within the context of the proposed hMC1R models.

Amino Acid Sequence↗

PubMed1995

Topographical modification of melanotropin peptide analogues with beta-methyltryptophan isomers at position 9 leads to differential potencies and prolonged biological activities.

We have introduced topographical constraints at the 9 position of a superpotent cyclic alpha-melanotropin analogue, Ac-Nle4-Asp5-His6-DPhe7-Arg8-Trp9-Lys10-NH2, by incorporating a methyl group at the beta-carbon of Trp9. These studies were performed on the Trp side chain pharmacophore to identify the bioactive topography of the indole moiety with melanocortin MC1 receptors. The four beta-MeTrp9 isomers, in addition to the stereochemical controls L- and DTrp9, were used to probe differential receptor molecular recognition of the tryptophan moiety in two bioassay systems. Approximately a 460-fold difference in potency was observed between the diastereoisomeric peptides in the frog skin bioassay, with only 33- and 10-fold efficacy differences observed in binding and intracellular cAMP accumulation, respectively, on the human melanocortin receptor, hMC1R. The relative orders of potencies in the frog skin bioassay were 2R,3S > 2S,3S = 2R,3R >> 2S,3R and for the hMC1R were 2S,3S > 2R,3R > 2R,3S >> 2S,3R. Of particular interest is the ability of these topographically constrained ligands to differentially affect prolonged biological activity. The 2R,3R diastereoisomeric peptide possessed superprolonged activity, whereas the 2S,3S peptide lacked any residual activity in the frog skin bioassay. However, on the melanocortin receptor, the 2S,3S diastereoisomeric peptide maintained slow dissociation rates (t1/2 = 7 h), while the other diastereoisomeric peptides possessed dissociation t1/2 rates of ca. 2 h. These data strongly implicate ligand-receptor interactions and kinetics as contributing to the observed prolonged biological activities and clearly illustrate topographical recognition differences between these two peripheral MC1 receptors involved in skin pigmentation. This study also demonstrates that topographical modifications of pharmacophore side chain residues, in addition to identifying preferential side chain orientation, can be a useful strategy for the design of peptides to increase the duration of biological activity, relative to the native ligand.

Amino Acid Sequence↗