An improved method for rapid and efficient radioiodination of iodine-123-IQNB.

UNLABELLED: The SPECT radioligand, 3-quinuclidinyl-4-[123I]iodobenzilate ([123I]IQNB), binds to muscarinic receptors and has generated interest as a potential agent for clinical SPECT. Unfortunately, cumbersome and inefficient radioiodination procedures have limited the practicality of [123I]IQNB SPECT imaging. METHODS: We report a rapid (5 min) and simple radioiodination procedure for preparing [123I]IQNB from a tri-n-butylstannyl precursor in a no-carrier-added reaction that yields high specific activity with radiochemical yield exceeding 60%. The radiochemical purity of the final product exceeds 95%. RESULTS: We have used this procedure to radioiodinate the four stereoisomers of [123I]IQNB. The procedure is highly reliable and reproducible. SPECT studies on a healthy human volunteer at 1, 2, 6 and 24 hr after injection of each of the four stereoisomers reveal expected differences in the kinetic and binding characteristics of the four stereoisomers. (R,S)-[123I]IQNB appears to be the SPECT agent of choice. CONCLUSION: Radioiodination of [123I]IQNB from our tri-n-butylstannyl precursor is simpler, more efficient and less expensive than previous techniques. The potential exists for a "kit" which would be practical in a typical clinical setting.

The in vitro pharmacological characterization of naloxone benzoylhydrazone.

On the basis of its in vivo activity and binding affinity, naloxone benzoylhydrazone has been characterized as a kappa 3-opioid receptor agonist and a mu-opioid receptor antagonist. This paper continues its pharmacological characterization with the help of isolated tissue preparations. Naloxone benzoylhydrazone was found to have partial agonist activity in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. As an antagonist, naloxone benzoylhydrazone is similar to naloxone, with pA2 values of 8.8, 7.8, and 7.8 for mu-, delta-, and kappa 1-opioid receptors, respectively. Its agonist activity in the guinea pig ileum preparation was not influenced by beta-funaltrexamine treatment but was reversed by the selective kappa-opioid receptor antagonist nor-binaltorphimine and by the irreversible kappa 1-opioid receptor blocker UPHIT (1S,2S)-trans-2-isothiocyanato-4,5-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide. The presence of kappa 3-opioid receptors could not be demonstrated by [3H]naloxone benzoylhydrazone binding in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. From these studies it is concluded that the partial agonist activity of naloxone benzoylhydrazone in this bioassay is probably due to the activation of the kappa 1-opioid receptors.

Opioid pharmacology of the antinociceptive effects of loperamide in mice.

Loperamide (0.1-3.2mg/kg i.p.) produced dose-dependent and complete suppression of writhing in the acetic acid-induced writhing assay in mice. Naltrexone (NTX; 0.1-10.0mg/kg s.c.) and its N-methylated derivative quaternary naltrexone (QNTX; 1.0 and 10.0mg/kg s.c.) were roughly equipotent in antagonizing the antinociceptive effects of loperamide. In contrast, NTX was approximately 100-fold more potent than QNTX in antagonizing the antinociceptive effects of the classical mu agonist morphine. Furthermore, the antinociceptive effects of loperamide were not antagonized by central administration of the selective mu antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP; 300ng i.c.v.), or by systemic administration of either the kappa selective antagonist nor-binaltorphimine (nor-BNI; 32.0mg/kg s.c.), or the delta antagonist naltrindole (NTI; 10.0mg/kg s.c.). These doses of CTAP, nor-BNI and NTI were effective antagonists of morphine, the kappa agonist U69,593 and the delta agonist BW 373U86 [(+/-)-4-((R*)-a-((2S*5R*)-4-allyl-2,5-dimethyl-1- piperazinal)-3-hydroxybenzyl)-N, N-diethylbenzamide dihydrochloride], respectively. These results indicate that the antinociceptive effects of loperamide in mice are mediated, at least in part, by opioid receptors; however, these receptors are distinct from the opioid receptors mediating the effects of morphine, U69,593 and BW 373U86. These results are consistent with the hypothesis that loperamide produces its antinociceptive effects by acting, at least in part, at peripheral opioid receptors.

Characterization of a (+)-azidophenazocine-sensitive sigma receptor on splenic lymphocytes.

A study was undertaken to structurally define and functionally assess sigma receptors on splenocytes using the highly selective sigma ligand (+)-azidophenazocine. Radioreceptor assays under reduced lighting show (+)-azidophenazocine can effectively block the binding of sigma ligands [3H]haloperidol (IC50 = 30 nM, Ki = 19.0 nM) and [3H](+)-pentazocine (IC50 = 40 nM, Ki = 350 nM), but not the dopamine (D2) ligand [3H]spiperone (IC50 > 5 microM) to splenic lymphocytes. [3H](+)-1-Propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-PPP) sites (Kd = 40.8 nM, Bmax = 2.32 pmol/mg) were also present on these lymphocytes. Additional studies using [3H](+)-azidophenazocine indicated the presence of saturable sites (Kd = 29.7 nM, Bmax = 760 fmol/mg) on splenic lymphocytes. There are no significant differences in affinity between sites found on T-enriched (Kd = 59 +/- 47 nM) and B-enriched lymphocytes (Kd = 23 +/- 5 nM). Photoaffinity labeling studies of splenocyte membranes with [3H](+)-azidophenazocine revealed a protein migrating at an apparent m.w. of 57 kDa under reducing and nonreducing conditions on SDS-PAGE. The labeling was specific because pretreatment with unlabeled haloperidol, (+)-PPP, 1,3 di(2-tolyl)guanidine, (+)-pentazocine, and (+)-azidophenazocine before cross-linking competed away > 75% of the radioactivity associated with the protein, whereas (-)-pentazocine and naloxone were significantly less effective. This data together with the observation that both (+)-azidophenazocine or haloperidol inhibit Con A-induced production of IFN by splenocytes, indicates that lymphocytes possess a biologically relevant sigma receptor.

Opioid thermal antinociception in rhesus monkeys: receptor mechanisms and temperature dependency.

The antinociceptive effects of the opioid agonists etonitazene and alfentanil, as well as the agonist/antagonists nalbuphine, [(1)-beta-2'-hydroxy-2,9-dimethyl-5-phenyl-6,7-benzomorphan (GPA 1657)] and profadol were studied in the warm water (48 degrees and 55 degrees C) tail-withdrawal assay in rhesus monkeys. Etonitazene and alfentanil produced dose-dependent increases in tail-withdrawal latency up to the maximum possible latency of 20 sec in 48 degrees and 55 degrees C water. Nalbuphine, GPA 1657 and profadol produced the maximum possible effect only at 48 degrees C, and were ineffective at 55 degrees C. The opioid antagonist quadazocine produced a dose-dependent antagonism of all agonists except profadol. In a Schild plot analysis, apparent pA2 values for quadazocine with alfentanil, etonitazene and nalbuphine were homogeneous (7.3-7.7 mol/kg), suggesting their effects were probably mediated by mu opioid receptors. The apparent pA2 value for GPA 1657 was significantly lower (6.2 mol/kg), suggesting GPA 1657 may have produced antinociception by a non mu receptor-mediated mechanism. The selective delta antagonist naltrindole (0.32-1.0 mg/kg) antagonized the antinociceptive effect of GPA 1657. The kappa-selective antagonist nor-binaltorphimine (nor-BNI, 3.2 mg/kg) caused a small rightward shift in the GPA 1657 dose-effect curve. Nalbuphine, GPA 1657 or profadol produced a rightward shift in the alfentanil dose-effect curve in 55 degrees C water, consistent with possible low-efficacy mu agonist effects of these compounds. These studies suggest agonists may be differentiated based on antinociceptive effectiveness, receptor selectivity and intrinsic efficacy in the rhesus monkey tail-withdrawal procedure.

Immunoregulatory properties of (+)-pentazocine and sigma ligands.

(+)-Pentazocine, phencyclidine, and other sigma ligands including 1,3-di-(o)-tolylguanidine (DTG), (+)-1-propyl-3-(3-hydroxyphenyl) piperidine [(+)-PPP] and haloperidol were investigated for their potential immunoregulatory properties. High concentrations (10(-5) M) of DTG and haloperidol were found to suppress in vitro murine splenocyte natural killer activity while equivalent concentrations of (+)-pentazocine, (-)-pentazocine and (+)-PPP were without effect. In a reciprocal fashion, lower doses (10(-9) M) of DTG enhanced natural killer activity. Sigma ligands were also found to affect in vitro polyclonal immunoglobulin production following mitogen stimulation. Specifically, high concentrations (10(-6) M) of haloperidol significantly (P < 0.001) suppressed pokeweed mitogen (PWM)-stimulated IgG and IgM production, yet enhanced lipopolysaccharide (LPS)-stimulated IgM production by murine splenocytes. Lower concentrations (10(-8) to 10(-10) M) enhanced (two- to fourfold) PWM-induced IgM production and LPS-stimulated IgG and IgM production. At high concentrations (10(-6)), (+)-pentazocine suppressed (P < 0.01) LPS-induced polyclonal IgG and IgM but enhanced (P < 0.01) PWM-induced IgM production. Both DTG and (-)-pentazocine (10(-8) to 10(-10) M) significantly augmented (two- to threefold) LPS-stimulated murine splenocyte production of polyclonal IgM. Intracellularly, (-)-pentazocine (10(-9) M), haloperidol (10(-7) M), DTG (10(-7) M) and (+)-PPP (10(-5) to 10(-9) M) enhanced forskolin (10(-6) M)-induced cAMP production in splenic lymphocytes while (+)-pentazocine was without effect. Collectively, the data suggest functional and biologically relevant sigma receptors on cells of the immune system.

Effects of drugs that bind to PCP and sigma receptors on punished responding.

Several arylcyclohexylamines and opioid benzomorphans that bind to phencyclidine (PCP) receptors were studied for their effects on punished and unpunished responding maintained under fixed-interval schedules of food presentation. All of these drugs increased both punished and unpunished responding, although higher doses decreased responding. The order of potency for increasing punished responding was MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzoa(a,d)-cyclohepten-5,1 0-imine] greater than [1-[1-(2-thienyl)cyclohexyl]piperidine] = PCP greater than (+)-N-allylnormetazocine = (-)-N-allynormetazocine. There was a high correlation (0.95) between the relative potency of these drugs in increasing punished responding and their relative affinity for PCP receptors. Because some of these drugs also bind to sigma receptors, drugs with a high affinity for sigma receptors, such as haloperidol, BD 737 [1S,2R-(-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N- methyl-2-(1-pyrrolidinyl) cyclohexylamine] and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, were also studied for their effects on punished and unpunished responding. These drugs produced only rate-decreasing effects. The correlation between the relative potency of drugs in increasing punished responding and their relative affinity for sigma receptors was low (-0.19). These data suggest that the PCP receptor is involved in some drug-induced increases in punished responding.

Enantioselective kappa opioid binding sites on the macrophage cell line, P388d1.

A kappa (kappa) opioid binding site has been characterized on the macrophage cell line, P388d1, using the kappa selective affinity ligand, [3H] (1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl) cyclohexyl] benzeneacetamide (-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((-)-U-50,488) blocks [3H](5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl]benzeneacetamide (U-69,593) binding to P388d1 cells with an IC50 = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((+)U-50,488) blocks [3H]U-69,593 binding to P388d1 cells with an IC50 = 7000 nM.

Corticotropin-releasing hormone augments natural killer cell activity through a naloxone-sensitive pathway.

Overnight treatment of murine leukocytes with corticotropin-releasing hormone (CRH) and arginine vasopressin enhances natural killer cell activity. Moreover, the opioid receptor antagonist, naloxone, as well as the delta-class opioid receptor antagonist, naltrindole, can block this effect. The responsivity of murine leukocytes to CRH is both dose- and time-dependent. The effector cells are both MAC-1 and Thy-1.2 antigen-positive. Whereas beta-endorphin is also shown to enhance natural killer cell activity in a naloxone-reversible manner, adrenocorticotropic hormone (ACTH) has a negligible effect. Macrophage depletion prior to incubation with CRH blocks the CRH-induced natural killer cell augmentation. These results suggest hypothalamic-releasing hormones such as CRH may have a biologically relevant role in the modulation of immune cells either directly or indirectly through the induction of neuropeptide hormones known to have immunomodulatory capabilities.

Anti-opioid receptor antibody recognition of a binding site on brain and leukocyte opioid receptors.

Opioid receptors reportedly exist on neuronal tissue of central and peripheral origin as well as on cells of the immune system. Previously, an opioid receptor has been purified from the neuroblastoma x glioma hybrid cell line, NG108-15 cells. In an effort to compare these results with opioid receptors isolated from primary neuronal tissue, we employed a methodology based on the molecular recognition theory to develop a monoclonal antibody which was used to isolate and biochemically characterize murine brain opioid receptors. We herein report the purification of an opioid receptor from mouse brain with a molecular weight of 65,000 daltons (range was 62-70 kD under reducing conditions) using a monoclonal antibody to an (the) opioid receptor. In situ labeling experiments with the delta-class selective opioid receptor affinity ligand, cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT) of brain membrane confirmed these observations. Moreover, SUPERFIT, when coupled to the binding site, could block the recognition of the receptor by the monoclonal antibody. However, the selective, mu-class opioid receptor affinity reagent, 2-(p-ethoxybenzyl)-1-N,N-diethylaminoethyl-5-isothiocyanatobenz imidazole was ineffective at masking the binding site from recognition by the monoclonal antibody. Likewise, opioid-like receptors were purified from murine leukocytes which migrated at a molecular weight of 58,000 daltons under nonreducing conditions and 70,000 daltons under reducing conditions. In addition, immunoaffinity-purified receptor is shown to specifically bind the delta-class-selective opioid ligand, cis-(+)-3-methylfentanylisothiocyanate as well as the endogenous opioid peptides, beta-endorphin and [Met]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of opioids on immunoglobulin production by lymphocytes isolated from Peyer's patches and spleen.

The mucosal immune system plays an important role in blocking the penetration of invasive organisms into various mucosal surfaces. Evidence now suggests neuroendocrine peptide hormones have immunomodulatory properties, including the ability to alter mucosal immunity. The potential for opioid compounds and corticotropic hormone (ACTH) to modulate mucosal immune function was investigated. We have found beta-endorphin, ACTH, and naltrindole (delta-class opioid receptor antagonist) to significantly suppress concanavalin A-stimulated Peyer's patch lymphocyte immunoglobulin production of IgA, IgG, and IgM isotypes. Oxymorphindole, a delta class opioid receptor agonist, significantly decreased IgM but not IgA or IgG production by the mitogen-stimulated Peyer's patch lymphocytes. Both oxymorphindole and naltrindole modestly reduced interleukin-2 receptor expression of concanavalin A- (Con A)-stimulated splenic and Peyer's patch lymphocytes. Neither compound appreciably affected immunoglobulin production by lipopolysaccharide-stimulated Peyer's patch lymphocytes. Collectively, these results indicate stress-related peptides such as ACTH and opioids may be involved in the regulation of immunoglobulin synthesis by Peyer's patch lymphocytes.

Opioid receptors on cells of the immune system: evidence for delta- and kappa-classes.

Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d1. A delta-class opioid receptor was found with an Mr of 58,000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T cell-and B cell-enriched populations. Using the central (brain) kappa-selective agonist, U-69,593, it was also determined that P388d1 cells possess kappa-like opioid receptors. Scatchard analysis of the binding of [3H]U-69,593 revealed a single population of sites with a dissociation constant of 17 +/- 3 (S.E.M.) nmol/l and a total number of binding sites of 53.8 +/- 1.0 (S.E.M.) fmol/10(6) cells. Moreover, the racemic kappa-selective agonist U-50,488H was able to displace 50% of [3H]U-69,593 binding at 8.0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (delta-selective) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (mu-selective) were ineffective displacers of [3H]U-69,593 except at high concentrations.

Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation.

Exposure to sunlight initiates the formation of vitamin D3 in skin as the UV B radiation in the solar spectrum causes the photoconversion of 7-dehydrocholesterol to previtamin D3. A heat-induced isomerization then converts previtamin D3 to vitamin D3 over a period of days. A number of irradiation products of vitamin D3 are known to form upon irradiation with high intensity UV radiation, but the effect of subsequent exposures to sunlight on the vitamin D3 formed in skin is not known. To investigate this phenomenon, human skin containing vitamin D3 was exposed to sunlight in Boston. A model system of [3H]vitamin D3 in methanol was also used to study the effects of sunlight on vitamin D3 throughout the year. Vitamin D3 proved to be exquisitely sensitive to sunlight, and once formed in the skin, exposure to sunlight resulted in its rapid photodegradation to a variety of photoproducts, including 5,6-transvitamin D3, suprasterol I, and suprasterol II.