Mitsuhiro Wakimasu

Evidence that dynorphin-(1–13) acts as an agonist on opioid κ-receptors

Abstract The study concerned the opioid-receptor subtype on which dynorphin-(1–13) acts in in vitro isolated preparations. The potency of dynorphin-(1–13) relative to that of ethylketocyclazocine (Mr 2266), a representative κ-receptor agonist, in inhibiting the electrically evoked contractions of the guinea-pig ileum was found to be similar to that found with either mouse vas deferens or rabbit ileum. Moreover, Mr 2266 was found to be several-fold more effective than naloxone to antagonize the agonist actions of both κ-receptor agonists such as ethylketocyclazocine, ketocyclazocine and bremazocine, and dynorphin-(1–13) either in the guinea-pig ileum, mouse vas deferens, or in rabbit ileum. Additionally, dynorphin-(1–13) was found to have a significant inhibitory action on the rabbit vas deferens which had been shown to contain κ-receptors exclusively. The data indicate that dynorphin-(1–13) acts as an endogenous agonist on κ-receptors.

Pharmacology of a non‐selective ETA and ETB receptor antagonist, TAK‐044 and the inhibition of myocardial infarct size in rats

1 The aims of the present study were to characterize the pharmacological profile of a new endothelin (ET) receptor antagonist, TAK‐044 and to consider whether it limits the extension of myocardial infarct size in rats. 2 Binding of [125I]‐ET‐1 to ET receptors on rabbit ventricular and cerebellar membrane fractions was inhibited by TAK‐044 with IC50 values of 3.8 nm and 130 nm, respectively. 3 It inhibited ET‐1, ET‐2 and ET‐3‐induced vasoconstriction of porcine isolated coronary arteries in a competitive (ET‐1, ET‐2) and a non‐competitive (ET‐3) manner. 4 In the rat in vivo, the ET‐1‐induced blood pressure changes including transient hypotension followed by sustained hypertension, were inhibited by TAK‐044 (0.1–10 mg kg−1, i.v.) in a dose‐dependent manner. 5 Acute myocardial infarction induced by 1 h coronary occlusion followed by 24 h reperfusion in rats caused an infarct size of 60 ± 2% (n = 12) of the area‐at‐risk by weight. 6 Intravenous injection of TAK‐044 10 min before coronary occlusion reduced the infarct size in a dose‐dependent manner: 32% and 54% reductions at 1 and 3 mg kg−1, respectively. 7 TAK‐044 administered 10 min before or 1 h after reperfusion (1 mg kg−1, i.v.) showed similar inhibitory effects: 34% and 23% reductions, respectively. 8 We conclude that TAK‐044 is an ETA/ETB receptor antagonist which shows strong inhibitory effects on the extension of myocardial infarct size after coronary artery occlusion‐reperfusion in rats.

Effects of a new endothelin antagonist, TAK-044, on post-ischemic acute renal failure in rats

Protective effects of a new endothelin (ET) receptor antagonist, TAK-044, were studied in a model of acute renal failure (ARF) in rats. ARF was induced by clamping the left renal pedicle for 45 minutes with contralateral nephrectomy and subsequent reperfusion of the left kidney. Plasma creatinine concentration (Pcr) increased to 2.28 mg/dl 24 hours after reperfusion of the ischemic kidney. Intravenous administration of TAK-044 (1-10mg/kg) prior to renal occlusion dose-dependently but partially attenuated the increase in Pcr and the morphological damages of the kidney. ET-1 and ET-3 increased perfusion pressure in isolated kidney preparations with similar potency, indicating that the renal vasoconstriction evoked by these ET isomers is mainly via ETB receptors, and TAK-044 (10nM) shifted the ET-1 dose-response curve to the right by a factor about 10. In a rat renal membrane fraction, ET-1 showed competitive inhibition of specific [125I]ET-1 binding with an IC50 value of 0.34nM and a Hill slope of 1.10. ET-3 did so with a higher IC50 value (3.3nM) and a lower Hill slope (0.56), suggesting that rat kidney contains both ETA and another receptor subtype, probably ETB. TAK-044 inhibited ET-1 binding with an IC50 value of 6.6nM and a Hill slope of 0.41. Plasma concentrations of immunoreactive TAK-044 were maintained over 7nM for 8 hours following i.v. injection of 10mg/kg TAK-044. These results suggest that endogenous ET is involved in the pathogenesis of post-ischemic ARF, at least, in part and that TAK-044 provided protective effects against ARF by blocking ET receptors, possibly both ETA and ETB receptors in renal vasculature and parenchymal cells.

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.

Monoclonal antibodies to human interferon-γ. I. Antibodies to a synthetic carboxyl-terminal peptide

Two types of hybridomas secreting monoclonal antibodies (MAB) against human interferon-gamma (HuIFN-gamma) were obtained by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with a conjugate of a synthetic carboxyl-terminal peptide (residues 131-146) of HuIFN-gamma and bovine thyroglobulin. One of the antibodies bound to recombinant HuIFN-gamma produced in E. coli as well as to natural HuIFN-gamma, while the others bound only to recombinant HuIFN-gamma. These 2 types of MAB did not neutralize the anti-viral activity of HuIFN-gamma. They were useful for effectively purifying recombinant HuIFN-gamma and quantitatively determining it by an enzyme-linked immunosorbent assay.

L-aspartyl-aminomalonic acid diester

A novel dipeptide, L-aspartyl-aminomalonic acid alkyl fenchyl diester, and its physiologically acceptable salts which are useful as a sweetener, and production thereof and sweetening compositions containing the dipeptide ester or its salt.

Identification of Bone Morphogenetic Protein-2 in Early Xenopus laevis Embryos

Polyclonal antibodies capable of reacting with amphibian bone morphogenetic protein (BMP-2 and -4) were raised in rabbits by immunization with a synthetic 21 amino acid peptide which corresponds to a sequence residing in the mature protein of Xenopus BMP-2 (xBMP-2). The antibodies recognized an embryonic BMP as well as mammalian and bacteria expressed recombinant xBMPs. The antibodies detected, under reducing conditions, a 30 kDa protein in the extract of oocytes and embryos during early development. Interestingly, acidification of the extract from each developmental stage yielded a protein band of smaller molecular weight of 18 kDa, which is similar in size to reduced form of mature BMPs purified from mammalian species. Two-dimensional electrophoresis employed to examine the molecular weight of unreduced forms using the antibody, revealed that both molecular forms are monomeric in the embryos. The result suggests that at least BMP-2 mRNA previously detected in early embryos, is translated into peptide but the dimerization may be incomplete or strictly limited in these embryos.

Large-scale synthesis of new cyclazines, 5-thia-1,8b-diazaacenaphthylene-3-carboxylic acid derivatives having the peripheral 12π-electron ring system

Abstract The 5-thia-1,8b-diazaacenaphthylenes ( 2 and its ester, 8 ) are new cyclazines, in which a paramagnetic ring is present in the peripheral 12π-electron ring system. Three convenient methods of preparing 8 have been developed. One involved thioglycolation of a new compound, 5-fluoroimidazo[1,2- a ]pyridine ( 6b ), followed by the Duff reaction gave 8 in 64% yield without chromatographic purification.

A Practical Synthesis of the Chronic Renal Disease Agent, 4,5-Dihydro-3H-1,4,8b-triazaacenaphthylen-3-one Derivatives, Using Regioselective Chlorination of Ethyl 5-methylimidazo[1,2-a]pyridine-3-carboxylate with N-Chlorosuccinimide

Abstract A convenient synthesis of the chronic renal disease agent, trifluoro-N-[4-(3-oxo-3,5-dihydro-4H-1,4,8b-triazaacenaphthylen-4-yl)butyl]methanesulfonamide (1a), for large scale has been developed via ethyl 5-(chloromethyl)imidazo[1,2-a]pyridine-3-carboxylate (3), which was given by the regioselective chlorination of ethyl 5-methylimidazo[1,2-a]pyridine-3-carboxylate (6) with N-chlorosuccinimide (NCS) using AcOEt as a solvent in 83% yield. The condensation of 3 and primary amines gave 4,5-dihydro-3H-1,4,8b-triazaacenaphthylen-3-one derivatives (1) in good yields. The present synthesis of 1a was accomplished in five steps from 2-amino-6-methylpyridine (4) without requiring a chromatographic method.

Reinforcing effects of the enkephalin analogs, EK-209 and EK-399, in rats

The reinforcing effects of two enkephalin analogs, Tyr-D-Ala-Gly-MePhe-NHNHCOCH2CH3.AcOH (EK-209) and Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3.AcOH (EK-399), were assessed by means of a self-administration technique with rats. The animals were trained to self-administer an intravenous dose of morphine by a lever-press response. A test drug was substituted for morphine after the rats had initiated and maintained its self-administration. When codeine, fentanyl, pentazocine or EK-209 was available, most of the rats increased the number of self-administrations as the unit dose of these drugs was decreased. When levallorphan or EK-399 was available, most of the rats did not increase responding; only one of 4 rats slightly increased the number of self-administrations as the unit dose of EK-399 was decreased. These results indicate that EK-209, like codeine, fentanyl, and pentazocine, possesses a reinforcing effect, whereas EK-399, like levallorphan, has a very weak effect, suggesting that the latter compound possesses low abuse liability.