Mechanisms of inhibitory action of TRK-130 (Naltalimide), a µ-opioid receptor partial agonist, on the micturition reflex.

PURPOSE: To clarify the mechanism of inhibitory action of TRK-130 (Naltalimide), a unique µ-opioid receptor partial agonist, on the micturition reflex. METHODS: The effect of TRK-130 on isovolumetric rhythmic bladder contractions (RBCs) was examined in guinea pigs, the effect of which was clarified by co-treatment with naloxone or in spinal cord transection. The effect of TRK-130 on urodynamic parameters was also observed in guinea pigs. In addition, the effect of TRK-130 on bladder contraction induced by peripheral stimulation of the pelvic nerve was investigated in rats. RESULTS: TRK-130 (0.001-0.01 mg/kg, iv) dose-dependently inhibited RBCs, which was dose-dependently antagonized by naloxone; however, the antagonism susceptibility was different from morphine (1 mg/kg, iv). The minimum effective dose (0.003 mg/kg) of TRK-130 remained similar in spinal cord-transected animals. TRK-130 (0.0025 mg/kg, iv) increased bladder capacity without changing the voiding efficiency, maximum flow rate, and intravesical pressure at the maximum flow rate, whereas oxybutynin (1 mg/kg, iv) increased the bladder capacity but affected the other parameters. TRK-130 (0.005 mg/kg, iv) did not produce significant changes on the bladder contractions induced by peripheral stimulation of the pelvic nerve, while oxybutynin (1 mg/kg, iv) significantly suppressed the bladder contractions. CONCLUSIONS: These results suggest that TRK-130 enhances the bladder storage function by modulating the afferent limb of the micturition reflex through µ-opioid receptors in the spinal cord. TRK-130 could be a more effective and safer therapeutic agent with a different fashion from antimuscarinics and conventional opioids for overactive bladder.

Novel pegylated interferon-ß as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer.

Malignant ascites manifests as an end-stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon-ß (IFN-ß) has been used to treat several cancer indications; however, little is known about the efficacy of IFN-ß on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN-ß, each conjugated with a polyethylene glycol molecule (PEG-hIFN-ß and PEG-mIFN-ß, respectively). We provide evidence that these IFN-ß molecules retain anti-viral potency comparable to unmodified IFN-ß in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG-mIFN-ß significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG-hIFN-ß directly suppresses VEGF165 -induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG-mIFN-ß enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN-ß in maintaining vascular integrity, and provide proof-of-mechanism for a novel and long-acting pegylated hIFN-ß for the therapeutic treatment of malignant ascites.

Characteristics of TRK-130 (Naltalimide), a novel opioid ligand, as a new therapeutic agent for overactive bladder.

We characterized TRK-130 (N-[(5R,6R,14S)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]phthalimide; naltalimide), an opioid ligand, to clarify the therapeutic potential for overactive bladder (OAB). In radioligand-binding assays with cells expressing human µ-opioid receptors (MORs), δ-opioid receptors (DORs), or κ-opioid receptors (KORs), TRK-130 showed high selectivity for MORs (Ki for MORs, DORs, and KORs = 0.268, 121, and 8.97 nM, respectively). In a functional assay (cAMP accumulation) with cells expressing each human opioid receptor subtype, TRK-130 showed potent but partial agonistic activity for MORs [EC50 (Emax) for MORs, DORs, and KORs = 2.39 nM (66.1%), 26.1 nM (71.0%), and 9.51 nM (62.6%), respectively]. In isovolumetric rhythmic bladder contractions (RBCs) in anesthetized guinea pigs, TRK-130 dose-dependently prolonged the shutdown time (the duration of complete cessation of the bladder contractions) (ED30 = 0.0034 mg/kg i.v.) without affecting amplitude of RBCs. Furthermore, TRK-130 ameliorated formalin-induced frequent urination at doses of higher than 0.01 mg/kg p.o. in guinea pigs under the freely moving condition. Meanwhile, TRK-130 showed only a negligible effect on the gastrointestinal transit at doses of up to 10 mg/kg s.c. in mice. These results indicate that TRK-130 is a potent and selective human MOR partial agonist without undesirable opioid adverse effects such as constipation and enhances the storage function by suppressing the afferent limb of the micturition reflex pathway, suggesting that TRK-130 would be a new therapeutic agent for OAB.

Discovery of potent transient receptor potential vanilloid 1 antagonists: design and synthesis of phenoxyacetamide derivatives.

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC50=411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC50=33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.

In vitro and in vivo pharmacological characterization of the main metabolites of nalfurafine hydrochloride.

Pharmacological characterization of the main metabolites of nalfurafine hydrochloride ((E)-N-[17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxymorphinan-6ß-yl]-3-(furan-3-yl)-N-methylprop-2-enamide monohydrochloride; a selective κ-opioid receptor agonist and an antipruritic for uremic pruritus in hemodialysis patients in Japan) such as 17-decyclopropylmethylated nalfurafine (de-CPM), 3-glucuronide of nalfurafine (NFA-G) and 3-glucuronide of 17-decyclopropylmethylated nalfurafine (de-CPM-G) was performed in vitro (human opioid receptor radioligand binding assay and forskolin-stimulated cyclic adenosine monophosphate (cAMP) assay) and in vivo (substance P-induced scratching behavior in mice). These main metabolites of nalfurafine showed the low affinities for human κ-, µ- and δ-opioid receptors except for the affinity of de-CPM to κ-opioid receptor (inhibition constant (Ki) values: 5.95nmol/l), which was 24 times lower than that of nalfurafine. Moreover, the main metabolites of nalfurafine had much lower agonistic activities than that of nalfurafine for three opioid receptors in forskolin-stimulated cAMP assays. In the substance P-induced mouse scratching behavior, the subcutaneous administration of each metabolite did not statistically significantly reduce the scratching behavior at doses up to 1000µg/kg which was 100 times higher than the effective dose of nalfurafine. These findings suggest that the main metabolites of nalfurafine do not make any contribution to its pharmacological actions including antipruritic effects in vivo.

Synthesis of new opioid derivatives with a propellane skeleton and their pharmacology: part 1.

The observation that 17-cyclopropylmethylmorphinan derivatives without the 4,5-epoxy ring showed more κ selectivity than those with a 4,5-epoxy ring led us to develop a working hypothesis: the position of the plane composed of the A and B rings would influence the opioid receptor type selectivity and that the decrease in the torsion angle C11-C12-C13-C14 could improve the κ selectivity. Consistent with our hypothesis, KNT-42 with an N-cyclopropylmethyl propellane structure, whose A and B rings were fixed in a torsion angle of approximately 0°, showed κ selective agonist activity.

Promotion of liver and lung tumorigenesis in DEN-treated cytoglobin-deficient mice.

Cytoglobin (Cygb) is a recently discovered vertebrate globin with molecular characteristics that are similar to myoglobin. To study the biological function of Cygb in vivo, we generated Cygb knockout mice and investigated their susceptibility to N,N-diethylnitrosamine (DEN)-induced tumorigenesis. Four-week-old male mice were administered DEN in drinking water at a dose of 25 ppm for 25 weeks or 0.05 ppm for 36 weeks. Cygb deficiency promoted the DEN-induced development of liver and lung tumors. All Cygb(+/-) and Cygb(-/-) mice treated with 25-ppm DEN exhibited liver tumors, compared with 44.4% of their wild-type counterparts. Lung tumors were present only in Cygb-deficient mice. More than 40% of Cygb(-/-) mice developed liver and lung tumors at the nontoxic dose of DEN (0.05 ppm), which did not induce tumors in wild-type mice. Cygb loss was associated with increased cancer cell proliferation, elevated extracellular signal-regulated kinase and Akt activation, overexpression of IL-1ß, IL-6, Tnfα, and Tgfß3 mRNAs, and hepatic collagen accumulation. Cygb-deficient mice also exhibited increased nitrotyrosine formation and dysregulated expression of cancer-related genes (cyclin D2, p53, Pak1, Src, Cdkn2a, and Cebpa). These results suggest that Cygb deficiency induces susceptibility to cancer development in the liver and lungs of mice exposed to DEN. Thus, globins such as Cygb will shed new light on the biological features of organ carcinogenesis.

Synthesis of a stable iminium salt and propellane derivatives.

The treatment of morphinan 1 with NaH and MsCl provided very stable iminium salt 7 possessing propellane skeleton. One of the synthesized iminium salts 7, isobutyl derivative 7b, was crystallized and its structure was determined by X-ray crystallography. The natural bond orbital analysis suggested that the stability of the iminium should result from the stereoelectronic effect (hyperconjugation) attributed to their own structures.

Catalytic aerobic oxidation of nor-binaltorphimine (nor-BNI) analogs without 4,5-epoxy bridge affords a more selective ligand for kappa opioid receptor than the representative kappa antagonist nor-BNI.

An analog of nor-binaltorphimine (nor-BNI) without the 4,5-epoxy bridge, 17,17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-6,6'-imino-14beta,14'alpha-dihydroxy-3,3'-dimethoxy-7,7'-bimorphinan (4), which was the precursor of the designed compound 1 as a selective kappa(3) opioid receptor antagonist, was catalytically oxidized with oxygen in the presence of platinum to give the 5'-oxo derivative 3 with some other oxidized products. Morphinan derivatives without the 4,5-epoxy moiety were labile to oxygen, although the corresponding 4,5-epoxymorphinan derivatives resisted aerobic oxidation. One of the oxidized nor-BNI analogs without 4,5-epoxy bridge, compound 18, showed high affinity and selectivity for kappa opioid receptor.