Lysosomal trapping of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine, a hydrophilic and weakly basic amine, in human aortic vascular smooth muscle cells.

Some weakly basic compounds lead to cell death accompanied by cellular vacuolation. The novel analgesic agent, 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), is a hydrophilic and weakly basic compound that induces vacuolation in the vascular smooth muscle cells in dogs. Here, we investigated the vacuolation mechanism and the potential cytotoxicity of DMIP using human aortic vascular smooth muscle cells. When cells were treated with DMIP (0.1, 0.3, and 1 mM) for 6, 24, and 48 h, clear cytoplasmic vacuolation was observed at 1 mM after 24 and 48 h, along with an increase in the intracellular DMIP concentration. The vacuolation and intracellular DMIP were markedly reduced by bafilomycin A1, a vacuolar H+-ATPase inhibitor. The late endosome marker Rab7 and lysosome marker LAMP-2 were highly expressed but the early endosome marker Rab5 and autophagosome marker LC3 were not expressed specifically on the vacuolar membranes. These results suggested that the most vacuoles were enlarged late endosomes/lysosomes, resulting from the accumulation of DMIP by ion trapping. Moreover, DMIP did not affect lysosomal membrane integrity and was less cytotoxic than chloroquine, an inducer of phospholipidosis. The current study provides further insight into the mechanisms of vacuolation and lysosomal trapping induced by the hydrophilic and weakly basic amine DMIP.

Cytoplasmic Vacuolation and Tapetal Changes Induced by a Novel Analgesic Agent in Beagle Dogs.

Drug-induced unique cytoplasmic vacuolation was found in the subchronic oral toxicity study of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), a potential therapeutic agent for neuropathic pain, in beagle dogs. In the first study, DMIP was administered at a dose of 250, 500, or 1,000 mg/kg/day once daily for 14 days. Discoloration of tapetum lucidum accompanied by tapetal swelling was observed at ≥250 mg/kg/day. The tapetal swelling was correlated to the light microscopic observation of cytoplasmic vacuolation in tapetal cells, and similar vacuolation was observed in several other tissues, including the coronary artery and aortal arch, in a dose-dependent manner. Immunohistochemistry for lysosomal-associated membrane protein 2 indicated that the vacuoles were enlarged lysosomes. However, the nature of these vacuoles was different from that of phospholipidosis because no lamellar bodies were observed. In the second study, DMIP was administered at a dose of 10, 50, or 250 mg/kg/day once daily for 14 days followed by a 14-day recovery period. Tapetal changes and systemic vacuolation were not observed at ≤50 mg/kg/day, and vacuolation observed at 250 mg/kg/day was reversible. A few reports have described the enlargement of lysosomes not attributable to phospholipid accumulation. Our findings provide further information about the toxicological implications of drug-induced lysosomal swelling.

Gene expression profiles in auricle skin as a possible additional endpoint for determination of sensitizers: A multi-endpoint evaluation of the local lymph node assay.

The murine local lymph node assay (LLNA) is widely used to test chemicals to induce skin sensitization. Exposure of mouse auricle skin to a sensitizer results in proliferation of local lymph node T cells, which has been measured by in vivo incorporation of H3-methyl thymidine or 5-bromo-2'-deoxyuridine (BrdU). The stimulation index (SI), the ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, is frequently used as a regulatory-authorized endpoint for LLNA. However, some non-sensitizing irritants, such as sodium dodecyl sulfate (SDS) or methyl salicylate (MS), have been reported as false-positives by this endpoint. In search of a potential endpoint to enhance the specificity of existing endpoints, we evaluated 3 contact sensitizers; (hexyl cinnamic aldehyde [HCA], oxazolone [OXA], and 2,4-dinitrochlorobenzene [DNCB]), 1 respiratory sensitizer (toluene 2,4-diisocyanate [TDI]), and 2 non-sensitizing irritants (MS and SDS) by several endpoints in LLNA. Each test substance was applied to both ears of female CBA/Ca mice daily for 3 consecutive days. The ears and auricle lymph node cells were analyzed on day 5 for endpoints including the SI value, lymph node cell count, cytokine release from lymph node cells, and histopathological changes and gene expression profiles in auricle skin. The SI values indicated that all the test substances induced significant proliferation of lymph node cells. The lymph node cell counts showed no significant changes by the non-sensitizers assessed. The inflammatory findings of histopathology were similar among the auricle skins treated by sensitizers and irritants. Gene expression profiles of cytokines IFN-γ, IL-4, and IL-17 in auricle skin were similar to the cytokine release profiles in draining lymph node cells. In addition, the gene expression of the chemokine CXCL1 and/or CXCL2 showed that it has the potential to discriminate sensitizers and non-sensitizing irritants. Our results suggest that multi-endpoint analysis in the LLNA leads to a better determination of the sensitizing potential of test substances. We also show that the gene expression of CXCL1 and/or CXCL2, which is involved in elicitation of contact hypersensitivity (CHS), can be a possible additional endpoint for discrimination of sensitizing compounds in LLNA.

Mechanism Underlying Induction of Hyperglycemia in Rats by Single Administration of Olanzapine.

Acute administration of olanzapine rapidly elevates blood glucose levels. However, the mechanism underlying the rapid development of hyperglycemia with the administration of olanzapine remains unclear. The aim of the present study was to clarify the mechanism underlying olanzapine-induced acute hyperglycemia. Male Wistar rats received an intravenous infusion of saline (control) or olanzapine 2.5, 5, or 10 mg/kg. Blood samples were obtained periodically after olanzapine infusion to determine serum concentrations of glucose, olanzapine, and several endogenous substances. In a separate experiment, rats received an intravenous injection of propranolol (2 mg/kg) 30 min before infusion of olanzapine (10 mg/kg). The intravenous infusion of olanzapine induced dose-dependent increases in the serum concentrations of glucose, epinephrine, and insulin. Pretreatment with propranolol suppressed olanzapine-induced elevations in the serum concentration of glucose, but did not affect the serum concentration of olanzapine or olanzapine-induced increase in the serum concentration of epinephrine. Although the serum concentration of corticosterone increased after administration of olanzapine, no significant differences were observed among the olanzapine dose groups. Furthermore, administration of olanzapine did not affect the serum concentration of glucagon or histamine. We developed a pharmacokinetic-pharmacodynamic model assuming that the olanzapine-induced secretion of epinephrine leads to elevated serum glucose concentrations. This model appeared to satisfactorily characterize olanzapine-induced hyperglycemia. In conclusion, a single intravenous dose of olanzapine dose-dependently increased the serum concentration of glucose in rats, and epinephrine plays a role in olanzapine-induced acute hyperglycemia.

Analgesic effect of GT-0198, a structurally novel glycine transporter 2 inhibitor, in a mouse model of neuropathic pain.

This study was conducted to identify the characteristic pharmacological features of GT-0198 that is phenoxymethylbenzamide derivatives. GT-0198 inhibited the function of glycine transporter 2 (GlyT2) in human GlyT2-expressing HEK293 cells and did not bind various major transporters or receptors of neurotransmitters in a competitive manner. Thus, GT-0198 is considered to be a comparatively selective GlyT2 inhibitor. Intravenous, oral, and intrathecal injections of GT-0198 decreased the pain-related response in a model of neuropathic pain with partial sciatic nerve ligation. This result suggests that GT-0198 has an analgesic effect. The analgesic effect of GT-0198 was abolished by the intrathecal injection of strychnine, a glycine receptor antagonist. Therefore, GT-0198 is considered to exhibit its analgesic effect via the activation of a glycine receptor by glycine following presynaptic GlyT2 inhibition in the spinal cord. In summary, GT-0198 is a structurally novel GlyT2 inhibitor bearing a phenoxymethylbenzamide moiety with in vivo efficacy in behavioral models of neuropathic pain.

The discovery of potent glycine transporter type-2 inhibitors: design and synthesis of phenoxymethylbenzamide derivatives.

We describe the discovery of phenoxymethylbenzamide derivatives as a novel class of glycine transporter type-2 (GlyT-2) inhibitors. We found hit compound 1 (human GlyT-2, IC50=4040 nM) in our library and converted its 1-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)pyrrolidin-3-yl group to an 1-(N,N-dimethylaminopropyl)piperidyl group and its tert-butyl group to a trifluoromethyl group to obtain N-(1-(3-(dimethylamino)propyl)piperidin-4-yl)-4-((4-(trifluoromethyl)phenoxy)methyl)benzamide (20). Compound 20 showed good inhibitory activity against human GlyT-2 (IC50=15.3 nM) and exhibited anti-allodynia effects in a mouse neuropathic pain model.

Plasma transferrin concentration as a nutritional marker in malnourished dogs with nutritional treatment.

Rapid turnover proteins, such as transferrin (Tf), are used as dynamic nutritional assessment proteins in human medicine. However, nutritional status in veterinary medicine is mostly assessed on the basis of classical static factors, such as body weight, body condition score and plasma albumin level. This study evaluated the clinical usefulness of Tf as a nutritional assessment marker by measuring plasma Tf concentrations in malnourished dogs before and after nutritional treatment. Posttreatment plasma Tf concentrations were significantly higher than the pretreatment concentrations, although the albumin concentration did not change significantly. The numbers of dogs that exhibited increases in plasma Tf concentrations were significantly related to weight gain. Furthermore, the survival rates at day 60 after treatment initiation were significantly higher in dogs with plasma Tf concentrations above the reference value (180 mg/dl) after the nutritional treatment than in those with a plasma Tf concentration<180 mg/dl. In conclusion, the plasma Tf concentration is related to nutritional condition and would be a candidate for a novel nutritional assessment marker in dogs.

Increased expression of fractalkine and its receptor CX3CR1 in canine inflammatory bowel disease and their possible role in recruitment of intraepithelial lymphocytes.

The interaction between fractalkine/CX(3)CL1 and its receptor CX(3)CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX(3)CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX(3)CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX(3)CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX(3)CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX(3)CR1(+) PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX(3)CR1(+) PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX(3)CR1(+) PBMCs. These results suggest that the fractalkine-CX(3)CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs.

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. Part 2: Synthesis of novel triplet drugs with the epoxymethano structure (capped homotriplet).

An improved synthetic method for triplet drugs with the 1,3,5-trioxazatriquinane skeleton was developed that used p-toluenesulfonylmethyl isocyanide (TosMIC) instead of 1,3-dithiane. Using the improved method, we synthesized compounds with two identical pharmacophore units and an epoxymethano group, that is, capped homotriplets. Among the synthesized capped homotriplets, KNT-123 showed high selectivity for the µ receptor over the κ receptor, and the µ selectivity was the highest among the reported µ selective nonpeptide ligands. KNT-123 administered subcutaneously induced a dose-dependent analgesic effect in the acetic acid writhing assay, and its potency was 11-fold more potent than that of morphine. KNT-123 may serve as a useful tool for the study of the pharmacological actions mediated specifically via the µ receptor.

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. 1: Synthesis of triplet drugs with morphinan skeletons.

We synthesized symmetrical and nonsymmetrical triplet drugs with 1,3,5-trioxazatriquinane skeletons. The isolation of key intermediates, oxazoline dimers, made it possible to effectively produce nonsymmetrical triplets. Among the synthesized triplets, KNT-93, composed of three identical opioid µ receptor agonists, showed dose-dependent antinociception via the µ receptor. The effect was 56-fold more potent than that of morphine, a representative µ agonist. The profound analgesic effect induced by KNT-93 might result from simultaneous occupation of three µ opioid receptors.

A rapid and simple method to obtain canine peripheral blood-derived macrophages.

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.

Synthesis of 6,14-epoxymorphinan derivatives and their pharmacologies.

A novel 6,14-epoxymorphinan benzamide derivative (NS22) that was previously reported showed opioid κ receptor agonistic activity and analgesic activity. The unsatisfactory κ selectivity of NS22 led us to synthesize its derivatives to improve the opioid κ receptor selectivity and the agonist activity. In the course of SAR of the various derivatives, 17-benzyl-6,14-epoxymorphinan derivatives (KNT-33, 53, 55, 80, 90, 133) were found to show high selectivities and affinities for the opioid κ receptor. In addition, KNT-33, 53, 55 showed dose-dependent analgesic effects in acetic acid writhing tests. Therefore, 17-benzyl substituents may play an important role for developing κ selectivity.

Design and synthesis of KNT-127, a δ-opioid receptor agonist effective by systemic administration.

We have reported previously the novel δ-opioid agonist, SN-28, which was more potent in in vitro assays than the prototype δ-agonists, TAN-67 and SNC-80. However, when administered by subcutaneous injection, this compound showed no analgesic effect at dosages greater than 30mg/kg in the acetic acid writhing test. We speculated that SN-28 was not effective in the test because the presence of the charged ammonium groups prevented its penetration through the blood-brain barrier. On the basis of our proposal, we designed the novel δ-agonist, KNT-127, which was effective with systemic administration.

Synthesis of pyrrolomorphinan derivatives as kappa opioid agonists.

We synthesized pyrrolomorphinan derivatives 6, 7, and 9 to examine whether the pyrrole ring would be an accessory site in the kappa opioid receptor selective antagonist, nor-binaltorphimine. Derivative 6 had an alpha,beta-unsaturated ketone substituent that strongly bound to the kappa receptor. The compound with the highest kappa receptor selectivity, 6e, produced a dose-dependent antinociceptive effect in the mouse acetic acid writhing test. However, derivatives 7 and 9, which did not have alpha,beta-unsaturated ketone substituents, showed less kappa receptor selectivity than compound 6. Based on structure-activity relationships, we proposed that these compounds adopted active structures for kappa selective agonist activity. The pyrrole ring would not function as an accessory site, but the ability of the side chain on the pyrrole ring to localize above the C-ring appeared to confer kappa selective agonist activity. These results will promote the design of novel kappa agonists.

Investigation of Beckett-Casy model 3: synthesis of novel naltrexone derivatives with contracted and expanded D-rings and their pharmacology.

Novel naltrexone derivatives 7 and 8 with contracted and expanded D-rings were synthesized to investigate the importance of orientation of lone electron pair on the nitrogen for binding abilities to the opioid receptor. Compound 7 showed almost no binding affinity, whereas compound 8 was comparable to naltrexone (6) in binding affinity. Conformational analyses and NOE experiments in D(2)O of compounds 6-8 suggested that the lone electron pairs of compounds 6 and 8 with respective six- and seven-membered D-rings would project in the pseudo-axial orientation, whereas compound 7 with five-membered D-ring would have the lone electron pair directing in pseudo-equatorial position. These results strongly supported the proposal that the axial orientation of the lone electron pair on nitrogen would provide sufficient binding abilities to the opioid receptor and that the 15-16 ethylene moiety in the morphine structure would play a role in fixation of the lone electron pair in the axial direction rather than interaction with the putative cavity in the Beckett-Casy model.

Investigation of Beckett-Casy model 2: synthesis of novel 15-16 nornaltrexone derivatives and their pharmacology.

We synthesized novel 15-16 nornaltrexone derivatives 9, 11 and 22 to examine the importance of the cavity in the Beckett-Casy model, which was proposed to interact with the 15-16 ethylene moiety in the morphine structure. All the synthesized compounds showed lower affinities for the opioid receptor than did the naltrexone (10). The binding affinities of 14-OH derivatives 11, in which the rotation of the 9-17 bond would be restricted by an intramolecular hydrogen bond, was improved compared to the corresponding 14-H derivatives 9. Compound 22 whose 9-17 bond was strictly fixed by the ethylene bridge hardly bound to the opioid receptor. Compound 26 also showed very weak binding affinity in spite of the existence of the 15-16 ethylene unit. We proposed an important role for the orientation of the lone electron pair on the 17-nitrogen rather than the significance of the cavity in the Beckett-Casy model.

Investigation of Beckett-Casy model 1: synthesis of novel 16,17-seco-naltrexone derivatives and their pharmacology.

Novel 16,17-seco-naltrexone derivatives 3 were synthesized using a 16-17 bond cleavage reaction of naltrexone as the key reaction to examine the Beckett-Casy model. All the prepared 16,17-seco-naltrexone derivatives 3 showed lower affinities for opioid receptors than naltrexone. Although the results of binding assay seem to support the existence of a cavity in the model, further investigation using 15,16-nornaltrexone derivatives 26 will be needed to confirm the model.

A double decarboxylation reaction of an oxazolidinone and carboxylic Acid: its application to the synthesis of a new opioid lead compound.

Treatment of oxazolidinone carboxylic acid 6 with potassium carbonate gave olefin 7 by a double decarboxylation reaction. The reaction was proposed to proceed via decarboxylation followed by E1cB-like mechanism. 15,16-Nornaltrexone derivative 17 prepared from double decarboxylation product 7 showed strong affinity for the mu opioid receptor, indicating it to be a new opioid lead compound.

Drug design and synthesis of a novel kappa opioid receptor agonist with an oxabicyclo[2.2.2]octane skeleton and its pharmacology.

A conformational analysis of kappa opioid receptor agonists, TRK-820 and U-50,488H indicated an active conformation of TRK-820 in which the C-ring was in the boat form with the 14-OH interacting with the amide nitrogen. Based on the obtained active conformation of TRK-820, we designed and synthesized a novel kappa agonist KNT-63 with oxabicyclo[2.2.2]octane skeleton. KNT-63 showed profound antinociceptive effects via the kappa receptor which were as potent as that of TRK-820.

Aerobic oxidation of indolomorphinan without the 4,5-epoxy bridge and subsequent rearrangement of the oxidation product to spiroindolinonyl-C-normorphinan derivative.

Aerobic oxidation of indolomorphinan 1 without a 4,5-epoxy bridge proceeded in the presence of platinum catalyst to give indoleninomorphinan 2 or quinolono-C-normorphinan 5. The 4-hydroxy group would play an important role in deciding the course of the reaction. Treatment of 2a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave spiroindolinonyl-C-normorphinan 3a whose structure resembles that of delta opioid receptor agonist spiroindanyloxymorphone (SIOM). Boron trichloride was effective for the reverse reaction from 3a to 2a without side reaction. This practical interconversion method between hydroxyindolenine and spiroindolinone would be useful for the design and construction of drug-like compound libraries. Although the compound 3b was expected to show the selectivity for delta opioid receptor because of the structural resemblance to SIOM, it was rather selective for 1 opioid receptor (1: K(i)=0.75nM; delta: K(i)=2.90nM; kappa: K(i)=13.4nM). The result suggests that the slight difference of the spatial location of the benzene rings in these compounds may definitively affect the binding affinity for delta opioid receptor.