Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

Possible mechanism for improving the endogenous immune system through the blockade of peripheral µ-opioid receptors by treatment with naldemedine.

BACKGROUND: It has been considered that activation of peripheral µ-opioid receptors (MORs) induces side effects of opioids. In this study, we investigated the possible improvement of the immune system in tumour-bearing mice by systemic administration of the peripheral MOR antagonist naldemedine. METHODS: The inhibitory effect of naldemedine on MOR-mediated signalling was tested by cAMP inhibition and ß-arrestin recruitment assays using cultured cells. We assessed possible changes in tumour progression and the number of splenic lymphocytes in tumour-bearing mice under the repeated oral administration of naldemedine. RESULTS: Treatment with naldemedine produced a dose-dependent inhibition of both the decrease in the cAMP level and the increase in ß-arrestin recruitment induced by the MOR agonists. Repeated treatment with naldemedine at a dose that reversed the morphine-induced inhibition of gastrointestinal transport, but not antinociception, significantly decreased tumour volume and prolonged survival in tumour-transplanted mice. Naldemedine administration significantly decreased the increased expression of immune checkpoint-related genes and recovered the decreased level of toll-like receptor 4 in splenic lymphocytes in tumour-bearing mice. CONCLUSIONS: The blockade of peripheral MOR may induce an anti-tumour effect through the recovery of T-cell exhaustion and promotion of the tumour-killing system.

Further investigation of the rapid-onset and short-duration action of the G protein-biased µ-ligand oliceridine.

TRV130 (oliceridine), a G protein-biased ligand for µ-opioid receptor, has recently been synthesized. It is considered to have strong antinociceptive effects and only minor adverse effects. However, whether or not oliceridine actually exhibits an ideal pharmacological profile as an analgesic has not yet been fully clarified in animal studies. This study examined the pharmacological profile of oliceridine in cells and animals. Oliceridine (10 µM) did not produce any µ-opioid receptor internalization in cells even though it increased impedance, which reflects the activation of Gi protein using the CellKey™ system, and inhibited the formation of cAMP. In mice, oliceridine (0.3-10 mg/kg) produced a dose-dependent antinociceptive effect with a rapid-onset and short-duration action in the hot-plate test, as well as antihyperalgesia after sciatic nerve ligation without the development of antinociceptive tolerance using the thermal hyperalgesia test. On the other hand, oliceridine inhibited gastrointestinal transit. Furthermore, oliceridine produced rapid-onset hyperlocomotion at antinociceptive doses; sensitization developed in mice and an emetic effect was observed in ferrets. These results indicate that, although oliceridine may produce dopamine-related behaviors even through selective stimulation of the G-protein-biased µ-opioid receptor pathway, it still offers advantages for breakthrough pain without antinociceptive tolerance with adequate doses.

Usefulness for the combination of G-protein- and ß-arrestin-biased ligands of µ-opioid receptors: Prevention of antinociceptive tolerance.

Background: µ-Opioid receptor internalization is considered to be critically linked to antinociceptive tolerance. Although µ-opioid receptor agonists have been administered simultaneously with other drugs to control pain, little information is available regarding opioid­opioid interactions. Therefore, the present study was designed to further investigate the utility of a new G protein-biased ligand for µ-opioid receptors, TRV130, which has an antinociceptive effect without ß-arrestin-dependent µ-opioid receptor internalization, and its combination with fentanyl using µ-opioid receptor-expressing cells and mice. Results: In the present study, we confirmed that fentanyl produced a profound increase in ß-arrestin-2 recruitment accompanied by µ-opioid receptor internalization, whereas TRV130 did not induce either the recruitment of ß-arrestin-2 or µ-opioid receptor internalization in µ-opioid receptor-expressing cells. Under these conditions, ß-arrestin-2 recruitment accompanied by µ-opioid receptor internalization induced by fentanyl was abolished by TRV130, whereas TRV130 did not alter the reduction of cyclic adenosine monophosphate formation by fentanyl in µ-opioid receptor-expressing cells. In a behavioral assay, TRV130 exerted an antinociceptive effect in a hot-plate test in mice. In a combination test, the antinociceptive effect of TRV130 was synergistically increased by fentanyl. Fentanyl induced antihyperalgesia and development of its tolerance under a neuropathic pain-like state following sciatic nerve ligation. However, treatment of mice with an antinociceptive dose of TRV130 did not induce the rapid development of tolerance to its antihyperalgesic effect under a neuropathic pain-like state. Furthermore, the rapid development of tolerance to the antihyperalgesic effect induced by fentanyl plus TRV130 in mice with sciatic nerve ligation was not observed, unlike in the case of fentanyl alone. Conclusions: These findings provide evidence that activation of the G protein-biased pathway through µ-opioid receptors can alter signaling in the ß-arrestin-2 pathway linked to the stimulation of µ-opioid receptors. Furthermore, the combination of G protein-biased and ß-arrestin-biased ligands of µ-opioid receptors exerts an ideal antinociceptive effect without the rapid development of antinociceptive tolerance.

Development of κ Opioid Receptor Agonists by Focusing on Phenyl Substituents of 4-Dimethylamino-3-phenylpiperidine Derivatives: Structure-Activity Relationship Study of Matrine Type Alkaloids.

A series of new κ opioid receptor (KOR) agonists were developed from the lead compound 4-dimethylamino-1-pentanoylpiperidine (3), a matrine-type alkaloid. Derivatives of 3 were synthesized with a variety of phenyl substituents and evaluated for their antinociceptive effects. Additionally, their selectivity for an opioid receptor was investigated for cis-4c and d, and trans-4g, all of which had high activity exerted through the KOR.

Design and Synthesis of a Piperidinone Scaffold as an Analgesic through Kappa-Opioid Receptor: Structure-Activity Relationship Study of Matrine Alkaloids.

The matrine-type alkaloid 4-dimethylamino-1-pentanoylpiperidine (3a) has an antinociceptive effect through its impact on the κ-opioid receptor (KOR). Derivatives of 3a were synthesized by altering its amide and tertiary amine groups, and were evaluated for their antinociceptive effects. The results indicated that the distance between these groups on 3a was optimal for the antinociceptive effect. The effects obtained with compounds 8 and 9 indicated that the relative configuration of the 3- and 4-substituents influenced the effect mediated through the KOR.

Differential substitution for the discriminative stimulus effects of 3,4-methylenedioxymethamphetamine and methylphenidate in rats.

Previous studies have demonstrated that methylphenidate, MDMA (3,4-methylenedioxymethamphetamine), and other psychostimulants exert stimulant-like subjective effects in humans. Furthermore, MDMA and methylphenidate substitute for the discriminative stimulus effects of psychostimulants, such as amphetamine and cocaine, in animals, which suggests that MDMA and methylphenidate may produce similar discriminative stimulus effects in rats. However, there is no evidence regarding the similarities between the discriminative stimulus effects of MDMA and methylphenidate. To explore this issue, cross-substitution, substitution, and combination tests were conducted in rats that had been trained to discriminate between MDMA (2.5 mg/kg) or methylphenidate (5.0 mg/kg) and saline. In the cross-substitution tests, MDMA and methylphenidate did not cross-substitute for each other. In the substitution test, methamphetamine substituted for the discriminative stimulus effects of methylphenidate, but not for those of MDMA. Furthermore, ephedrine and bupropion, which activate dopaminergic and noradrenergic systems, substituted for the discriminative stimulus effects of methylphenidate. On the other hand, serotonin (5-HT) receptor agonists 5-HT1A and 5-HT2 fully substituted for the discriminative stimulus effects of MDMA. These results suggest that activation of the noradrenergic and dopaminergic systems is important for the discriminative stimulus effects of methylphenidate, whereas activation of the serotonergic system is crucial for the discriminative stimulus effects of MDMA. Even though MDMA, like psychostimulants, exerts stimulant-like effects, our findings clearly indicate that the discriminative stimulus effects of MDMA are distinctly different from those of other psychostimulants in rats.

Effect of p-aminophenols on tyrosinase activity.

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure-activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver-Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.

Efficient microwave-assisted prenylation of pinostrobin and biological evaluation of its derivatives as antitumor agents.

Pinostrobin (5-hydroxy-7-methoxyflavanone) obtained in relatively large amounts from fingerroot (Boesenbergia pandurata) was converted to its C-6 and C-8 prenylated derivatives. The Mitsunobu reaction, europium(III)-catalyzed Claisen-Cope rearrangement, and Claisen reaction coupled with cross-metathesis were used as the key steps. Using a sealed-vessel microwave reactor, the Mitsunobu and Claisen/Cope reactions occurred smoothly with short reaction times and in satisfactory yields. The target compounds and five new intermediary substances showed cytotoxic activity toward SK-BR-3, MCF-7, PC-3, and Colo-320DM human tumor cell lines, and all of them had significantly lower IC(50) (microM) values than pinostrobin.

[Overview of 43 Years of Studies on Asymmetric Synthesis].

Our investigations of various aspects of organic chemistry over the past 43 years are reviewed in this paper. The following subjects are discussed: 1) the diastereoselective addition reaction to chiral imines and oxazolidines of organometallic reagents and its applications and 2) the reaction and synthesis of fluorine-containing compounds.

p-Dodecylaminophenol derived from the synthetic retinoid, fenretinide: antitumor efficacy in vitro and in vivo against human prostate cancer and mechanism of action.

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR) is an aminophenol-containing synthetic retinoid derivative of all-trans-retinoic acid, which is a potent chemopreventive and antiproliferative agent against various cancers. Clinical studies of 4-HPR have shown side effects consisting of night blindness and ocular toxicity. To maintain potent anticancer activity without side effects, p-dodecylaminophenol (p-DDAP) was designed based on structure-activity relationships of 4-HPR. In our study, we investigate whether p-DDAP shows anticancer activity against human prostate cancer cell line PC-3 when compared with 4-HPR. p-DDAP inhibited PC-3 cell growth progressively from low to high concentration in a dose-dependent manner. p-DDAP was the most potent antiproliferative agent in vitro among 6 p-alkylaminophenols and 3 4-hydroxyphenyl analogs examined including 4-HPR. Cells treated with p-DDAP were shown to undergo apoptosis, based on condensation nuclei, cytofluorimetric analysis, propidium iodide staining and the expression of bcl-2 and caspase 3. p-DDAP arrested the S phase of the cell cycle, while 4-HPR arrested the G(0)/G(1) phase. In addition, both the i.v. and i.p. administration of p-DDAP suppressed tumor growth in PC-3-implanted mice in vivo. p-DDAP showed no effects on blood retinol concentrations, in contrast to reductions after 4-HPR administration. These results indicate that p-DDAP exhibits excellent anticancer efficacy against hormonal independent prostate cancer in vitro and in vivo, and it may have great potential for clinical use in the treatment of prostate cancer with reduced side effects.

Changes in the rewarding effects induced by tramadol and its active metabolite M1 after sciatic nerve injury in mice.

INTRODUCTION: The present study was designed to investigate the rewarding effects induced by tramadol and its active metabolite O-desmethyltramadol (M1) under a neuropathic pain-like state. RESULTS: In opioid receptor binding and G protein activation, we confirmed that M1, but not tramadol, showed mu-opioid receptor (MOR) agonistic activity. Furthermore, we found that the subcutaneous (s.c.) injection of tramadol and M1 each produced a significant place preference in mice, and these effects were significantly suppressed by pretreatment with the MOR antagonist beta-funaltrexamine. The dopamine level in the mouse nucleus accumbens was significantly increased by s.c. injection of either tramadol or M1. Mice with sciatic nerve ligation exhibited a marked decrease in the latency of paw withdrawal in response to a thermal stimulus only on the ipsilateral side. Under these neuropathic pain-like conditions, the rewarding effect induced by s.c. injection of either tramadol or M1 was dramatically inhibited after sciatic nerve ligation. Furthermore, the M1-induced G protein activation in the lower midbrain area was suppressed after sciatic nerve ligation. DISCUSSION: Our present data support the notion that the rewarding effect induced by tramadol is mediated mainly through metabolism to its active metabolite M1 via MOR. Furthermore, the suppression of the M1-induced G protein activation in the lower midbrain area caused by sciatic nerve ligation may be responsible for inhibiting the rewarding effects induced by s.c. injection of tramadol and M1 under a neuropathic pain-like state.

Concise total synthesis of (-)-deoxocuscohygrine and (-)-dihydrocuscohygrine.

The concise enantioselective total synthesis of C(2)-asymmetrical (-)-deoxocuscohygrine and (-)-dihydrocuscohygrine are described. Double-diastereoselective additions of normal Grignard reagent to bis(1,3-oxazolidine) have been deployed to construct chiral diamine fragments as a key step.

Hydroxyethylated cationic cholesterol derivatives in liposome vectors promote gene expression in the lung.

Three cationic cholesterol derivatives (CCDs), which differ in their types of amine and bear a hydroxyethyl group at the amine group, were synthesized and formulated into liposomes and nanoparticles as gene delivery vectors. In vitro transfection into A549 cells proved that liposomes formulated with CCDs and dioleoylphosphatidylethanolamine (DOPE) of 1/2 molar ratio were more effective than the corresponding nanoparticles with CCDs and Tween 80 at charge ratios (+/-) of 1/2, 3/1 and 5/1. Among the liposomal formulations, non-hydroxyethylated CCDs were more effective than hydroxyethylated ones in vitro. However, gene transfection in the lung through intratracheal injection showed opposite results to those in vitro, with liposomes containing hydroxyethylated CCDs being more potent than those containing non-hydroxyethylated CCDs. Transfection by liposomes with N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (MHAPC) showed the highest luciferase activity, resulting in 2- and 60-fold higher gene expression than jet-PEI and naked DNA, respectively. The distribution of MHAPC lipoplex after intratracheal injection was heterogeneous, and luciferase was expressed in epithelial cells lining the bronchi and bronchioles. All the lipoplexes led to higher TNF-alpha levels in the lung compared to the nanoplex and jet-PEI, but our findings suggested that modification of the cationic cholesterol with a hydroxyethyl group at the tertiary amine terminal, MHAPC, promoted gene expression in the lung without increasing the toxicity compared with other CCDs. This work firstly proved that liposomes containing hydroxyethylated CCDs could promote gene expression in the lung through intratracheal injection.

Lack of development of behavioral sensitization to methylphenidate in mice: correlation with reversible astrocytic activation.

Methamphetamine is a powerfully addictive psychostimulant that dramatically affects the mammalian central nervous system. Methylphenidate has been shown to have psychostimulus effects similar to methamphetamine. In the present study, we compared several effects of methylphenidate to those of methamphetamine. The subcutaneous administration of either methamphetamine or methylphenidate increased extracellular dopamine levels in the nucleus accumbens of mice. Interestingly, methamphetamine, but not methylphenidate, also increased the extracellular serotonin levels in this area. Further, repeated treatment with methamphetamine induced the development of sensitization to hyperlocomotion, whereas methylphenidate failed to induce behavioral sensitization. Moreover, in vitro treatment with methamphetamine, but not methylphenidate, caused long-lasting astrocytic activation in limbic neuron/glia co-cultures. These findings suggest that, unlike methamphetamine, methylphenidate shows a lack of development of behavioral sensitization to its hyperlocomotion and induces reversible astrocytic activation.

Narcolepsy-like sleep disturbance in orexin knockout mice are normalized by the 5-HT1A receptor agonist 8-OH-DPAT.

RATIONALE: Orexin knockout (KO) mice exhibit a phenotype that is similar to human narcolepsy, and monoamine-related compounds, such as psychostimulants and 5-HT uptake inhibitors, have been used for the treatment of narcoleptic disorders. However, little information is available regarding the pathophysiological features of orexin KO mice, particularly with respect to their narcoleptic-like disorder and how it is affected by monoamine-related compounds. OBJECTIVES: The present study was designed to investigate both the nature of the neuronal changes in orexin KO mice and the therapeutic effects of monoamine-related compounds on the sleep disorder in orexin KO mice. RESULTS: A decrease in locomotor activity in the dark phase was observed in orexin KO mice, and psychostimulants and 5-HT-related compounds, such as 8-OH-DPAT (5-HT1A receptor agonist) and DOI (5-HT2 receptor agonist), inhibited this hypolocomotion. We also found that 5-HT1A receptor mRNA levels, but not those for 5-HT2 or dopamine receptors, were significantly decreased in the prefrontal cortex of orexin KO mice in the dark period and were accompanied by compromising the increase in 5-HT metabolite levels. In addition, the sleep disorder in orexin KO mice, as analyzed by a polysomnography during the dark period, was completely normalized by 8-OH-DPAT. CONCLUSION: These results suggest that a dysfunction of 5-HT1A receptors is involved in the narcoleptic-like sleep dysfunction in orexin KO mice, and such dysfunction may participate in orexin deficiency-induced sleep disorders. Further, the use of 5-HT1A receptor agonist could be useful for treating the sleep disorder under a deficiency of orexin.

Highly regioselective hydrogenolysis of bis(alpha-methylbenzyl)amine derivatives affected by the trifluoromethyl substituent on the aromatic ring.

[reaction: see text] The highly regioselective hydrogenolysis of bis(alpha-methylbenzyl)amine derivatives proceeded with influence not from the electronic effect but from the steric effect of the trifluoromethyl substituent on the aromatic ring to provide a practical asymmetric synthesis of trifluoromethyl-substituted alpha-phenylethylamines.

Effect of 1-O-ethyl-3-butylcyclohexanol on the skin permeation of drugs with different physicochemical characteristics.

The effects of 1-O-ethyl-3-butylcyclohexanol (OEBC) on the in vitro skin permeation of ten model drugs with different physicochemical properties across excised rat skin were evaluated. The results showed that the addition of OEBC significantly improved the in vitro skin permeation of the model drugs compared with the control (without OEBC). To clarify the promoting mechanism of OEBC, a multiple regression analysis was employed. When the permeation study was performed without OEBC, the permeability coefficient was quantitatively predicted as a linear function of molecular weight (log MW) and their lipophilicity (partition coefficient of drugs between octanol and water (log K(o/w)) with a sufficiently high correlation coefficient (r=0.842). It was suggested that skin permeation of drugs without OEBC was explained as a function of diffusion of drugs through the skin and partitioning of drugs to the skin. Although OEBC was administered, the permeability coefficient of drugs cannot be predicted as a linear function of log MW and log K(o/w) (r=0.572).

[Structure activity relation study of matrine-type alkaloids. Part III].

6-Acyldecahydro [1,6]naphthyridines were synthesized as derivatives of matrine-type and allomatrine-type alkaloids, and the structure-activity relations were examined by the acetic acid-induced abdominal contraction test. All synthesized derivatives produced the antinociception in mice. The antinociceptive potencies of 15a-c and 16a-c were lower than those of 17a-c, 18a-c, 19a-c and 20a-c. Furthermore, those of the matrine-type derivatives 17b and 17c are greater than other derivatives. These findings suggest that less hindered tertiary amine and highly lipophilic acyl group are better functional groups for the greater antinociceptive potencies. Furthermore, these findings suggest that A or B ring of 1 and 2 are not essential for the antinociceptive effect.

A combination of liposomal sunitinib plus liposomal irinotecan and liposome co-loaded with two drugs enhanced antitumor activity in PC12-bearing mouse.

Pheochromocytomas are highly angiogenic neuroendocrine tumors. The side effects of treatment with cytotoxic agents frequently outweigh the benefits. Neuroendocrine tumors are highly angiogenic, dependent on vascular endothelial growth factor and receptor (VEGFR) activation. Sunitinib has antitumor and antiangiogenic activities that target VEGFRs. We investigated the antitumor activity of liposomal sunitinib and irinotecan alone and in combination. Liposomal sunitinib and irinotecan, and liposomes co-loaded with both drugs were prepared, and antitumor activity and biodistribution were examined in nude mice bearing PC12 tumors. Liposomal sunitinib increased in life span (ILS, 14.3%) compared with free sunitinib (-17.1% ILS) with moderate tumor growth suppression, whereas liposomal irinotecan suppressed tumor growth significantly without a survival benefit compared with free irinotecan (-21.7 and -13.3% ILSs, respectively). The combination of liposomal sunitinib plus liposomal irinotecan, and liposomes co-loaded with both drugs, induced significant inhibition of tumor growth and increased life-span more than the combination of free drugs. Accumulation of irinotecan in tumors by the combination of the two liposomal drugs and liposomes co-loaded with both drugs was significantly increased compared with the combination of free drugs. This study provides novel formulations of sunitinib and irinotecan in combination for the treatment of pheochromocytoma.