Immune complexome analysis of serum samples from non-small-cell lung cancer patients identifies predictive biomarkers for nivolumab therapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have achieved important outcomes in cancer treatment. However, current clinical biomarker tests are not suitable for some patients because they require tumor tissues and have poor predictive value for treatment responses. Therefore, the identification of biomarkers that enable screening tests in all patients is necessary. METHODS: We performed an immune complexome analysis of non-small cell lung cancer patients treated with nivolumab to comprehensively identify and compare antigens incorporated into immune complexes (IC-antigens) in serum samples from the responders (n = 15) and non-responders (n = 20). Additionally, combinations of IC-antigens characteristic to the responder group were evaluated by logistic regression analysis and receiver operating characteristics curves to examine their predictiveness for ICI treatment responses. RESULTS: The combination of predictive biomarkers detected before treatment was profilin-1, purine nucleoside phosphorylase, alpha-enolase, and nucleoside diphosphate kinase A [p = 0.0043, odds ratio = 2.26, 95% confidence interval (CI) = 1.19-4.28, area under the curve = 0.76]. The combination of predictive biomarkers detected after treatment was peptidyl-prolyl cis-trans isomerase A, ubiquitin-like modifier-activating enzyme 1, complement component C8 beta chain, and apolipoprotein L1 (p = 0.0039, odds ratio = 2.56, 95% CI = 1.25-5.23, area under the curve = 0.77). CONCLUSION: Combinations of serum IC-antigens may predict the therapeutic effect of nivolumab in non-small cell lung cancer patients.

Determination of Gemcitabine in Plasma of Bladder Cancer Patients by Hydrophilic Interaction Chromatography with Ultraviolet Detection.

Gemcitabine is a deoxycytidine analog that has been used for a broad spectrum of tumor, such as nonsmall-cell lung cancer, bladder cancer and pancreatic cancer. Because gemcitabine is hydrophilic, hydrophilic interaction liquid chromatography (HILIC), where analytes are retained on a polar column according to their hydrophilicity, should be adequate for separation analysis of gemcitabine. In the present study, we proposed a hydrophilic interaction chromatography with ultraviolet (HILIC-UV) method with liquid-liquid extraction and adding tetrahydrouridine to plasma samples for gemcitabine analysis of clinical samples with respect to daily and wide usage. The method successfully determined gemcitabine in 56 plasma samples of 30 unique patients. Mean plasma concentration of gemcitabine was 15.0 ± 6.0 µg/mL (mean ± standard deviation). The concentration range is consistent with the data from previous literatures. Our proposed HILIC-UV method is simple and easy handling, and is widely and clinically usable for determination of gemcitabine in human plasma.

Proteomic analysis and ATP assay reveal a positive effect of artificial cerebral spinal fluid perfusion following microdialysis sampling on repair of probe-induced brain damage.

BACKGROUND: Microdialysis (MD) is conventionally used to measure the in vivo levels of various substances and metabolites in extracellular and cerebrospinal fluid of brain. However, insertion of the MD probe and subsequent perfusion to obtain samples cause damage in the vicinity of the insertion site, raising questions regarding the validity of the measurements. NEW METHOD: We used fluorogenic derivatization liquid chromatography-tandem mass spectrometry, that quantifies both high and low abundance proteins, to differentiate the effects of perfusion from the effects of probe insertion on the proteomic profiles of expressed proteins in rat brain. RESULTS: We found that the expression levels of five proteins were significantly lower in the perfusion group than in the non-perfusion group. Three of these proteins are directly involved in ATP synthesis. In contrast to decreased levels of the three proteins involved in ATP synthesis, ATP assays show that perfusion, following probe insertion, even for a short time (3 h) increased ATP level up to 148% that prior to perfusion, and returned it to normal state (before probe insertion). COMPARISON WITH EXISTING METHOD: There is essentially no information regarding which observed changes are due to probe insertion and which to perfusion. CONCLUSIONS: Our findings partially demonstrate that the influence of whole MD sampling process may not significantly compromise brain function and subsequent analytical results may have physiological equivalence to normal, although energy production is transiently damaged by probe insertion.

Computational study of the competitive binding of valproic acid glucuronide and carbapenem antibiotics to acylpeptide hydrolase.

The efficacy of the antiepileptic drug VPA is decreased by co-administered carbapenems (CBPMs). The mechanism of CBPM selective inhibition of acylpeptide hydrolase (APEH) hydrolysis of VPA-glucuronide (VPA-G) to VPA is unclear due to the lack of APEH structural information. Here we performed homology modeling of the three-dimensional structure of APEH and subsequent docking simulations with a modeled structure to understand this mechanism. Docking simulations indicated that four groups of binding structures were involved in the binding of VPA-G, panipenem, and meropenem to APEH, but only one or two binding structures were involved in the binding of meropenem with an open ß-lactam ring structure and other antibiotics involving ampicillin. One of the four VPA-G binding structures was close enough to the APEH catalytic triad to facilitate VPA-G hydrolysis. This binding structure was also the most stable binding structure for panipenem, suggesting potential inhibition of VPA-G hydrolysis by panipenem. Fragment molecular orbital calculations of interaction energies of amino acid residues of APEH with VPA-G, panipenem, and meropenem indicated that the binding structure for panipenem closest to the catalytic triad is stabilized upon APEH interaction. These data suggest that APEH binding characteristics with CBPMs may help explain the selective inhibition of APEH by CBPMs.

Investigation of protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders using an electrophysiological method.

PURPOSE: The purpose of this study was to investigate the protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders caused by antiglaucomatous eyedrops using an electrophysiological method. METHODS: Three kinds of antiglaucomatous eyedrops, including benzalkonium chloride (BAC) as an ophthalmic preservative, a BAC-free antiglaucomatous eyedrop, and a sodium hyaluronate eyedrop, were used in this study. Eyedrops were applied to excised rabbit corneas, and the electrophysiological property of the cornea was monitored using an Ussing chamber with a turnover system that mimics human tear turnover. With this system, changes in transepithelial electrical resistance (TER) in the corneal surface were recorded. RESULTS: The corneal TER after applying antiglaucomatous eyedrops tended to decrease concomitantly with increasing the concentration of the BAC included as a preservative. On the other hand, there was no significant change in the corneal TER for the initial 60 min after applying sodium hyaluronate eyedrop compared with those of the control. Moreover, the pretreatment with a sodium hyaluronate eyedrop reduced the extent of decrease in the corneal TER observed after application of antiglaucomatous eyedrops alone. CONCLUSION: Those results indicate that a sodium hyaluronate eyedrop has the potential to protect the corneal surface from antiglaucomatous eyedrops, including BAC as an ophthalmic preservative.

Population pharmacokinetic investigation for optimization of amiodarone therapy in Japanese patients.

BACKGROUND: Optimal use of amiodarone (AMD) requires information regarding the drug's pharmacokinetics and the influence of various factors on the drug's disposition. This study was conducted to establish the role of patient characteristic in estimating doses of AMD using nonlinear mixed effects modeling in Japanese patients treated with oral therapy. METHODS: Serum concentrations of AMD were determined by high-performance liquid chromatography. The 151 serum trough concentrations from 23 patients receiving repetitive oral AMD were collected. Analysis of the pharmacokinetics of AMD was accomplished using a 1-compartment open pharmacokinetic model. The effect of a variety of developmental and demographic factors on AMD disposition was investigated. RESULTS: Estimates generated by nonlinear mixed effects modeling indicated that the clearance of AMD was influenced by the demographic variables: total body weight (TBW), daily dosage of AMD (DD), body mass index (BMI), gender (GEN), duration of AMD dosing (DUR), and patient clearance factor (Conc(θ); Conc = serum trough concentration of AMD). The final pharmacokinetic parameters were CL/F (L/h) = 0.072·TBW·Conc(-1.01)·1.95(DD≥200)·0.931(BMI≥25)·1.37(GEN)·DUR(-0.016), and Vd/F (L) = 78.4·TBW, where CL is total body clearance and Vd is volume of distribution. As all doses were given orally, it was impossible to assess the bioavailability (F). DD ≧200 is an indicator variable that has a value of 1 if the patient is receiving more than 200 mg daily dosage of AMD, and 0 otherwise. BMI ≧25 is an indicator variable that has a value of 1 if the BMI is 25 kg/m² and over, and 0 otherwise. GEN is an indicator variable that has a value of 1 if the patient is woman, and 0 otherwise. CONCLUSIONS: The authors developed new population pharmacokinetic parameters. Clinical application of the findings in the present study to patient care may permit selection of an appropriate initial maintenance dose, thus enabling the clinician to achieve a desired therapeutic effect.

Cross-sectional study on relationship between constipation and medication in consideration of sleep disorder.

Constipation can be caused by adverse drug reactions as a result of many drugs and might be induced by sleep disorders; however, the relative risk of its occurrence with individual drugs and the influence of sleep conditions have not been clarified. To clarify the relationship between constipation and various drugs in consideration of sleep disorders, we investigated the self-reported bowel habits, use of laxatives, and the Athens Insomnia Scale (AIS, a self-administered psychometric instrument to measure insomnia) in 344 inpatients on admission. They were divided into a constipation group (self-reported bowel habits of "Constipation" or "Occasional constipation" and/or use of laxatives, n=161) and a non-constipation group (both "Normal" and the non-use of laxatives, n=183). A comparison of the backgrounds of the two patient groups revealed significant differences in age, gender, number of used drugs, AIS score, hypothyroidism, chronic obstructive pulmonary disease, use of diuretics, coronary vasodilators, thyroid hormones, non-steroidal anti-inflammatory drugs, proton pump inhibitors, antidepressants, anti-anxiety drugs, and hypnotics. Multiple logistic regression analysis using these fourteen factors as autonomous variables showed that age (odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01-1.04; p=0.007), female gender (OR, 1.96; 95% CI, 1.21-3.18; p=0.006), the AIS score (OR, 1.10; 95% CI, 1.02-1.18; p=0.010), and the use of hypnotics (OR, 2.33; 95% CI, 1.30-4.16; p=0.004) were significantly related to constipation; therefore, as hypnotics appear more likely to cause constipation than other drugs, they should be used with caution.

Case-controlled study on risk factors for the development of constipation in hospitalized patients.

Constipation is a common problem in hospitalized patients; however, the relative risks of its development with various factors have not been clarified. To clarify the risk factors associated with constipation, we performed a case-controlled study of 165 hospitalized patients who were not laxative users on admission. They were divided into case (n=35) and control (n=130) groups according to laxative administration during hospitalization. Comparison of the patient backgrounds in the two groups revealed significant differences in the activities of daily living, length of fasting, rest level on admission, cerebrovascular disease, and administration of hypnotics. Multiple logistic regression analysis using these five factors as autonomous variables showed that administration of hypnotics (odds ratio, 2.79; 95% confidence interval, 1.10-7.06; p=0.031) was significantly related to laxative use. Therefore, the administration of hypnotics may be the principal cause of constipation development in hospitalized patients and they should be used with caution.

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection.

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.

Determination of paclitaxel in human and rat blood samples after administration of low dose paclitaxel by HPLC-UV detection.

A simple and sensitive HPLC-UV method was developed for the determination of paclitaxel (TXL) in human and rat blood samples. 4-Hydroxybenzoic acid n-hexyl ester was used as an internal standard. TXL was extracted by a liquid-liquid extraction with tert-butylmethyl ether. The disturbing peaks in the case of serum sample were removed by pre-extraction with hexane. The separation of TXL was achieved within 25 min using an ODS column with 50% acetonitrile aqueous solution as a mobile phase at a flow rate of 1.0 mL/min. The eluent was monitored at 230 nm, and the resulted retention times of TXL and IS were 11.2 and 20.4 min. The detection limits of TXL for human plasma, serum and rat plasma samples at a signal-to-noise ratio of 3 were 10, 9.5 and 7.5 ng/mL, respectively. The proposed methods were applicable to the determination of TXL in human patients' plasma ranging from 15 to 27 ng/mL. Furthermore, monitoring of the time course of TXL after its single administration to rat could be demonstrated.

Pentazocine monitoring in rat hair and plasma by HPLC-fluorescence detection with DIB-Cl as a labelling reagent.

Pentazocine (PZ) in rat hair and plasma was determined by HPLC-fluorescence detection with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelling reagent and cyclazocine (CZ) as an internal standard (IS). PZ and IS extracted from hair or plasma sample were derivatized with DIB-Cl and the resulted solution was cleaned up with solid phase extraction. The isocratic separation of DIB-PZ and -CZ within 20 min could be achieved by a Wakopak Handy-ODS column (250 x 4.6 mm i.d.) using a mobile phase composed of 0.1 mol/L acetate buffer (pH 6.2):acetonitrile (25:75, v/v). The detection limits of PZ at a signal-to-noise ratio of 3 for rat hair and plasma were 0.18 ng/mg and 0.57 ng/mL, respectively. Reproducible and precise results could be obtained by an IS method with RSD values less than 6.6% for within- and between-day measurements. The method was successfully applied for the monitoring of PZ levels in Zucker rat hair and plasma samples after a single administration of 25 mg/kg PZ. Moreover, incorporation rates of PZ into black and white hair of Zucker rat were evaluated.

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label.

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.

Determination of donepezil hydrochloride in human and rat plasma, blood and brain microdialysates by HPLC with a short C30 column.

A simple and sensitive HPLC method with fluorescence (FL) detection for determination of donepezil (DP) in plasma and microdialysate samples was developed. A rapid isocratic separation of DP could be achieved by a short C30 column using mobile phases of 25 mM citric acid/50 mM Na2HPO4 (pH 6.0)-CH3CN (73:27%, v/v) containing 3.5 mM sodium 1-octanesulfonate for plasma and H2O-CH3CN-CH3OH (80:17:3%, v/v/v) containing 0.01% acetic acid for microdialysate. The eluate was monitored at 390 nm with an excitation at 325 nm. The detection limits (S/N = 3) of DP for human plasma, rat plasma and rat brain or blood microdialysates were 0.2, 1.0 and 2.1 ng/ml, respectively. Reproducible results could be obtained by using (+/-)-2-[(1-benzyl-piperidine-4-yl)ethyl]-5,6-dimethoxyindan-1-one hydrochloride as an internal standard (IS). The method was successfully applied for monitoring of DP levels in rat plasma, blood and brain microdialysates and patient plasma.

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.

Capillary electrochromatography of caffeine and its metabolites in rat brain microdialysate.

A capillary electrochromatography (CEC) method has been developed for the separation of caffeine and its two metabolites 1-methylxanthine (1-MX) and 1,7-dimethylxanthine (1,7-DX). The stationary phase was 3-(1,8-naphthalimido) propyl-modified silyl silica gel (NAIP) and the best separations were achieved with 4.0 mM citrate buffer (pH 5.0) containing 80% methanol at an applied voltage of 25 kV. The compounds were completely separated in less than 3.5 min with good repeatability, which was approximately 3-times less than that in high-performance liquid chromatography (HPLC) with NAIP. The proposed method coupled with microdialysis was successfully applied to the monitoring of caffeine concentration in rat brain with detection limits of 1.11 microg/mL.

Hair analysis for fenfluramine and norfenfluramine as biomarkers for N-nitrosofenfluramine ingestion.

In this paper, a high performance liquid chromatographic method with fluorescence detection (HPLC-FL) for the determination of fenfluramine (Fen) and norfenfluramine (Norf) in human hair as biomarker metabolites of N-nitrosofenfluramine (N-Fen) is described. Washed and cut hair segments were extracted by ultrasonication for 1h at room temperature in methanol. The extract was evaporated and applied for derivatization with the fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). An HPLC-FL analysis was performed using an ODS column with mobile phase composition of acetonitrile and water (65:35, v/v) and monitored at 430 nm (excitation 325 nm). The method was sensitive with detection limits of 36 and 16 pg/mg hair for Fen and Norf, respectively. The linearity was assessed in the range 0.036-144 ng/mg for Fen and 0.016-127 ng/mg for Norf with correlation coefficients larger than 0.999. The method was successfully used for the segmental determination of Fen and Norf in hair samples obtained from hospitalized patients diagnosed with hepatotoxicity and suspected to ingest N-Fen. Both Fen and Norf could be detected in these patients' hair samples in the ranges 43-1389 pg/mg for Fen and 18-680 pg/mg for Norf and the results showed that the patients might ingest N-Fen for a period of not less than 5 months. As well, the method was applied for the determination of Fen and Norf in rats that possess pigmented and non-pigmented hair after an intraperitoneal administration of Fen. Both compounds were determined in black as well as in white hair.

Pharmacokinetic interactions between phenylpropanolamine, caffeine and chlorpheniramine in rats.

As the mechanism involved in the serious adverse effects associated with phenylpropanolamine (PPA) has not yet been clarified, and as PPA in usual cases is not being ingested without other drugs combination, the aim of this study was to characterize the possibility of pharmacokinetic interactions between PPA and most often combined drugs existing in the same dosage. The pharmacokinetics of PPA in rat brain and blood were evaluated when administered alone (group I), combined with caffeine (group II), combined with chlorpheniramine (group III), combined with both caffeine and chlorpheniramine (group IV) and finally when existed in one of the available OTC products (group V). This product contains multiple ingredients of PPA, caffeine and chlorpheniramine. In brain the pharmacokinetic parameters of PPA were significantly affected with the combined administration of caffeine and/or chlorpheniramine. The single intraperitoneal administration of caffeine (5 mg/kg) with PPA (2.5 mg/kg) to rats caused 1.6-fold increase in the AUC of PPA in brain compared to the single administration of PPA, and was comparable to the 1.5-fold increase caused by chlorpheniramine (0.4 mg/kg). The multiple combinations caused an increase in the AUC by 1.9-fold, which is comparable to the increase in the AUC of PPA obtained from the OTC product (2.2-fold). On the other hand, there was no significant difference in the pharmacokinetics of PPA in blood between the groups except for the C(max) of PPA in groups I and IV. The observed adverse effects associated with PPA use could be related to the significant increase in its levels in the brain.

High performance liquid chromatography with fluorescence detection for the determination of phenylpropanolamine in human plasma and rat's blood and brain microdialysates using DIB-Cl as a label.

A high performance liquid chromatographic method for the determination of phenylpropanolamine (PPA) in human plasma and rat's brain and blood microdialysates using fluorescence (FL) detection after precolumn derivatization with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) is described. PPA was extracted from plasma samples by a liquid-liquid extraction method with ethyl acetate followed by derivatization with DIB-Cl, while the blood and brain microdialysates were directly subjected for derivatization. The DIB-derivatives of PPA and the internal standard, ephedrine (EP), were then separated using an isocratic HPLC-FL set at excitation and emission wavelengths of 325 and 430 nm, respectively, on an ODS column. Calibration curves of PPA in spiked human plasma were linear over the concentration range of 5-5000 nM (0.755-755 ng/ml) and those in spiked blood and brain microdialysates were linear over the range of 25-5000 nM (3.775-755 ng/ml) with limits of detection of 17, 48 and 40 fmol on column in plasma and blood and brain microdialysates, respectively. As well, the intra- and the inter-assay precisions were lower than 12% for human plasma and the microdialysates. The method was successfully applied for the monitoring of PPA levels in rat's brain and blood microdialysates administered with a single oral dose of PPA (2.5 mg/kg).

Disability of development of tolerance to morphine and U-50,488H, a selective kappa-opioid receptor agonist, in neuropathic pain model mice.

We examined the analgesic and anti-allodynic effects of morphine and U-50,488H (trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide methanesulfonate salt), a selective kappa-opioid receptor agonist, and the development of tolerance to their effects in neuropathic pain model mice induced by sciatic nerve ligation (SNL). In the tail-pinch method, morphine at 10 mg/kg, s.c. produced a weak analgesic effect in SNL mice; however, U-50,488H at 5 mg/kg, s.c. produced an analgesic effect equipotent to that in normal mice. In contrast, morphine produced an adequate analgesic effect when given either intracerebroventricularly (i.c.v.) or intrathecally (i.t.), but U-50,488H only produced analgesia when given i.t. Repeated administration of morphine (either i.c.v. or i.t.) or U-50,488H (either s.c. or i.t.), did not induce tolerance to the effect. In the static allodynia test with an application of von Frey filaments, both compounds given s.c. suppressed the allodynic effect, but in the dynamic allodynia test involving lightly stroking the plantar surface with a cotton bud, only U-50,488H produced an anti-allodynic effect. Repeated administrations of both compounds did not develop tolerance to these anti-allodynic effects. Thus, U-50,488H was found to be a highly effective at blocking hyperalgesia and allodynia in nerve injury, and these findings suggest that kappa-opioid receptor agonists are attractive pharmacological targets for the control of patients with neuropathic pain.

Decrease in serotonin concentration in raphe magnus nucleus and attenuation of morphine analgesia in two mice models of neuropathic pain.

The alleviation of neuropathic pain cannot be satisfactorily achieved by treatment with opioids. There is much evidence to indicate that the active site of morphine for inducing effective analgesia is in the raphe magnus nucleus, where serotonin (5-HT, 5-hydroxytryptamine) acts as a primary transmitter. Therefore, we developed the hypothesis that 5-HT released in the raphe magnus nucleus could be related to the effectiveness of morphine in two mice models of neuropathic pain, diabetic (DM)-induced neuropathy and sciatic nerve ligation (SL). Two weeks after a single administration of streptozotocin, or 10 days after sciatic nerve ligation, mice were subcutaneously (s.c.) injected with morphine at 3, 5 and 10 mg/kg. The antinociceptive effect of morphine was estimated in the tail-pinch test; 5-HT content was measured after induction of neuropathic pain by microdialysis followed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Morphine produced as insufficient antinociceptive effect in SL mice at all doses compared with that in sham-operated mice, while in DM mice, morphine given s.c. at 5 and 10 mg/kg produced antinociceptive effects compared with those in non-diabetic mice, but not at 3 mg/kg. The 5-HT content of dialysates, expressed as AUC for 75 min, in SL and DM mice was less than that in control mice. However, morphine given s.c. at 5 mg/kg did not significantly affect 5-HT levels in both mice models compared to their controls. These results suggest that the decrease in 5-HT levels in the raphe magnus nucleus may be related to attenuation of the analgesic effect of morphine caused by the abnormal pain state found in diabetes and partial peripheral nerve injury.