前田 康博

We previously showed that castration of rats reduced erectile function over time; when testosterone replacement therapy was started 4 weeks after castration, erectile function improved. In this study, we examined the mechanism of improvement in erectile function following testosterone replacement therapy in rats. Thirty 12-week-old rats were divided into castrated (Cast), castrated with subcutaneous administration of testosterone (Cast + T), and sham (Sham) groups. Erectile function and mRNA and protein expression were evaluated in the rats by using standard methods. To assess erectile function, we measured the intracavernosal pressure, mean arterial pressure, mRNA expression of endothelial growth factors, and protein expression of endothelial nitric oxide synthase (eNOS). The intracavernosal pressure/mean arterial pressure ratio was significantly lower in the Cast group, and testosterone administration significantly improved (P = 0.017). Compared to the Cast group, the Cast+T group exhibited significantly increased mRNA expressions of vascular endothelial growth factor A (VEGF-A), intercellular adhesion molecule 1 (ICAM-1), transforming growth factor-β (TGF-β), nerve growth factor (NGF), α-smooth muscle actin (α-SMA), caveolae associated protein 1 (Cavin-1), Cavin-2, Cavin-3, sirtuin 1 (Sirt-1), sphingosine-1-phosphate 1 (S1P1), S1P2, and S1P3 and eNOS protein expression. Testosterone replacement therapy improved erectile function in castrated rats by increasing growth factors and eNOS protein.

BACKGROUND: Testosterone is an important hormone for the physical and mental health of men; however testosterone administration has also been suggested to adversely affect the cardiovascular system. AIM: To investigate the effects of excessive testosterone administration on vascular endothelial and erectile function in rats. METHODS: A total of seventy-five 12-week-old rats were divided into the following groups: Sham, castrated (Cast), castrated with subcutaneous administration of 100 mg/kg/month testosterone (Cast + T1), and castrated with subcutaneous administration of 100 mg/kg/week testosterone (Cast + T4). To observe the changes in testosterone level after the administration, rats were further divided into the following groups: control; T(6.25), wherein the rats were subcutaneously injected with 6.25 mg/kg testosterone; T(25) per week, wherein the rats were subcutaneously injected with 25 mg/kg testosterone per week; and T(100), wherein the rats were subcutaneously injected with 100 mg/kg testosterone per week. The relaxation responses of aorta were measured in these rats using standardized methods, and their erectile function was also evaluated. Statistical analysis of the obtained data was performed using two-way analysis of variance (ANOVA), Tukey-Kramer's multiple comparison test, or Student's t-test. OUTCOMES: At the end of the study period, endothelial function was evaluated through measurement of isometric tension, while erectile function was assessed using intracavernosal pressure (ICP), mean arterial pressure (MAP), and the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), sirtuin 1 (Sirt1) and vascular endothelial growth factor A. RESULTS: The ICP/MAP ratio in the Cast group (0.42 ± 0.04) was significantly lower than that in the Sham group (0.79 ± 0.07). The ICP/MAP ratio in the Cast + T1 group (0.73 ± 0.06) was significantly higher than that in the Cast group (P < .01) and that of the Cast + T4 (0.38 ± 0.01) group was unchanged (P > .05). The T(25) and T(100) groups exhibited significantly lower responses to ACh than the control group at 4 weeks (P < .01). Meanwhile, the ICP/MAP ratios in the T(25) group (0.44 ± 0.07) and T(100) group (0.47 ± 0.03) were significantly lower than that in the control group (0.67 ± 0.05) at stimulation frequencies of 16 Hz (P < .05). The expression of androgen receptor, Sirt1, and eNOS were significantly lower while that of iNOS was higher in the T(25) group compared with the control group (P < .05). CLINICAL TRANSLATION: The results based on this animal model indicate that extremely high testosterone levels may affect endothelial and erectile function. STRENGTHS AND LIMITATIONS: We found that high-dose testosterone administration decreased endothelial function in aorta and erectile function in rats. A major limitation of this study is that the blood concentration may not be representative of that in humans, and further research is needed. CONCLUSION: The findings suggest that high doses of testosterone may cause endothelial dysfunction in the aorta and erectile dysfunction in rats and that the blood concentration should be monitored after testosterone administration. Kataoka T, Fukamoto A, Hotta Y, et al. Effect of High Testosterone Levels on Endothelial Function in Aorta and Erectile Function in Rats. Sex Med 2022;XX:XXXXXX.

BACKGROUND: A platinum-containing anti-cancer agent, oxaliplatin (L-OHP), is known to induce peripheral neuropathy, including erectile dysfunction (ED) as a side effect, while Gosha-jinki-gan (GJG) is a traditional Japanese herbal medicine mainly used for peripheral neuropathy. AIM: To investigate the effect of GJG on L-OHP-induced ED in rats. METHODS: Twelve-week-old male Wister/ST rats were categorized into the following groups: Sham, Sham+GJG, L-OHP, and L-OHP+GJG (each n = 10). The L-OHP and L-OHP+GJG groups were injected intravenously with L-OHP (4 mg/kg) for 2 consecutive days in the first week. Statistical significance was determined using Bonferroni's multiple comparison test. OUTCOMES: At the end of the study period, erectile function was evaluated by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) after cavernous nerve stimulation. Western blot analysis was used to assess the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) levels, and quantitative polymerase chain reaction was used to assess the expression of phosphodiesterase-5 (PDE-5) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1. RESULTS: The ICP/MAP ratio of L-OHP rats (0.34 ± 0.06) was significantly lower than that of Sham rats (0.67 ± 0.03, P < .01), however, the ICP/MAP ratio of L-OHP+GJG rats (0.55 ± 0.01) was significantly higher than that of L-OHP rats (P < .01). There were no significant differences in the nNOS and eNOS protein expression between both groups (P > .05). GJG administration significantly decreased PDE-5 and NADPH oxidase-1 messenger RNA expressions in the L-OHP+GJG group. CLINICAL TRANSLATION: This animal model study suggests that GJG might be effective for erectile function in cancer survivors. STRENGTHS & LIMITATIONS: Our study identified that GJG had no notable side effects in the treated group. Further investigation of the cavernous nerve would also help elucidate the mechanism of GJG effect, which is a limitation of this study. CONCLUSION: We found that GJG administration improved L-OHP-induced ED by improving transcriptional PDE-5 expression. Kataoka T, Kawaki Y, Kito Y, et al. Gosha-Jinki-Gan Improved Erectile Dysfunction Caused by Anti-Cancer Agent Oxaliplatin by Decreasing Transcriptional Expression of Phosphodiesterase-5 in Rats. Sex Med 2022;10:100484.

BACKGROUND: Patients with cancer are treated with chemotherapeutics that cause adverse effects, including erectile dysfunction (ED). OBJECTIVES: We investigated erectile function in rats after the administration of anticancer agents based on data retrieved through mining of the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database. MATERIALS AND METHODS: The statistical signal strength for the association between anticancer drugs and ED was calculated using the reporting odds ratio (ROR). A drug-event combination was detected when the lower limit of the 95% confidence interval (CI) of the ROR exceeded 1.00. Rats were administered anticancer agents detected in the FDA AERS analysis. Erectile function was assessed using intracavernous pressure (ICP) and mean arterial pressure (MAP) analysis after electrical stimulation of the cavernous nerve. Statistical significance was determined using Welch's t-test or two-way ANOVA. RESULTS: Melphalan (L-PAM; ROR = 4.72, 95% CI = 2.78-8.00), vincristine (VCR; ROR = 2.47, 95% CI = 1.54-3.97), docetaxel (DTX; ROR = 2.25, 95% CI = 1.28-3.95), methotrexate (MTX; ROR = 1.96, 95% CI = 1.39-2.75), and doxorubicin (DOX; ROR = 1.82, 95% CI = 1.07-3.19) enhanced ED risk. L-PAM and MTX decreased the ICP/MAP ratio 1 week after administration. VCR and DOX decreased erectile function 4 weeks after administration. DTX decreased erectile function at all assessed time points. DISCUSSION AND CONCLUSION: Certain anticancer agents should be considered risk factors for ED. Our results provide possible treatment strategies for maintaining erectile function in cancer survivors, including careful erectile function monitoring after treatment.

BACKGROUND: Because androgen replacement therapy (ART) is not performed immediately after the onset of androgen deficiency, the treatment is considered to be late. AIM: To investigate the effects of late ART, starting 4 weeks after castration of rats, on erectile function and structural changes in the corpus cavernosum. METHODS: Rats were subjected to ART for 4 (Late-ART [4w]) or 8 (Late-ART [8w]) weeks. In either case, rats were assigned to the following groups: castrated (Cast), castrated with subcutaneous administration of testosterone (3 mg/kg/day; Cast+T), and sham (Sham). Cast + T rats received daily subcutaneous doses of testosterone starting 4 weeks after castration for 4 or 8 weeks whereas Sham and Cast rats received only the vehicle. OUTCOMES: Erectile function was assessed by evaluating intracavernosal pressure (ICP) and mean arterial pressure (MAP) after electrical stimulation of the cavernous nerve, corporal veno-occlusive function using dynamic infusion cavernosometry, and histology using Masson's trichrome staining. RESULTS: No increase in the ICP was observed in Cast+T rats in the Late-ART (4w) group (0.47 ± 0.02, P > .05), whereas, in Cast+T rats in the Late-ART (8w) group, there was a significant increase in the ICP/MAP ratio (0.60 ± 0.02, P < .05), drop rate, and smooth muscle/collagen ratio. CLINICAL TRANSLATION: The present study provides scientific evidence for the effect of late ART on erectile function. STRENGTHS AND LIMITATIONS: This study provides insights into the influence of late ART on erectile function through improvements in the structure of corpus cavernosum. The major limitation of this study is the difference in the time required for healing between the humans and rats, which might have a bearing on the translational relevenace of the results. CONCLUSIONS: Late ART could improve erectile function. However, as improvement requires a considerable time period, it is necessary to persist with therapy patiently for optimal results. Kataoka T, Hotta Y, Yamamoto Y, et al. Effect of Late Androgen Replacement Therapy on Erectile Function Through Structural Changes in Castrated Rats. Sex Med 2021;XX:XX-XXX.

BACKGROUND: Chemotherapeutics, one of the standard treatment options for cancer worldwide, have various adverse effects, including erectile dysfunction (ED). AIM: To investigate erectile function in an animal model after administration of the anticancer agent oxaliplatin (L-OHP). METHODS: Male Wistar/ST rats were divided into 2 groups: L-OHP rats (n = 21), which were intravenously administered L-OHP (4 mg/kg; twice a week for 4 weeks), and Control rats (n = 21), which were injected with the same volume of 5% glucose solution, using the same dosing schedule. At the end of the study period, erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) after cavernous nerve stimulation (n = 9-10). Endothelial function was evaluated with an isometric tension study using corpus cavernosum strips (n = 11). Western blot analysis was used to assess neuronal nitric oxide (nNOS) and endothelial NO synthase (eNOS) protein levels (n = 7). Real-time quantitative polymerase chain reaction (qRT-PCR) was used to assess the expression of inflammation- and oxidative stress-related markers (nicotinamide adenine dinucleotide phosphate oxidase-1, p22phox, interleukin [IL]-6, and nuclear factor-kappa B) (n = 6). Statistical significance was determined using the Student's t-test. OUTCOMES: The L-OHP group had a significantly lower ICP:MAP ratio than the control group (P < .05). Compared to the Control group, the L-OHP group exhibited significantly lower responses to ACh and eNOS protein levels and significantly higher inflammatory biomarker levels. CLINICAL TRANSLATION: The results based on this animal model indicate that use of the anticancer agent L-OHP should be considered as a risk factor for ED occurring via reduction of NO bioavailability in humans; our results provide possible treatment strategies for maintaining the erectile function of cancer survivors. STRENGTHS AND LIMITATIONS: Our study showed that the anticancer agent L-OHP has the propensity to cause ED in rats. A major limitation of this study is the lack of an established cure for ED associated with L-OHP and the lack of clinical evidence. CONCLUSIONS: L-OHP causes ED in rats via reduction of NO bioavailability caused by endothelial dysfunction. Kataoka T, Mori T, Suzuki J, et al. Oxaliplatin, an Anticancer Agent, Causes Erectile Dysfunction in Rats due to Endothelial Dysfunction. J Sex Med 2021;18:1337-1345.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme associated with immunomodulation through its regulation of the tryptophan-kynurenine (Kyn) pathway in advanced cancers, including metastatic renal cell carcinoma (mRCC). However, the failure of IDO1 inhibitors when used in combination with immune checkpoint inhibitors (ICIs), as observed in clinical trials, raises a number of questions. This study aimed to investigate the association of tryptophan 2,3-dioxygenase (TDO) and IDO1 with cancer development and resistance to immunotherapy in patients with RCC. In our analysis of RCC tissue samples, tissue Kyn levels were elevated in advanced-stage RCC and correlated well with TDO expression levels in RCC tumor cells. In patients with mRCC, TDO rather than IDO1 was expressed in RCC tumor cells, showing a strong association with Kyn expression. Furthermore, immunohistochemical staining of TDO was strongly associated with the staining intensity of forkhead box P3, as well as ICI therapy response and survival in patients with mRCC. Our study is the first to show that TDO expression in tumor tissues is associated with progression and survival, confirming its potential as a predictive biomarker of primary resistance to immunotherapy in patients with mRCC. Our findings suggest that strategies aimed at inhibiting TDO, rather than IDO1, in combination with ICI therapy may aid in the control of mRCC progression.

The enzyme, indoleamine 2,3-dioxygenase 1 (IDO), catabolizes tryptophan (Trp) in the kynurenine (Kyn) pathway, and is important in suppressing antitumor immune responses in the tumor microenvironment. With regard to previously untreated patients with follicular lymphoma (FL), we sought to establish the prognostic significance of Trp catabolism in this disease. Serum Trp and Kyn levels in 110 patients with FL were quantified, and their relationship to different clinical parameters studied. IDO expression in the lymph nodes of affected patients was studied. Study participants included 54 males and 56 females (age range 39-86, median 62 years), showing a 5-year overall survival (OS) rate of 78.5%. Patients with a high Kyn level (5-year OS, 65.0% vs. 81.7%; p = 0.026), high Kyn/Trp ratio (71.1% vs. 81.7%; p = 0.002), and low hemoglobin (Hb) level (<12.0 g/dL; p = 0.001; a component of FL international prognostic indexes) demonstrated a significantly shorter OS. Multivariate analysis included the following 10 variables: age, sex, serum β2-microglobulin, Hb, longest diameter of the largest involved node, Ann Arbor stage, serum lactate dehydrogenase, histologic grading, B symptoms, and serum Kyn/Trp ratio; a lower Hb level and a high Kyn/Trp ratio (HR, 3.239; 95% CI, 1.296-8.096) led to a significantly inferior OS. In the microenvironment, some CD11c-positive myeloid dendritic cells but not FL tumor cells were found to produce IDO. Overall, measuring levels of serum Kyn and Trp in individual patients with FL contributed to predicting their prognosis.

Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype-guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP-related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP-related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss-of-function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP-related high toxicity (P = .003). Although the availability of screening of these rare loss-of-function variants is still unknown, our data provide useful information that may help to alleviate FP-related toxicity in Japanese patients with cancer.

Phosphodiesterase 5 inhibitors are widely used to treat erectile dysfunction and lower urinary tract symptoms with benign prostatic hyperplasia. Recent studies have indicated the renoprotective effects of this class of compounds. Whether renoprotection depends on blood pressure reduction remains controversial. In this study, we investigated the renoprotective effects of the phosphodiesterase 5 inhibitor, tadalafil, in a rat model of high-salt induced kidney injury with hypertension. Dahl salt-sensitive rats were fed a normal diet, high-salt (8% sodium chloride) diet, or high-salt diet with oral administration of either low- or high-dose tadalafil (1 and 10 mg kg-1  day-1 , respectively). Serum creatinine, urinary protein, and blood pressure were measured at baseline and after 8 weeks, at which point the rats were examined for glomerular injury and fibrosis. PAI1 mRNA levels were also evaluated. After 8 weeks, blood pressure, serum creatinine, and urinary protein levels were significantly higher in the high-salt group than those in the normal-salt group. Serum creatinine and urinary protein were significantly lower in both tadalafil groups than those in the high-salt group, while only high-dose tadalafil affected blood pressure. In addition, glomerulosclerosis and α-smooth muscle actin expression significantly decreased in both tadalafil treatment groups. PAI1 mRNA increased significantly in the high-salt group but decreased in both tadalafil-treated groups. Our results indicated that both low- and high-dose tadalafil prevented fibrosis and glomerular injury in a chronic kidney disease rat model. Mechanistically, these effects may be associated with PAI1 expression and glomerular structure protection.

BACKGROUND: High salt intake is a risk factor for hypertension, which can potentially lead to erectile dysfunction (ED); however, the underlying pathological mechanisms remain unclear. AIM: To investigate whether erectile function is directly impaired by high salt intake and whether selective inhibition of mineralocorticoid receptor (MR) could provide protection from ED. METHODS: 6-week-old male Dahl salt-sensitive rats were randomly divided into 3 groups: normal diet (0.3% NaCl; control, n = 8), high-salt diet (8% NaCl; HS, n = 8), and high-salt diet plus eplerenone (HS + EPL, n = 11). HS + EPL rats were orally administered daily doses of EPL (75 mg/kg) for 6 weeks; control and HS rats received purified water on the same schedule. OUTCOMES: At the end of the study period, erectile function was evaluated by measuring intracavernosal pressure and mean arterial pressure after cavernous nerve stimulation. Serum levels of asymmetric dimethylarginine and L-arginine were determined using ultraperformance liquid chromatography-tandem mass spectrometry. Quantitative PCR was used to assess the expression of MR, inflammation, and oxidative stress markers (nicotinamide adenine dinucleotide phosphate oxidase-1/4, p22phox, interleukin-6, and superoxide dismutase-1), and protein arginine N-methyltransferase-1. RESULTS: The intracavernosal pressure/mean arterial pressure ratio was significantly lower, whereas systolic blood pressure, MR expression, serum asymmetric dimethylarginine levels, oxidative stress, and levels of inflammatory biomarkers were significantly higher in HS rats than in control rats (P < .05). EPL administration significantly improved each of these parameters except systolic blood pressure and MR expression. No significant intergroup differences were observed for L-arginine and superoxide dismutase-1 levels. CLINICAL TRANSLATION: Our results provide a rationale for the need of salt restriction and the use of selective MR inhibitors in prophylaxis or treatment of ED in men consuming a high-salt diet. STRENGTHS & LIMITATIONS: We are the first to report that the adverse impact of high salt intake on erectile function is mediated via MR activation, independent of its effect on blood pressure. A major limitation of this study is that responses of salt-resistant rats were not studied. CONCLUSIONS: High salt intake directly impaired erectile function in Dahl salt-sensitive rats, whereas selective MR inhibition ameliorated this effect. Kishimoto T, Kataoka T, Yamamoto Y, et al. High Salt Intake Impairs Erectile Function in Salt-Sensitive Rats Through Mineralocorticoid Receptor Pathway Beyond Its Effect on Blood Pressure. J Sex Med 2020;17:1280-1287.

有機酸代謝異常症の新生児マススクリーニングは血中アシルカルニチンをフローインジェクション-タンデムマス(FI-MS/MS)法によって分析するため、イソ吉草酸血症の指標であるイソバレリルカルニチン(C5-1)と、ピボキシル基を含む薬剤の服用で生成するピバロイルカルニチン(C5-P)の様な異性体は区別できない。従って、C5アシルカルニチン(C5)高値の場合には、尿中有機酸分析などの特殊検査が必要となる。アシルカルニチンエステラーゼ(ACE、カイノス社)はアシルカルニチンを加水分解し遊離カルニチンを生成する酵素であるが、C5-Pの様に嵩高いアシル基を有するアシルカルニチンに対する活性が低い。この酵素活性の違いを利用し、イソ吉草酸血症の偽陽性の原因となるC5-Pの判別法を検討した。C5-1にACEを反応させると99%以上が加水分解されたが、C5-Pは全く加水分解されなかった。以上から、C5高値の検体をACEで処理後FI-MS/MS法によって分析し、C5のピークが消失した場合はイソ吉草酸血症の可能性が高く、ピーク強度に変化がない場合には薬剤の影響によるC5-Pの蓄積であると判定できる。この方法はスクリーニングで用いた検体を再利用しFI-MS/MS法で分析できるため、C5高値における2次検査法として有用である。(著者抄録)

OBJECTIVE: To investigate whether arginine methylation is altered in patients with amyotrophic lateral sclerosis (ALS) and how it affects disease severity, progression, and prognosis. METHODS: We compared the immunoreactivity of protein arginine methyltransferase 1 (PRMT1) and its products, asymmetric dimethylated proteins (ASYM), in postmortem spinal cord. We also measured the concentrations of total l-arginine and methylated arginine residues, including asymmetric dimethyl l-arginine (ADMA), symmetric dimethyl arginine, and monomethyl arginine, in CSF samples from 52 patients with ALS using liquid chromatography-tandem mass spectrometry, and we examined their relationship with the progression and prognosis of ALS. RESULTS: The immunoreactivity of both PRMT1 (p < 0.0001) and ASYM (p = 0.005) was increased in patients with ALS. The concentration of ADMA in CSF was substantially higher in patients with ALS than in disease controls. The ADMA/l-arginine ratio was correlated with the change of decline in the ALS Functional Rating Scale at 12 months after the time of measurement (r = 0.406, p = 0.010). A Cox proportional hazards model showed that the ADMA/l-arginine ratio was an independent predictor for overall survival. Moreover, a high ADMA/l-arginine ratio predicted poor prognosis, even in a group with normal percentage forced vital capacity. CONCLUSION: There was an enhancement of arginine dimethylation in patients with ALS, and the ADMA/l-arginine ratio predicted disease progression and prognosis in such patients.

Dihydropyrimidinase deficiency is a rare autosomal recessive disease affecting the second step of pyrimidine degradation. It is caused by mutations in the DPYS gene. Only approximately 30 cases have been reported to date, with a phenotypical variability ranging from asymptomatic to severe neurological illness. We report a case of dihydropyrimidinase deficiency incidentally detected by urine metabolome analysis. Gas chromatography-mass spectrometry-based urine metabolomics demonstrated significant elevations of dihydrouracil and dihydrothymine, which were subsequently confirmed by a quantitative analysis using liquid chromatography-tandem mass spectrometry. Genetic testing of the DPYS gene revealed two mutations: a novel mutation (c.175G > T) and a previously reported mutation (c.1469G > A). Dihydropyrimidinase deficiency is probably underdiagnosed, considering its wide phenotypical variability, nonspecific neurological presentations, and an estimated prevalence of 2/20,000. As severe 5-fluorouracil-associated toxicity has been reported in patients and carriers of congenital pyrimidine metabolic disorders, urinary pyrimidine analysis should be considered for those who will undergo 5-fluorouracil treatment.

We conducted data mining using the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database on spontaneously reported adverse events to evaluate the association between anticancer drug therapy and hepatitis B infection. Reports of hepatitis B infection were retrieved from the FAERS database. The reporting odds ratio (ROR) was used to estimate the association between hepatitis B infection and various anticancer agents and drug combinations. We detected statistically significant risk signals of hepatitis B for 33 of 64 anticancer agents by ROR (26 cytotoxicity drugs and seven molecular-targeted drugs). We focused on molecular-targeted drugs and assessed the risk of hepatitis B from specific anticancer drug combinations. The frequency of hepatitis B infection was significantly high for drugs such as rituximab, bortezomib, imatinib, and everolimus. The addition of cyclophosphamide, doxorubicin, and fludarabine to drug combinations additively enhanced the frequency of hepatitis B infection. There were no reports on hepatitis B infection associated with trastuzumab or azacitidine monotherapy. However, trastuzumab-containing regimens (e.g., combinations with docetaxel or paclitaxel) were correlated with the incidence of hepatitis B infection, similar to azacitidine monotherapy. Our findings suggest that the concomitant use of anticancer drugs, such as trastuzumab, taxane, and azacitidine, may contribute to the risk of hepatitis B infection. The unique signals detected from the public database might provide clues to eliminate the threat of HBV in oncology.

Indoleamine 2,3-dioxygenase 1 (IDO) is an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway. The purpose of the present study was to determine the clinical significance of Trp catabolism in newly diagnosed Hodgkin lymphoma (HL) patients. We quantified serum Trp and Kyn in 52 HL patients, and analyzed their associations with different clinical parameters including serum soluble CD30 concentration. The IDO expression was evaluated in the patients' affected lymph nodes. The cohort comprised 22 male and 30 female patients (age range, 15-81 years; median, 45 years), with a 5-year overall survival (OS) of 88.6%. The OS was significantly shorter for patients with a high Kyn/Trp ratio (OS at 5 years, 60.0% vs 92.2%), for those with stage IV disease, and for those with lymphocytopenia (<600/mm3 and/or <8% white blood cell count). The latter two parameters are components of the international prognostic score for advanced HL. In contrast, there were no significant differences in OS according to age, serum albumin, hemoglobin, sex, white blood cell count, or serum soluble CD30 (≥ or <285.6 ng/mL). Multivariate analysis using the three variables stage, lymphocytopenia, and serum Kyn/Trp ratio showed that only the latter significantly affected OS. Indoleamine 2,3-dioxygenase 1 was produced by macrophages/dendritic cells, but not by HL tumor cells, and IDO levels determined by immunohistochemistry had a significant positive correlation with the serum Kyn/Trp ratio. In conclusion, quantification of serum Kyn and Trp is useful for predicting prognosis of individual HL patients.

Oseltamivir has a hypothermic effect in mice when injected intraperitoneally (i.p.) and intracerebroventricularly (i.c.v.). Here we show that the hypothermia evoked by i.c.v.-oseltamivir is inhibited by non-selective dopamine receptor antagonists (sulpiride and haloperidol) and the D2-selective antagonist L-741,626, but not by D1/D5-selective and D3-selective antagonists (SCH-23390 and SB-277011-A, respectively). The hypothermic effect of i.p.-administered oseltamivir was not inhibited by sulpiride, haloperidol, L-741,626 and SCH-23390. In addition, neither sulpiride, haloperidol nor SCH-23390 blocked hypothermia evoked by i.c.v.-administered oseltamivir carboxylate (a hydrolyzed metabolite of oseltamivir). These results suggest that oseltamivir in the brain induces hypothermia through activation of dopamine D2 receptors.

BACKGROUND: Testosterone is believed to mediate the penile erectile response by producing adequate nitric oxide; therefore, testosterone deficiency results in erectile dysfunction through decreased nitric oxide bioavailability. However, the mechanisms underlying endothelial dysfunction in testosterone deficiency remain unclear. AIM: To investigate the mechanism of endothelial dysfunction in a rat model of testosterone deficiency. METHODS: Rats were distributed into 3 groups: castrated (Cast), castrated and supplemented with testosterone (Cast + T), and sham (Sham). In the Cast + T group, castrated rats were treated daily with subcutaneous testosterone (3 mg/kg daily) for 4 weeks; Sham and Cast rats received only the vehicle. OUTCOMES: Erectile function using intracavernosal pressure and mean arterial pressure measurements after electrical stimulation of the cavernous nerve, endothelial function using isometric tension, asymmetric dimethylarginine (ADMA) levels using ultra-performance liquid chromatography and tandem mass spectrometry, and inflammatory biomarker expression were performed 4 weeks after the operation. RESULTS: In the Cast group, the ratio of intracavernosal pressure to mean arterial pressure significantly decreased, acetylcholine-induced relaxation was lower, and serum ADMA, oxidative stress, and inflammation biomarker levels were significantly increased (P < .01). Testosterone injection significantly improved each of these parameters (P < .01). CLINICAL TRANSLATION: The present results provide scientific evidence of the effect of testosterone deficiency on erectile function and the effect of testosterone replacement therapy. STRENGTHS AND LIMITATIONS: This study provides evidence of the influence of testosterone deficiency on endothelial function by investigating ADMA and oxidative stress. A major limitation of this study is the lack of a direct link of increased ADMA by oxidative stress to inflammation. CONCLUSION: Testosterone deficiency increased not only ADMA levels but also oxidative stress and inflammation in castrated rats, which can cause damage to the corpus cavernosum, resulting in erectile dysfunction. Kataoka T, Hotta Y, Maeda Y, Kimura K. Testosterone Deficiency Causes Endothelial Dysfunction via Elevation of Asymmetric Dimethylarginine and Oxidative Stress in Castrated Rats. J Sex Med 2017;14:1540-1548.

We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5 p,mol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3-7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan. (C) 2017 Elsevier B.V. All rights reserved.

PURPOSE: To investigate whether relaxation of the rat penile corpus cavernosum could be controlled with NOBL-1, a novel, light-controllable nitric oxide (NO) releaser. MATERIALS AND METHODS: Fifteen-week-old male Wistar-ST rats were used. The penile corpus cavernosum was prepared and used in an isometric tension study. After noradrenaline (10(-5) M) achieved precontraction, the penile corpus cavernosum was irradiated by light (470-500 nm) with and without NOBL-1 (10(-6) M). In addition, we noted rats' responses to light with vardenafil (10(-6) M), a phosphodiesterase-5 (PDE-5) inhibitor. Next, responses to light in the presence of a guanylate cyclase inhibitor, ODQ (1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one) (10(-5) M), were measured. All measurements were performed in pretreated L-NAME (10(-4) M) conditions to inhibit endogenous NO production. RESULTS: Corpus cavernosal smooth muscle, precontracted with noradrenaline, was unchanged by light irradiation in the absence of NOBL-1. However, in the presence of NOBL-1, corpus cavernosal smooth muscle, precontracted with noradrenaline, relaxed in response to light irradiation. After blue light irradiation ceased, tension returned. In addition, the light response was obviously enhanced in the presence of a PDE-5 inhibitor. CONCLUSIONS: This study showed that rat corpus cavernosal smooth muscle relaxation can be light-controlled using NOBL-1, a novel, light sensitive NO releaser. Though further in vivo studies are needed to investigate possible usefulness, NOBL-1 may be prove to be a useful tool for erectile dysfunction therapy, specifically in the field of penile rehabilitation.

We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10-12-week-old, male Sprague-Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms.

PURPOSE: Indoleamine 2,3-dioxygenase 1 (IDO1: IDO) is an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway, and is an important micro-environmental factor suppressing antitumor immune responses. We investigated the prognostic significance of Trp catabolism in adult T-cell leukemia/lymphoma (ATL). EXPERIMENTAL DESIGN: We quantified serum Trp and Kyn in 96 ATL patients, 38 human T-cell lymphotropic virus type-1 asymptomatic carriers (HTLV-1 ACs), and 40 healthy adult volunteers. The relationships between various clinical parameters were analyzed. IDO expression was evaluated in the affected lymph nodes of ATL patients. RESULTS: Serum Kyn concentrations and Kyn/Trp ratios were significantly higher in HTLV-1 ACs than in healthy controls. Both increased significantly with progression from HTLV-1 AC to ATL. There were no significant differences in serum Trp concentrations between ATL patients, HTLV-1 ACs and controls. IDO was possibly produced by ATL and/or cells in the microenvironment. Multivariate analyses demonstrated a high serum Kyn/Trp ratio and high Kyn level, but not a high Trp level, to be significant independent detrimental prognostic factors in ATL and aggressive variant ATL. CONCLUSIONS: Quantification of serum Kyn and Trp is prognostically useful for individual ATL patients. Furthermore, ATL is an appropriate disease for testing novel cancer immunotherapies targeting IDO.

Oseltamivir, an anti-influenza virus drug, induces marked hypothermia in normal mice. We have proposed that the hypothermic effect arises from inhibition of the nicotinic acetylcholine receptor function of sympathetic ganglion neurons which innervate the brown adipose tissue (a heat generator). It has been reported that local anesthetics inhibit nicotinic acetylcholine receptor function by acting on its ionic channels, and that bupropion, a nicotinic antagonist, induces hypothermia. In this study, we compared the effects of oseltamivir, procaine and bupropion on body temperature, cardiovascular function and neuromuscular transmission Intraperitoneal administration of oseltamivir (100 mg/kg), procaine (86.6 mg/kg) and bupropion (86.7 mg/kg) lowered the core body temperature of normal mice. At lower doses (10-30 mg/kg oseltamivir, 8.7-26 mg/kg procaine and bupropion), when administered subcutaneously, the three drugs antagonized the hypothermia induced by intraperitoneal injection of nicotine (1 mg/kg). In anesthetized rats, intravenous oseltamivir (30-100 mg/kg), procaine (10 mg/kg) and bupropion (10 mg/kg) induced hypotension and bradycardia. Oseltamivir alone (100 mg/kg) did not inhibit neuromuscular twitch contraction of rats, but at 3-30 mg/kg it augmented the muscle-relaxing effect of d-tubocurarine. Similar effects were observed when lower doses of procaine (10-30 mg/kg) and bupropion (3-10 mg/kg) were administered, suggesting that systemic administration of oseltamivir inhibits muscular nicotinic acetylcholine receptors. These results support the idea that the hypothermic effect of oseltamivir is due to its effects on sympathetic ganglia which innervate the brown adipose tissue, and suggest that oseltamivir may exert non-selective ion channel blocking effects like those of ester-type local anesthetics. (C) 2015 Elsevier B.V. All rights reserved.

PURPOSE: Indoleamine 2,3-dioxygenase 1 (IDO1: IDO), an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway, is increasingly being recognized as an important microenvironmental factor suppressing antitumor immune responses. The purpose of the present study was to determine the prognostic significance of Trp catabolism in adult T-cell leukemia/lymphoma (ATL). EXPERIMENTAL DESIGN: We quantified serum Trp and Kyn in 96 ATL patients, 38 human T-cell lymphotropic virus type-1 asymptomatic carriers (HTLV-1 ACs), and 40 healthy adult volunteer controls. The relationships between various clinical parameters including overall survival were analyzed. IDO expression was evaluated in the affected lymph nodes of ATL patients. RESULTS: Serum Kyn concentrations and Kyn/Trp ratios were significantly higher in HTLV-1 ACs than healthy controls. Both increased significantly with progression from HTLV-1 AC to ATL. However, there were no significant differences in the serum Trp concentrations between ATL patients, HTLV-1 ACs, and controls. IDO was possibly produced by ATL and/or cells of the microenvironment. Multivariate analyses demonstrated that a high serum Kyn/Trp ratio and high Kyn level, but not a high Trp level, were significantly independent detrimental prognostic factors in ATL, as well as in that subset of patients with aggressive variant ATL. CONCLUSIONS: Quantification of serum Kyn and Trp is useful for predicting prognosis of an individual ATL patient. Furthermore, ATL, especially in patients with a high serum Kyn/Trp ratio, is an appropriate disease for testing novel cancer immunotherapies targeting IDO.

The effects of oseltamivir, a neuraminidase inhibitor, were tested on the function of neuronal nicotinic acetylcholine receptors (nAChRs) in a neuroblastoma cell line IMR32 derived from human peripheral neurons and on recombinant human 34 nAChRs expressed in HEK cells. IMR32 cells predominately express 34 nAChRs. Nicotine (nic, 30m)-evoked currents recorded at -90mV in IMR32 cells using the whole-cell patch clamp technique were reversibly blocked by oseltamivir in a concentration-dependent manner. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) at 30m had little effect on the nic-evoked currents. Oseltamivir also blocked nic-evoked currents derived from HEK cells with recombinant 34 nAChRs. This blockade was voltage-dependent with 10, 30 and 100 m oseltamivir inhibiting similar to 50% at -100, -60 and -40mV, respectively. Non-inactivating currents in IMR32 cells and in HEK cells with 34 nAChRs, which were evoked by an endogenous nicotinic agonist, ACh (5m), were reversibly blocked by oseltamivir. These data demonstrate that oseltamivir blocks nAChRs, presumably via binding to a site in the channel pore.

OBJECTIVES: To investigate the efficacy of oral L-citrulline for erectile dysfunction and penile structure disruption in a rat model. METHODS: Male Wistar-ST rats aged 15 weeks were randomly divided into three groups as follows: sham-operated rats (control group), surgically castrated rats (castrated group) and surgically castrated rats subsequently treated with 2% L-citrulline water (castrated + L-citrulline). At 4 weeks postoperative, erectile function was assessed based on intracavernous pressure changes, followed by electrostimulation of cavernous nerves and calculation of maximum intracavernous pressure/mean arterial pressure. Penile structure was evaluated by Masson's trichrome staining and the smooth muscle-to-collagen ratio was calculated. The serum bioavailable testosterone, L-arginine, L-citrulline, N(G),N(G) -dimethylarginine and nitrogen oxide levels were evaluated. RESULTS: The bioavailable testosterone concentrations were decreased in the castrated and castrated + L-citrulline groups compared with the control group at 4 weeks after surgery. The intracavernous pressure-to-mean arterial pressure and smooth muscle-to-collagen ratios were significantly decreased in the castrated group compared with the control group, but significantly increased in the castrated + L-citrulline group compared with the castrated group. The serum L-citrulline, L-arginine and N(G),N(G)-dimethylarginine levels, and the L-arginine-to-N(G),N(G)-dimethylarginine ratios were significantly increased in the castrated +L-citrulline group compared with the castrated group. The serum nitrogen oxide levels were increased in the castrated + L-citrulline group compared with the castrated group. CONCLUSIONS: Oral L-citrulline can improve the erectile response to electric stimulation of cavernous nerve and penile structure in castrated rats.

Introduction: Type 2 diabetes mellitus (T2DM) has become a major public health issue and is considered a risk factor for erectile dysfunction (ED). T2DM is also associated with androgen deficiency. However, there have been few basic studies on androgen replacement therapy (ART) for ED treatment in T2DM animal models, and the mechanism underlying the effect of ART on T2DM-induced ED is unclear. Aim: To investigate the effect of ART on ED in T2DM rats by examining inflammatory and nitric oxide (NO)-related factors. Methods: Otsuka Long-Evans Tokushima Fatty (OLETF) rats and their controls, Long-Evans Tokushima Otsuka (LETO) rats, were distributed into three groups: LETO, OLETF, and ART. In the ART group, OLETF rats were treated daily with testosterone (3mg/kg/day, subcutaneously) from 20 to 25 weeks of age LETO and OLETF rats received vehicle only. Main Outcome Measures: We measured erectile function by using measurements of the ratio between intracavernosal pressure (ICP) and mean arterial pressure (MAP) following electrical stimulation of the cavernous nerve and by evaluating the endothelial function of the corpus cavernosum in an isometric tension study. Expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS), sirtuin-1 (Sirt1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA was detected using polymerase chain reaction. Results: The ICP/MAP ratio in the OLETF group was significantly decreased and that in the ART group was significantly improved (P<0.01). The response to acetylcholine was significantly decreased in the OLETF group and improved in the ART group (P<0.01). Although expression of eNOS and Sirt1 mRNA was decreased and that of iNOS, IL-6, and TNF-α mRNA was increased in the OLETF group, ART improved mRNA expression. Conclusions: ART suppressed inflammation in rats with T2DM and metabolic disorders and improved their endothelial and erectile functions. ART could be effective for T2DM-induced ED and may be considered a potential ED treatment method. Kataoka T, Hotta Y, Maeda Y, and Kimura K. Assessment of androgen replacement therapy for erectile function in rats with type 2 diabetes mellitus by examining nitric oxide-related and inflammatory factors. © 2014 International Society for Sexual Medicine.

Some studies suggest that high-estrogen levels lead to erectile dysfunction (ED); high-estrogen levels are known to decrease testosterone levels. However, no study has examined whether testosterone replacement can improve the ED induced by high-estrogen levels. We investigated the effects of testosterone on ED caused by high-estrogen levels in rats. Rats were distributed in the following groups: (1) control (vehicle for 2 weeks), (2) the estrogen-treated group (ES; estradiol (3 μg kg -1 day -1) for 2 weeks), and (3) the estrogen- and testosterone-treated group (ES+TE; estradiol (3 μg kg -1 day -1) and testosterone (3 mg kg -1 day -1) for 2 weeks). We measured smooth muscle function via isometric tension and erectile function by measuring the intracavernosal pressure on cavernous nerve stimulation. In the ES group, the contraction of the corpus cavernosum smooth muscle increased in response to noradrenalin, and its relaxation decreased in response to the nitric oxide donor, sodium nitroprusside. Further, the erectile function was significantly decreased. In the ES+TE group, neither smooth muscle function nor erectile function was significantly improved. In conclusion, a high-estrogen milieu affected erectile function in rats, and testosterone treatment did not improve the ED caused by high-estrogen levels. © 2013 Macmillan Publishers Limited. All rights reserved.

INTRODUCTION: Oral L-citrulline supplementation increases serum L-arginine levels more efficiently than L-arginine itself and increases nitric oxide (NO) production. AIM: To investigate whether oral L-citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction (ED). METHODS: We divided 8-week-old male Wistar-ST rats into 3 groups: sham-operated rats (control group), arteriogenic ED rats who underwent ligation of both internal iliac arteries (ligation group), and arteriogenic ED rats receiving oral 2% L-citrulline water supplementation (citrulline group). Citrulline water was given to arteriogenic ED rats for 3 weeks from 1 week after surgery. Erectile function was evaluated by maximum intracavernous pressure/mean arterial pressure (ICP/MAP) ratios via cavernous nerve stimulation at 4 weeks after surgery. Then, the penises were resected, stained with Masson's trichrome, and observed microscopically. Serum nitrogen oxides (NOx) levels were measured by high-performance liquid chromatography. Bonferroni's multiple t-test was used for statistical analysis. MAIN OUTCOME MEASURES: The main outcome measures were changes in ICP/MAP, smooth muscle (SM)/collagen ratios, and NOx levels following L-citrulline supplementation. RESULTS: The ICP/MAP ratio in the ligation group was significantly lower than that in the control group (P<0.05), denoting ED. The ICP/MAP ratio of the citrulline group was significantly higher than that of the ligation group (P<0.05), indicating ED amelioration. Levels of NOx in the ligation group were significantly lower than in the control group (P<0.05), while those in the citrulline group were significantly higher than in the ligation group (P<0.05). SM/collagen ratios in the ligation group were significantly lower than in the control group (P<0.05), while ratios in the citrulline group were significantly higher than those in the ligation group (P<0.05). CONCLUSIONS: Oral L-citrulline supplementation improved ICP/MAP and SM/collagen ratios and increased NOx. Therefore, oral L-citrulline supplementation might be a useful novel therapy for acute arteriogenic ED.

Oseltamivir, an anti-influenza virus drug, has strong antipyretic effects in mice (Biological and Pharmaceutical Bulletin, 31, 2008, 638) and patients with influenza. In addition, hypothermia has been reported as an adverse event. The prodrug oseltamivir is converted to oseltamivir carboxylate (OC), an active metabolite of influenza virus neuraminidase. In this study, core body temperature was measured in mice, and oseltamivir and OC were administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p). Low i.c.v. doses of oseltamivir and OC dose-dependently produced hypothermia. Zanamivir (i.c.v.), another neuraminidase inhibitor, did not produce hypothermia. These results suggested that the hypothermic effects of oseltamivir (i.p. and i.c.v.) and OC (i.c.v.) are not due to neuraminidase inhibition. OC (i.p.) did not lower body temperature. Although mecamylamine (i.c.v.) blocked the hypothermic effect of nicotine-administered i.c.v., the hypothermic effects of oseltamivir and OC (i.c.v.) were not blocked by mecamylamine (i.c.v.). The effect of oseltamivir (i.p.) was markedly increased by s.c.-pre-administered mecamylamine and also hexamethonium, a peripherally acting ganglionic blocker, suggesting their potentiating interaction at peripheral sites. The hypothermic effect of nicotine (i.c.v.) was decreased by lower doses of oseltamivir (i.c.v.), suggesting the anti-nicotinic action of oseltamivir. These results suggest that oseltamivir (i.p.) causes hypothermia through depression of sympathetic temperature regulatory mechanisms via inhibition of nicotinic receptor function and through unknown central mechanisms.

INTRODUCTION: In our previous study, chronic vardenafil treatment improved erectile function soon after the end of the treatment in rats with acute arteriogenic erectile dysfunction (ED). AIM: The aim of this study is to evaluate whether the effects of chronic vardenafil treatment persist after the end of treatment using rats with acute arteriogenic ED. METHODS: Rats were randomly divided into three groups: (i) control; (ii) ligation; and (iii) vardenafil + no treatment. Rats in the ligation and vardenafil + no treatment groups underwent ligature of the bilateral internal iliac arteries to induce acute arteriogenic ED and were subsequently treated with vehicle or vardenafil (4.0 mg/kg/day), respectively, for 3 weeks. Subsequently, all rats were kept for a further 2 weeks with no treatment. Rats in the control group underwent sham surgery. MAIN OUTCOME MEASURES: Erectile function was assessed by changes in intracavernous pressure (ICP). Smooth muscle (SM)/collagen ratios in corpus cavernosum were analyzed by Masson trichrome staining. Transforming growth factor-β1 (TGF-β1 ) mRNA and protein levels in corpus cavernosum (CC) were, respectively, evaluated by real-time polymerase chain reaction (PCR) analysis and Western blotting analysis. RESULTS: ICP/mean arterial pressure (MAP) in the ligation group remained significantly lower than that in control group (P < 0.01). Despite no treatment for 2 weeks, ICP/MAP in the var + no treatment group remained significantly higher than that in ligation group (P < 0.05). SM/collagen ratio in the ligation group remained significantly lower when compared with the control group (P < 0.01). The ratio in the var + no treatment group remained significantly higher when compared with the ligation group at 2 weeks after the end of treatment (P < 0.05). TGF-β(1) mRNA and protein levels did not differ among the groups. CONCLUSIONS: The effects of chronic vardenafil treatment on erectile function and penile structure persist, even after the end of treatment, in acute arteriogenic ED rats.

We aimed to elucidate the changes in the relaxation function of corpus cavernosal smooth muscle (CCSM) and erectile function using acute penile ischemic rabbits. Relaxation response to acetylcholine (Ach) was unchanged at 3 days after ischemia. The response to ACh had significantly decreased at 1 week, but had recovered completely at 4 weeks. The reaction to sodium nitroprusside and electrical field stimulation was unchanged by ischemia at all time points. Erectile function was changed in the same manner as the response of ACh. The endothelium-dependent relaxing responses of CCSM and erectile function were found to be initially decreased, but subsequently improved completely. © The Japanese Pharmacological Society.

Background: Valproate (VPA) is a simple fatty acid and a substrate for the fatty acid beta-oxidation pathway. Previous data suggested that the toxicity of VPA may be provoked by carnitine deficiency and the inhibition of mitochondrial beta-oxidation. Objective: The aim of the present study was to elucidate the effect of VPA treatment on carnitine and isomer-differentiated acylcarnitine disposition, and determined the relationships between acylcarnitines and blood VPA levels in long-term treated patients with VPA and/or other antiepileptic drugs. Methods: Serum samples were obtained from children aged 1-15 years old treated for at least 6 months with VPA alone (n = 28) or VPA combined with other anticonvulsants (a = 23) and untreated controls (a = 23). Serum acylcarnitines were separated from their isomers and quantified using high-performance liquid chromatography tandem mass spectrometry. Results: We found higher 3-hydroxyisovalerylcarnitine levels and trace amounts of valproylcarnitine in both VPA monotherapy and polytherapy patients. Other acylcarnitines, hexanoylcarnitine, C12, C14:1-carnitines and the ratio of long-chain acylcarnitine to free carnitine were also higher in VPA polytherapy individuals than in controls. VPA monotherapy does not result in decreases in free carnitine or in the accumulation of long-chain acylcarnitines. Blood VPA concentrations correlated positively with hexanoylcarnitine, C12, C14:1, C16:1, C18:1-carnitines in all VPA-treated children (a = 51). Conclusion: Long-term VPA treatment in pediatric patients could affect some specific acylcarnitines, which is enhanced by the concomitant use of other anticonvulsants, and the formation of valproylcarnitine alone seems insufficient to develop severe carnitine deficiency at therapeutic doses of VPA. (C) 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

INTRODUCTION: Chronic phosphodiesterase type 5 inhibitor treatment may be useful in reversing erectile dysfunction (ED). However, the mechanisms of this improvement remain unknown. AIM: The aim of this article was to determine the mechanisms of the improvement by chronic vardenafil treatment for acute arteriogenic ED in rats. METHODS: Eight-week-old male Wistar-ST rats were divided into four groups: sham-operated rats (Control group) and rats with acute arteriogenic ED induced by ligating bilateral internal iliac arteries (Ligation group), subsequently treated with low-dose (0.4 mg/kg/day; VL group) or high-dose (4.0 mg/kg/day; VH group) vardenafil for 20 days from 1 week after ligature. MAIN OUTCOME MEASURES: Erectile function was assessed based on changes of intracavernous pressure (ICP) followed by electrostimulation of the cavernous nerves and was evaluated by the area under the curve of ICP/area under the curve of mean arterial pressure (area of ICP/MAP). Transforming growth factor (TGF)-β(1), vascular endothelial growth factor-A, endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS mRNA expression levels in penile corpus cavernosum were determined by real-time PCR. Western blotting for TGF-β(1) protein levels and Masson trichrome staining of penile tissues were performed in each at group 4 weeks after surgery. RESULTS: In the VH group, area of ICP/MAP was significantly improved when compared with the Ligation group (P < 0.01). The smooth muscle (SM)/collagen ratio in the VH group was significantly higher than in the Ligation group (P < 0.05), and was comparable with that in the Control group. TGF-β(1) mRNA and protein levels in the VH group were significantly lower when compared with the Ligation group (P < 0.05). CONCLUSIONS: Chronic vardenafil administration ameliorates impairment of penile hemodynamics and maintains normal SM to collagen ratio in cavernous tissues after acute arterial injury in rats.

Some oral antibiotics contain a pivalate ester, because molecules with a pivalate entity show enhanced absorption in the intestine. Upon absorption, such a "prodrug" is broken down into the active form of a given antibiotic and a pivalate molecule, the latter of which is converted to pivaloylcarnitine through pivaloyl-CoA and is excreted in the urine. Long-term administration of drugs containing pivalate decreases blood carnitine level and causes defects in fatty acid oxidation. Here, we used liquid chromatography tandem mass spectrometry to measure carnitine and pivaloylcarnitine levels in two patients (Patient 1: 16-month-old boy and Patient 2: 18-month-old boy) with secondary carnitine deficiency and hypoglycemic convulsions caused by pivalate-containing antibiotics. Both patients were administered excessive doses of pivalate for the long-term treatment of recurrent infection, and consequently, the serum free carnitine levels were very low (Patient 1: 1.0 micromol/L and Patient 2: 0.4 micromol/L), compared to normal range of 33.3-43.0 micromol/l, while the serum pivaloylcarnitine levels were elevated from normally undetectable level (Patient 1: 3.7 micromol/L and Patient 2: 1.6 micromol/L). Patient 1 recovered immediately after the glucose infusion, whereas Patient 2 remained symptomatic even after blood glucose level was normalized and fully recovered after carnitine supplementation. The urine pivaloylcarnitine level in Patient 2 was increased during carnitine supplementation (from 821.4 to 12,200 micromol/g creatinine) even after discontinuing the antibiotics, indicating that a considerable amount of pivalate was accumulated in the tissues. In conclusion, long-term administration of pivalate-containing antibiotics should be avoided particularly in children.

Holocarboxylase synthetase (HCS) deficiency is an inborn error of biotin metabolism, leading to a multiple carboxylases deficiency. As the affected fetus sometimes presents with enlargement of the cerebral ventricles and intrauterine growth retardation (IUGR), prenatal administration of biotin has been attempted in some pregnancies. We present herein the case of a Japanese neonate with HCS deficiency who received maternal administration of biotin (10mg/day) from 33 weeks' gestation. After biotin administration, the fetal body weight increased and gestation was continued to full term. However, lactic acidemia and metabolic acidosis were observed after birth. To evaluate the effects of prenatal therapy, we collected serum samples and measured the acylcarnitine profiles using high-performance liquid chromatography electrospray ionization tandem mass spectrometry. At birth, levels of propionylcarnitine and 3-hydroxyisovalerylcarnitine had already increased. At 2h after birth, these levels of acylcarnitines were further increased. At 3.5h after the start of biotin, these chemical findings were slightly improved. In conclusion, we considered that prenatal biotin therapy at 10mg/day may have been inadequate to avoid neonatal acidotic crisis in this case.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is rare among Asian individuals, and the clinical course and biochemical findings remain unclear. We report herein a 3-year-old Japanese girl with MCADD. The diagnosis was suggested by acylcarnitine profiles and confirmed by enzyme activity and genetic analysis after clinical presentation. Our described method with high-performance liquid chromatography/tandem mass spectrometry allows quantification of levels of n-octanoylcarnitine (C8-N) and other isomers (e.g. valproylcarnitine). We examined the patient's acylcarnitine profiles in serum and urine samples during carnitine loading and 14-hr fasting tests with/without carnitine supplementation. Under hypocarnitinemia, serum level of C8-N was 0.16 micromol/l and C8-N/decanoylcarnitine (C10) ratio was 1.8, which did not correspond to the diagnostic criteria for MCADD. However, intravenous carnitine loading test (100 mg/kg/day for 3 days and 50 mg/kg/day for 1 day) led to increased serum C8-N levels and urinary excretion was obvious, strongly suggesting MCADD. In the fasting test with carnitine supplementation, marked production of acylcarnitines (C8-N > C2 >> C6 > C10) was found, compared to the fasting test without carnitine supplementation. These results indicate that carnitine supplementation may be useful for detoxification of accumulated acylcarnitines even in an asymptomatic state. Moreover, the one-point examination for serum C8-N level and/or C8-N/C10 ratio may make the diagnosis of MCADD difficult, particularly in the presence of significant hypocarnitinemia. To avoid this pitfall, attention should be given to serum levels of free carnitine, and carnitine loading may be demanded in hypocarnitinemia.

Tandem mass spectrometry (MS/MS) has become a prominent method for screening newborns for diseases such as organic acidemia and fatty acid oxidation defects, although current methods cannot separate acylcarnitine isomers. Accurate determination of dicarboxylic acylcarnitines such as methylmalonylcarnitine and glutarylcarnitine has not been carried out, because obtaining standards of these acylcarnitines is difficult. We attempted the individual determinations of acylcarnitines with isomers and dicarboxylic acylcarnitines by applying high-performance liquid chromatography (HPLC). Chromatographic separation was performed by gradient elution using a mixture of 0.08% aqueous ion-pairing agent and acetonitrile as the mobile phase. Mass transitions of m/z 161.8 -&gt; 84.8 for carnitine and m/z 164.8 -&gt; 84.8 for deuterated carnitine were monitored in positive ion electrospray ionization mode. One carnitine and 16 acylcarnitines were quantified. The limit of quantitation (LOQ) was 0.1 mu mol/L for methylmalonylcarnitine and 0.05 mu mol/L for the other acylcarnitines. Intra-day and inter-day coefficients of variance (CVs) were &lt; 8.3% and &lt; 8.8%, respectively, for all acylcarnitines in serum, and both were &lt; 9.2% in urine. Mean recoveries were &gt; 90% for all acylcarnitines. Human samples were quantified by this method. After addition of deuterated acylcarnitines as internal standards, acylcarnitines in serum or urine were extracted using a solid-phase extraction cartridge. In healthy adult individuals, isobutyryl-, 2-methylbutyryl- and isovalerylcarnitine were detected in serum and urine. Dicarboxylic acylcarnitines were detected in urine. High concentrations of methylmalonylcarnitine and propionylcarnitine were found in both the serum and the urine of a patient with methylmalonic acidemia. The described HPLC/MS/MS method could separate most acylcarnitine isomers and quantify them, potentially allowing detailed diagnoses and follow-up treatment for those diseases. Copyright (c) 2007 John Wiley & Sons, Ltd.