ストレス防御神経ステロイドの精密捕捉と向精神病薬開発への展開

金沢大学大学院自然科学研究科神経ステロイドは脳内で独自に合成され,種々の膜受容体を介して精神活動へ関与するとともに抗不安・抗ストレス活性を有することから,神経ステロイド自身あるいはその生合成制御物質が新規向精神病薬の有力候補と期待されている.本研究ではそれの開発を究極の目標として,神経ステロイドの高感度・精密分析システムの構築を手始めに,そのストレス,薬物投与による脳内レベル変動を解析した.得られた成果は次の通りである.1)神経ステロイド解析手法として信頼性に優れるLC-MS/MSを選択し,十分な感度が得られないステロイドに対して種々の誘導体化法を開発した.2)代表的なストレス応答神経ステロイドである5α-還元型プレグナン系神経ステロイドの一斉LC-MS/MS分析法を開発し,epiallopregnanoloneが脳特有の産物であることを見出した.3)ラット脳内3-オキソ-4-エン-ステロイドの一斉分析法を開発し,ストレス負荷に伴うレベル変動がプレグナン系ステロイドとアンドロスタン系のそれでは大きく異なることを発見した.4)ラット脳内アンドロゲンレベルの精密測定法を構築し,拘束ストレス負荷,エタノール投与による変動を解析した.その結果,5α-dihydrotestosteroneが末梢から輸送されたtestosteroneを原料として脳内で独自に合成されることを明らかとした.さらに,GABA_A受容体のモジュレータとされる3α,5α-androstanediolの生理的条件下における脳内レベルも初めて明らかとした.5)代表的なコルチコイドであるcorticosterone及びその還元型代謝物のLC-MS/MS分析法を開発し,それらのラット脳内存否を初めて明らかとするとともに,ストレス負荷に伴う変動や性差に関する重要な知見を得た.Neurosteroids bind to the γ-aminobutyric acid type A (GABA_A) receptors with a high affinity, positively modulate the action of GABA at these receptors and thus elicit marked anesthetic, sedative and anxiolytic effects. The quantitative analysis of the endogenous neurosteroids in the brain and serum is very important for characterization of their physiological functions and the mechanism by which they affect the brain functions. The brain and serum assays of the neurosteroids can also contribute to the development of new antipsychotic agents targeting neurosteroidogenesis. Based on this background information, sensitive liquid chromatography-tandem mass spectrometric methods that enable the quantification of trace neurosteroids (5α-redduced pregnane neurosteroids, 3-oxo-4-ene-neurosteroids, neuroavtive androgens and tetrahydrocorticosterone isomers) in rat brain and serum were developed and validated. Some methods employed derivatization procedures, which were very effective in increas ing the detection responses of steroids.New discoveries from the animal studies using the developed methods were as follows. 1) The brain allopregnanolone, epiallopregnanolone and 5α-dihydroprogesterone levels are rapidly elevated by immobilization stress and ethanol administration. Epiallopregnanolone is the brain-specific product. 2) The stress-induced level changes in the brain testosterone and androstenedione (androstane steroids) are much lower than those of progesterone and its metabolites (pregnane steroids). 3) Most of the brain testosterone is derived from the peripheral organs, while a fair amount of 5α-dihydrotestosterone is continuously synthesized in the brain. The GABAergic neuroactive androgen, 5α-androstane-3α,17β-diol, is always present in the male rat brain and its level is not influenced by the stress. 4) 3α,5α-Tetrahydrocorticosterone is present in the brain of both male and female rats after being subjected to the immobilization stress, but there is a sex difference in its brain level.研究課題/領域番号:18590031, 研究期間(年度):2006 – 2007出典：研究課題「ストレス防御神経ステロイドの精密捕捉と向精神病薬開発への展開」課題番号18590031（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-18590031/185900312007kenkyu_seika_hokoku_gaiyo/）を加工して作

24,25-ジヒドロキシビタミンD[3]の酵素免疫測定法の開発

取得学位：博士(薬学)，学位授与番号：博乙第184号，学位授与年月日：平成11年3月25日,学位授与年：199

FZD10-targeted α-radioimmunotherapy with 225Ac-labeled OTSA101 achieves complete remission in a synovial sarcoma model

Synovial sarcomas are rare tumors arising in adolescents and young adults. The prognosis for advanced disease is poor, with an overall survival of 12-18 months. Frizzled homolog 10 (FZD10) is overexpressed in most synovial sarcomas, making it a promising therapeutic target. The results of a phase 1 trial of β-radioimmunotherapy (RIT) with the 90Y-labeled anti-FZD10 antibody OTSA101 revealed a need for improved efficacy. The present study evaluated the potential of α-RIT with OTSA101 labeled with the α-emitter 225Ac. Competitive inhibition and cell binding assays showed that specific binding of 225Ac-labeled OTSA101 to SYO-1 synovial sarcoma cells was comparable to that of the imaging agent 111In-labeled OTSA101. Biodistribution studies showed high uptake in SYO-1 tumors and low uptake in normal organs, except for blood. Dosimetric studies showed that the biologically effective dose (BED) of 225Ac-labeled OTSA101 for tumors was 7.8 Bd higher than that of 90Y-labeled OTSA101. 90Y- and 225Ac-labeled OTSA101 decreased tumor volume and prolonged survival. 225Ac-labeled OTSA101 achieved a complete response in 60% of mice, and no recurrence was observed. 225Ac-labeled OTSA101 induced a larger amount of necrosis and apoptosis than 90Y-labeled OTSA101, although the cell proliferation decrease was comparable. The BED for normal organs and tissues was tolerable; no treatment-related mortality or obvious toxicity, except for temporary body weight loss, was observed. 225Ac-labeled OTSA101 provided a high BED for tumors and achieved a 60% complete response in the synovial sarcoma mouse model SYO-1. RIT with 225Ac-labeled OTSA101 is a promising therapeutic option for synovial sarcoma

Hepatitis B virus markers in patients with schistosomiasis, liver cirrhosis and hepatocellular carcinoma in Khartoum, Sudan.

Markers of hepatitis A and B virus were tested in 88 adult Sudanese subjects in Khartoum, Sudan. The subjects consisted of 25 control hospitalized patients, 21 volunteer blood donors, 23 patients with hepatosplenic schistosomiasis, 13 patients with liver cirrhosis and 6 patients with hepatocellular carcinoma (HCC). Antibody to hepatitis A virus was detected in 96% of the total. Hepatitis B surface antigen (HBsAg) was positive in 4, 24, 22, 31, and 67% of the subject groups, respectively. Antibody against hepatitis B core antigen (HBcAb) of undiluted serum was positive in 60, 57, 65, 77 and 83%, and there was no difference in incidence among the groups. It was positive in 200X diluted serum in 4, 24, 17, 23 and 60%. HBsAg and HBcAb (200X) were detected more often in HCC patients than in the control subjects (p less than 0.01). Hepatitis B virus is an important factor in the etiology of HCC in the Sudan.</p

Evaluation of transporter-mediated hepatobiliary transport of newly developed ¹⁸F-labeled pitavastatin derivative, PTV-F1, in rats by PET imaging

Quantitative evaluations of the functions of uptake and efflux transporters directly in vivo is desired to understand an efficient hepatobiliary transport of substrate drugs. Pitavastatin is a substrate of organic anion transporting polypeptides (OATPs) and canalicular efflux transporters; thus, it can be a suitable probe for positron-emission tomography (PET) imaging of hepatic transporter functions. To characterize the performance of [¹⁸F]PTV-F1, an analogue of pitavastatin, we investigated the impact of rifampicin (a typical OATP inhibitor) coadministration or Bcrp (breast cancer resistance protein) knockout on [¹⁸F]PTV-F1 hepatic uptake and efflux in rats by PET imaging. After intravenous administration, [¹⁸F]PTV-F1 selectively accumulated in the liver, and the radioactivity detected in plasma, liver, and bile mainly derived from the parent PTV-F1 during the PET study (∼40 min). Coadministration of rifampicin largely decreased the hepatic uptake of [¹⁸F]PTV-F1 by 73%. Because of its lower clearance in rats, [¹⁸F]PTV-F1 is more sensitive for monitoring changes in hepatic OATP1B function that other previously reported OATP1B PET probes. Rifampicin coadministration also significantly decreased the biliary excretion of radioactivity by 65%. Bcrp knockout did not show a significant impact on its biliary excretion.[¹⁸F]PTV-F1 enables quantitative analysis of the hepatobiliary transport system for organic anions

Hyperbilirubinemia and enhanced mitosis of hepatocytes in phalloidin-treated female rats.

Cholestasis with htperbilirubinemia was induced in female, but not male, Sprague-Dawley rats by daily treatment with phalloidin for 7 days. Increases in serum direct bilirubin level and alkaline phosphatase (Al-Pase) activity were observed concomitantpy with diminished bile flow and a decreased output of bile acid and cholesterol. Kidht microscope findings of the liver revealed proliferated bile ductules and enhanced mitosis of hepatocytes.</p

Reduction of 223Ra retention in the Large Intestine During Targeted Alpha Therapy with 223RaCl2 by Oral BaSO4 Administration in Mice

Background: Targeted alpha therapy with 223RaCl2 is used to treat skeletal metastases of hormone-refractory prostate cancer. The intravenous injection of 223RaCl2 causes gastrointestinal disorders such as nausea, abdominal discomfort, and diarrhea as frequent clinical adverse events caused by radiation. BaSO4 is known to display Ra2+ ion uptake in its structure and is clinically used as a contrast agent for X-ray imaging following oral administration. Here, we investigated the feasibility of a method to reduce 223Ra retention in the large intestine with BaSO4 by biodistribution studies in mice. Methods: 223RaCl2 biodistribution was examined in ddY mice after intravenous administration (10 kBq/mouse).BaSO4 (100 mg/mouse) was orally administered 1 h before 223RaCl2 injection. We also investigated the effect of laxative treatment on BaSO4 activity, since laxatives are clinically used with BaSO4 to avoid impaction in the large intestine. Results: BaSO4 significantly reduced 223Ra retention in the large intestine after 223RaCl2 injection in mice when compared with the control without BaSO4 administration (P &lt; 0.05). Excretion of 223Ra into the feces was significantly increased by BaSO4 administration (P &lt; 0.05). Laxative treatment did not affect BaSO4 activity in reducing 223Ra retention, although no additional effect of laxative treatment to 223Ra excretion was observed in mice. Conclusions. BaSO4 administration was effective in reducing 223Ra retention in the large intestine during 223RaCl2 therapy, and laxative treatment did not attenuate BaSO4 activity. This method could be useful in reducing adverse events caused by radiation exposure to the large intestine during 223RaCl2 therapy