エンドサイト-シス機構を利用した脳へのペプチドデリバリ-

金沢大学薬学部本研究では、アルツハイマ-の有望な治療薬として注目を集めている副腎皮質刺激ホルモン(ACTH)様ペプチドebiratide(構造式1)とダイノルフィン様鎮痛ペプチドDLAP(構造式2)をモデル基質として、in vitro及びin vivo実験系を用いて中枢作用型ペプチドのエンドサイト-シス機構の解明を行なった。両ペプチドはクロラミンT法で^Iラベルして実験に用いた。牛脳毛細血管(B-cap)を用いたmild acid wash法でebiratideの内皮細胞内への内在化量と表面結合量を測定した。EbiratideのB-capへの取り込みは時間依存的、温度依存的、濃度依存的、浸透圧依存的であった。さらに、エンドサイト-シス阻害剤のdansylcadaverinによって著しく阻害された。また、ACTH及び塩基性ペプチドのpoly-L-lysine,protamineによって阻害された。以上のことから、既に報告したDLAPと同様に、ebiratideは血液-脳関門をabsorptive-mediatedエンドサイト-シスすることが明らかになった。一方、in vivo系においても極めて安定なDLAPについて脳移行ル-トについて検討した。毛細血管沈澱法を用いたところDLAP脳毛細血管分画に比べて実質細胞分画の方に多量に存在した。さらに、脳微小透析法を用いて脳細胞液中DLAPの濃度を測定したところ脳脊髄液中濃度の100倍も高かった。従って、DLAPは血液-脳脊髄関門ではなく、血液-脳関門を介して脳内へ移行していることが明らかになった。以上の検討結果から、absorptive-mediatedエンドサイト-シス機構の利用は脳へのペプチド・デリバリ-において有用であることが示された。本研究成果をもとに、今後、中枢作用型ペプチドのデリバリ-研究が飛躍的に進歩するものと思われる。構造式1 H-Met(O_2)-Glu-His-Phe-D-Lys-Phe-NH-(CH_2)_8-NH_2構造式2 H_3C-Tyr-Gly-Gly-Phe-Leu-Arg-CH_3Arg-(D)Leu-NHC_2H_5研究課題/領域番号:01571176, 研究期間(年度):1989出典：研究課題「エンドサイト-シス機構を利用した脳へのペプチドデリバリ-」課題番号01571176（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/grant/KAKENHI-PROJECT-01571176/）を加工して作

アフリカツメガエル卵母細胞発現系を用いた薬物輸送機構の分子論的解析

東北大学 / 金沢大学薬学部本研究はアフリカツメガエル卵母細胞を発現系として用い、小腸上皮細胞における輸送担体の実体を明らかにすることを目的とした。特に、核酸輸送系に焦点を絞った。mRNA注入卵母細胞はヘキソース及アミノ酸(Aシステム)輸送系が発現していることが確認された。mRNA注入卵母細胞においてNa^十依存的なウリジンの取り込みが見られた。GTC-CsCl法、Phenol/Chloroform法、GTC-Phenol/Chloroform法を用いてmRNAを抽出したところ、いずれの方法においてもNa^十依存的ウリジン輸送系の発現に差は見られなかった。mRNA注入卵母細胞におけるウリジンのNa^十依存的取り込みに飽和性が見られ、ミカエリス定数Ktは6.7μM、最大輸送速度Jmaxは3.40μ1/0.5hr/oocyteとなった。このKt値は、家兎小腸刷子縁膜小胞系(BBMV)で報告されている値(6.4μM)と極めてよく一致していた。さらに、ウリジンの取り込みは構造類似体であるアデノシン及びチミジンで顕著に阻害された。イノシンによって阻害傾向が見えたが、アデニン及びウラシルで阻害されなかった。以上の結果より、アフリカツメガエル卵母細胞に家兎小腸上皮細胞のmRNAを注入することでウリジン輸送担体が発現することが明らかになった。発現した輸送担体の特性はBBMVを用いて解明された輸送特性と一致していたことから、家兎小腸上皮細胞刷子縁膜に存在する輸送担体と同一であることが示唆された。薬物の細胞膜透過性に関する機構論的研究において、輸送担体の存在あるいは共通性についてしばしば問題となる。本実験系は透過現象の実体が膜蛋白であることを輸送機能の発現によって証明出来る点で優れている。この実験法を小腸のみなず種々の臓器における薬物輸送機構の解析に応用することで、標的組織に選択的に移行する薬物の合理的な分子設計が可能になると考えられる。研究課題/領域番号:04671409, 研究期間(年度):1992出典：研究課題「アフリカツメガエル卵母細胞発現系を用いた薬物輸送機構の分子論的解析」課題番号04671409（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/grant/KAKENHI-PROJECT-04671409/）を加工して作

透析ファイバー移植法を用いた脳内薬物動態機構の解析

金沢大学薬学部研究課題/領域番号:02771763, 研究期間(年度):1990出典：研究課題「透析ファイバー移植法を用いた脳内薬物動態機構の解析」課題番号02771763（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/grant/KAKENHI-PROJECT-02771763/）を加工して作

中枢作用型塩基性薬物の血液-脳関門透過機構の解明

金沢大学薬学部研究課題/領域番号:63772014, 研究期間(年度):1988出典：研究課題「中枢作用型塩基性薬物の血液-脳関門透過機構の解明」課題番号63772014（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/grant/KAKENHI-PROJECT-63772014/）を加工して作

初代培養脳毛細血管内皮細胞系を用いた薬物輸送

金沢大学薬学部研究課題/領域番号:03771792, 研究期間(年度):1991出典：研究課題「初代培養脳毛細血管内皮細胞系を用いた薬物輸送」課題番号03771792（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/grant/KAKENHI-PROJECT-03771792/）を加工して作

1α,25-Dihydroxyvitamin D3 enhances cerebral clearance of human amyloid-β peptide(1-40) from mouse brain across the blood-brain barrier

<p>Abstract</p> <p>Background</p> <p>Cerebrovascular dysfunction has been considered to cause impairment of cerebral amyloid-β peptide (Aβ) clearance across the blood-brain barrier (BBB). Further, low levels of vitamin D are associated with increased risk of Alzheimer's disease, as well as vascular dysfunction. The purpose of the present study was to investigate the effect of 1α,25-dihydroxyvitamin D₃(1,25(OH)₂D3), an active form of vitamin D, on cerebral Aβ clearance from mouse brain.</p> <p>Methods</p> <p>The elimination of [¹²⁵I]hAβ(1-40) from mouse brain was examined by using the Brain Efflux Index method to determine the remaining amount of [¹²⁵I]hAβ(1-40) radioactivity after injection into the cerebral cortex. [¹²⁵I]hAβ(1-40) internalization was analyzed using conditionally immortalized mouse brain capillary endothelial cells (TM-BBB4).</p> <p>Results</p> <p>Twenty-four hours after intraperitoneal injection of 1,25(OH)₂D3 (1 μg/mouse), [¹²⁵I]hAβ(1-40) elimination from mouse brain was increased 1.3-fold, and the level of endogenous Aβ(1-40) in mouse brain was reduced. These effects were observed at 24 h after i.p. injection of 1,25(OH)₂D3, while no significant effect was observed at 48 or 72 h. Vitamin D receptor (VDR) mRNA was detected in mouse brain capillaries, suggesting that 1,25(OH)₂D3 has a VDR-mediated genomic action. Furthermore, forskolin, which activates mitogen-activated protein kinase kinase (MEK), enhanced [¹²⁵I]hAβ(1-40) elimination from mouse brain. Forskolin also enhanced [¹²⁵I]hAβ(1-40) internalization in TM-BBB4 cells, and this enhancement was inhibited by a MEK inhibitor, suggesting involvement of non-genomic action.</p> <p>Conclusions</p> <p>The active form of vitamin D, 1,25(OH)₂D3, appears to enhance brain-to-blood Aβ(1-40) efflux transport at the BBB through both genomic and non-genomic actions. Compounds activating these pathways may be candidate agents for modulating Aβ(1-40) elimination at the BBB.</p

Major role of organic anion transporter 3 in the transport of indoxyl sulfate in the kidney

Major role of organic anion transporter 3 in the transport of indoxyl sulfate in the kidney.BackgroundIndoxyl sulfate is a uremic toxin that accumulates in the body because of the patient's inability to excrete it and it induces a number of uremic symptoms and leads to chronic renal failure. The functional failure of the excretion system for indoxyl sulfate causes its accumulation in blood. The purpose of the present study was to characterize the transport mechanism responsible for the renal excretion of indoxyl sulfate.MethodsThe [3H]indoxyl sulfate transport mechanism was investigated using an in vivo tissue-sampling single-injection technique, the kidney uptake index (KUI) method. Rat organic anion transporter 3 (rOAT3)-expressing Xenopus laevis oocyte system was used for measuring [3H]indoxyl sulfate uptake activity.ResultsProbenecid showed a concentration-dependent inhibitory effect on the uptake of [3H]indoxyl sulfate using the KUI method, and uptake was inhibited by organic anions such as para-aminohippuric acid (PAH) and benzylpenicillin, by weak base such as cimetidine, and by uremic toxins, such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and hippuric acid (HA). However, salicylic acid, indomethacin, 3,5,3′-triiodo-L-thyronine and indole acetic acid (IA) had no effect on the uptake. rOAT3-expressing oocytes exhibited uptake of [3H]indoxyl sulfate by rOAT3 (Km = 158 μmol/L). Moreover, a number of uremic toxins inhibited the uptake of [3H]indoxyl sulfate by rOAT3.ConclusionsThese results suggest that rOAT3 is responsible for the renal uptake of indoxyl sulfate, and uremic toxins share the transport mechanism for indoxyl sulfate. Mutual inhibition of these uremic toxins via OAT3 may accelerate their accumulation in the body and, thereby, the progression of nephrotoxicity in uremia

Attenuation of prostaglandin E2 elimination across the mouse blood-brain barrier in lipopolysaccharide-induced inflammation and additive inhibitory effect of cefmetazole

<p>Abstract</p> <p>Background</p> <p>Peripheral administration of lipopolysaccharide (LPS) induces inflammation and increases cerebral prostaglandin E₂(PGE₂) concentration. PGE₂is eliminated from brain across the blood-brain barrier (BBB) in mice, and this process is inhibited by intracerebral or intravenous pre-administration of anti-inflammatory drugs and antibiotics such as cefmetazole and cefazolin that inhibit multidrug resistance-associated protein 4 (Mrp4/Abcc4)-mediated PGE₂transport. The purpose of this study was to examine the effect of LPS-induced inflammation on PGE₂elimination from brain, and whether antibiotics further inhibit PGE₂elimination in LPS-treated mice.</p> <p>Methods</p> <p>[³H]PGE₂elimination across the BBB of intraperitoneally LPS-treated mice was assessed by the brain efflux index (BEI) method. Transporter protein amounts in brain capillaries were quantified by liquid chromatography-tandem mass spectrometry.</p> <p>Results</p> <p>The apparent elimination rate of [³H]PGE₂from brain was lower by 87%, in LPS-treated mice compared with saline-treated mice. The Mrp4 protein amount was unchanged in brain capillaries of LPS-treated mice compared with saline-treated mice, while the protein amounts of organic anion transporter 3 (Oat3/Slc22a8) and organic anion transporting polypeptide 1a4 (Oatp1a4/Slco1a4) were decreased by 26% and 39%, respectively. Either intracerebral or intravenous pre-administration of cefmetazole further inhibited PGE₂elimination in LPS-treated mice. However, intracerebral or intravenous pre-administration of cefazolin had little effect on PGE₂elimination in LPS-treated mice, or in LPS-untreated mice given Oat3 and Oatp1a4 inhibitors. These results indicate that peripheral administration of cefmetazole inhibits PGE₂elimination across the BBB in LPS-treated mice.</p> <p>Conclusion</p> <p>PGE₂elimination across the BBB is attenuated in an LPS-induced mouse model of inflammation. Peripheral administration of cefmetazole further inhibits PGE₂elimination in LPS-treated mice.</p

Kinetic evidence for active efflux transport across the blood-brain barrier of quinolone antibiotics.

ABSTRACT A distributed model has been used to clarify the mechanism of the restricted and differential distribution of the quinolone antibiotics in the rat central nervous system (CNS). The symmetrical permeability clearances across the blood-brain barrier (BBB), PS BBB , and across the blood-cerebrospinal fluid barrier (BCSFB), PS CSF , and the active efflux clearances across the BBB, PS BBB,eff , were obtained from a nonlinear least squares regression analysis combined with the fast inverse Laplace transforming program for in vivo data. The values of PS BBB,eff were 10-to 260-fold greater than those of PS BBB , providing kinetic evidence to support the hypothesis that a significant efflux transport across the BBB is responsible for the limited distribution of quinolones in brain tissue. Moreover, by simulation studies, we could demonstrate the concentration profiles in the brain as a function of the distance from the ependymal surface. However, active efflux transport across the BCSFB has been suggested to have only a slight effect on the apparent elimination from the cerebrospinal fluid. Comparing the apparent brain tissue-to-unbound serum concentration ratio at steady state, it has been suggested that the net flux across the BBB, i.e., the ratio of PS BBB to the sum of PS BBB and PS BBB,eff , is a determinant for the differential distribution of these quinolones in brain tissue. Such a putative active efflux transport system would play a significant role in decreasing the brain interstitial fluid concentration of quinolones. The side effect of quinolone antimicrobial agents (quinolones) on the CNS such as confusion, hallucinations, anxiety, agitation, depression and convulsive seizures is one of the most serious problems associated with their use as chemotherapeutic agents The present study investigated the process governing the restricted distribution of quinolones in the CNS based on the distributed model. In this study, we determined the initia