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

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

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

Thermoelectric phase diagram of the SrTiO3-SrNbO3 solid solution system

Thermoelectric energy conversion - the exploitation of the Seebeck effect to convert waste heat into electricity - has attracted an increasing amount of research attention for energy harvesting technology. Niobium-doped strontium titanate (SrTi1-xNbxO3) is one of the most promising thermoelectric material candidates, particularly as it poses a much lesser environmental risk in comparison to materials based on heavy metal elements. Two-dimensional electron confinement, e.g. through the formation of superlattices or two-dimensional electron gases, is recognized as an effective strategy to improve the thermoelectric performance of SrTi1-xNbxO3. Although electron confinement is closely related to the electronic structure, the fundamental electronic phase behavior of the SrTi1-xNbxO3 solid solution system has yet to be comprehensively investigated. Here, we present a thermoelectric phase diagram for the SrTi1-xNbxO3 (0.05 =< x =< 1) solid solution system, which we derived from the characterization of epitaxial films. We observed two thermoelectric phase boundaries in the system, which originate from the step-like decrease in carrier effective mass at x ~ 0.3, and from a local minimum in carrier relaxation time at x ~ 0.5. The origins of these phase boundaries are considered to be related to isovalent/heterovalent B-site substitution: parabolic Ti 3d orbitals dominate electron conduction for compositions with x < 0.3, whereas the Nb 4d orbital dominates when x > 0.3. At x ~ 0.5, a tetragonal distortion of the lattice, in which the B-site is composed of Ti4+ and Nb4+ ions, leads to the formation of tail-like impurity bands, which maximizes the electron scattering. These results provide a foundation for further research into improving the thermoelectric performance of SrTi1-xNbxO3.Comment: 20 pages, 6 figure