PMC:6194691 / 44989-73998 JSONTXT

{"project":"MyTest","denotations":[{"id":"30340614-20468051-30706066","span":{"begin":588,"end":591},"obj":"20468051"},{"id":"30340614-20468051-30706067","span":{"begin":698,"end":701},"obj":"20468051"},{"id":"30340614-27799072-30706068","span":{"begin":962,"end":963},"obj":"27799072"},{"id":"30340614-3468856-30706069","span":{"begin":1577,"end":1580},"obj":"3468856"},{"id":"30340614-27799072-30706070","span":{"begin":1610,"end":1611},"obj":"27799072"},{"id":"30340614-5499038-30706071","span":{"begin":1740,"end":1743},"obj":"5499038"},{"id":"30340614-8592149-30706072","span":{"begin":1760,"end":1763},"obj":"8592149"},{"id":"30340614-25481827-30706073","span":{"begin":3429,"end":3431},"obj":"25481827"},{"id":"30340614-19395337-30706074","span":{"begin":3433,"end":3436},"obj":"19395337"},{"id":"30340614-10696512-30706075","span":{"begin":4721,"end":4724},"obj":"10696512"},{"id":"30340614-25675910-30706076","span":{"begin":4726,"end":4729},"obj":"25675910"},{"id":"30340614-7392035-30706077","span":{"begin":5707,"end":5710},"obj":"7392035"},{"id":"30340614-11259830-30706078","span":{"begin":6742,"end":6745},"obj":"11259830"},{"id":"30340614-9466345-30706079","span":{"begin":6972,"end":6975},"obj":"9466345"},{"id":"30340614-15051171-30706079","span":{"begin":6972,"end":6975},"obj":"15051171"},{"id":"30340614-21374607-30706079","span":{"begin":6972,"end":6975},"obj":"21374607"},{"id":"30340614-10696512-30706080","span":{"begin":7514,"end":7517},"obj":"10696512"},{"id":"30340614-9466345-30706081","span":{"begin":7524,"end":7527},"obj":"9466345"},{"id":"30340614-9466345-30706082","span":{"begin":7929,"end":7932},"obj":"9466345"},{"id":"30340614-15051171-30706083","span":{"begin":7934,"end":7937},"obj":"15051171"},{"id":"30340614-9466345-30706084","span":{"begin":8291,"end":8294},"obj":"9466345"},{"id":"30340614-15051171-30706085","span":{"begin":8296,"end":8299},"obj":"15051171"},{"id":"30340614-10696512-30706086","span":{"begin":8497,"end":8500},"obj":"10696512"},{"id":"30340614-15051171-30706087","span":{"begin":8502,"end":8505},"obj":"15051171"},{"id":"30340614-9466345-30706088","span":{"begin":9033,"end":9036},"obj":"9466345"},{"id":"30340614-14709630-30706089","span":{"begin":9489,"end":9492},"obj":"14709630"},{"id":"30340614-9886238-30706090","span":{"begin":9816,"end":9819},"obj":"9886238"},{"id":"30340614-21374607-30706091","span":{"begin":9882,"end":9885},"obj":"21374607"},{"id":"30340614-26189671-30706092","span":{"begin":9887,"end":9890},"obj":"26189671"},{"id":"30340614-25675910-30706093","span":{"begin":9958,"end":9961},"obj":"25675910"},{"id":"30340614-26305616-30706094","span":{"begin":10470,"end":10473},"obj":"26305616"},{"id":"30340614-16306934-30706095","span":{"begin":11766,"end":11769},"obj":"16306934"},{"id":"30340614-19429473-30706095","span":{"begin":11766,"end":11769},"obj":"19429473"},{"id":"30340614-21060791-30706095","span":{"begin":11766,"end":11769},"obj":"21060791"},{"id":"30340614-21707071-30706095","span":{"begin":11766,"end":11769},"obj":"21707071"},{"id":"30340614-25186741-30706095","span":{"begin":11766,"end":11769},"obj":"25186741"},{"id":"30340614-28188910-30706095","span":{"begin":11766,"end":11769},"obj":"28188910"},{"id":"30340614-21349151-30706096","span":{"begin":11785,"end":11787},"obj":"21349151"},{"id":"30340614-16365086-30706097","span":{"begin":11789,"end":11791},"obj":"16365086"},{"id":"30340614-25703048-30706098","span":{"begin":11793,"end":11795},"obj":"25703048"},{"id":"30340614-21707071-30706099","span":{"begin":11797,"end":11800},"obj":"21707071"},{"id":"30340614-17619998-30706100","span":{"begin":11802,"end":11805},"obj":"17619998"},{"id":"30340614-18619525-30706100","span":{"begin":11802,"end":11805},"obj":"18619525"},{"id":"30340614-19094056-30706100","span":{"begin":11802,"end":11805},"obj":"19094056"},{"id":"30340614-18219561-30706100","span":{"begin":11802,"end":11805},"obj":"18219561"},{"id":"30340614-21792245-30706100","span":{"begin":11802,"end":11805},"obj":"21792245"},{"id":"30340614-21291474-30706100","span":{"begin":11802,"end":11805},"obj":"21291474"},{"id":"30340614-23650139-30706100","span":{"begin":11802,"end":11805},"obj":"23650139"},{"id":"30340614-24702234-30706100","span":{"begin":11802,"end":11805},"obj":"24702234"},{"id":"30340614-26661193-30706100","span":{"begin":11802,"end":11805},"obj":"26661193"},{"id":"30340614-28954515-30706100","span":{"begin":11802,"end":11805},"obj":"28954515"},{"id":"30340614-27799072-30706101","span":{"begin":11828,"end":11829},"obj":"27799072"},{"id":"30340614-23298398-30706102","span":{"begin":11831,"end":11833},"obj":"23298398"},{"id":"30340614-21349151-30706103","span":{"begin":11835,"end":11837},"obj":"21349151"},{"id":"30340614-19664713-30706104","span":{"begin":11839,"end":11841},"obj":"19664713"},{"id":"30340614-25481827-30706105","span":{"begin":11843,"end":11845},"obj":"25481827"},{"id":"30340614-17619998-30706106","span":{"begin":11850,"end":11853},"obj":"17619998"},{"id":"30340614-6361813-30706107","span":{"begin":11855,"end":11858},"obj":"6361813"},{"id":"30340614-11734619-30706107","span":{"begin":11855,"end":11858},"obj":"11734619"},{"id":"30340614-16111480-30706107","span":{"begin":11855,"end":11858},"obj":"16111480"},{"id":"30340614-15717058-30706107","span":{"begin":11855,"end":11858},"obj":"15717058"},{"id":"30340614-19393264-30706107","span":{"begin":11855,"end":11858},"obj":"19393264"},{"id":"30340614-21118088-30706107","span":{"begin":11855,"end":11858},"obj":"21118088"},{"id":"30340614-23789951-30706107","span":{"begin":11855,"end":11858},"obj":"23789951"},{"id":"30340614-25561720-30706107","span":{"begin":11855,"end":11858},"obj":"25561720"},{"id":"30340614-26187753-30706107","span":{"begin":11855,"end":11858},"obj":"26187753"},{"id":"30340614-27503090-30706107","span":{"begin":11855,"end":11858},"obj":"27503090"},{"id":"30340614-18619525-30706108","span":{"begin":11896,"end":11899},"obj":"18619525"},{"id":"30340614-25703048-30706109","span":{"begin":11918,"end":11920},"obj":"25703048"},{"id":"30340614-16365086-30706110","span":{"begin":11953,"end":11955},"obj":"16365086"},{"id":"30340614-21349151-30706111","span":{"begin":11966,"end":11968},"obj":"21349151"},{"id":"30340614-25481827-30706112","span":{"begin":11987,"end":11989},"obj":"25481827"},{"id":"30340614-26661193-30706113","span":{"begin":12018,"end":12021},"obj":"26661193"},{"id":"30340614-27503090-30706114","span":{"begin":12035,"end":12038},"obj":"27503090"},{"id":"30340614-23506860-30706115","span":{"begin":12379,"end":12382},"obj":"23506860"},{"id":"30340614-7392035-30706116","span":{"begin":12744,"end":12747},"obj":"7392035"},{"id":"30340614-7910522-30706117","span":{"begin":12989,"end":12992},"obj":"7910522"},{"id":"30340614-2563168-30706118","span":{"begin":13069,"end":13072},"obj":"2563168"},{"id":"30340614-2463300-30706118","span":{"begin":13069,"end":13072},"obj":"2463300"},{"id":"30340614-1975805-30706118","span":{"begin":13069,"end":13072},"obj":"1975805"},{"id":"30340614-1356979-30706118","span":{"begin":13069,"end":13072},"obj":"1356979"},{"id":"30340614-12486090-30706118","span":{"begin":13069,"end":13072},"obj":"12486090"},{"id":"30340614-16361063-30706118","span":{"begin":13069,"end":13072},"obj":"16361063"},{"id":"30340614-1968065-30706119","span":{"begin":13454,"end":13457},"obj":"1968065"},{"id":"30340614-7910961-30706119","span":{"begin":13454,"end":13457},"obj":"7910961"},{"id":"30340614-24624364-30706119","span":{"begin":13454,"end":13457},"obj":"24624364"},{"id":"30340614-19325113-30706119","span":{"begin":13454,"end":13457},"obj":"19325113"},{"id":"30340614-19325113-30706120","span":{"begin":13558,"end":13561},"obj":"19325113"},{"id":"30340614-18619525-30706121","span":{"begin":14380,"end":14383},"obj":"18619525"},{"id":"30340614-23789951-30706122","span":{"begin":14385,"end":14388},"obj":"23789951"},{"id":"30340614-12438926-30706123","span":{"begin":14390,"end":14393},"obj":"12438926"},{"id":"30340614-12706238-30706123","span":{"begin":14390,"end":14393},"obj":"12706238"},{"id":"30340614-12958161-30706123","span":{"begin":14390,"end":14393},"obj":"12958161"},{"id":"30340614-15126373-30706123","span":{"begin":14390,"end":14393},"obj":"15126373"},{"id":"30340614-17196184-30706123","span":{"begin":14390,"end":14393},"obj":"17196184"},{"id":"30340614-18619525-30706124","span":{"begin":14462,"end":14465},"obj":"18619525"},{"id":"30340614-23789951-30706125","span":{"begin":14467,"end":14470},"obj":"23789951"},{"id":"30340614-16361063-30706126","span":{"begin":14472,"end":14475},"obj":"16361063"},{"id":"30340614-17196184-30706127","span":{"begin":14477,"end":14480},"obj":"17196184"},{"id":"30340614-15314169-30706127","span":{"begin":14477,"end":14480},"obj":"15314169"},{"id":"30340614-15501592-30706127","span":{"begin":14477,"end":14480},"obj":"15501592"},{"id":"30340614-18619525-30706128","span":{"begin":14722,"end":14725},"obj":"18619525"},{"id":"30340614-21291474-30706129","span":{"begin":14727,"end":14730},"obj":"21291474"},{"id":"30340614-17196184-30706130","span":{"begin":14732,"end":14735},"obj":"17196184"},{"id":"30340614-9489736-30706131","span":{"begin":14737,"end":14740},"obj":"9489736"},{"id":"30340614-9681461-30706131","span":{"begin":14737,"end":14740},"obj":"9681461"},{"id":"30340614-10421622-30706131","span":{"begin":14737,"end":14740},"obj":"10421622"},{"id":"30340614-12817896-30706131","span":{"begin":14737,"end":14740},"obj":"12817896"},{"id":"30340614-18680196-30706131","span":{"begin":14737,"end":14740},"obj":"18680196"},{"id":"30340614-11879359-30706132","span":{"begin":14823,"end":14826},"obj":"11879359"},{"id":"30340614-15101825-30706132","span":{"begin":14823,"end":14826},"obj":"15101825"},{"id":"30340614-16190927-30706132","span":{"begin":14823,"end":14826},"obj":"16190927"},{"id":"30340614-25481827-30706133","span":{"begin":15010,"end":15012},"obj":"25481827"},{"id":"30340614-21118088-30706134","span":{"begin":15014,"end":15017},"obj":"21118088"},{"id":"30340614-23789951-30706135","span":{"begin":15019,"end":15022},"obj":"23789951"},{"id":"30340614-26187753-30706136","span":{"begin":15024,"end":15027},"obj":"26187753"},{"id":"30340614-12535575-30706137","span":{"begin":15034,"end":15037},"obj":"12535575"},{"id":"30340614-15134482-30706137","span":{"begin":15034,"end":15037},"obj":"15134482"},{"id":"30340614-16025095-30706137","span":{"begin":15034,"end":15037},"obj":"16025095"},{"id":"30340614-16337012-30706137","span":{"begin":15034,"end":15037},"obj":"16337012"},{"id":"30340614-16714484-30706137","span":{"begin":15034,"end":15037},"obj":"16714484"},{"id":"30340614-25481827-30706138","span":{"begin":17620,"end":17622},"obj":"25481827"},{"id":"30340614-21707071-30706139","span":{"begin":17624,"end":17627},"obj":"21707071"},{"id":"30340614-27503090-30706140","span":{"begin":17629,"end":17632},"obj":"27503090"},{"id":"30340614-17196184-30706141","span":{"begin":17634,"end":17637},"obj":"17196184"},{"id":"30340614-12883891-30706142","span":{"begin":17639,"end":17642},"obj":"12883891"},{"id":"30340614-15717059-30706142","span":{"begin":17639,"end":17642},"obj":"15717059"},{"id":"30340614-16357150-30706142","span":{"begin":17639,"end":17642},"obj":"16357150"},{"id":"30340614-22013971-30706142","span":{"begin":17639,"end":17642},"obj":"22013971"},{"id":"30340614-21854228-30706142","span":{"begin":17639,"end":17642},"obj":"21854228"},{"id":"30340614-24013810-30706142","span":{"begin":17639,"end":17642},"obj":"24013810"},{"id":"30340614-23343976-30706142","span":{"begin":17639,"end":17642},"obj":"23343976"},{"id":"30340614-23506880-30706142","span":{"begin":17639,"end":17642},"obj":"23506880"},{"id":"30340614-23789957-30706142","span":{"begin":17639,"end":17642},"obj":"23789957"},{"id":"30340614-27320645-30706142","span":{"begin":17639,"end":17642},"obj":"27320645"},{"id":"30340614-27783531-30706142","span":{"begin":17639,"end":17642},"obj":"27783531"},{"id":"30340614-22013971-30706143","span":{"begin":18415,"end":18418},"obj":"22013971"},{"id":"30340614-14579113-30706144","span":{"begin":18598,"end":18601},"obj":"14579113"},{"id":"30340614-9399971-30706145","span":{"begin":19157,"end":19160},"obj":"9399971"},{"id":"30340614-12684544-30706146","span":{"begin":19231,"end":19234},"obj":"12684544"},{"id":"30340614-15504935-30706147","span":{"begin":19303,"end":19306},"obj":"15504935"},{"id":"30340614-16639426-30706148","span":{"begin":19347,"end":19350},"obj":"16639426"},{"id":"30340614-12684544-30706149","span":{"begin":19394,"end":19397},"obj":"12684544"},{"id":"30340614-9694947-30706150","span":{"begin":19448,"end":19451},"obj":"9694947"},{"id":"30340614-9694947-30706151","span":{"begin":19473,"end":19476},"obj":"9694947"},{"id":"30340614-11105986-30706152","span":{"begin":19513,"end":19516},"obj":"11105986"},{"id":"30340614-11105986-30706153","span":{"begin":19545,"end":19548},"obj":"11105986"},{"id":"30340614-11032880-30706154","span":{"begin":19604,"end":19607},"obj":"11032880"},{"id":"30340614-11408557-30706155","span":{"begin":19699,"end":19702},"obj":"11408557"},{"id":"30340614-15292460-30706156","span":{"begin":19759,"end":19762},"obj":"15292460"},{"id":"30340614-15292460-30706157","span":{"begin":19839,"end":19842},"obj":"15292460"},{"id":"30340614-12679720-30706158","span":{"begin":19916,"end":19919},"obj":"12679720"},{"id":"30340614-12358729-30706159","span":{"begin":19969,"end":19972},"obj":"12358729"},{"id":"30340614-23297298-30706160","span":{"begin":20020,"end":20023},"obj":"23297298"},{"id":"30340614-23297298-30706161","span":{"begin":20101,"end":20104},"obj":"23297298"},{"id":"30340614-17365275-30706162","span":{"begin":20165,"end":20168},"obj":"17365275"},{"id":"30340614-9103519-30706163","span":{"begin":20251,"end":20254},"obj":"9103519"},{"id":"30340614-9103519-30706164","span":{"begin":20334,"end":20337},"obj":"9103519"},{"id":"30340614-24136970-30706165","span":{"begin":20477,"end":20480},"obj":"24136970"},{"id":"30340614-11120756-30706166","span":{"begin":20516,"end":20518},"obj":"11120756"},{"id":"30340614-16639426-30706167","span":{"begin":20552,"end":20555},"obj":"16639426"},{"id":"30340614-16639426-30706168","span":{"begin":20659,"end":20662},"obj":"16639426"},{"id":"30340614-16639426-30706169","span":{"begin":20697,"end":20700},"obj":"16639426"},{"id":"30340614-16639426-30706170","span":{"begin":20740,"end":20743},"obj":"16639426"},{"id":"30340614-16639426-30706171","span":{"begin":20782,"end":20785},"obj":"16639426"},{"id":"30340614-16639426-30706172","span":{"begin":21059,"end":21062},"obj":"16639426"},{"id":"30340614-18619525-30706173","span":{"begin":22554,"end":22557},"obj":"18619525"},{"id":"30340614-6033532-30706174","span":{"begin":23584,"end":23587},"obj":"6033532"},{"id":"30340614-10696507-30706174","span":{"begin":23584,"end":23587},"obj":"10696507"},{"id":"30340614-12843411-30706174","span":{"begin":23584,"end":23587},"obj":"12843411"},{"id":"30340614-18726697-30706174","span":{"begin":23584,"end":23587},"obj":"18726697"},{"id":"30340614-26794270-30706175","span":{"begin":24261,"end":24263},"obj":"26794270"},{"id":"30340614-2600816-30706176","span":{"begin":24352,"end":24355},"obj":"2600816"},{"id":"30340614-26794270-30706177","span":{"begin":24387,"end":24389},"obj":"26794270"},{"id":"30340614-18726697-30706178","span":{"begin":24391,"end":24394},"obj":"18726697"},{"id":"30340614-15722162-30706179","span":{"begin":24396,"end":24399},"obj":"15722162"},{"id":"30340614-9443901-30706180","span":{"begin":24407,"end":24410},"obj":"9443901"},{"id":"30340614-7680372-30706181","span":{"begin":24477,"end":24480},"obj":"7680372"},{"id":"30340614-8227337-30706182","span":{"begin":24562,"end":24565},"obj":"8227337"},{"id":"30340614-7680372-30706183","span":{"begin":24801,"end":24804},"obj":"7680372"},{"id":"30340614-7514652-30706184","span":{"begin":24806,"end":24809},"obj":"7514652"},{"id":"30340614-25481827-30706185","span":{"begin":25403,"end":25405},"obj":"25481827"},{"id":"30340614-25340933-30706186","span":{"begin":25407,"end":25409},"obj":"25340933"},{"id":"30340614-19395337-30706187","span":{"begin":25411,"end":25414},"obj":"19395337"},{"id":"30340614-10696507-30706188","span":{"begin":25416,"end":25419},"obj":"10696507"},{"id":"30340614-2688350-30706189","span":{"begin":25426,"end":25429},"obj":"2688350"},{"id":"30340614-15180331-30706189","span":{"begin":25426,"end":25429},"obj":"15180331"},{"id":"30340614-16777679-30706189","span":{"begin":25426,"end":25429},"obj":"16777679"},{"id":"30340614-17619996-30706189","span":{"begin":25426,"end":25429},"obj":"17619996"},{"id":"30340614-26687840-30706189","span":{"begin":25426,"end":25429},"obj":"26687840"},{"id":"30340614-29067674-30706189","span":{"begin":25426,"end":25429},"obj":"29067674"},{"id":"30340614-10696507-30706190","span":{"begin":25542,"end":25545},"obj":"10696507"},{"id":"30340614-12843411-30706191","span":{"begin":25547,"end":25550},"obj":"12843411"},{"id":"30340614-16777679-30706192","span":{"begin":25552,"end":25555},"obj":"16777679"},{"id":"30340614-8912898-30706193","span":{"begin":25557,"end":25560},"obj":"8912898"},{"id":"30340614-14597140-30706194","span":{"begin":25562,"end":25565},"obj":"14597140"},{"id":"30340614-11240028-30706195","span":{"begin":26102,"end":26105},"obj":"11240028"},{"id":"30340614-16354907-30706195","span":{"begin":26102,"end":26105},"obj":"16354907"},{"id":"30340614-19636712-30706195","span":{"begin":26102,"end":26105},"obj":"19636712"},{"id":"30340614-22202501-30706195","span":{"begin":26102,"end":26105},"obj":"22202501"},{"id":"30340614-23978455-30706195","span":{"begin":26102,"end":26105},"obj":"23978455"},{"id":"30340614-22838637-30706195","span":{"begin":26102,"end":26105},"obj":"22838637"},{"id":"30340614-28624473-30706195","span":{"begin":26102,"end":26105},"obj":"28624473"},{"id":"30340614-3224280-30706196","span":{"begin":26131,"end":26133},"obj":"3224280"},{"id":"30340614-11238745-30706197","span":{"begin":26188,"end":26190},"obj":"11238745"},{"id":"30340614-11238745-30706198","span":{"begin":26658,"end":26660},"obj":"11238745"},{"id":"30340614-3694713-30706199","span":{"begin":26662,"end":26665},"obj":"3694713"},{"id":"30340614-3224280-30706200","span":{"begin":26855,"end":26857},"obj":"3224280"},{"id":"30340614-1450923-30706201","span":{"begin":26859,"end":26862},"obj":"1450923"},{"id":"30340614-9593852-30706202","span":{"begin":26864,"end":26867},"obj":"9593852"},{"id":"30340614-14580938-30706203","span":{"begin":27058,"end":27061},"obj":"14580938"},{"id":"30340614-25315861-30706204","span":{"begin":27804,"end":27807},"obj":"25315861"},{"id":"30340614-26687231-30706204","span":{"begin":27804,"end":27807},"obj":"26687231"},{"id":"30340614-29054412-30706204","span":{"begin":27804,"end":27807},"obj":"29054412"},{"id":"30340614-3224280-30706205","span":{"begin":28121,"end":28123},"obj":"3224280"},{"id":"30340614-9593852-30706206","span":{"begin":28305,"end":28308},"obj":"9593852"},{"id":"30340614-11238745-30706207","span":{"begin":28372,"end":28374},"obj":"11238745"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"The blood–brain barrier\nThe blood–brain barrier is more selective than the perivascular pathway in what can and cannot permeate. This selectivity arises from the properties of the endothelial cells surrounding the microvessels. The brain is highly vascularized and cells within the parenchyma are usually within 20 µm of a microvessel [148]. Diffusion over distances this short is rapid. To reach the microvessel, substances must also cross the surrounding layer composed of glial endfeet. This is normally possible because the gaps between the endfeet are not sealed by tight junctions [149, 150]. Even the almost complete coverage of the endothelial cells by glial endfeet proposed by Mathiisen [149] leaves sufficient gaps (see Footnote 7). Thus normally it is the endothelial cells that are the site for the rate limiting steps in efflux across the blood–brain barrier. The current state of knowledge about the role of the endfeet was considered further in [4].\n\nPassive, non-specific transfer across the blood–brain barrier\nThere are two possible routes for passive, non-specific transfer across the microvascular endothelial layer, through the cells or around them. The paracellular pathway is “blocked” by the presence of tight junctions but this pathway may still be the principal route for the passive fluxes of small solutes that are barred from the transcellular route by being too polar (mannitol, sucrose and inulin are considered in Appendix B). In addition to neutral molecules like mannitol, the paracellular pathway may be measurably permeable to Na+ and Cl− [151]. As discussed in detail in [4] and in Sect. 5.6 evidence for this includes the observation that the tracer fluxes of Na+ and Cl− are not affected by ouabain [152] or bumetanide [153], agents that specifically inhibit ion transporters known to be involved in transcellular fluxes of these ions.\nAlmost all of the passive, non-selective permeability of the blood–brain barrier to molecules more lipophilic than mannitol is the result of their ability to diffuse across both the cell membranes and the interior of the endothelial cells. Strong indications that such a physical mechanism applies are the observations: that transport does not saturate, that it is not inhibited by competition by other transported substances, and that no specific inhibitors have been found. Small neutral substances that are able to enter and leave the brain parenchyma by this mechanism include water, methanol, ethanol, isopropanol, glycerol, ethylene glycol, urea and thiourea (see Fig. 8).\nFig. 8 Plot of log(PS/mL g−1 min−1), versus log(Kn-octanol/water MW−1/2) for the substances indicated along the abscissa. PS is the product of permeability and surface area for the blood–brain barrier, Kn-octanol/water is the octanol/water partition coefficient and MW is the molecular weight of the substance. The slope of 1 for the heavy blue line indicates PS proportional to Kn-octanol/water MW−1/2. A closer fit to the data can be obtained by allowing the slope to vary, shown as the thin red line, but the improvement in fit is not statistically significant (F = 2.33, p = 0.11, n = 43, extra sum of squares F test [640])\n(Data read from Figure 8 of [159])\nMost studies of the passive permeability of the blood–brain barrier have focussed on influx, because it is easier to measure and has obvious importance for the delivery of agents and drugs to the CNS (see e.g. [57, 154]). However, passive permeability allows both influx and efflux and thus these studies are directly relevant to understanding how substances are eliminated from the parenchyma.\nIn the simplest view the rate limiting steps in the transcellular, passive, unmediated transfer of substances can be thought of as occurring by dissolution in a liquid hydrophobic core of the membranes and diffusion through it. For molecules not much larger than those of the solvent the diffusion constant for the various compounds is taken to be inversely proportional to the square root of their molecular weights [155–157]. The exact relationship assumed is not critical because the dominant factor determining the relative permeabilities is the free energy cost of the transfer from water into the core of the membrane, ΔGmembrane/water. This cost determines the relative concentrations in the membrane and the aqueous phase,5 cmembranecwater=Kmembrane/water=e-ΔGmembrane/water/RTwhere Kmembrane/water is the partition coefficient, R the universal gas constant, and T the absolute temperature. The free energy cost and the partition coefficient are usually estimated by assuming that the membrane core can be described as being like a layer of n-octanol (see [158, 159] and for more recent discussions [160, 161]), and thus6 cmembranecwater∝Kn-octanol/water=e-ΔGn-octanol/water/RT.\nIt is likely that n-octanol rather than, say, n-octane is appropriate as a model for the membrane interior because the –OH group can participate in hydrogen bonds.\nFenstermacher [159] reviewed the studies up to 1984 with the result summarized in a plot of log[PS] versus log[Kn-octanol/water MW−1/2] (see Fig. 8) where PS is the permeability surface area product for brain capillaries. For the substances listed in the figure, which have simple structures and molecular weights less than 200, the slope of the loglog plot is not significantly different from 1, i.e. PS appears to be proportional to Kn-octanol/water MW−1/2.\nThere have been many other reports based on studies using more complicated or larger molecules. These have usually reported a linear relation between log(PS) and either log[Kn-octanol/water] or log[Kn-octanol/water MW−1/2] but often with a slope substantially less than 1 (see e.g. [162, 163]). It should be emphasized that slope not equal to 1 means that the fluxes are not proportional to Kn-octanol/water MW−1/2 and thus, for at least some of the substances tested, simple diffusion and partition into an environment that looks like n-octanol are not the only important factors that need to be considered. The appropriate factors are considered further in Appendix C.\nCorrelating the passive permeabilities for substances at the blood–brain barrier with their partition coefficients for transfer from water to n-octanol has the virtue of focussing attention on the most critical aspect of the passive permeation process, the free energy cost of removing the solute from water and inserting it into a relatively hydrophobic environment. However, these correlations have been thought too imprecise to use as a criteria for selecting candidates to consider in a drug discovery setting. There have been many attempts to do better, some in terms of a set of rules analogous to the “rule of 5” for intestinal absorption [164], some using better estimates of the free energy cost for solutes to reach the rate limiting step of the transport, and some using a mixture of both.\nTo obtain better estimates of the free energy, Abraham and colleagues (see [165–168]) have employed linear free energy relations, LFER, to calculate correlations based on a two step process. First quantitative “descriptors” of the molecules under consideration are chosen without regard to the process of interest. Then, once the descriptors have been chosen, the relevant free energy changes for processes such as partition into a solvent or permeability across the blood–brain barrier, are calculated as linear sums of the descriptors with coefficients that depend on the process but not on the molecules (see e.g. [160, 165, 166]. Having used data for some substances to calculate the LFER coefficients, these can then be used for other substances. This approach has been applied with considerable success to partition into solvents for many more molecules than are needed to calculate the coefficients [165]. It has also allowed closer prediction of blood–brain barrier permeabilities than the simple solubility-diffusion model [166, 167] (see Appendix C).\nThere is, however, a danger in adopting this approach to the prediction of permeability. The use of linear free energy relations reveals correlations between the descriptors and the rate of transport, but unless used carefully it can obscure important features of the mechanism. For instance in the correlations reported for log(PS) [166, 167], the strongest correlation was a positive correlation between molecular volume and permeability, i.e. this approach seems to say that increases in molecular size result in increased permeability [160, 167]. However, the idea that bigger objects will be more permeable because they are bigger is completely counter-intuitive. The likely explanation for this paradox is simple. For the molecules considered in the correlations, increases in molecular volume were associated with large increases in lipophilicity as measured by Kn-octanol/water and it is plausible that it was the increase in lipophilicity that increased the permeability. Indeed as shown in Appendix C Abraham’s descriptor approach predicts for the compounds tested [166] that log[PS/Kn-octanol/water] varies much less than log[PS] and furthermore that it decreases when molecular volume is increased. In terms of Fig. 8, because large values of Kn-octanol/water are associated with large molecules, slopes less than 1 are expected if increasing molecular size has some effect that decreases permeability in addition to its effect that increases permeability by virtue of increasing Kn-octanol/water (see Appendix C).\nLiu [169] investigated the utility of many different descriptors for predicting log(PS) for neutral molecules and settled on three, log(D), TPSA and vas_base where D is Kn-octanol/water measured specifically at pH 7.4, TPSA is the polar surface area of a molecule, which correlates with the ability to form hydrogen bonds (compare [170]), and vas_base is the surface area of basic groups.\nAbraham [168, 171] has presented the extension of the LFER approach to ions.11\nFong [161] has reviewed many of the attempts to predict permeabilities of the blood–brain barrier to solutes. He concludes that the most important factors for neutral solutes are: the free energy required to remove the solute from water; the free energy gained from the interactions of the solute with the membrane core, usually modelled by its interaction with n-octanol; the dipole moment of the solute; and lastly its molecular volume. Increases in molecular volume per se decrease permeability. Geldenhuys et al. [172] has provided many useful references in a review prepared from the perspective of the utility of predictions in high-throughput screening.\n\nTransporters at the blood–brain barrier\nThe membranes of the endothelial cells that constitute the blood–brain barrier possess transporters for many different types of solutes. These transporters may be present on luminal, abluminal or both surfaces of the endothelial cells. Prominent among them are transporters for common nutrients and waste products of metabolism: GLUT1 for glucose, MCT1 for lactic acid and other small monocarboxylic acids, a range of transporters for amino acids, and several for nucleosides. There are also ion transporters involved in maintenance of the ionic composition of the brain fluids. Many of the transporters are specific and are involved in moving the normal constituents of brain extracellular fluid. Some of these are considered in Sect. 5. In addition there are also less specific transporters. Many of these can mediate efflux of a variety of other substrates including many exogenous substances and toxic occasional products of metabolism.\nEvidence concerning the presence and identity of many of these transporters has been reviewed elsewhere with studies being conducted primarily at the level of transcript [173–178], protein [31, 44, 58, 176, 179–188] and/or function [4, 20, 31, 46, 55–57, 179, 189–200]. The reports by Roberts et al. [180] and Kubo et al. [58] and reviews by Hawkins et al. [44], Redzic [31], Campos-Bedolla [57], Worzfeld and Schwaninger [187] and Nalecz [200] have been useful as sources of information about the localization of transporters to the luminal or abluminal membranes.\nThis review will not seek to provide yet another comprehensive survey. Extensive lists of transporters and substrates are available in many of the cited references and for SLC transporters at the BioParadigms website [201, 202].\n\nABC efflux transporters\nIt has long been appreciated that the brain represents a pharmacological sanctuary and is selectively “protected” from the toxic effects of many chemotherapeutic agents. These include vincristine and doxorubicin (aka adriamycin), which fail to penetrate the blood–brain barrier as well as their lipid solubilities would suggest [162]. A major part of this failure to penetrate has since been attributed to the presence of the multidrug transporter, P-glycoprotein. Absence of this transporter in knock-out mice was shown to allow entry of toxic agents including ivermectin [203]. P-glycoprotein was found to be located in the luminal membrane (see e.g. [204–209]) of the endothelial cells and is believed to act there to transport substrates out of the cells so rapidly that little remains to penetrate the abluminal membrane and enter the brain.\nIt is believed by many that P-glycoprotein, a transmembrane protein, acts by removing its lipophilic substrates from the lipid layer of the cell membrane, depositing them back into the blood [210–213]. Its structure has been investigated in both substrate-free and inhibitor bound conformations [213] and binding sites for various of its many substrates identified within the large cavity seen in the substrate-free conformation. It is the binding and hydrolysis of ATP that provides the motive force leading to a large conformational change in the P-glycoprotein and the transfer and expulsion of its substrates. There are two ATP binding sites located on the cytoplasmic side of the protein.\nP-glycoprotein, otherwise called ABCB1, is a member of the ABC (ATP-Binding Cassette) family of proteins many of which are primary active transporters that utilize the hydrolysis of ATP to fuel substrate transport. Since its discovery, other ABC active transporters with broad substrate profiles have been found in the luminal membrane of the endothelial cells. These include Breast Cancer Resistance Protein, BCRP (ABCG2) [180, 197, 214–218] and Multidrug Resistance Proteins, MRPs 4 and 5 (ABCC4 and 5) [180, 197, 209, 218–221]. MRP1 (ABCC1) has also been implicated but levels of this transporter are thought to be low in brain endothelial cells in situ and only increase in cultured brain endothelial cells once they are removed from the brain microenvironment [180, 184, 218, 222–226]. MRP1 and MRP2 are apparently upregulated and clearly expressed in epilepsy [227–229].\nThe role of efflux from endothelial cell to blood by ABC transporters in preventing influx of many substances from blood into the brain has been extensively reviewed (see e.g. [57, 196, 197, 199, 221, 230–234]. The regulation of P-glycoprotein, BCRP and MRP2 at the blood–brain barrier has been reviewed by Miller [221].\nThe role of ABC transporters in efflux from the brain parenchyma differs depending on the nature of the substrate. As described in Fig. 9, for substances that are sufficiently lipid soluble to cross the endothelial cell membranes rapidly by passive transport, the presence of ABC efflux transporters can greatly reduce blood-to-brain influx, as observed experimentally. However, as also explained in Fig. 9 the ABC transporters in the luminal membrane will have only a modest effect, e.g. a doubling, on the rate of brain-to-blood efflux. This may be of little consequence as the rate of efflux for lipid soluble substances is already high.\nFig. 9 The influence of ABC transporters on the movements of lipophilic substances. The substance is presumed to be able to enter and leave the endothelial cells by diffusion with rate constant kdiff., which for simplicity in this example is assumed to be same on both sides. The substance is expelled from the cell by ABC transporters on the luminal side at a rate, kactiveccell. With these assumptions the effect of the ABC transporters on influx can be calculated by setting cisf = 0 and the effect on efflux by setting cplasma = 0. In both sets of equations, the first line states that at steady-state the net flux into the cell on one side must equal the net flux out of the cell on the other. From the next to the last lines of both sets of equations, if the rate of ABC mediated expulsion from the cell is small or zero, the rate constants for both influx and efflux are (kdiff./2). By contrast from the last lines if the rate of ABC mediated expulsion is large, influx to the brain, J→, becomes very small, while efflux from the brain, J←, is doubled compared to the efflux with no ABC transporter\nThe role of ABC transporters for solutes with low passive permeability across the membranes is considered in the next section.\n\nEfflux mediated in part by SLC solute transporters\nMany of the SLC (solute carrier) transporters (see [202] for a list) are present in the membranes of the endothelial cells of the blood–brain barrier. Some are considered in connection with the transport of specific solutes in Sect. 5. Others, primarily from the SLC21 (OATPs, organic anion transporting polypeptides) and SLC22 (OATs and OCTs, organic anion transporters and organic cation transporters) families are associated with transport of a variety of organic anions and cations. These have been reviewed frequently and extensively [57, 176, 200, 218, 235–246]. (Uppercase labels, e.g. SLC or OAT, strictly refer to human sequences and proteins, while mixed-case labels, e.g. Slc or Oat, refer to any other species. In this review uppercase is also used when there is no intention to specify species).\nThere is little quantitative data on the efflux of organic anions and cations from the parenchyma in humans though many are known to be transported. In rodents more information is available for transfer of organic anions than cations. Table 1 lists some examples of organic anions/neutral molecules for which brain-to-blood transport rate constants have been determined. These are all believed to be substrates for Oat3 (Slc22a8) and/or one or more of the Oatp transporters present at the blood–brain barrier. In broad terms [238], small hydrophobic anions are substrates for Oats (Slc22 family) while larger amphipathic anions are substrates for Oatps (Slc21 family, whose member names start with Slco, see [247]). For comparison Table 1 also lists rate constants and clearances for examples of markers for perivascular efflux. It is clear that the rates of elimination of the Slc substrates are considerably greater than could be supported by perivascular efflux alone.\nTable 1 Comparison of rate constants for efflux and clearances for Slc22 and Slco substrates and the perivascular markers inulin, mannitol and sucrose\nkeff/min−1 t1/2/min Vd/mL/g CL/µL g−1 min−1 Ref # Notes\nSlc substrates Rat unless stated otherwise\n p-Aminohippuric acid (PAH) 0.059 12 0.80 47 [558] Influx much slower than efflux\n p-Aminohippuric acid (PAH) 0.039 18 [559] Abl. Oat3 based on inhibitors, lum. Mrp4 possible based on kidney [560]\n p-Aminohippuric acid (PAH) 0.0175 40 [131]\n Penicillin G, benzylpenicillin 0.043 16 [559] Oat3 based on inhibitors\n Taurocholate 0.023 30 [561]\n BQ-123 0.0078 100 [561]\n Estrone sulfate 0.066 9.9 1.1 75 [562]\n Estrone 0.061 11 3.3 227 [562]\n Dehydroepiandrosterone sulfate (DHEAS) 0.027 26 118 [563] Influx much slower than efflux, Oatp2\n Estradiol-17beta-glucuronide (E217betaG) 0.037 19 [564] Oatp2, 40% and Oat3 20%\n Pravastatin 0.060 12 0.99 59 [565] Oat3 (Slc22a8) and Oatp2 (Slco1a4) + others\n Pitavastatin 0.026 27 14 364 [565] Oat3, Oatp2 (Slco1a4) +others +diffusion\n Homovanillic acid 0.017 40.8 [566] Oat3 from inhibitors\n Indoxyl sulfate 0.011 64 [567] Oat3 and others\n Pemetrexed 0.018 39 0.62 11 [568] Mouse. Oat3 and unknown (not Mrp2 not Bcrp)\n Methotrexate 0.024 29 0.85 20 [568] Oat3 and Bcrp suggested\n Buprenorphine 0.025 27.5 6.1 154 [569] Pgp and unknown, possibly diffusion\n AZT, (3′-azido-3′-deoxythymidine) 0.032 22 [570] Oat3 from benzylpenicillin inhibition\n DDI, (2′,3′-dideoxyinosine) 0.253 2.8 [570] Oat3 from benzylpenicillin inhibition + diffusion\nMarkers for perivascular elimination\n Inulin 0.006 awake0.016 asleep 11543 0.2* 1.23.2 [128] Mouse\n Inulin 0.003 230 0.2* 0.6 [62] Mouse\n Inulin 0.005 135 0.2* 1 [131] Rat\n Mannitol 0.004 170 0.2* 0.8 [25] Mouse\n Sucrose 0.0028 awake0.0043 anesth. 245160 0.2* 0.560.86 [131] Rat\n Albumin 0.006 115 0.2* 1.2 [131] Rat\n Dextran-10K 0.0035 197 0.2* 0.7 [131] Rat\n Dextran-70K 0.004 170 0.2* 0.8 [131] Rat\nkeff rate constant for efflux determined from the time course of the decrease in concentration after injection of solute into the parenchyma (brain efflux index for the Slc substrates relative to inulin; these underestimate keff for values less than ~ 0.01 min−1 see [131]); t1/2 = 0.69/(rate constant) is the half-life; Vd, volume of distribution in the parenchyma determined using brain slices (ISF volume for inulin and mannitol); CL = Vd × keff, the clearance. For the Slc substrates more than one transporter in each membrane is likely to be involved in the transport\n* Assumed equal to ISF volume\nAs indicated in Fig. 10 transport from the parenchyma into the endothelial cells occurs via one or more of the SLC transporters, while exit from the endothelial cells to plasma occurs via either SLC or ABC transporters. For many of the anions efflux from brain to blood is clearly an active uphill process suggesting that the ABC route is dominant (for a caveat see.12) Transport across either membrane can be rate limiting and in many cases transport across each can occur by more than one route. As a consequence demonstration that a specific inhibitor of a transporter reduces the rate of efflux is evidence for involvement of that transporter, but failure to inhibit is relatively uninformative.\nFig. 10 Transport of organic anions across the blood–brain barrier. Organic anion transporters at the blood–brain barrier. The principal known transporters in the rat are shown. In human OAT3 is abluminal, while both OATP1A4 and OATP2B1 are present on both membranes. The ABC efflux pumps, P-gp, BCRP, MRP4 and MRP5, are all localized to the luminal, plasma facing, membrane. The Oat and Oatp transporters are exchangers (see Footnote 12). Localizations from [180] and the references in Table 1\nFor the SLC substrates in Table 1 the half-lives are shorter than the 1–2 h characteristic of markers eliminated from the parenchyma by perivascular efflux (see Sect. 3). As noted earlier, shorter half-lives imply that there are mechanisms for elimination other than perivascular. This is reinforced by noting that the clearances for those solutes for which volumes of distribution are available are much greater than the clearance associated with the perivascular route (see Sect. 3.2). There is ample further evidence (see the references for the entries in Table 1) for the importance of the Oat and Oatp transporters in the elimination of these solutes from the parenchyma including saturation, competition, the availability of transport inhibitors, and the rapid appearance of effluxed material in venous blood draining the head.\n\nEfflux by transcytosis\nTranscytosis is much less prevalent across the endothelial cells of the blood–brain barrier than across those of peripheral capillaries [248–251]. Nevertheless both adsorptive mediated transcytosis (AMT) and receptor mediated transcytosis (RMT) are still likely to be important mechanisms for the transfer of some large substrates across the blood–brain barrier. The initial event in AMT is the adsorption of usually positive substrates onto the surfaces of caveolae, while that for RMT is binding of the substrate to specific receptors that are in or become incorporated into clathrin coated pits. In both cases at the blood–brain barrier this leads to endocytosis followed by delivery of a substantial fraction of the contents of the resulting vesicles to the opposite membrane for exit, possibly by exocytosis [49, 63, 252]. AMT is thought to account for much of the influx into the brain of histones [253], “cell penetrating peptides” [49, 251, 254], HIV [255, 256], and cargos conjugated to the lectin wheat germ agglutinin [257] and to underlie the increase in “generalized permeability” caused by protamine [258]. The downsides of AMT are that it is relatively non-selective for substrates [256] and that it occurs in many cells throughout the body. In addition there is little if any evidence that it occurs in the direction from brain to blood [257, 259]. While RMT also occurs throughout the body, transport by this mechanism depends on interaction of the substrates with specific receptors that may be found primarily in specific locations such as the blood–brain barrier. In addition there is evidence that RMT can occur in either direction, i.e. from brain to blood as well as from blood to brain.\nAMT and RMT in the direction from blood to brain have been studied extensively as routes of entry to the brain for endogenous substrates, but even more in the context of mechanisms for drug delivery. These studies have been reviewed frequently [57, 64, 154, 249, 252, 260–266]. However, even so, the steps occurring after the initial endocytosis remain only partially understood [63, 249, 250, 262, 267, 268] including even the answer to the important question of whether the cargo is released within the cell or delivered to the far side by exocytosis. By contrast evidence for transport via transcytosis in the direction brain to blood has been reported for only a few systems including transport of amyloid-β peptides via interaction with LRP1 (low density lipoprotein receptor related protein 1) and LRP2 (low density lipoprotein receptor related protein 2) (see Sect. 5.7), of IgG antibodies via interaction with an unidentified receptor [269–275] and of transferrin [60] via interaction with the transferrin receptor (TfR) [61] (see below).\nTransport of transferrin is closely related to transfer of iron. Iron in plasma and in brain extracellular fluid is present almost entirely complexed to transferrin i.e. as holo-transferrin. It has long been known that iron and transferrin enter the brain across the blood–brain barrier and it was originally hypothesised that they are transferred together by endocytosis followed by exocytosis, i.e. direct transcytosis, of holo-transferrin (see e.g. [61, 276]). Yet there have been arguments against this idea arising from dual labelling experiments showing that far more labelled iron than labelled transferrin accumulates in the brain, see e.g. [60, 277, 278]. In addition it has been argued that release of holo-transferrin from TfR is unlikely to occur as there needs to be prior dissociation of iron for release of transferrin from its receptor [279]. So though there is general agreement that holo-transferrin interacts with TfR, which then mediates endocytosis of the iron/transferrin/receptor complex into the endothelial cells, there has been controversy over the subsequent steps in the transfers of transferrin and iron into the brain. Assuming that holo-transferrin is indeed directly transcytosed across the blood–brain barrier, then the limited net entry observed of transferrin to the brain implies that there must be transcytosis of transferrin without iron, apo-transferrin, back out of the brain. Alternatively if the iron is dissociated from the transferrin within the endothelial cells, it is likely that there is exocytosis of apo-transferrin on both sides of the cells (see [280–282] and the footnote13 for further discussion).\nLittle is known about transport of transferrin out of the brain. There have been reports that labelled apo-transferrin injected into the brain can be transported from brain to blood, but it is not clear how important this is under normal conditions. Banks et al. [60] found that the apo-transferrin was removed from the brain faster than albumin, implying the existence of a route other than washout via CSF. However, subsequently Moos and Morgan [278] did not confirm this result. By contrast Zhang and Pardridge [61] found an early component of loss of injected apo-transferrin, half-life 39 min, which was much faster than that for loss of injected 70 kDa-dextran. Furthermore this rapid component was inhibited by cold apo-transferrin, i.e. there was competition, with an apparent dissociation constant of less than 30 nM implying interaction with a specific receptor which was presumed to be the receptor protein detected by OX26, i.e. TfR. As these studies on transferrin efflux are substantially older than the studies on iron uptake linked to transferrin, further investigation of transferrin transport from brain to blood might be informative."}

{"project":"2_test","denotations":[{"id":"30340614-20468051-30706066","span":{"begin":588,"end":591},"obj":"20468051"},{"id":"30340614-20468051-30706067","span":{"begin":698,"end":701},"obj":"20468051"},{"id":"30340614-27799072-30706068","span":{"begin":962,"end":963},"obj":"27799072"},{"id":"30340614-3468856-30706069","span":{"begin":1577,"end":1580},"obj":"3468856"},{"id":"30340614-27799072-30706070","span":{"begin":1610,"end":1611},"obj":"27799072"},{"id":"30340614-5499038-30706071","span":{"begin":1740,"end":1743},"obj":"5499038"},{"id":"30340614-8592149-30706072","span":{"begin":1760,"end":1763},"obj":"8592149"},{"id":"30340614-25481827-30706073","span":{"begin":3429,"end":3431},"obj":"25481827"},{"id":"30340614-19395337-30706074","span":{"begin":3433,"end":3436},"obj":"19395337"},{"id":"30340614-10696512-30706075","span":{"begin":4721,"end":4724},"obj":"10696512"},{"id":"30340614-25675910-30706076","span":{"begin":4726,"end":4729},"obj":"25675910"},{"id":"30340614-7392035-30706077","span":{"begin":5707,"end":5710},"obj":"7392035"},{"id":"30340614-11259830-30706078","span":{"begin":6742,"end":6745},"obj":"11259830"},{"id":"30340614-9466345-30706079","span":{"begin":6972,"end":6975},"obj":"9466345"},{"id":"30340614-15051171-30706079","span":{"begin":6972,"end":6975},"obj":"15051171"},{"id":"30340614-21374607-30706079","span":{"begin":6972,"end":6975},"obj":"21374607"},{"id":"30340614-10696512-30706080","span":{"begin":7514,"end":7517},"obj":"10696512"},{"id":"30340614-9466345-30706081","span":{"begin":7524,"end":7527},"obj":"9466345"},{"id":"30340614-9466345-30706082","span":{"begin":7929,"end":7932},"obj":"9466345"},{"id":"30340614-15051171-30706083","span":{"begin":7934,"end":7937},"obj":"15051171"},{"id":"30340614-9466345-30706084","span":{"begin":8291,"end":8294},"obj":"9466345"},{"id":"30340614-15051171-30706085","span":{"begin":8296,"end":8299},"obj":"15051171"},{"id":"30340614-10696512-30706086","span":{"begin":8497,"end":8500},"obj":"10696512"},{"id":"30340614-15051171-30706087","span":{"begin":8502,"end":8505},"obj":"15051171"},{"id":"30340614-9466345-30706088","span":{"begin":9033,"end":9036},"obj":"9466345"},{"id":"30340614-14709630-30706089","span":{"begin":9489,"end":9492},"obj":"14709630"},{"id":"30340614-9886238-30706090","span":{"begin":9816,"end":9819},"obj":"9886238"},{"id":"30340614-21374607-30706091","span":{"begin":9882,"end":9885},"obj":"21374607"},{"id":"30340614-26189671-30706092","span":{"begin":9887,"end":9890},"obj":"26189671"},{"id":"30340614-25675910-30706093","span":{"begin":9958,"end":9961},"obj":"25675910"},{"id":"30340614-26305616-30706094","span":{"begin":10470,"end":10473},"obj":"26305616"},{"id":"30340614-16306934-30706095","span":{"begin":11766,"end":11769},"obj":"16306934"},{"id":"30340614-19429473-30706095","span":{"begin":11766,"end":11769},"obj":"19429473"},{"id":"30340614-21060791-30706095","span":{"begin":11766,"end":11769},"obj":"21060791"},{"id":"30340614-21707071-30706095","span":{"begin":11766,"end":11769},"obj":"21707071"},{"id":"30340614-25186741-30706095","span":{"begin":11766,"end":11769},"obj":"25186741"},{"id":"30340614-28188910-30706095","span":{"begin":11766,"end":11769},"obj":"28188910"},{"id":"30340614-21349151-30706096","span":{"begin":11785,"end":11787},"obj":"21349151"},{"id":"30340614-16365086-30706097","span":{"begin":11789,"end":11791},"obj":"16365086"},{"id":"30340614-25703048-30706098","span":{"begin":11793,"end":11795},"obj":"25703048"},{"id":"30340614-21707071-30706099","span":{"begin":11797,"end":11800},"obj":"21707071"},{"id":"30340614-17619998-30706100","span":{"begin":11802,"end":11805},"obj":"17619998"},{"id":"30340614-18619525-30706100","span":{"begin":11802,"end":11805},"obj":"18619525"},{"id":"30340614-19094056-30706100","span":{"begin":11802,"end":11805},"obj":"19094056"},{"id":"30340614-18219561-30706100","span":{"begin":11802,"end":11805},"obj":"18219561"},{"id":"30340614-21792245-30706100","span":{"begin":11802,"end":11805},"obj":"21792245"},{"id":"30340614-21291474-30706100","span":{"begin":11802,"end":11805},"obj":"21291474"},{"id":"30340614-23650139-30706100","span":{"begin":11802,"end":11805},"obj":"23650139"},{"id":"30340614-24702234-30706100","span":{"begin":11802,"end":11805},"obj":"24702234"},{"id":"30340614-26661193-30706100","span":{"begin":11802,"end":11805},"obj":"26661193"},{"id":"30340614-28954515-30706100","span":{"begin":11802,"end":11805},"obj":"28954515"},{"id":"30340614-27799072-30706101","span":{"begin":11828,"end":11829},"obj":"27799072"},{"id":"30340614-23298398-30706102","span":{"begin":11831,"end":11833},"obj":"23298398"},{"id":"30340614-21349151-30706103","span":{"begin":11835,"end":11837},"obj":"21349151"},{"id":"30340614-19664713-30706104","span":{"begin":11839,"end":11841},"obj":"19664713"},{"id":"30340614-25481827-30706105","span":{"begin":11843,"end":11845},"obj":"25481827"},{"id":"30340614-17619998-30706106","span":{"begin":11850,"end":11853},"obj":"17619998"},{"id":"30340614-6361813-30706107","span":{"begin":11855,"end":11858},"obj":"6361813"},{"id":"30340614-11734619-30706107","span":{"begin":11855,"end":11858},"obj":"11734619"},{"id":"30340614-16111480-30706107","span":{"begin":11855,"end":11858},"obj":"16111480"},{"id":"30340614-15717058-30706107","span":{"begin":11855,"end":11858},"obj":"15717058"},{"id":"30340614-19393264-30706107","span":{"begin":11855,"end":11858},"obj":"19393264"},{"id":"30340614-21118088-30706107","span":{"begin":11855,"end":11858},"obj":"21118088"},{"id":"30340614-23789951-30706107","span":{"begin":11855,"end":11858},"obj":"23789951"},{"id":"30340614-25561720-30706107","span":{"begin":11855,"end":11858},"obj":"25561720"},{"id":"30340614-26187753-30706107","span":{"begin":11855,"end":11858},"obj":"26187753"},{"id":"30340614-27503090-30706107","span":{"begin":11855,"end":11858},"obj":"27503090"},{"id":"30340614-18619525-30706108","span":{"begin":11896,"end":11899},"obj":"18619525"},{"id":"30340614-25703048-30706109","span":{"begin":11918,"end":11920},"obj":"25703048"},{"id":"30340614-16365086-30706110","span":{"begin":11953,"end":11955},"obj":"16365086"},{"id":"30340614-21349151-30706111","span":{"begin":11966,"end":11968},"obj":"21349151"},{"id":"30340614-25481827-30706112","span":{"begin":11987,"end":11989},"obj":"25481827"},{"id":"30340614-26661193-30706113","span":{"begin":12018,"end":12021},"obj":"26661193"},{"id":"30340614-27503090-30706114","span":{"begin":12035,"end":12038},"obj":"27503090"},{"id":"30340614-23506860-30706115","span":{"begin":12379,"end":12382},"obj":"23506860"},{"id":"30340614-7392035-30706116","span":{"begin":12744,"end":12747},"obj":"7392035"},{"id":"30340614-7910522-30706117","span":{"begin":12989,"end":12992},"obj":"7910522"},{"id":"30340614-2563168-30706118","span":{"begin":13069,"end":13072},"obj":"2563168"},{"id":"30340614-2463300-30706118","span":{"begin":13069,"end":13072},"obj":"2463300"},{"id":"30340614-1975805-30706118","span":{"begin":13069,"end":13072},"obj":"1975805"},{"id":"30340614-1356979-30706118","span":{"begin":13069,"end":13072},"obj":"1356979"},{"id":"30340614-12486090-30706118","span":{"begin":13069,"end":13072},"obj":"12486090"},{"id":"30340614-16361063-30706118","span":{"begin":13069,"end":13072},"obj":"16361063"},{"id":"30340614-1968065-30706119","span":{"begin":13454,"end":13457},"obj":"1968065"},{"id":"30340614-7910961-30706119","span":{"begin":13454,"end":13457},"obj":"7910961"},{"id":"30340614-24624364-30706119","span":{"begin":13454,"end":13457},"obj":"24624364"},{"id":"30340614-19325113-30706119","span":{"begin":13454,"end":13457},"obj":"19325113"},{"id":"30340614-19325113-30706120","span":{"begin":13558,"end":13561},"obj":"19325113"},{"id":"30340614-18619525-30706121","span":{"begin":14380,"end":14383},"obj":"18619525"},{"id":"30340614-23789951-30706122","span":{"begin":14385,"end":14388},"obj":"23789951"},{"id":"30340614-12438926-30706123","span":{"begin":14390,"end":14393},"obj":"12438926"},{"id":"30340614-12706238-30706123","span":{"begin":14390,"end":14393},"obj":"12706238"},{"id":"30340614-12958161-30706123","span":{"begin":14390,"end":14393},"obj":"12958161"},{"id":"30340614-15126373-30706123","span":{"begin":14390,"end":14393},"obj":"15126373"},{"id":"30340614-17196184-30706123","span":{"begin":14390,"end":14393},"obj":"17196184"},{"id":"30340614-18619525-30706124","span":{"begin":14462,"end":14465},"obj":"18619525"},{"id":"30340614-23789951-30706125","span":{"begin":14467,"end":14470},"obj":"23789951"},{"id":"30340614-16361063-30706126","span":{"begin":14472,"end":14475},"obj":"16361063"},{"id":"30340614-17196184-30706127","span":{"begin":14477,"end":14480},"obj":"17196184"},{"id":"30340614-15314169-30706127","span":{"begin":14477,"end":14480},"obj":"15314169"},{"id":"30340614-15501592-30706127","span":{"begin":14477,"end":14480},"obj":"15501592"},{"id":"30340614-18619525-30706128","span":{"begin":14722,"end":14725},"obj":"18619525"},{"id":"30340614-21291474-30706129","span":{"begin":14727,"end":14730},"obj":"21291474"},{"id":"30340614-17196184-30706130","span":{"begin":14732,"end":14735},"obj":"17196184"},{"id":"30340614-9489736-30706131","span":{"begin":14737,"end":14740},"obj":"9489736"},{"id":"30340614-9681461-30706131","span":{"begin":14737,"end":14740},"obj":"9681461"},{"id":"30340614-10421622-30706131","span":{"begin":14737,"end":14740},"obj":"10421622"},{"id":"30340614-12817896-30706131","span":{"begin":14737,"end":14740},"obj":"12817896"},{"id":"30340614-18680196-30706131","span":{"begin":14737,"end":14740},"obj":"18680196"},{"id":"30340614-11879359-30706132","span":{"begin":14823,"end":14826},"obj":"11879359"},{"id":"30340614-15101825-30706132","span":{"begin":14823,"end":14826},"obj":"15101825"},{"id":"30340614-16190927-30706132","span":{"begin":14823,"end":14826},"obj":"16190927"},{"id":"30340614-25481827-30706133","span":{"begin":15010,"end":15012},"obj":"25481827"},{"id":"30340614-21118088-30706134","span":{"begin":15014,"end":15017},"obj":"21118088"},{"id":"30340614-23789951-30706135","span":{"begin":15019,"end":15022},"obj":"23789951"},{"id":"30340614-26187753-30706136","span":{"begin":15024,"end":15027},"obj":"26187753"},{"id":"30340614-12535575-30706137","span":{"begin":15034,"end":15037},"obj":"12535575"},{"id":"30340614-15134482-30706137","span":{"begin":15034,"end":15037},"obj":"15134482"},{"id":"30340614-16025095-30706137","span":{"begin":15034,"end":15037},"obj":"16025095"},{"id":"30340614-16337012-30706137","span":{"begin":15034,"end":15037},"obj":"16337012"},{"id":"30340614-16714484-30706137","span":{"begin":15034,"end":15037},"obj":"16714484"},{"id":"30340614-25481827-30706138","span":{"begin":17620,"end":17622},"obj":"25481827"},{"id":"30340614-21707071-30706139","span":{"begin":17624,"end":17627},"obj":"21707071"},{"id":"30340614-27503090-30706140","span":{"begin":17629,"end":17632},"obj":"27503090"},{"id":"30340614-17196184-30706141","span":{"begin":17634,"end":17637},"obj":"17196184"},{"id":"30340614-12883891-30706142","span":{"begin":17639,"end":17642},"obj":"12883891"},{"id":"30340614-15717059-30706142","span":{"begin":17639,"end":17642},"obj":"15717059"},{"id":"30340614-16357150-30706142","span":{"begin":17639,"end":17642},"obj":"16357150"},{"id":"30340614-22013971-30706142","span":{"begin":17639,"end":17642},"obj":"22013971"},{"id":"30340614-21854228-30706142","span":{"begin":17639,"end":17642},"obj":"21854228"},{"id":"30340614-24013810-30706142","span":{"begin":17639,"end":17642},"obj":"24013810"},{"id":"30340614-23343976-30706142","span":{"begin":17639,"end":17642},"obj":"23343976"},{"id":"30340614-23506880-30706142","span":{"begin":17639,"end":17642},"obj":"23506880"},{"id":"30340614-23789957-30706142","span":{"begin":17639,"end":17642},"obj":"23789957"},{"id":"30340614-27320645-30706142","span":{"begin":17639,"end":17642},"obj":"27320645"},{"id":"30340614-27783531-30706142","span":{"begin":17639,"end":17642},"obj":"27783531"},{"id":"30340614-22013971-30706143","span":{"begin":18415,"end":18418},"obj":"22013971"},{"id":"30340614-14579113-30706144","span":{"begin":18598,"end":18601},"obj":"14579113"},{"id":"30340614-9399971-30706145","span":{"begin":19157,"end":19160},"obj":"9399971"},{"id":"30340614-12684544-30706146","span":{"begin":19231,"end":19234},"obj":"12684544"},{"id":"30340614-15504935-30706147","span":{"begin":19303,"end":19306},"obj":"15504935"},{"id":"30340614-16639426-30706148","span":{"begin":19347,"end":19350},"obj":"16639426"},{"id":"30340614-12684544-30706149","span":{"begin":19394,"end":19397},"obj":"12684544"},{"id":"30340614-9694947-30706150","span":{"begin":19448,"end":19451},"obj":"9694947"},{"id":"30340614-9694947-30706151","span":{"begin":19473,"end":19476},"obj":"9694947"},{"id":"30340614-11105986-30706152","span":{"begin":19513,"end":19516},"obj":"11105986"},{"id":"30340614-11105986-30706153","span":{"begin":19545,"end":19548},"obj":"11105986"},{"id":"30340614-11032880-30706154","span":{"begin":19604,"end":19607},"obj":"11032880"},{"id":"30340614-11408557-30706155","span":{"begin":19699,"end":19702},"obj":"11408557"},{"id":"30340614-15292460-30706156","span":{"begin":19759,"end":19762},"obj":"15292460"},{"id":"30340614-15292460-30706157","span":{"begin":19839,"end":19842},"obj":"15292460"},{"id":"30340614-12679720-30706158","span":{"begin":19916,"end":19919},"obj":"12679720"},{"id":"30340614-12358729-30706159","span":{"begin":19969,"end":19972},"obj":"12358729"},{"id":"30340614-23297298-30706160","span":{"begin":20020,"end":20023},"obj":"23297298"},{"id":"30340614-23297298-30706161","span":{"begin":20101,"end":20104},"obj":"23297298"},{"id":"30340614-17365275-30706162","span":{"begin":20165,"end":20168},"obj":"17365275"},{"id":"30340614-9103519-30706163","span":{"begin":20251,"end":20254},"obj":"9103519"},{"id":"30340614-9103519-30706164","span":{"begin":20334,"end":20337},"obj":"9103519"},{"id":"30340614-24136970-30706165","span":{"begin":20477,"end":20480},"obj":"24136970"},{"id":"30340614-11120756-30706166","span":{"begin":20516,"end":20518},"obj":"11120756"},{"id":"30340614-16639426-30706167","span":{"begin":20552,"end":20555},"obj":"16639426"},{"id":"30340614-16639426-30706168","span":{"begin":20659,"end":20662},"obj":"16639426"},{"id":"30340614-16639426-30706169","span":{"begin":20697,"end":20700},"obj":"16639426"},{"id":"30340614-16639426-30706170","span":{"begin":20740,"end":20743},"obj":"16639426"},{"id":"30340614-16639426-30706171","span":{"begin":20782,"end":20785},"obj":"16639426"},{"id":"30340614-16639426-30706172","span":{"begin":21059,"end":21062},"obj":"16639426"},{"id":"30340614-18619525-30706173","span":{"begin":22554,"end":22557},"obj":"18619525"},{"id":"30340614-6033532-30706174","span":{"begin":23584,"end":23587},"obj":"6033532"},{"id":"30340614-10696507-30706174","span":{"begin":23584,"end":23587},"obj":"10696507"},{"id":"30340614-12843411-30706174","span":{"begin":23584,"end":23587},"obj":"12843411"},{"id":"30340614-18726697-30706174","span":{"begin":23584,"end":23587},"obj":"18726697"},{"id":"30340614-26794270-30706175","span":{"begin":24261,"end":24263},"obj":"26794270"},{"id":"30340614-2600816-30706176","span":{"begin":24352,"end":24355},"obj":"2600816"},{"id":"30340614-26794270-30706177","span":{"begin":24387,"end":24389},"obj":"26794270"},{"id":"30340614-18726697-30706178","span":{"begin":24391,"end":24394},"obj":"18726697"},{"id":"30340614-15722162-30706179","span":{"begin":24396,"end":24399},"obj":"15722162"},{"id":"30340614-9443901-30706180","span":{"begin":24407,"end":24410},"obj":"9443901"},{"id":"30340614-7680372-30706181","span":{"begin":24477,"end":24480},"obj":"7680372"},{"id":"30340614-8227337-30706182","span":{"begin":24562,"end":24565},"obj":"8227337"},{"id":"30340614-7680372-30706183","span":{"begin":24801,"end":24804},"obj":"7680372"},{"id":"30340614-7514652-30706184","span":{"begin":24806,"end":24809},"obj":"7514652"},{"id":"30340614-25481827-30706185","span":{"begin":25403,"end":25405},"obj":"25481827"},{"id":"30340614-25340933-30706186","span":{"begin":25407,"end":25409},"obj":"25340933"},{"id":"30340614-19395337-30706187","span":{"begin":25411,"end":25414},"obj":"19395337"},{"id":"30340614-10696507-30706188","span":{"begin":25416,"end":25419},"obj":"10696507"},{"id":"30340614-2688350-30706189","span":{"begin":25426,"end":25429},"obj":"2688350"},{"id":"30340614-15180331-30706189","span":{"begin":25426,"end":25429},"obj":"15180331"},{"id":"30340614-16777679-30706189","span":{"begin":25426,"end":25429},"obj":"16777679"},{"id":"30340614-17619996-30706189","span":{"begin":25426,"end":25429},"obj":"17619996"},{"id":"30340614-26687840-30706189","span":{"begin":25426,"end":25429},"obj":"26687840"},{"id":"30340614-29067674-30706189","span":{"begin":25426,"end":25429},"obj":"29067674"},{"id":"30340614-10696507-30706190","span":{"begin":25542,"end":25545},"obj":"10696507"},{"id":"30340614-12843411-30706191","span":{"begin":25547,"end":25550},"obj":"12843411"},{"id":"30340614-16777679-30706192","span":{"begin":25552,"end":25555},"obj":"16777679"},{"id":"30340614-8912898-30706193","span":{"begin":25557,"end":25560},"obj":"8912898"},{"id":"30340614-14597140-30706194","span":{"begin":25562,"end":25565},"obj":"14597140"},{"id":"30340614-11240028-30706195","span":{"begin":26102,"end":26105},"obj":"11240028"},{"id":"30340614-16354907-30706195","span":{"begin":26102,"end":26105},"obj":"16354907"},{"id":"30340614-19636712-30706195","span":{"begin":26102,"end":26105},"obj":"19636712"},{"id":"30340614-22202501-30706195","span":{"begin":26102,"end":26105},"obj":"22202501"},{"id":"30340614-23978455-30706195","span":{"begin":26102,"end":26105},"obj":"23978455"},{"id":"30340614-22838637-30706195","span":{"begin":26102,"end":26105},"obj":"22838637"},{"id":"30340614-28624473-30706195","span":{"begin":26102,"end":26105},"obj":"28624473"},{"id":"30340614-3224280-30706196","span":{"begin":26131,"end":26133},"obj":"3224280"},{"id":"30340614-11238745-30706197","span":{"begin":26188,"end":26190},"obj":"11238745"},{"id":"30340614-11238745-30706198","span":{"begin":26658,"end":26660},"obj":"11238745"},{"id":"30340614-3694713-30706199","span":{"begin":26662,"end":26665},"obj":"3694713"},{"id":"30340614-3224280-30706200","span":{"begin":26855,"end":26857},"obj":"3224280"},{"id":"30340614-1450923-30706201","span":{"begin":26859,"end":26862},"obj":"1450923"},{"id":"30340614-9593852-30706202","span":{"begin":26864,"end":26867},"obj":"9593852"},{"id":"30340614-14580938-30706203","span":{"begin":27058,"end":27061},"obj":"14580938"},{"id":"30340614-25315861-30706204","span":{"begin":27804,"end":27807},"obj":"25315861"},{"id":"30340614-26687231-30706204","span":{"begin":27804,"end":27807},"obj":"26687231"},{"id":"30340614-29054412-30706204","span":{"begin":27804,"end":27807},"obj":"29054412"},{"id":"30340614-3224280-30706205","span":{"begin":28121,"end":28123},"obj":"3224280"},{"id":"30340614-9593852-30706206","span":{"begin":28305,"end":28308},"obj":"9593852"},{"id":"30340614-11238745-30706207","span":{"begin":28372,"end":28374},"obj":"11238745"}],"text":"The blood–brain barrier\nThe blood–brain barrier is more selective than the perivascular pathway in what can and cannot permeate. This selectivity arises from the properties of the endothelial cells surrounding the microvessels. The brain is highly vascularized and cells within the parenchyma are usually within 20 µm of a microvessel [148]. Diffusion over distances this short is rapid. To reach the microvessel, substances must also cross the surrounding layer composed of glial endfeet. This is normally possible because the gaps between the endfeet are not sealed by tight junctions [149, 150]. Even the almost complete coverage of the endothelial cells by glial endfeet proposed by Mathiisen [149] leaves sufficient gaps (see Footnote 7). Thus normally it is the endothelial cells that are the site for the rate limiting steps in efflux across the blood–brain barrier. The current state of knowledge about the role of the endfeet was considered further in [4].\n\nPassive, non-specific transfer across the blood–brain barrier\nThere are two possible routes for passive, non-specific transfer across the microvascular endothelial layer, through the cells or around them. The paracellular pathway is “blocked” by the presence of tight junctions but this pathway may still be the principal route for the passive fluxes of small solutes that are barred from the transcellular route by being too polar (mannitol, sucrose and inulin are considered in Appendix B). In addition to neutral molecules like mannitol, the paracellular pathway may be measurably permeable to Na+ and Cl− [151]. As discussed in detail in [4] and in Sect. 5.6 evidence for this includes the observation that the tracer fluxes of Na+ and Cl− are not affected by ouabain [152] or bumetanide [153], agents that specifically inhibit ion transporters known to be involved in transcellular fluxes of these ions.\nAlmost all of the passive, non-selective permeability of the blood–brain barrier to molecules more lipophilic than mannitol is the result of their ability to diffuse across both the cell membranes and the interior of the endothelial cells. Strong indications that such a physical mechanism applies are the observations: that transport does not saturate, that it is not inhibited by competition by other transported substances, and that no specific inhibitors have been found. Small neutral substances that are able to enter and leave the brain parenchyma by this mechanism include water, methanol, ethanol, isopropanol, glycerol, ethylene glycol, urea and thiourea (see Fig. 8).\nFig. 8 Plot of log(PS/mL g−1 min−1), versus log(Kn-octanol/water MW−1/2) for the substances indicated along the abscissa. PS is the product of permeability and surface area for the blood–brain barrier, Kn-octanol/water is the octanol/water partition coefficient and MW is the molecular weight of the substance. The slope of 1 for the heavy blue line indicates PS proportional to Kn-octanol/water MW−1/2. A closer fit to the data can be obtained by allowing the slope to vary, shown as the thin red line, but the improvement in fit is not statistically significant (F = 2.33, p = 0.11, n = 43, extra sum of squares F test [640])\n(Data read from Figure 8 of [159])\nMost studies of the passive permeability of the blood–brain barrier have focussed on influx, because it is easier to measure and has obvious importance for the delivery of agents and drugs to the CNS (see e.g. [57, 154]). However, passive permeability allows both influx and efflux and thus these studies are directly relevant to understanding how substances are eliminated from the parenchyma.\nIn the simplest view the rate limiting steps in the transcellular, passive, unmediated transfer of substances can be thought of as occurring by dissolution in a liquid hydrophobic core of the membranes and diffusion through it. For molecules not much larger than those of the solvent the diffusion constant for the various compounds is taken to be inversely proportional to the square root of their molecular weights [155–157]. The exact relationship assumed is not critical because the dominant factor determining the relative permeabilities is the free energy cost of the transfer from water into the core of the membrane, ΔGmembrane/water. This cost determines the relative concentrations in the membrane and the aqueous phase,5 cmembranecwater=Kmembrane/water=e-ΔGmembrane/water/RTwhere Kmembrane/water is the partition coefficient, R the universal gas constant, and T the absolute temperature. The free energy cost and the partition coefficient are usually estimated by assuming that the membrane core can be described as being like a layer of n-octanol (see [158, 159] and for more recent discussions [160, 161]), and thus6 cmembranecwater∝Kn-octanol/water=e-ΔGn-octanol/water/RT.\nIt is likely that n-octanol rather than, say, n-octane is appropriate as a model for the membrane interior because the –OH group can participate in hydrogen bonds.\nFenstermacher [159] reviewed the studies up to 1984 with the result summarized in a plot of log[PS] versus log[Kn-octanol/water MW−1/2] (see Fig. 8) where PS is the permeability surface area product for brain capillaries. For the substances listed in the figure, which have simple structures and molecular weights less than 200, the slope of the loglog plot is not significantly different from 1, i.e. PS appears to be proportional to Kn-octanol/water MW−1/2.\nThere have been many other reports based on studies using more complicated or larger molecules. These have usually reported a linear relation between log(PS) and either log[Kn-octanol/water] or log[Kn-octanol/water MW−1/2] but often with a slope substantially less than 1 (see e.g. [162, 163]). It should be emphasized that slope not equal to 1 means that the fluxes are not proportional to Kn-octanol/water MW−1/2 and thus, for at least some of the substances tested, simple diffusion and partition into an environment that looks like n-octanol are not the only important factors that need to be considered. The appropriate factors are considered further in Appendix C.\nCorrelating the passive permeabilities for substances at the blood–brain barrier with their partition coefficients for transfer from water to n-octanol has the virtue of focussing attention on the most critical aspect of the passive permeation process, the free energy cost of removing the solute from water and inserting it into a relatively hydrophobic environment. However, these correlations have been thought too imprecise to use as a criteria for selecting candidates to consider in a drug discovery setting. There have been many attempts to do better, some in terms of a set of rules analogous to the “rule of 5” for intestinal absorption [164], some using better estimates of the free energy cost for solutes to reach the rate limiting step of the transport, and some using a mixture of both.\nTo obtain better estimates of the free energy, Abraham and colleagues (see [165–168]) have employed linear free energy relations, LFER, to calculate correlations based on a two step process. First quantitative “descriptors” of the molecules under consideration are chosen without regard to the process of interest. Then, once the descriptors have been chosen, the relevant free energy changes for processes such as partition into a solvent or permeability across the blood–brain barrier, are calculated as linear sums of the descriptors with coefficients that depend on the process but not on the molecules (see e.g. [160, 165, 166]. Having used data for some substances to calculate the LFER coefficients, these can then be used for other substances. This approach has been applied with considerable success to partition into solvents for many more molecules than are needed to calculate the coefficients [165]. It has also allowed closer prediction of blood–brain barrier permeabilities than the simple solubility-diffusion model [166, 167] (see Appendix C).\nThere is, however, a danger in adopting this approach to the prediction of permeability. The use of linear free energy relations reveals correlations between the descriptors and the rate of transport, but unless used carefully it can obscure important features of the mechanism. For instance in the correlations reported for log(PS) [166, 167], the strongest correlation was a positive correlation between molecular volume and permeability, i.e. this approach seems to say that increases in molecular size result in increased permeability [160, 167]. However, the idea that bigger objects will be more permeable because they are bigger is completely counter-intuitive. The likely explanation for this paradox is simple. For the molecules considered in the correlations, increases in molecular volume were associated with large increases in lipophilicity as measured by Kn-octanol/water and it is plausible that it was the increase in lipophilicity that increased the permeability. Indeed as shown in Appendix C Abraham’s descriptor approach predicts for the compounds tested [166] that log[PS/Kn-octanol/water] varies much less than log[PS] and furthermore that it decreases when molecular volume is increased. In terms of Fig. 8, because large values of Kn-octanol/water are associated with large molecules, slopes less than 1 are expected if increasing molecular size has some effect that decreases permeability in addition to its effect that increases permeability by virtue of increasing Kn-octanol/water (see Appendix C).\nLiu [169] investigated the utility of many different descriptors for predicting log(PS) for neutral molecules and settled on three, log(D), TPSA and vas_base where D is Kn-octanol/water measured specifically at pH 7.4, TPSA is the polar surface area of a molecule, which correlates with the ability to form hydrogen bonds (compare [170]), and vas_base is the surface area of basic groups.\nAbraham [168, 171] has presented the extension of the LFER approach to ions.11\nFong [161] has reviewed many of the attempts to predict permeabilities of the blood–brain barrier to solutes. He concludes that the most important factors for neutral solutes are: the free energy required to remove the solute from water; the free energy gained from the interactions of the solute with the membrane core, usually modelled by its interaction with n-octanol; the dipole moment of the solute; and lastly its molecular volume. Increases in molecular volume per se decrease permeability. Geldenhuys et al. [172] has provided many useful references in a review prepared from the perspective of the utility of predictions in high-throughput screening.\n\nTransporters at the blood–brain barrier\nThe membranes of the endothelial cells that constitute the blood–brain barrier possess transporters for many different types of solutes. These transporters may be present on luminal, abluminal or both surfaces of the endothelial cells. Prominent among them are transporters for common nutrients and waste products of metabolism: GLUT1 for glucose, MCT1 for lactic acid and other small monocarboxylic acids, a range of transporters for amino acids, and several for nucleosides. There are also ion transporters involved in maintenance of the ionic composition of the brain fluids. Many of the transporters are specific and are involved in moving the normal constituents of brain extracellular fluid. Some of these are considered in Sect. 5. In addition there are also less specific transporters. Many of these can mediate efflux of a variety of other substrates including many exogenous substances and toxic occasional products of metabolism.\nEvidence concerning the presence and identity of many of these transporters has been reviewed elsewhere with studies being conducted primarily at the level of transcript [173–178], protein [31, 44, 58, 176, 179–188] and/or function [4, 20, 31, 46, 55–57, 179, 189–200]. The reports by Roberts et al. [180] and Kubo et al. [58] and reviews by Hawkins et al. [44], Redzic [31], Campos-Bedolla [57], Worzfeld and Schwaninger [187] and Nalecz [200] have been useful as sources of information about the localization of transporters to the luminal or abluminal membranes.\nThis review will not seek to provide yet another comprehensive survey. Extensive lists of transporters and substrates are available in many of the cited references and for SLC transporters at the BioParadigms website [201, 202].\n\nABC efflux transporters\nIt has long been appreciated that the brain represents a pharmacological sanctuary and is selectively “protected” from the toxic effects of many chemotherapeutic agents. These include vincristine and doxorubicin (aka adriamycin), which fail to penetrate the blood–brain barrier as well as their lipid solubilities would suggest [162]. A major part of this failure to penetrate has since been attributed to the presence of the multidrug transporter, P-glycoprotein. Absence of this transporter in knock-out mice was shown to allow entry of toxic agents including ivermectin [203]. P-glycoprotein was found to be located in the luminal membrane (see e.g. [204–209]) of the endothelial cells and is believed to act there to transport substrates out of the cells so rapidly that little remains to penetrate the abluminal membrane and enter the brain.\nIt is believed by many that P-glycoprotein, a transmembrane protein, acts by removing its lipophilic substrates from the lipid layer of the cell membrane, depositing them back into the blood [210–213]. Its structure has been investigated in both substrate-free and inhibitor bound conformations [213] and binding sites for various of its many substrates identified within the large cavity seen in the substrate-free conformation. It is the binding and hydrolysis of ATP that provides the motive force leading to a large conformational change in the P-glycoprotein and the transfer and expulsion of its substrates. There are two ATP binding sites located on the cytoplasmic side of the protein.\nP-glycoprotein, otherwise called ABCB1, is a member of the ABC (ATP-Binding Cassette) family of proteins many of which are primary active transporters that utilize the hydrolysis of ATP to fuel substrate transport. Since its discovery, other ABC active transporters with broad substrate profiles have been found in the luminal membrane of the endothelial cells. These include Breast Cancer Resistance Protein, BCRP (ABCG2) [180, 197, 214–218] and Multidrug Resistance Proteins, MRPs 4 and 5 (ABCC4 and 5) [180, 197, 209, 218–221]. MRP1 (ABCC1) has also been implicated but levels of this transporter are thought to be low in brain endothelial cells in situ and only increase in cultured brain endothelial cells once they are removed from the brain microenvironment [180, 184, 218, 222–226]. MRP1 and MRP2 are apparently upregulated and clearly expressed in epilepsy [227–229].\nThe role of efflux from endothelial cell to blood by ABC transporters in preventing influx of many substances from blood into the brain has been extensively reviewed (see e.g. [57, 196, 197, 199, 221, 230–234]. The regulation of P-glycoprotein, BCRP and MRP2 at the blood–brain barrier has been reviewed by Miller [221].\nThe role of ABC transporters in efflux from the brain parenchyma differs depending on the nature of the substrate. As described in Fig. 9, for substances that are sufficiently lipid soluble to cross the endothelial cell membranes rapidly by passive transport, the presence of ABC efflux transporters can greatly reduce blood-to-brain influx, as observed experimentally. However, as also explained in Fig. 9 the ABC transporters in the luminal membrane will have only a modest effect, e.g. a doubling, on the rate of brain-to-blood efflux. This may be of little consequence as the rate of efflux for lipid soluble substances is already high.\nFig. 9 The influence of ABC transporters on the movements of lipophilic substances. The substance is presumed to be able to enter and leave the endothelial cells by diffusion with rate constant kdiff., which for simplicity in this example is assumed to be same on both sides. The substance is expelled from the cell by ABC transporters on the luminal side at a rate, kactiveccell. With these assumptions the effect of the ABC transporters on influx can be calculated by setting cisf = 0 and the effect on efflux by setting cplasma = 0. In both sets of equations, the first line states that at steady-state the net flux into the cell on one side must equal the net flux out of the cell on the other. From the next to the last lines of both sets of equations, if the rate of ABC mediated expulsion from the cell is small or zero, the rate constants for both influx and efflux are (kdiff./2). By contrast from the last lines if the rate of ABC mediated expulsion is large, influx to the brain, J→, becomes very small, while efflux from the brain, J←, is doubled compared to the efflux with no ABC transporter\nThe role of ABC transporters for solutes with low passive permeability across the membranes is considered in the next section.\n\nEfflux mediated in part by SLC solute transporters\nMany of the SLC (solute carrier) transporters (see [202] for a list) are present in the membranes of the endothelial cells of the blood–brain barrier. Some are considered in connection with the transport of specific solutes in Sect. 5. Others, primarily from the SLC21 (OATPs, organic anion transporting polypeptides) and SLC22 (OATs and OCTs, organic anion transporters and organic cation transporters) families are associated with transport of a variety of organic anions and cations. These have been reviewed frequently and extensively [57, 176, 200, 218, 235–246]. (Uppercase labels, e.g. SLC or OAT, strictly refer to human sequences and proteins, while mixed-case labels, e.g. Slc or Oat, refer to any other species. In this review uppercase is also used when there is no intention to specify species).\nThere is little quantitative data on the efflux of organic anions and cations from the parenchyma in humans though many are known to be transported. In rodents more information is available for transfer of organic anions than cations. Table 1 lists some examples of organic anions/neutral molecules for which brain-to-blood transport rate constants have been determined. These are all believed to be substrates for Oat3 (Slc22a8) and/or one or more of the Oatp transporters present at the blood–brain barrier. In broad terms [238], small hydrophobic anions are substrates for Oats (Slc22 family) while larger amphipathic anions are substrates for Oatps (Slc21 family, whose member names start with Slco, see [247]). For comparison Table 1 also lists rate constants and clearances for examples of markers for perivascular efflux. It is clear that the rates of elimination of the Slc substrates are considerably greater than could be supported by perivascular efflux alone.\nTable 1 Comparison of rate constants for efflux and clearances for Slc22 and Slco substrates and the perivascular markers inulin, mannitol and sucrose\nkeff/min−1 t1/2/min Vd/mL/g CL/µL g−1 min−1 Ref # Notes\nSlc substrates Rat unless stated otherwise\n p-Aminohippuric acid (PAH) 0.059 12 0.80 47 [558] Influx much slower than efflux\n p-Aminohippuric acid (PAH) 0.039 18 [559] Abl. Oat3 based on inhibitors, lum. Mrp4 possible based on kidney [560]\n p-Aminohippuric acid (PAH) 0.0175 40 [131]\n Penicillin G, benzylpenicillin 0.043 16 [559] Oat3 based on inhibitors\n Taurocholate 0.023 30 [561]\n BQ-123 0.0078 100 [561]\n Estrone sulfate 0.066 9.9 1.1 75 [562]\n Estrone 0.061 11 3.3 227 [562]\n Dehydroepiandrosterone sulfate (DHEAS) 0.027 26 118 [563] Influx much slower than efflux, Oatp2\n Estradiol-17beta-glucuronide (E217betaG) 0.037 19 [564] Oatp2, 40% and Oat3 20%\n Pravastatin 0.060 12 0.99 59 [565] Oat3 (Slc22a8) and Oatp2 (Slco1a4) + others\n Pitavastatin 0.026 27 14 364 [565] Oat3, Oatp2 (Slco1a4) +others +diffusion\n Homovanillic acid 0.017 40.8 [566] Oat3 from inhibitors\n Indoxyl sulfate 0.011 64 [567] Oat3 and others\n Pemetrexed 0.018 39 0.62 11 [568] Mouse. Oat3 and unknown (not Mrp2 not Bcrp)\n Methotrexate 0.024 29 0.85 20 [568] Oat3 and Bcrp suggested\n Buprenorphine 0.025 27.5 6.1 154 [569] Pgp and unknown, possibly diffusion\n AZT, (3′-azido-3′-deoxythymidine) 0.032 22 [570] Oat3 from benzylpenicillin inhibition\n DDI, (2′,3′-dideoxyinosine) 0.253 2.8 [570] Oat3 from benzylpenicillin inhibition + diffusion\nMarkers for perivascular elimination\n Inulin 0.006 awake0.016 asleep 11543 0.2* 1.23.2 [128] Mouse\n Inulin 0.003 230 0.2* 0.6 [62] Mouse\n Inulin 0.005 135 0.2* 1 [131] Rat\n Mannitol 0.004 170 0.2* 0.8 [25] Mouse\n Sucrose 0.0028 awake0.0043 anesth. 245160 0.2* 0.560.86 [131] Rat\n Albumin 0.006 115 0.2* 1.2 [131] Rat\n Dextran-10K 0.0035 197 0.2* 0.7 [131] Rat\n Dextran-70K 0.004 170 0.2* 0.8 [131] Rat\nkeff rate constant for efflux determined from the time course of the decrease in concentration after injection of solute into the parenchyma (brain efflux index for the Slc substrates relative to inulin; these underestimate keff for values less than ~ 0.01 min−1 see [131]); t1/2 = 0.69/(rate constant) is the half-life; Vd, volume of distribution in the parenchyma determined using brain slices (ISF volume for inulin and mannitol); CL = Vd × keff, the clearance. For the Slc substrates more than one transporter in each membrane is likely to be involved in the transport\n* Assumed equal to ISF volume\nAs indicated in Fig. 10 transport from the parenchyma into the endothelial cells occurs via one or more of the SLC transporters, while exit from the endothelial cells to plasma occurs via either SLC or ABC transporters. For many of the anions efflux from brain to blood is clearly an active uphill process suggesting that the ABC route is dominant (for a caveat see.12) Transport across either membrane can be rate limiting and in many cases transport across each can occur by more than one route. As a consequence demonstration that a specific inhibitor of a transporter reduces the rate of efflux is evidence for involvement of that transporter, but failure to inhibit is relatively uninformative.\nFig. 10 Transport of organic anions across the blood–brain barrier. Organic anion transporters at the blood–brain barrier. The principal known transporters in the rat are shown. In human OAT3 is abluminal, while both OATP1A4 and OATP2B1 are present on both membranes. The ABC efflux pumps, P-gp, BCRP, MRP4 and MRP5, are all localized to the luminal, plasma facing, membrane. The Oat and Oatp transporters are exchangers (see Footnote 12). Localizations from [180] and the references in Table 1\nFor the SLC substrates in Table 1 the half-lives are shorter than the 1–2 h characteristic of markers eliminated from the parenchyma by perivascular efflux (see Sect. 3). As noted earlier, shorter half-lives imply that there are mechanisms for elimination other than perivascular. This is reinforced by noting that the clearances for those solutes for which volumes of distribution are available are much greater than the clearance associated with the perivascular route (see Sect. 3.2). There is ample further evidence (see the references for the entries in Table 1) for the importance of the Oat and Oatp transporters in the elimination of these solutes from the parenchyma including saturation, competition, the availability of transport inhibitors, and the rapid appearance of effluxed material in venous blood draining the head.\n\nEfflux by transcytosis\nTranscytosis is much less prevalent across the endothelial cells of the blood–brain barrier than across those of peripheral capillaries [248–251]. Nevertheless both adsorptive mediated transcytosis (AMT) and receptor mediated transcytosis (RMT) are still likely to be important mechanisms for the transfer of some large substrates across the blood–brain barrier. The initial event in AMT is the adsorption of usually positive substrates onto the surfaces of caveolae, while that for RMT is binding of the substrate to specific receptors that are in or become incorporated into clathrin coated pits. In both cases at the blood–brain barrier this leads to endocytosis followed by delivery of a substantial fraction of the contents of the resulting vesicles to the opposite membrane for exit, possibly by exocytosis [49, 63, 252]. AMT is thought to account for much of the influx into the brain of histones [253], “cell penetrating peptides” [49, 251, 254], HIV [255, 256], and cargos conjugated to the lectin wheat germ agglutinin [257] and to underlie the increase in “generalized permeability” caused by protamine [258]. The downsides of AMT are that it is relatively non-selective for substrates [256] and that it occurs in many cells throughout the body. In addition there is little if any evidence that it occurs in the direction from brain to blood [257, 259]. While RMT also occurs throughout the body, transport by this mechanism depends on interaction of the substrates with specific receptors that may be found primarily in specific locations such as the blood–brain barrier. In addition there is evidence that RMT can occur in either direction, i.e. from brain to blood as well as from blood to brain.\nAMT and RMT in the direction from blood to brain have been studied extensively as routes of entry to the brain for endogenous substrates, but even more in the context of mechanisms for drug delivery. These studies have been reviewed frequently [57, 64, 154, 249, 252, 260–266]. However, even so, the steps occurring after the initial endocytosis remain only partially understood [63, 249, 250, 262, 267, 268] including even the answer to the important question of whether the cargo is released within the cell or delivered to the far side by exocytosis. By contrast evidence for transport via transcytosis in the direction brain to blood has been reported for only a few systems including transport of amyloid-β peptides via interaction with LRP1 (low density lipoprotein receptor related protein 1) and LRP2 (low density lipoprotein receptor related protein 2) (see Sect. 5.7), of IgG antibodies via interaction with an unidentified receptor [269–275] and of transferrin [60] via interaction with the transferrin receptor (TfR) [61] (see below).\nTransport of transferrin is closely related to transfer of iron. Iron in plasma and in brain extracellular fluid is present almost entirely complexed to transferrin i.e. as holo-transferrin. It has long been known that iron and transferrin enter the brain across the blood–brain barrier and it was originally hypothesised that they are transferred together by endocytosis followed by exocytosis, i.e. direct transcytosis, of holo-transferrin (see e.g. [61, 276]). Yet there have been arguments against this idea arising from dual labelling experiments showing that far more labelled iron than labelled transferrin accumulates in the brain, see e.g. [60, 277, 278]. In addition it has been argued that release of holo-transferrin from TfR is unlikely to occur as there needs to be prior dissociation of iron for release of transferrin from its receptor [279]. So though there is general agreement that holo-transferrin interacts with TfR, which then mediates endocytosis of the iron/transferrin/receptor complex into the endothelial cells, there has been controversy over the subsequent steps in the transfers of transferrin and iron into the brain. Assuming that holo-transferrin is indeed directly transcytosed across the blood–brain barrier, then the limited net entry observed of transferrin to the brain implies that there must be transcytosis of transferrin without iron, apo-transferrin, back out of the brain. Alternatively if the iron is dissociated from the transferrin within the endothelial cells, it is likely that there is exocytosis of apo-transferrin on both sides of the cells (see [280–282] and the footnote13 for further discussion).\nLittle is known about transport of transferrin out of the brain. There have been reports that labelled apo-transferrin injected into the brain can be transported from brain to blood, but it is not clear how important this is under normal conditions. Banks et al. [60] found that the apo-transferrin was removed from the brain faster than albumin, implying the existence of a route other than washout via CSF. However, subsequently Moos and Morgan [278] did not confirm this result. By contrast Zhang and Pardridge [61] found an early component of loss of injected apo-transferrin, half-life 39 min, which was much faster than that for loss of injected 70 kDa-dextran. Furthermore this rapid component was inhibited by cold apo-transferrin, i.e. there was competition, with an apparent dissociation constant of less than 30 nM implying interaction with a specific receptor which was presumed to be the receptor protein detected by OX26, i.e. TfR. As these studies on transferrin efflux are substantially older than the studies on iron uptake linked to transferrin, further investigation of transferrin transport from brain to blood might be informative."}