>> GOOD EVENING AND WELCOME TO ANOTHER SESSION OF PRINCIPLES OF CLINICAL FAR PHARMACOLOGY. WISHING ALL OF YOU A HAPPY NEW YEAR. TONIGHT WE'RE DELIGHTED TO HAVE TWO SPEAKERS THAT WILL ADDRESS THE IMPORTANT TOPIC OF DRUG TRANSPORT. SOME SPECIAL ISSUES IN DRUG TRANSPORT. AND FIRST WE WILL HAVE DR. MICHAEL GOTTESMAN WHO IS THE DEPUTY DIRECTOR FOR INTRAMURAL RESEARCH AT THE NATIONAL INSTITUTES OF HEALTH. DR. GOTTESMAN IS A GRADUATE OF HARVARD COLLEGE AND HARVARD MEDICAL SCHOOL. AND SUBSEQUENTLY TRAINED IN INTERNAL MEDICINE AND BEGAN HIS RESEARCH TRAINING ALSO AT HARVARD IN THE LABORATORIES OF WILLIAM BECK AND BUTTER BALANCE -- BERT BALLARD. HE JOIN THE NATIONAL CANCER INSTITUTE IN 1976 BECOMING THE CHIEF OF THE MOLECULAR CELL GENETIC SECTION OF THE LABORATORY OF MOLECULAR BIOLOGY IN 1980. AND ALSO BEING CHIEF OF THE LABORATORY OF CELL BIOLOGY STARTING IN 1990. HE BECAME DEPUTY DIRECTOR FOR INTRAMURAL RESEARCH IN NOVEMBER OF 1993. HIS RESEARCH INTERESTS HAVE RAN FROM HOW DNA IS REPLICATED IN BACTERIA, TO HOW CANCER CELLS AND CHEMOTHERAPY. HE HAS PUBLISHED EXTENSIVELY ON THESE SUBJECTS. HE WAS AMONG THE FIRST TO USE TECHNIQUE OF DNA TRANSFER TO DEMONSTRATE THE ROLE OF CYCLIC AND INDEPENDENT KINASE IN GROWTH REGULATION AND TO STUDY THE EFFECT OF MICROTUBULE DEFECTS ON MITOSIS. FOR OVER A DECADE, HE WORKED IN CLOSE COLLABORATION WITH DR. IRA PASSEN AND IDENTIFIED THE HUMAN GENE RESPONSIBLE FOR RESISTANCE OF CANCER CELLS TO MANY OF THE MOST COMMON ANTI-CANCER DRUGS. AND HAS SHOWN THAT THIS -- CANCER DRUGS OUT OF DRUG RESISTENT HUMAN CANCERS. AND HE WILL OF COURSE TELL YOU A LOT ABOUT THAT DURING HIS PRESENTATION. HE WAS ELECTED A FELLOW OF THE AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, AND ALSO RECEIVED NUMEROUS AWARDS INCLUDING THE ROSENFELD FOUNDATION AWARD AND THE AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS AWARD IN 1997. OUR SECOND SPEAKER TONIGHT IS DR. MATTHEW HALL, WHO IS CURRENTLY GROUP LEADER OF BIOLOGY AT THE NATIONAL CENTER FOR THE ADVANCEMENT OF TRANSLATIONAL SCIENCES OR N-CAPS. WHERE HE IS WORKING TO DEVELOP HIGH THROUGHPUT SCREENINGS FOR DISCOVERING AND DEVELOPING BIOACTIVE MOLECULE FOR A RANGE OF DISEASES AND BIOLOGICAL PROCESSES. DR. HALL OBTAINED HIS DOCTORAL DEGREE FROM THE UNIVERSITY OF SYDNEY AUSTRALIA AND SERVE THE ONE YEAR AS A FELLOW AT JAWNLS JAWNLS -- JOHNS HOPKINS SCHOOLS OF PUBLIC HEALTH BEFORE A POST FELLOWSHIP IN DR. GOTTESMAN'S LABORATORY. HE BECAME A STAFF SCIENTIST IN THAT LABORATORY IN 2013. AND HE'S AN ADJUNCT FELLOW AT JOHNS HOPKINS UNIVERSITY. SO DR. GOTTESMAN WILL BEGIN AND THEN DR. HALL WILL CONTINUE IN THE SECOND HALF OF THE SESSION. THANK YOU. >> THANK YOU VERY MUCH FOR THAT KIND INTRODUCTION. IT'S ALWAYS A PLEASURE TO BE HERE AND IT'S PARTICULARLY A PLEASURE THAT IT'S NOT SNOWING TONIGHT. I DON'T KNOW. WE'VE DONE THIS LECTURE ABOUT A DOZEN TIMES AND ALMOST INVARIABLY IT'S SNOWING. IT DOESN'T LOOK LIKE IT'S GOING TO SNOW THIS WINTER SO WE'RE GOING TO HAVE A NICE WINTER. LET'S SEE. HOW DO I CHANGE THE SLIDES? SO LET ME TELL YOU GENERALLY WHAT MY LABORATORY'S INTERESTED IN AND I'M GOING TO BE ZEROING IN IN MY INTRODUCT TWREA COMMENTS I --INTRODUCTION COMMENTS ON PHARMACOLOGY OF ATPITY PNT TRANSPORTERS. WE'RE GENERALLY INTERESTED IN DEFINING RESISTANCE TO MORE TRADITIONAL ANTI-CANCER DRUGS. THESE ARE NOT TARGETED DRUGS BUT DRUGS THAT HAVE A BROUGHT SPECTRUM OF TOXICITY AND ARE QUITE EFFECTIVE IN KILLING CANCER CELLS. THESE INCLUDE NATURAL PRODUCTS LIKE ANTHRO PSYCH LIEN LIKE TAX IAN AND PLATINUM COMPOUNDS. WE'RE INTERESTED IN THE CLINICAL RELEVANCE OF WHAT WE LEARNED FROM OUR TISSUE CULTURE STUDIES. AND WE WANT TO USE THIS INFORMATION TO EXPLOIT OR CIRCUMVENT RESISTANCE MECHANISMS. AND ULTIMATELY LEARN MORE ABOUT THE CELLULAR PHARMACOLOGY AND THE PHARMACOKINETICS OF DRUGS. SO, THIS SLIDE INTRODUCES THE FACT THAT DRUG RESISTANCE IS A VERY COMPLEX PHENOMENA. AS YOU KNOW, CONFERENCE CELLS MUTATE AT HIGH FREQUENCY. THEY'RE QUITE HETEROGENEOUS. AND THEY'RE VERY EXPERT AT COMING UP WITH WAYS TO CIRCUMVENT THE EFFECTS OF ANTI-CANCER DRUGS. FOR DRUGS THAT ARE VERY SPECIFIC TARGETED TO SPECIFIC GROWTH PROMOTING PATHWAYS IN CELLS, IT'S FAIRLY STRAIGHTFORWARD AND EASY FOR THE CELLS TO DEVELOP. MUTATIONS IN THOSE TARGETS THAT RESULT IN RESISTANCE. IN ADDITION, THE CELLS THEMSELVES HAVE A VERY COMPLEX PHARMACOLOGY. AS YOU'LL SEE THAT TAKE UPS IN E EFFLUX DRUGS AND METABOLIZED DRUGS. PHARMACOLOGY CAN HAVE PROFOUND EFFECT TO THE SENSITIVITY OF DRUGS ANTI-CANCER COMPOUNDS. THERE ARE ADDITIONAL WAYS WHICH I WON'T BE GOING INTO IN ANY DETAIL TODAY, INCLUDING THE FACT THAT CANCER CELLS THEMSELVES CAN CHANGE THE DIFFERENTIATION PATTERNS THAT CAN TURN ON AND OFF LARGE SETS OF DIFFERENT GENES. THE EXAMPLE OF THIS WHICH IS BEST KNOWN IS WHAT'S CALLED EPITHELIAL AND PARENCHYMAL TRANSITION WHERE AN EVEN TELEANNUAL -- EPITHELIAL -- THEN THERE'S NO QUESTION THAT THE LOCAL ENVIRONMENT, THE OTHER CELL TYPES, THE NATURE OF THE SUBSTRATE U IN WHICH IT'S SITTING, THE OXYGEN TENSION AND SO ON. ALL OF THOSE ASPECTS HAVE BELOW FOUND EFFECTS ON RESISTANCE. ON THIS LECTURE, BECAUSE IT'S A PHARMACOLOGY COURSE, I'LL BE FOCUSING ON THESE CHANGES IN CELLULAR PHARMACOLOGY. SO THIS IS A CELL. AS I MENTIONED, IN ORDER FOR ANTI-CANCER DRUG TO BE EFFECTIVE, IT HAS TO GET INTO THE CELL. AND WE NOW KNOW THAT THERE ARE OVER 400 OR SO SAHL YOUTUBE CARRIERS -- SOLUTE CHARACTERS AND PROBABLY 40 TO 50 OF THEM INVOLVED SPECIFIC DRUGS. THIS IS AN EXAMPLE OF WHERE A DRUG PIGGYBACKS ON TRANSPORT MECHANISM FOR GETTING THINGS INTO CELLS. IN ADDITION, CELLS ARE QUITE GOOD AT RIDDING THEMSELVES OF MATERIALS THAT THEY DON'T WANT. AND THERE ARE A SET OF THESE ATP DEPENDENT TRANSPORTERS EFFLUX PUMPS FOR BINDING TRANSPORTERS. MOST OF WHAT I WILL BE TALKING ABOUT FOCUSES ON THESE TRANSPORTERS BECAUSE THEY'RE IMPORTANT NOT ONLY IN THE CELLULAR PHARMACOLOGY OF ANTI-CANCER DRUG BUT TURN OUT TO BE REALLY IMPORTANT FOR PHARMACOLOGY OF MANY OF THE DRUGS THAT ARE IN COMMON USE IN THE CLINIC. NOW, IF THE DRUG HAPPENS TO GET INTO THE CELL, AND THAT OF COURSE IS THE GOAL OF MOST PHARMACOLOGISTS TO GET DRUGS INTO CELLS. THERE ARE A VARIETY OF ALTERATIONS WITHIN THE CELL, ALTERATIONS IN THE EVEN TAUGHTIC PATHWAYS -- EP ALSO TARGETS REPAIRED DAMAGE AND COMPARTMENTIZATION THAT RENDERS CELLS MORE RESISTENT TO THESE COMPOUNDS. BUT I WILL BE FOCUSING ON THE EFFLUX PUMPS. WHY DO WE STUDY THEM IN WELL PARTLY BECAUSE WE'RE INTERESTED IN MULTIDRUG RESISTANCE IN CANCER AND THE INTEREST THAT I MENTION IN DRUG PHARMACOKINETICS. THESE TRANSPORTERS ARE EXPRESSED NOT ONLY IN CANCER CELLS BUT IN MANY IMPORTANT CELLS IN THE BODY INVOLVED IN DRUG METABOLISM. THEY PROBABLY PLAY AN IMPORTANT ROLE IN DRUG TOXICITY. THE PRESENCE OR ABSENCE OF AN EFFLUX PUMP IN A CELL CAN DETERMINE ITS SENSITIVITY TO ATTACK THIS DRUG. THEY ARE INVOLVED WITH DEVELOPMENT. THERE ARE MANY EXAMPLES. THEY ARE HIGHLY EXPRESSED, MANY OF THEM STEM CELL AND THEY'RE INVOLVED IN MORPH GENESIS OF A NUMBER OF DIFFERENT ORGANISMS INCLUDING PROBABLY THE HUMAN. AND THEY ARE, STUDYING THEM IN DETAIL WE LEARN A LOT ABOUT THE BIOLOGY OF TRANSPORT SYSTEMS. SO THIS PARTICULAR FAMILY THAT WE'RE INTERESTED IN IS ONE OF THE LARGEST FAMILIES OF TRANSPORTERS THAT ARE KNOWN. TO DATE, WELL OVER 2000 MEMBERS HAVE BEEN IDENTIFIED IN EVERY KNOWN ORGANISM. SUBSTRTS FOSUBSTRATES FOR TRANSPORT ARE ION, SUGARS -- PROTEINS, TOXINS, ANTIBIOTICS AND OF COURSE THE NATURAL PRODUCT CANS DRUG. STRUCTURALLY THEY CONSIST OF VARIOUS COMBINATIONS OF ATP BINDING CASSETTES. THESE ARE PARTS OF THE MOLECULE THAT FIND ATP AND PRODUCE THE ENERGY NEEDED FOR TRANSPORT. AND SEGMENTS, TRANSMEMBERRING SEGMENTS WHICH HAVE SIX DOMAINS. SO IF YOU LOOK AT THE EVOLUTION OF THESE TRANS PORTERS. AS I MENTIONED, THEY ARE PRESENT IN ALL ORGANISMS. THERE ARE THREE OR FOUR DIFFERENT FAIRLY CLOSELY RELATED FAMILIES SHOWN HERE IN GREEN. THIS IS A FAMILY WHICH INCLUDES -- INSECT FAMILIES, FAMILIES OF VERTEBRATES AND INCLUDING FISH AS WELL HERE. AND THEN OTHER KINDS OF ORGANISMS THAT ALL OF WHICH EXPRESS THESE TRANSPORTERS. INTERESTINGLY THE -- TRANSPORTERS ARE MORE CLOSELY RELATED TO THE VERTEBRATE ONE THAN THE SINGLE CELL ORGANISMS. AS I MENTIONED THERE ARE VARIOUS COMBINATIONS OF ATP BINDING CASSETTES. AND TRANSPORT MECHANISMS THAT ARE INVOLVED. AND THERE ARE IN THE HUMAN, THERE ARE 48 ABC GENES IN DIFFERENT FAMILIES LABELED A, B, C, D, E, F AND G. AS YOU CAN SEE BY THESE CARTOONS HERE, THEY HAVE VARIOUS FORMATS. I'LL GET INTO THIS IN A FEW MOMENTS IN A LITTLE MORE DETAIL. BUT FOR EXAMPLE THE ABCD FAMILY INCLUDES MEMBERS THAT HAVE TWO SEGMENTS OF SIX TRANSMEMBRANES, ATP BINDING CASSETTES OR SINGLE SIX DOMAIN REGIONS WITH ATP BINDING. WHEN YOU SEE THIS FORMAT IN EITHER MAMMALIAN OR BACTERIAL SYSTEM IT'S ALWAYS PRESENT AS A DIMER IN THE MOLECULE. AND SO WE THINK THIS IS THE FORMAT TO TRANSMEMBRANE REGIONS AND TWO ATP BINDING WHICH IS MOST COMMON IN THESE ORGANISMS. NOW, THERE ARE MEMBERS OF THIS FAMILY THAT INCLUDE ONLY ABC TRANSPORTERS AND THEY FUNCTION IN PROCESSES LIKE THE TRANSLOCATION IN RIBOSOMES AND SO ON. AND SEEM TO BE ENERGY FACTORIES FOR TRANSLOCATION OF THINGS OTHER THAN SUBSTRATES ACROSS MEMBRANES. NOW, THERE ARE THREE TRANSPORTERS THAT ARE PARTICULAR INTEREST TO US IN THIS FAMILY. ABCD1 WHICH IS ALSO CALLED G GLYCOPROTEIN NAMED BY -- WHO FOUND IT TO BE PRESENT IN HIGH LEVELS OF DRUG RESISTENT CELLS. P LYCO PROTEIN. P STANDS FOR PERMEABILITY. INITIALLY IT WAS THOUGHT TO KEEP DRUGS FROM GETTING IN AND IT WAS THE DISCOVERED TO BE ENERGY DEPENDENT EFFLUX. ABC1 OR MRP1. MRP STANDS FOR MUST BE TIE DRUG RESISTENT RELATED PROTEIN. AND ABCG2 OR BCRP WHICH IS BREAST CANCER RESISTANCE LOW TEEN OR -- RESISTANCE PROTEIN. WE WERE THE FIRST PEOPLE TO CLONE IN THE HUMAN. THE ABCD1 GENE AND SEVERAL YEARS LATER THESE OTHER MULTIDRUG TRANSPORTERS WERE DISCOVERED CLONED AND CHARACTERIZED. NOW, IN THE NORMAL BODY, THESE TRANSPORTERS ARE EXPRESSED AT BARRIER SITES. AND DR. HALL WILL TELL YOU IN MUCH MORE DETAIL HOW THESE FUNCTION AND WHAT THEIR IMPORTANCE IS. BUT JUST IN GENERAL, WHEN THEY ARE PRESENT FOR EXAMPLE IN THE PLACENTA, IN THE BRAIN AND IN THE CAPILLARIES IN THE BRAIN, IN THE TESTES AND THE OVARIES, THEIR FUNCTION IS TO BE PART OF THE BLOOD BARRIER IN THOSE PARTICULAR ORGANS TO MAKE SURE THAT THINGS THAT ARE IN THOSE ORGANS GET POUND BACK INT PUMPED BACK INTO THE BLOOD STREAM. THEY HAVE A AN EXCREE TREE FUNCTION WHERE THEY PUMP MATERIALS OUT OF THE BODY SO THEY CAN BE EXCRETED. THEY ARE BOTH A BARRIER FUNCTION WITHIN THE ORGANISM THAT EFFECTS THE DISTRIBUTION OF DRUGS IN THE BODY BUT THEY ALSO HAVE AN EXCRETORY FUNCTION. AND THIS IS JUST TO POINT OUT THAT THERE ARE MANY DRUGS IN COMMON USE THAT ARE SUBSTRATES FOR ONE OR MORE OF THESE TRANSPORTERS. AND EXAMPLES ARE SHOWN HERE. YOU STILL PROVIDE THE SLIDES? OKAY. SO YOU CAN STUDY THIS. I'M NOT GOING TO READ THROUGH THESE BUT I'M SURE SOME OF YOUR FAVORITE DRUGS ARE LISTED HERE. NOW, IT TURNS OUT THAT MANY OF THESE ABC TRANSPORTERS ARE INVOLVED IN DAILY GENETIC DISORDERS IN WHICH KNOCKING OUT A SINGLE GENE PRODUCES A DISEASE STATE. MOST CASES THESE ARE NOT THAT HEAL DISEASES BUT THEY ARE QUITE SIGNIFICANT DISEASES. WE MENTIONED ALREADY IN CANCER THAT THESE MULTIDRUG TRANSPORTERS ARE INVOLVED IN DRUG RESISTANCE IN CANCER. AND CYSTIC FIBROSIS, ANOTHER MEMBER OF THIS FAMILY, ABC7, THE FTR GENE IS THE -- ARE RESPONSIBLE FOR THE CHLORIDE CHANNELS IN CYSTIC FIBROSIS. THERE'S A DEFECT IN ADULT MAC INCLUDE -- MAC INCLUDE DEGENERATION WHICH IS A RENT NOID TRANSPORTER. THIS DISEASE IS QUITE INTERESTING. HIGH DENSITY LYCO PROTEIN AND IT'S TWO TO A DEFECT AND SO ON AND SO FORTH. YOU CAN TAKE A LOOK AT THESE. THESE ARE QUITE INTERESTING DISEASES AND I THINK IT POINTS OUT THE CENTRAL ROLE THAT THESE TRANSPORTERS PLAY IN THE MOVING OF NORMAL MATERIALS AS WELL AS DRUGS IN THE BODY. SO THE THREE TRANSPORTERS OF INTEREST TO US ARE SHOWN HERE. AND YOU CAN SEE ABCD1 HAS THE CONICAL 12 TRANS 34E7 MEMBRANE REGION. IT HAS AN INTERNAL EXTENSION. WE KNOW IF YOU DELETE THIS PART OF THE MOLECULE YOU STILL GOT A TRANSPORTER. WE'RE NOT EXACTLY SURE. ABCG2 IS ONE OF THE TEST MOLECULES WHICH FUNCTIONS AS A HOMO DIMER. THE THREE TRANSPORTERS DON'T HAVE IDENTICAL SUBSTRATES. THIS IS ADDITIONALLY COMPLICATED BY THE FACT THAT EVERY ORGANISM THAT YOU STUDY, EVERY MAMMAL THAT YOU STUDY THAT HAS THESE THREE TRANSPORTERS HAS A SLIGHTLY DIFFERENT SUBSTRATE FOR ANY ONE OF THEM. BUT IN THE HUMAN, WHAT YOU CAN SEE IN THE SEN ZEN DIAGRAM, THESE ARE ALL AN SIGH CANCER DRUGS THEY ARE TRANSPORTED BY ALL THREE OF THE TRANSPORTERS. THERE ARE SOME DRUGS THAT ARE VERY SPECIFIC. FOR EXAMPLE, PAX ILL -- ARE ONLY TRANSPORTERS BY ABCB1 AND SO ON AND SO FORTH. SO THE SPECIFICITY IS NOT ABSOLUTE. BUT EACH HAS A VERY BROAD RING RING -- RANGE OF ABILITY TO TRANSPORT DRUGS AND SOME OF THE DRUGS ARE SHARED AS TRANSPORT SUBSTRATES BY ALL OF THE TRANSPORTERS. NOW, WHAT MAKES US THINK THAT THESE TRANSPORTERS INVOLVE DRUG RESISTANCE? WELL THE SIMPLEST EXPERIMENT IN WHICH YOU TAKE A CULTURED CANCER CELL WITH RESISTANCE TO ANY SPECIFIC DRUG. VERY FREQUENTLY RESULTS IN OVER EXPRESSION OF THESE TRANSPORTERS. AND BY TRANSFERRING BY GENETIC MEANS INTO A SENSITIVE CELL ONE OF THESE TRANSPORTERS YOU GET RESISTANCE. AMONG THE 48 TRANSPORTERS, AT LEAST 12 ARE INVOLVED IN RESISTANCE TO THIS, ONE OR MORE OF THESE ANTI-CANCER DRUGS. SO THERE'S NO QUESTION THAT THESE TRANSPORTERS ARE CAPABLE OF COMPARING RESISTANCE. THE QUESTION FINANCIAL BEFORE US IS ARE THEY ACTUALLY RESPONSIBLE FOR RESISTANCE IN CANCER AND I'LL SAY MORE ABOUT THIS IN A MOMENT. SO, ABCB1 OR P GLYCOPROTEIN IS 1280 AMINO ACIDS. IF YOU SPREAD THEM OUT IN A LINEAR WAY YOU CAN SEE THE 12 TRANSMEMBRANE DOMAINS. THE TWO ATB BINDING CASSETTES. AND THE YELLOW SPOTS HERE SHOW MUTATIONS THAT HAVE EITHER BEEN MADE OR OCCURRED NATURALLY WITHIN P LYCO PROTEIN. IT CHANGES SUBSTRATES. THESE EARLY STUDIES LED US TO BELIEVE IT'S IN THE TRANSMEMBRANE REGION THAT ARE INVOLVED IN INTERACTION. BECAUSE MOST OF THE SUBSTRATES ALTERING MUTATIONS WERE IN THE TRANSMEMBRANE MUTATION. IF YOU INTO IT TO THE BINDING SIDE YOU LOSE TRANSPORT ABILITY. SO YOU NEED BOTH SIDES TO GET TRANSPORT. WE WERE REALLY QUITE CURIOUS EARLY ON ABOUT HOW IT WAS POSSIBLE FOR ONE TRANSPORTER TO RECOGNIZE LITERALLY DOZENS AND PROBABLY HUNDREDS OF DIFFERENT SUBSTRATES. MAYBE THOUSANDS, ACTUALLY. AND THE AT THAT POINT, BASED ON SOME EXPERIMENTAL RESULTS, HYPOTHESIZED WHAT WAS HAPPENING HERE IS THE TRANSPORTER WAS NOT NECESSARILY MOVING THINGS FROM THE INSIDE OF THE CELL TO THE OUTSIDE BUT WAS RECOGNIZING THE MOSTLY HYDROPHOBIC DRUGS WITHIN THE MEMBRANE, BINDING THEM TO A BINDING SITE WHICH YOU CAN DEFINE GENETICALLY FOREBY -- STUDIES AND IN THE FOR INSTANCE OF ATP WHICH TURNS OUT BINDS TO BOTH OF THESE ATP BINDING SITES GETS HYDROIZED AND THERE ARE CHANGES IN THE PROTEIN OF THE EXTRUSION OF THE DRUG FROM A THE CELL. ONE OF THE REASONS FOR THE SO-CALLED LACK OF SPECIFICITY IS THAT MOST OF THE INTERACTIONS WITH THE DRUGS ARE HYDROPHOBIC. AND THE DRUGS ARE PRESENT IN HIGH CONCENTRATIONS IN THE MEMBRANE AND LOWER CONCENTRATIONS IN THE SIT TOE CYTOPLASM -- USING THE TRANSPORT SYSTEM TO PROTECT THEM FROM THE HYDROPHOBIC ENVIRONMENT. THERE'S A FEW CRYSTAL STRUCTURES OF P LYCO PROTEIN. THE FIRST ONE WAS THE STRUCTURE PUBLISHED BY ALLER AND CHANG SEVERAL YEARS AGO WHICH SHOWS THE ATP SITES APART. WHEN THEY WERE HYDROLYZED THEY INTERSECT WITH EACH OTHER. IT BINDS TO PART OF THE ATP SITES HERE AND THE REST OF THE ATP SITES HERE. THIS IS SLIGHTLY NON-PHYSIOLOGICAL KIND OF STRUCTURE. BUT THIS PARTICULAR MOLECULE IS SHOWN IN THE PRESENCE OF ONE OF THE HYDROPHOBIC SUBSTRATES THAT ARE RECOGNIZED IN THEM. YOU CAN SEE THE SUBSTRATE BINDS WITHIN THE BI-LAYER WHICH IS ONE OF OUR INITIAL HYPOTHESES. THERE ARE BETTER CRYSTAL STRUCTURES OF SOME OF THE MICROBIAL TRANSPORTERS, AND THIS IS AN EXAMPLE THAT, OF A STAFF -- MOLECULE IN WHICH THE TWO ATP SITES ARE IN FACT TOGETHER. AND ATP BINDS IN THIS REGION AND IS ABLE TO TRANSPORT DRUGS AND OTHER COMPOUNDS THIS WAY. THE PRECISE DETAILS OF HOW THESE TRANSPORTERS WORK ARE NOT ACTUALLY KNOWN. BUT THE IDEA IS THAT DRUGS BIND WHEN THE ATP SITES ARE FAR APART. THE BINDINGS -- THE MOVING OF THE TWO ATP SITES TOGETHER. ATP IS HYDROLYZED. THERE'S A CHANGE IN THE OARCHTATIOORIENTATION THAT ARE BINDING THE DRUGS AND THE DRUGS ARE HE CAN TRUDED. -- EXTRUDED. WHAT IS THE NORMAL PHYSIOLOGICAL ROLE OF P LYCO PROTEIN? GIVEN THE LOCATION IN THE INTESTINE AND THE LIVER, THE KIDNEY. WE'RE PRETTY SURE IT HAS EXCRAW TREE FUNCTION -- EXCRETORY FUNCTION. YOU'LL HEAR MORE ABOUT THIS FROM DR. HALL. FOR CELL IN THE INTERSTITIAL SPACE BY CANCER CELLS THAT EXPRESS P LYCO PROTEIN IT EXPRESSES THOSE CELLS BY ANTI-CANCER DRUGS. FINALLY, LET ME JUST SAY THAT THERE IS A FAIR AMOUNT OF EVIDENCE THAT THESE ABC TRANSPORTERS CAN CONTRIBUTE TO DRUG RESISTANCE. BUT NOT A LOT OF EVIDENCE THAT THEY ARE THE MAJOR CAUSE OF RESISTANCE AND THE MAIN REASON FOR TREATMENT FAILURE. AND LET ME JUST SORT OF SUMMARIZE THE EVIDENCE. THE FIRST POINT IS THAT QUITE A LOT OF HUMAN CANCERS EXPRESS THESE TRANSPORTERS PARTICULARLY P LYCO PROTEIN AT LEVELS THAT WE KNOW ARE SUFFICIENT TO MAKE THEM RESISTENT. SO THAT'S SORT OF A BIT OF CIRCUMSTANTIAL EVIDENCE THAT CORRELATES THE EXPRESSION OF PGP WITH THE EXTENT OF RESISTANCE IN CERTAIN TUMORS. THERE ARE TUMORS WHICH HAVE RELATIVELY LOW LEVELS OF PGP, LEUKEMIAS, MILO MAS, INFOMAS AND BREAST AND OVARIAN CANCER IN WHICH TREATMENT WITH CHEMOTHERAPY AND RECURRENCE OF THE TUMOR IS ASSOCIATED WITH INCREASED LEVELS WITH PGP. ON CORRELATIVE EXPRESSION BETWEEN PGP EXPRESSION AND DRUG RESISTANCE. THERE ARE A LOT OF OTHER TUMORS THAT ARE INTRINSICALLY RESISTENT, TUMORS OF THE COLON, THESE ARE THE ORGAN THAT NORMALLY, PRESS PGP. THEY ARE EXCRETORY ORGANS. SO THEIR ABILITY TO HANDLE DRUGS SEEMS TO BE RELATED TO THEIR NORMAL EXCRETORY FUNCTION. THEY CONTINUE TO EXPRESS THESE TRANSPORTERS AND EXTRUDE DRUGS FROM THE CELLS. THE EXPERIMENTAL MODELS FOR STUDYING THIS ARE MOSTLY ANIMAL MODELS TUMORS. XENO GRAPHS IN MODELS AND MODELS BEFORE THIS WAS DEVELOPED IN WHICH TUMORS THAT ARE NATURALLY DERIVED FROM GENETICALLY ENGINEERED LIKE BREAST CONNORS THAT ARE BRCA MINUS AND P53 MINUS CAN BE SHOWN TO DEVELOP DRUG RESISTANCE AFTER EXPOSURE TO ANTI-CANCER DRUGS THAT ARE SUBSTRATES FOR ABC TRANSPORTERS. AND THEY VERY COMMONLY EXPRESS PGP. THIS IS IN A MOUSE MODEL IN A NATURALLY OCCURRING TUMOR. IF YOU INHIBIT PGP THEY BECOME DRUG SENSITIVE FOR A WHILE AND ULTIMATELY BECOME MORE RESISTENT. SO BASED ON THESE KINDS OF EXPERIMENTS WE THINK PGP IS CERTAINLY A CONTRIBUTING CAUSE OF RESISTANCE BUT NOT THE ENTIRE CAUSE OF RESISTANCE. AND UNFORTUNATELY, ALTHOUGH EFFORTS HAVE BEEN MADE TO INHIBIT P FLY CO-PROTEIN AND THESE OTHER MULTIDRUG TRANSPORTERS BY GENERATION OF INHIBITORS, THE REALITY, THE UNFORTUNATE REALITY IS THAT THERE MAY BE TRANSJUDGMENT TRANS-- TRANSIE NT IN PATIENTS THAT I SHOWED YOU IN MY FIRST SLIDE. IF YOU'RE INTERESTED IN STUDYING DRUG RESISTANCE, IT SEEMS TO BE A LIFETIME PURSUIT. THERE'S AMPLE WORK STILL TO BE DONE, AND I ENCOURAGE YOU TO BECOME INTERESTED IN PURSUING. I THINK THAT'S MY LAST SLIDE. YES. SO LET ME THANK YOU AND INTRODUCE DR. HALL WHO WILL TALK MORE SPECIFICALLY ABOUT THE FARM -- PHARMACOLOGICAL FUNCTION OF THESE TRANSPORTERS. >> SO THANKS EVERYONE. I UNTIL VERY RECENTLY WORKED AT THE NATIONAL CANCER INSTITUTE. I WORK NOW AT N CAP BUT I'VE VERY MUCH TAKEN MY INTERESTS IN THE BLOOD BRAIN BARRIER WITH ME. I'LL TELL YOU A LITTLE BIT ABOUT FOLLOWING UP ON SOME OF THE THINGS DR. GOTTESMAN SAID. I'LL BE TELLING YOU ON HOW ATP TRANSPORTERS WORK PLACES LIKE THE BLOOD BRAIN BARRIER BUT OTHER PROTECTIVE SLIDES SUCH AS THE PLACENTA. I'LL GIVE YOU AN EXAMPLE AND TELL YOU SOME OF THE RESEARCH DR. GOTTESMAN LABORATORY AND ALSO COLLABORATED WITH THE NATIONAL INSTITUTES FOR MENTAL HEALTH. DEVELOPING IMAGES FOR PETS AND OTHER IMAGING MOAFLGHTSITIES STUDYING THESE BARRIERS. THE CRITICAL POINT TO MAKE ABOUT THEM ALL IS THAT AS WELL AS THEY PLAY A ROLE IN CONTROLLING DRUG ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION. ALL OF THE CRITICAL COMPONENTS YOU WILL BE HEARING ABOUT IN THIS COURSE. DELIVERING DRUGS TO THE BRAIN IS A HUGE CHALLENGE, IT'S A WELL-KNOWN CHALLENGE IN THE PHARMACEUTICAL INDUSTRY. ONE OF THE REASONS FOR THAT IS IT'S RECOGNIZED BUT WELL OVER 98% OF MOLECULES DRUGS DO NOT CROSS THE BLOOD BRAIN BAIR YUR. IT'S A STUMBLING BLOCK IN DRUG DEVELOPMENT AND EVEN MORE SO NOW WITH THE EMERGENCE OF ANTI-BODY DIRECTED THERAPEUTIC. 100% OF LARGE MOLECULE SUCH AS 57B9 BODIES CANNOT CROSS THE BLOOD BRAIN BARRIER. THERE ARE A LOT OF CHALLENGES HERE. THIS RADIO GRAM IS BEING TAKEN BY INJECTING A RAT WITH 14C HISTAMINE IN THE PERIPHERY THE TAIL PROBABLY AND LETTING IT CIRCULATE. THE ANIMAL WAS SACRIFICED, FIXED AND SLICED. THIS IS ON FILM. YOU CAN SEE WHERE THIS MOLECULE HAS GONE YOU GET STRONG DARK IMAGE. YOU CAN SEE IT'S DISTRIBUTED PRETTY MUCH EVERYWHERE IN THE BODY EXCEPT THE BRAIN AND SPINAL CORD. THIS IS A RECAPITULATION TO DISCOVER THE BLOOD BRAIN BARRIER THEY INFECTED A RABBIT WITH A BLUE DIE AND THEY INTERSECTED IT THEY NOTICED ALL THE BLUE TISSUE, ALL THE TISSUE WAS STAINED BLUE IN THE ANIMAL EXCEPT THE BRAIN AND SPINAL CORD. IF THEY INJECTED IT DIRECTLY INTO THE BRAIN THEY FOUND THE BRAIN AND SPINAL CORD WAS STAYING BLUE BUT THE PERIPHERY WAS NOT. THERE'S SOME BARRIER BETWEEN THEM AND THAT EARLY OBSERVATION WITH DIE AND LABELED MOLECULE HODES TRUE. THIS IS A GOOD EXAMPLE OF SOME OF THE FACTORS THAT CAN CONTROL WHETHER OR NOT DRUGS GET INTO THE BRAIN. THIS IS HEROIN. A GOOD EXAMPLE. IT'S GOT TWO GROUPS HERE AND IT HAS REALLY GOOD BRAIN UP TAKE. THIS IS MEASURED BY THE BRAIN UP TAKE INDEX. HOWEVER, HEROIN, SOME OF YOU MAY KNOW, SOME OF YOU MAY NOT, IT'S ACTUALLY A PRO DRUG. IT'S NOT AN ACTIVE MOLECULE. ONCE HEROIN CAN CROSS THE BLOOD BRAIN BARRIER BECAUSE IT'S -- IT'S METABOLIZED AND THE TWO GROUPS, THE HYDROXYL GROUPS INTO MORPHINE AND IT BINDS THE OPIOID RECEPTOR AND THE NEUROLOGICAL ACTIVITY THAT HEROIN IS ASSOCIATED WITH. SO HEROIN IS A -- MORPHINE ITSELF ISN'T AS COMMON A DRUG BECAUSE IT DOESN'T HAVE STRONG ABILITY TO CROSS THE BLOOD BRAIN BARRIER. IT HAS BRAIN UP TAKE. THERE'S COATING -- THAT HAVE BEEN NOT FIDE TO GET -- IT'S GOT ONE MENTAL GROUP IN THE HIGH DRUKSAL GROUP AND INTERMEDIATE BRAIN UP TAKE -- SO GETTING MOLECULES INTO THE BRAIN FROM A PHARMACOLOGIC POINT OF VIEW IS CHALLENGING BUT IT'S CERTAINLY ACHIEVABLE. THIS IS ONE OF TWO SORT OF CLASSIC PIECES OF DATA I'LL SHOW YOU TO GIVE YOU A SENSE. THE PHYSICAL CHEMICAL PROPERTIES -- THE NUMBER ONE RULE OF THUMB OUTSIDE DRUG TRANSPORTERS IS -- AS YOU CAN TELL IT'S FROM AN OLDER PAPER WHICH IS WHY THE QUALITY OF THE SCAN IS THE WAY IT IS. BUT YOU CAN SEE ACROSS THE BOTTOM HERE GOING FROM 1 UP TO 4. AND THIS IS FOR A SERIES OF DOPAMINE RECEPTOR AGONISTS. YOU CAN SEE UP TAKE INTO THE BRAIN THAT'S FAIRLY LOW AND CONSIDERED TO BE BECAUSE THEY'RE NOT VERY PERMEABLE. BRAIN UP TAKE INCREASES TO ABOUT 2.5. WHEN IT GETS MUCH HIGHER IT GOES DOWN AGAIN. THERE ARE A COUPLE REASONS FOR THAT HIGH PROTEIN BINDING AND THE BLOOD PLASMA AS WELL AS JUST A GENERAL BELIEF THAT THERE MIGHT BE A HIGHER RESIDENCE KIND OF MEMBRANES THAT STOP THESE MOLECULES CROSSING. SO THIS INVERT PARABOLIC RELATIONSHIP IS BEING SHOWN AGAIN AND AGAIN. THE SERIES OF MOLECULES AND STRUCTURE ACTIVITY STUDIES. THE REASON IT'S SO IMPORTANT FOR BRAIN UP TAKE IS BECAUSE PLACES LIKE THE BLOOD BRAIN BARRIER IS CONSIDERED TO BE ONE OF THE MOST IMPORTANT WAYS THAT MOLECULES ENTER. SO 2.5 IS OPTIMAL FOR THAT. I'LL SHOW YOU IN A FEW SLIDES. THERE ARE UP TAKE TRANSPORTERS THAT OCCASIONALLY DRUGS THAT CAN FIT INTO HIJACK AND THE EFFLUX TRANSPORT ISSUES WE'LL BE TALKING ABOUT. I PUT THESE DOWN FOR YOU TO GO HOME AND GOOGLE IF YOU'RE INTERESTED. AS I MENTIONED -- DEFUSION IS A CRITICALLY IMPORTANT PART OF DRUG ENTRY INTO CELLS. HOWEVER THERE'S BEEN AN EMERGING VIEW OVER IT IN THE LAST FIVE TO TEN YEARS. BUT IN FACT -- DIFFUSION DOES NOT EXIST AND IT'S A TRICK AND IT'S A MYTH AND THAT ONLY TRANSPORTER MEDIATEDDIAN TAKE INTO CELLS IS THE PATHWAY FOR DRUGS TO ENTER. AND SO THE HYPOTHESES BEHIND THESE REVIEWS AND YOU CAN READ THEM IS THAT MOST DRUGS WILL INTERCELLS NOT, AT VERY LOW SPECIFICITY THROUGH MANY DRUG UP TAKE TRANSPORTERS. AND THERE'S NO -- THERE'S A SERIES OF ARGUMENTS AND EXPERIMENTS BASED ON THE -- DOESN'T EXIST. ON THE OTHER SIDE THESE GUYS GOT TOGETHER WRITING A BUNCH OF PAPERS SAYING YOU BETTER BE JOKING IT ABSOLUTELY IS -- AND THEY HAVE A SERIES OF REALLY EXCELLENTLY PERFORMED STUDIES TO PROVE -- IS REALLY IMPORTANT. THEY'VE GONE BACK AND FORTH QUITE A FEW TIMES. WHEN YOU DO CITATIONS THEY GET QUITE PERSONAL BUT THEY'RE FUN TO READ. HAVING SAID THAT -- UPTAKE INTO THE BLOOD BRAIN BARRIER SEEMS TO BE DRIVEN PARTLY BY -- DIFFUSION. THIS WAS A STUDY IN 1980 THAT FOCUSED PEOPLE ON WHAT THE BIOLOGICAL PROCESSES MUST BE REALLY REGULATED BRAIN ENTRY. A WHOLE BUNCH OF COMPOUNDS ARE TESTED HERE. THEY'RE LOOKING AT PERMEABILITY INTO THE BRAIN WHICH IS BASICALLY BRAIN UPTAKE OVER HERE. YOU CAN SEE FROM VERY LOW P LEVELS HYDROPHILIC MOLECULES -- PERMEABILITY INTO THE BRAIN INCREASES. THERE WERE OUTLIERS AND THESE OUTLIERS NOW MAKE SENSE TO US. ONE WAS THAT GLUCOSE HAD MUCH HIGHER ENTRY INTO THE BRAIN THAN YOU WOULD EXPECT BASED ON THE -- AND ITS CORRELATION. WE NOW KNOW THE REASON FOR THAT IS VERY HIGH LEVELS OF GLUCOSE TRANSPORT EXPRESSED THE BLOOD BRAIN BARRIER COLLECTIVELY BRING GLUCOSE INTO THE BRAIN. THERE'S A WHOLE BUNCH OF MOLECULES ALL OF WHICH NATURAL PRODUCT ARRIVES SMALL MOLECULES USED IN COP -- THAN YOU WOULD ANTICIPATES. THE REASON IS THE EFFLUX OUT OF THE BLOOD BRAIN BARRIER. WHAT DOES THE BLOOD BRAIN BARRIER LOOK LIKE. THIS IS A CROSS-SECTION OF THE TAP LARRY IN THE BRAIN AND YOU CAN SEE THE ENDOTHELIAL CELLS HERE AND THEY HAVE TIGHT JUNCTIONS WHICH IS IMPORTANT. A LOT OF DRUGS CAN INTER-- PASSING BETWEEN VASCULAR VELZ. THAT CANNOT OCCUR HERE BECAUSE OF THE TIGHT JUNCTIONS THAT FORM ANDIVE UP THESE CELLS. THESE ARE ENTERED BACK INTO 9 BLOOD FOR THE ABC TRANSPORTERS THAT DR. GOTTESMAN TOLD YOU A LITTLE BIT ABOUT IN THE FIRST HALF OF THIS PRESENTATION. THERE ARE ALSO UP TAKE TRANSPORTERS LIKE GLUCOSE AND AMINO ACID TRANSPORTERS TO BRING NUTRIENTS INTO THE BRAIN. THIS IS EFFECTIVELY A HIGHLY REGULATED SYSTEM THAT ONLY ALLOWS VERY FEW MOLECULES TO CROSS THE BLOOD BRAIN BARRIER. I DON'T WANT TO GO INTO TOO MUCH DETAIL BUT I WILL MENTION THIS HERE IN THE BRAIN RESPONSIBLE FOR PRODUCING CFF. IT DOES THAT BY FACILITATING THE MOVEMENT OF A SMALL NUMBER OF SOLUTES ALONG WITH WATER OF COURSE TO CREATE CFF. THERE ARE OF ABC TRANSPORTERS EXPRESSED AS WELL THAT ARE INVOLVED IN REGULATING THE PASSAGE OF DRUGS FROM THE VASCULAR INTO THE CFS. IF THEY DIDN'T EXIST THE CFS WOULD BE BACK DOOR INTO THE BRAIN AND THAT'S TIGHTLY REGULATED AS WELL. THIS IS THE MODEL DR. GOTTESMAN SHOWED YOU TO SHOW YOU THESE ARE -- MOLECULES AND THESE AGAIN -- THIS IS DRAWN TO SCALE IN THIS DIAGRAM SO IT GIVES YOU THE SENSE OF THE SIZE OF THE TRANSMOARLTERS AND THE SIZE OF THE SUBSTRADDLE THAT THEY TEND TO INTERACT WITH. AGAIN DR. GOTTESMAN SHOWED A DIAGRAM LIKE THIS. THE REASON IT'S HERE IS TO MAKE THE POINTS THAT THE THREE ABC TRANSPORTER MEMBERS STRONGLY ASSOCIATED -- ARE ALL EXPRESSED AT THE BLOOD BRAIN BARRIER. SO THIS OVERLAPPING REDUNDANCY IN SUBSTRATE RECOGNITION MEANS WE HAVE A MOLECULAR DEFENSE SYSTEM THAT EXISTS IN THE BRAIN. IT'S OBVIOUSLY A CRITICAL ORGAN. THAT'S WHY THESE TRANSPORTERS ARE TO BE HERE AND TO PROTECT US. THEY'VE ALSO BEEN ASSOCIATED, THESE TRANSPORTERS WITH THE RANGE OF PATHOLOGY WHICH IS PART OF THE REASON WE GOT INTERESTED IN THEM. DRUG RESISTENT EPILEPSY -- HIV INFECTION OF THE BRAIN. MULTIDRUG RESISTANCE IN CANCER INCLUDING BRAIN CANCERS WHICH I'LL MENTION AGAIN. AND ALZHEIMER'S IS ALSO BEING ASSOCIATED WITH ABC -- IN FACT IT'S BEING SHOWN THAT PGP CAN TRANSPORT THE BETA PROTEIN THAT MAY BE RESPONSIBLE FOR THE PROGRESSION OF ALZHEIMER'S DISEASE. WE'VE DONE SOME WORK LOOKING AT DATA AND LITERATURE ON THE BRAIN. AND WE'VE TAKEN SOME REALLY NICE QUANTITATIVE DATA AND ASKED WHAT ARE THE MOST HIGHLY EXPRESSED TRANS PORTERS OF THE BLOOD BRAIN BARRIER. ON THE UP TAKE TRANSPORTER SIDE YOU CAN SEE THERE ARE MORE UP TAKE TRANSPORTERS THE GLUCOSE THAT ARE EASILY THE MOST HIGHLY EXPRESSIONED TRANSPORTERS. GLUCOSE IS NUMBER ONE. NUMBER TWO IS UP TAKE OF AMINO ACID TRANSPORTERS. I PROMISE I'M NOT NERVOUS IT'S JUST HARD TO KEEP IT STUDY ON THE SCREEN. THERE ARE A RANGE OF OTHERS AS WELL. LOOKING AT THE ABC TRANSPORTERS RESPONSIBLE FOR PROTECTION THE THREE WE REFERRED TO -- REMEMBER THESE ARE ENZYMES SO YOU NEED LITTLE EXPRESSION TO GET VERY HIGH FUNCTIONAL CAPACITY. YOU DON'T NEED CONCENTRATIONS LIKE YOU WOULD GLUCOSE UP TAKE TRANSPORT. THIS IS A UTILIZED IMAGE OF A PLASTIC MOLD OF A HUMAN BRAIN AND THE BRAIN IS MELTED AWAY LEAVING BEHIND THE VASCULAR. YOU CAN SEE THE VAC IS INCREDIBLY DENSE AND ABOUT 5% OF THE BRAIN IS OCCUPIED BY VASCULAR -- ELECTRON M MICROSCOPY. IT'S AN EXCELLENT SUPPLY OF OXYGEN AND GLUCOSE TO EVERY CELL IN THE BRAIN THROUGH THESE DENSE CAPILLARY VASCULAR. AND ALL OF THESE CAPILLARIES ARE LINED WITH ENDOTHELIAL CELL THAT FORM THE BLOOD BRAIN BARRIER. WHEN YOU PASS DOWN AND LOOK AT THE CROSS-SECTION OF A CAPILLARY. THIS IS CREATED FROM AN ELECTRON MICROSCOPE IMAGE, YOU CAN SEE THAT ABOUT 10% OF THIS CROSS-SECTION OF THIS TAPE LARRY, ACTUALLY THE CAP CAP LEERS. 2% ARE OCCUPIED BY ENDOTHELIAL CELLS. SO IT'S OCCUPIED BY ONLY 2% OF THE CELLS THAT EXIST IN THE BRAIN. INCREDIBLY EFFICIENT SYSTEM. QUITE REMARKABLE. AND AS I'LL SHOW YOU IN SOME OF OUR IMAGES EXTREMELY EFFICIENT IN STOPPING THINGS FROM GETTING IN. ONE OF THE THING TO MAKE FROM A PATHOLOGY POINT OF VIEW IS THAT BRAIN TUMORS DO NOT CONSTITUTE BRAIN MATTER. THEY ARE FOREIGN CELLS THAT ARE MUTATED SO THEY DON'T CREATE A BLOOD BRAIN BARRIER BY VASCULAR. THEY TEND TO BE QUITE FENESTRATED. THAT'S THE REASON WHEN PEOPLE ARE INJECTED -- FOR MRI, THE BRAIN TUMOR WILL SHOW UP WITH QUITE HIGH CONTRAST BECAUSE THERE'S NO TIGHT JUNCTIONS THERE LIMITING THE INTRALS T INTRASES TO THAT AGENT. WHAT YOU SEE IS THE LACK OF ACCUMULATION. AND VERY HIGH CONTRAST REVEALS IN THIS CASE -- THIS PARTICULAR IMAGE WE SELECTED FROM THE SIGHT HAS A LOW -- WHICH SEEMS TO BE HIDING FROM THE BLOOD BRAIN BARRIER. IT DOESN'T RESULT IN HIGH CONTRAST. SO THESE -- FOR DIFFERENT REASONS. PRESENT REALLY BIG CHALLENGES FROM A CANCER DRUG DEVELOPMENT POINT OF VIEW BECAUSE THEY RESIDE IN THE BRAIN AND DELIVER THE DRUGS TO THEM IS STILL REALLY QUITE CHALLENGING. SO WE WANT -- WE THOUGHT ABOUT HOW WE WOULD DO THIS USING PET IMAGING FOR EXAMPLE. THE IDEA WITH THE SUBSTRATE IS IT COULDN'T INTERTHE BRAIN. WHAT YOU EXPECT TO SEE WHEN YOU DO A PET IMAGE OF A SUBSTRATE ON A TRANSPORT IN THIS CASE IN A MOUSE BRAIN IS NOTHING. THAT'S EXACTLY WHAT WE SEE, NOTHING. IF WE CO-INJECTED THESE ANIMALS WITH THE PHARMACOLOGIC INHIBITORS, THE SUBSTRATE OF PGP COULD NOW ENTER THE BRAIN BECAUSE THE EFFLUX TRANSPORTER IS RESPONSIBLE FOR EXTRUDING AND SURE ENOUGH -- AND I'LL SHOW YOU THAT'S THE CASE. AGAIN IT COMES FROM DIAGNOSTIC MEDICAL IMAGING, MULTIDRUG RESISTANCE CANCER. THIS IS MY SUSAN -- UNTIL RECENTLY HERE AT THE NATIONAL CANCER INSTITUTE. THIS IS A PATIENT WITH A METASTASIS TO THE THIGH INJECTED WITH A RADIAL LIGANDS FOR IMAGING AND YOU CAN SEE FAIRLY LOW ACCUMULATION THAT WASHES AWAY. HOWEVER WHEN THE SAME PATIENT WAS INJECTED WITH A BLOCKER PGP YOU GET MUCH HIGHER INTO THE TUMOR AND THESE SET OF IMAGES DIAGNOSE THE PATIENT AS HAVING A MULTIDRUG RESISTANCE. SO WE HAVE TO TAKE ADVANTAGE OF. PET IMAGING THERE WAS SOME VERY DISCREET RULES WE HAVE TO FOLLOW. YOU NEED TO BE RADIALLY CHEMICAL IN THE BRAIN SO YOU HAVE TO UNDERSTAND -- WE DECIDED THAT THE RADIO CHOICES WE UTILIZED HAD TO BE SELECTED FOR ONE ATP TRANSPORTER. IF YOU SAW FROM THE DIAGRAM THAT'S CERTAINLY POSSIBLE IT WOULD BE REALLY HARD TO UNDERSTAND THE FUNCTION OF AN ABC TRANSPORT. YOU'RE UNDERSTANDING THE PROTECTIVE CAPACITY OF THE ENTIRE BLOOD BRAIN BARRIER BUT NOT LEARNING ABOUT INDIVIDUAL TRANSPORTERS. WE NEED TO HAVE A HIGH MAGNITUDE AND I WANT TO TELL YOU THE WAY WE FELT WE WERE GOING TO DEAL WITH THAT ESPECIALLY GIVEN OUR BASELINE WOULD HAVE A VERY LOW MAG TMAGNITUDE. WE SETTLED ON A METABOLITE CALLED -- IT IS ALSO AN OPIOID AGONIST. I MENTIONED HEROIN AND MORPHINE EARLIER. THIS FALLS INTO THE SAME CLASS EXCEPT YOU CAN BUY IT OVER THE COUNTER BECAUSE IT DOESN'T GET INTO THE BRAIN THEREFORE CANNOT GET YOU HIGH AND BECOME A DRUG ABUSE. IT DOESN'T GET INTO THE BRAIN. WE DIDN'T KNOW ABOUT SPECIFICITY. WE DIDN'T KNOW IF OTHER TRANSPORTERS COULD BE IDENTIFIED MORE RECENTLY. WE'RE REASON FOR TRANSPORTING ITEMS SO WE ASSESS THIS IN A FEW WAYS. THE FIRST WAS SIMPLE UP TAKE, PERIMENTS. YOU TAKE CELLS THAT DO TAKE -- THERE'S QUITE A LARGE -- HOWEVER WITH CELLS WITH -- WE DIDN'T SEE ANY ACCUMULATION DIFFERENCE. SO IT HELPED US WITH SOME OTHER DATA. IT'S SHOWN HERE TO BELIEVE THAT IT'S A VERY SPECIFIC SUBSTRATES FOR PGP WHICH WOULD GIVE IT QUITE A LOT OF POWER. WE CAN ALSO HAVE ACCESS TO GENETICALLY LACKING AGENTS FOR TRANSPORTERS I'VE BEEN TALKING ABOUT. ONLY WHEN PGP WAS DELETED DO WE GET A VERY HIGH AND STABLE LEVEL OF SIGNAL IN THE MOUSE BRAIN. SO AGAIN, SUGGESTING THAT THIS IS A VERY SPECIFIC PGP SUBSTRATE IN BOTH HUMAN TRANSPORTERS AND ANIMALS. IT DOESN'T GET INTO THE BRAIN IF YOU INHIBIT THE BLOOD BRAIN BARRIER -- YOU GET HIGH BRAIN SIGNAL. WE DID THE SAME THING IN THE MONKEY. GOT EXACTLY THE SAME EFFECTS. SO WE HAVE THIS VERY SPECIFIC SUBSTRATE THAT WE CAN USE TO STUDYING FUNCTION. I WANTED TO SHOW YOU A FEW FEATURES WHAT WE'RE LOOKING AT THESE BRAIN IMAGES. ONE POINT YOU MAY HAVE NOTICED THIS SHORT OF HOT SPOT IN THE BRAIN RENAL. THIS IS A PITUITARY WHICH IS OUTSIDE THE BLOOD BRAIN BARRIER. WE GET A VERY STRONG SIGNAL FROM RADIAL LIGAND ACCUMULATION BECAUSE PGP IS NOT THERE TO PREVENT ITS ENTRY. WHEN WE BLOCK PGP WE GET MUCH HIGHER SIGNAL AND THAT OCCURS OVER TIME. WE'RE USING A NUMBER OF PHARMACOLOGIC INHIBITORS INCLUDING PGP2. IT'S IMPORTANT THAT YOU UNDERSTAND THE SPECIFICITY OF YOUR INHIBITORS. WE DUMPED EXPERIMENTS EARLIER ON NOT UNDERSTANDING THE DATA ONLY TO FIND OUR -- FOUR THE TRANSPORTERS AS WELL. THERE'S TWO SIDES TO THE COIN WHEN YOU START COMBINING THESE MOLECULES. YOU NEED TO UNDERSTAND WHAT BOTH OF THEM DO. LOOK THE A WHOLE BODY IMAGE. IT'S INTERESTING TO WATCH AT 320 AND 100 MINUTES YOU CAN SEE VERY EARLY TIME POINTS, VERY HIGH KIDNEY LEVEL ASSOCIATED OF COURSE WITH EXCRETION THAT'S BACKED UP AT LATER TIME THE YOU'RE ANYWHERE BLADDEURINARY BLADDER IS E XCRETING. WE ALSO INCIDENTALLY OR ACCIDENTALLY DISCOVERED THAT THIS MOLECULE IS KEPT -- SO YOU DO GET IN LYSOSOME SUCH AS THE SPLEEN -- AS A RESULT. IT'S ACTUALLY THE REASON FOR HAT STABLE TRAPPING IN THE BRAIN THAT YOU WOULD HAVE SEEN IN THE TIME ACTIVITY CURVE. I DON'T HAVE A MOUSE WITH ME SO I DON'T KNOW HOW TO PLAY THIS VIDEO. IT'S A PICTURE THAT WOULD HAVE SPUN AROUND BUT YOU CAN SEE IN THIS ONE IMAGE THE DRAINAGE OF THE VASCULAR. HERE WE GO. THE -- THAT I REFERRED TO WHICH IS AGAIN OUTSIDE THE BLOOD BRAIN BARRIER -- THE POOCH TREE DOWN HERE -- PENSION TRE WE DON'T UNDERSTAND BU T THERE'S A BLOOD RETINAL BARRIER AS WELL JUST LIKE THE BLOOD BRAIN BARRIER. LOOKING AT THIS SECTION I WANT TO MAKE THE POINT AGAIN YOU CAN SEE THIS LINING UP QUITE STRONGLY. AND ON MRI MERGE IT'S ALWAYS UTILIZED SO WE CAN UNDERSTAND WHERE THESE OCCUR. YOU CAN SEE IN THE HUMAN WE FINALLY GOT TO THE POINT WHERE WE INJECTED LORDOSIS OF THE INHIBITOR. WE CAN SHOW WE WERE INDEED LIMITING THE BLOOD BRAIN BEAR WHY YOU -- SO THIS IS REALLY INFORMATIVE DATA FOR US IN THE CONTEXT OF PERHAPS HOPING TO HELP DELIVER DRUGS TO THE BRAIN THAT WOULD BE ACTIVE IF THEY COULD CROSS THE BLOOD BRAIN BARRIER -- TO FACILITATE DELIVERY TO THE BRAIN. SO I TALKED ABOUT PGP. THE OTHER TRANSPORTER WE WERE INTERESTED IN IS -- WE REALLY WANTED TO DEVELOP FOR ABC2 FUNCTION BUT WE COULDN'T FIND A MOLECULE -- THAT JUST INVOLVES GOOGLING FOR A LONG LONG TIME AND BEING DISAPPOINTED. WE DID A LOT OF LITERATURE SEARCHING AND A LOT OF EXPERIMENTS AND WE COULDN'T FIND ANYTHING. IT NEEDS TO BE SPECIFIC BUT IT WAS AMENABLE TO RADIAL LABELING. SO IT WAS A CHEMICAL CHALLENGE AS WELL AS A ME MOLECULAR CHALLENGE FOR US. AFTER A LOT OF READING IT DOESN'T GET INTO THE BRAIN VERY WELL. YOU CAN SEE IN THE DISTRIBUTION STUDY OF LOOSE FERON. IT WAS ASSOCIATED WITH LOW SIGNAL IN BIOLUMINESCENT STUDIES -- I'M SURE YOU WILL KNOW THE DIFFERENCE IN A RESEARCH TOOL. IN THE PRESENCE OF ATP AND OXYGEN IS CONVERTED -- SO WE DECIDED WE'D TRY TO WORK OUT A MOAFLGHTSITY THAT WOUL WOULDMODALITY THAT WOULD BE USEFUL. THIS LOOKS LIKE IT WOULD BE USEFUL FOR IMAGING POINT OF VIEW. PEOPLE USE IT FOR ANIMAL IMAGING. IS IT A SPECIFIC SUBSTRATE. COULD WE GET IT RADIAL LABELED? WE COULD. WE WERE READING THE LITERATURE THAT THIS PRODUCES -- AWAY FROM THE ENZYME, IT'S FLUORESCENT MOLECULE WHICH IS A REALLY USEFUL TOOL FOR US. YOU CAN SEE HERE THE -- OF THE MOLECULE. THIS IS PUBLISHED IN 19 ALS ALSO 58 SO BE SURE TO READ -- THE FURTHER BACK YOU READ -- THIS IS ACTUALLY, THIS OBSERVATION THAT WE WOULDN'T HAVE MADE THAT LOOSE FERON -- PUT IT ON THEM AND IT JUST DOESN'T GET INTO THE CELLS. ADD AN INHIBITOR OF ABCG2 AND WE USE ONE THAT I'LL SHOW YOU A FEW TIMES DURING THE NEXT SLIDES. YOU CAN GET QUITE HIGH FLUENCENT IN THESE CELLS. WE HAVE THE EXFLUX TRANSPORTER AND IT GETS INTO THE CELLS. IT'S PLAYING A ROLE THAT WE ANTICIPATED BUT IS IT SPECIFIC. WE ENDED UP DECIDING TO USE FLORESCENCE IN NO CYTOMETRY. EXPRESSING ABCG2 AND YOU GET VERY LOW FLORESCENCE INTENSITY. IF YOU INHIBIT IT, YOU GET MUCH HIGHER ACCUMULATION. THIS IS JUST A QUANTITATIVE VERSION. TAKE A -- NOT EXPRESSING G2. WITHOUT AN INHIBITOR YOU GET VERY HIGH ACCUMULATION. THERE'S NO TRANSPORTER TAKING PLACE. IF YOU DO THIS A LOT, YOU CAN QUANTIFY THE DATA YOU'RE GETTING A CREATE A HISTOGRAM AND YOU CAN SEE THAT THE ACCUMULATION IN THE G2 EXPRESSING CELL LINE IS MUCH LOWER THAN THE PARENT LINE. IF YOU ADD INHIBITOR YOU GET HIGH ACCUMULATION. MUCH LIKE THE RADIUM LABELED DATA I SHOWED YOU, THE SAME IS TRUE FOR PGP -- IT'S NOT A SUBTRACK OF THOSE TRANSPORTERS AND SO WE MANAGE TO END UP WITH A SPECIFIC SUBSTRATE OF ABCG2. THE CHALLENGE FOR US. ULTIMATELY WHAT WE IDENTIFIED WAS A MOUSE MODEL THAT ONLY EXPRESSED LOOSE FERON IN THE BRAIN. SO THE THEORY WAS WITH THE MOUSE MODEL WE COULDN'T USE ONE THAT WOULD EXPRESS LOOSE FERON THROUGHOUT THE ENTIRE BODY BECAUSE YOU GET THIS FROM THE WHOLE ANIMAL. WE NEED AN EXPRESSION -- THE IDEA IS YOU WOULD INJECT THE MOUSE WITH LOOSE FERON. ABCG2 WOULD STOP IT FROM CROSSING THE BLOOD BRAIN BARRIER. YOU WOULD EXPECT LOW SIGNALS. BUT IF YOU INJECTED AN INHIBITOR -- GIVE YOU A CENTRAL THAT YOU CAN QUANTIFY. WE SET ABOUT SCEUFLTING EXACTLY THAT. YOU CAN SEE HERE A MOUSE ON THE BASELINE INJECTED WITH LOOSE FERON GAVE US QUITE LOW SIGNALS. WE HAD ANTICIPATED THAT. IT SHOOJT BE ABLE TO CROSS THE BLOOD BRAIN BARRIER -- FARM COLLAGIC BLOCKER, YOU CAN SEE WE WERE GETTING INCREASING SIGNALS. THIS IS THE QUALITATIVE IMAGE. THIS IS EXACTLY WHAT YOU'RE LOOKING AT IN THE ANIMAL FACILITY. YOU COLLECT THE SIGNAL OVER TIME -- JUST AS YOU WOULD IN PET IMAGING YOU CAN SEE THAT BASELINE CONDITIONS YOU GET LOW SIGNAL. I CAN ALSO SAY WHEN WE INJECTED THINGS LIKE A PGP INHIBITOR WE DIDN'T GET ANY INCREASING SIGNAL. SO THERE WASN'T A NON-SPECIFIC EFFECT TAKING PLACE. AND WITH INCREASING CONCENTRATIONS OF THE INHIBITOR WE GOT INCREASING SIGNALS. WE CAN INTEGRATE THE SIGNAL TO GET THE AREA AROUND TO THE CURVE AND GET A DOSE EFFECT FROM THAT -- EFFECTIVE DOSE OF 50% OF THE MAXIMAL RESPONSE OF ABOUT 6 ON.5 MILLIGRAMS PER KILOGRAM WHICH IS THE FIRST ONE ANYBODY'S BEEN ABLE TO GET INSIGHT OF WHAT EFFECTIVE DOSE FOR ANIMAL STUDIES WOULD BE. THAT'S THE USE AND PURPOSES OF THESE IMAGING STUDIES IS TO CHARACTERIZE AND VALIDATE ABC TRANSPORT INHIBITORS. SOMETHING PEOPLE OFTEN ASK ME IS HOW DO YOU KNOW THIS IS ACTUALLY BRAIN SIGNAL. THIS IS TOMOGRAPHIC LIKE PET THIS IS THE ONLY IMAGE YOU GET. YOU CAN SEE HERE WHEN YOU PULL THE BRAIN OUT OF THE ANIMAL ALL OF THE BIOLUMINESCENT SIGNAL COMES FROM THE BRAIN HERE AND THERE'S NO SIGNAL COMING FROM THAT. THERE'S NO -- E SIGNAL OR SOMETHING LIKE THAT OF A TARGET IT'S JUST WHAT WE'RE MEASURING IN THESE ANIMAL STUDIES. ANOTHER ASPECT OF THIS I MENTIONED IN THE BEGINNING WAS THE BLAH -- MOVING ON FROM TAKING ADVANTAGE OF THIS TRANSPORT MINUTING MODALITY. WE CAN'T GO TO HUMANS BECAUSE WE'RE NOT ALLOWED TO DO GENE THERAPY UNFORTUNATELY SO WE HAD TO THINK OF INVENTIVE WAYS TO UTILIZE MASS MODELS TO UNDERSTAND IT WITH THIS TRANSGENIC SYSTEM. THE PLACENTA BARRIER AND THE PLACENTA IT SELL IS AN IMPORTANT PART OF DEVELOPMENT AND CONTROLLING THE MOVEMENT OF NUTRIENTS ACROSS THE PLACENTA TO THE FETUS. AND METABOLIZE AND MOLECULES TO BE EXCRETED BACK OUT ACROSS THE PLACENTA. AT THE THE BARRIER THERE'S A CIVIL HER SET OF CELLS THEY EXPRESS ABC TRANSPORTERS AND OUR THEORY HERE WE NEEDED TO FIND A WAY TO GET -- EARLY BEHIND THE PLACENTA SO WE COULD STUDY IT. WE ENDED UP, IT TOOK A LONG TIME TO HAVE A PRETTY SIMPLE IDEA. WE WOULD TAKE -- MOUSE MODEL WHICH IS 4EU7B BEHIND THE -- AND WE WOULD CROSS IT WITH A WILD TYPE FEMALE THAT DIDN'T EXPRESS -- WE END UP WITH A PREGNANT WILD TYPE MOUSE CARRYING PUPS THAT WERE EXPRESSING -- SO THE THEORY IS AGAIN WITH THE FLOOD BRAIN BARRIER THAT -- HOWEVER A LITTLE BIT WOULD. YOU GET SO BACKGROUND SIGNAL WHICH INDEED WE SAW. HOWEVER IF WE USE PHARMACOLOGIC -- GETS IN AND WE GET THIS SIGNAL FROM THE MOTHER AS A RESULT OF IT PRODUCING BIOLUMINESCENCE WITH A SUB STRAIGHT. THIS IS RAW DATA YOU WOULD GET. THIS IS A NON-PREGNANT FEMALE. THIS IS A MALE. THIS IS A PREGNANT FEMALE AND YOU CAN NOW SEE SHE'S LIGHT TERM BUT YOU CAN SEE THE BIOLUMINESCENT SIGNALS AND YOU CAN SEE BY OPENING YOU ALL OF THE FETUSES LINED UP HERE. WHAT YOU SEE ALONG THE WAY ARE ACTUALLY THE PLACENTA. WE GOT STRONG SIGNALS FROM THE PLACENTA BECAUSE THAT'S THE FIRST PLACE IT GOT AS FAR AS FLOWING THROUGH THE VASCULAR TO BE THROUGH THE PLACENTA ON ITS WAY TO THE FETUS. WE DEVELOPED WHAT ENDED UP BEING A SLIGHTLY MORE COMPLICATED IMAGING PROTOCOL BECAUSE EVERY SINGLE FEMALE MOUSE HAD A DIFFERENT NUMBER OF PUPS AND A DIFFERENT SIZE, DIFFERENT SIZE PLACENTA AND EVERY DAY THE SIGNAL WOULD BE STRONGER THAN THE DAY BEFORE AS THE FETUSES GREW AND BASICALLY THE TOTAL BLINDNESS INCREASED. SO WE HAD TO INTRODUCE THE NORMALIZATION STEP BUT HE'D INJECT WITH -- YOU CAN SEE A BASELINE LEVEL OF SIGNAL THAT WOULD BE ASSOCIATED WITH THAT. WE WOULD TAKE THEM OUT OF THE IMAGING BOX AND EITHER INJECT THEM WITH -- OR IN THIS CASE THROUGH ABCG2 INHIBITORS AND WE PUT THEM BACK INTO THE BOX. AND THE -- THAT WAS CIRCULATING COULD START CROSSING THE BLOOD PLACENTA BARRIER. CARRIER -- WAS OUR BEST INHIBITOR IN THE BLOOD BRAIN BARRIER STUDIES AND THAT WAS THE CASE HERE AS WELL. YOU CAN SEE HERE ONE OF THE THING WE SHOWED BY DOING THIS BUT FOR THE FIRST TIME ABCG2 DOES PLAY A PROTECTIVE ROLE. I PROBABLY SHOULD HAVE SAID EARLIER BOTH THE BLOOD BRAIN AND BLAH ISN'T TAU BARRIERS WHETHER -- PLAYED A ROLE IS REALLY IN DISPUTE. THERE ARE HUMANS WHO DON'T EXPRESS ABCG2 WE DON'T FULLY UNDERSTAND THE IMPLICATIONS OF THAT. IT WAS ONLY IDENTIFIED A FEW YEARS AGO. BECAUSE IT'S SUCH A POWERFUL DRUG TRANSPORTER WE DON'T KNOW WHETHER IT PLAYED ANY ROLE. IT CERTAINLILY DOES. WE SEE THAT HERE. IF WE INJECT THE PGP INHIBITOR IT DOESN'T HAVE ANY EFFECT BEHIND THE BLOOD PLACENTA BARRIER. AS A FINAL POINT I THOUGHT I WOULD MENTION AND GIVE YOU EXAMPLES OF THE MODELS AND TISSUES. NOW WHERE WE USE FAIRLY LARGE SCALE AUTOMATION TO SCREEN HUNDREDS OF THOUSANDS OF COMPOUNDS ON AING SINGLE TARGET AND IN MY GROUP AT THE MOMENT AT DIFFERENT STAGES WE HAVE ABOUT 50 PROJECTS. AND 10 OF THOSE ARE TARGETS THAT ARE IN THE BRAIN. THEY'RE BEHIND THE BLOOD BRAIN BARRIER AND I TOLD YOU IN THE FIRST OR SECOND SLIDE 98% OF MOLECULES CAN'T CROSS THE BLOOD BRAIN BARRIER. IT'S REALLY MORE OF LOOKING FROM ASSAYS AS WELL TO BE ABLE TO PREDICT WHICH OF THOSE HITS MIGHT CROSS THE BLOOD BRAIN BARRIER AND PICK THOSE ONES FOR FOLLOW UP BECAUSE YOU DON'T WANT TO SPEND TWO YEARS DEVELOPING A LEAD THAT WON'T GET INTO THE BRAIN WHEN YOU FINALLY GET TO AN ANIMAL MODEL. ONE OF THE WAYS WE'RE SETTING UP AT N CAP WHICH IS POPULAR IN PHARMA IS TO TAKE -- WHICH YOU INSERT INTO A WELL AND YOU SEE CELLS DOWN HERE THAT EXPRESS ABC TRANSPORTERS. THEY FORM TIGHT JUNCTIONS AND THEY EXPRESS PGP OR -- AND THEY ORIENT -- YOU ADD THEM ON TO ONE SIDE AND WHETHER OR NOT TO MOVE THEM TO THE OTHER. YOU CAN QUANTIFY. IF YOU SEE MOVEMENT FROM ONE SIDE TO THE OTHER YOU KNOW THEY ARE STILL PERMEABLE WHICH IS JUST AS IMPORTANT GENERALLY AND B COMPARED TO CONTROL CELLS WITHOUT TRANSPORTERS YOU GET A SENSE WHETHER THEY LOOK LIKE THEY HAVE TRANSPORTER SUSCEPTIBILITY. THE WHOLE REASON TO DO THIS IS TO TRIAGE THOSE MOLECULES THAT PGP SUBSTRATES NOT LOSE THEM THROUGH ASSAY DEVELOPMENT MOVING TOWARDS ANIMAL MODELS AND AS I SAY BECOMING EXTREMELY DISAPPOINTED WITH A BUNCH OF REALLY GREAT CELL BASED PROBES THAT HAVE NO EFFECT AT ALL WHEN YOU GET INTO ANIMALS. SO THAT'S HOW PEOPLE ACTUALLY TACKLE THIS AT A NON-ANIMAL MODEL LEVEL AND IT'S VERY INFORMATIVE AND CORRELATES REALLY WELL WITH ACTUAL BRAIN PENETRANTS. SO THE SUMMARY HERE IS JUST SOME TAKE HOMES ABOUT THE FACT OF USING NON-INVASIVE IMAGING CAN BE A REALLY USEFUL WAY TO UNDERSTAND DRUG TRANSPORTER FUNCTION. MOUSE MODELS AND SO ON WE'VE BEEN TAKING ADVANTAGE OF HAVE BEEN CRITICAL. I WILL SAY NEARLY ALL OF THE BLIND BASE EXPERIMENTS TRANSLATED TO ANIMALS. IF YOU CAN YOU CAN SEE HERE I TOLD YOU A LITTLE BIT IS ABOUT ABCG2 AND THE FACT WE KNOW IN MULTIPLE DRUG TRANSPORTERS THAT ARE PLAYING ACTIVE SEPARATE FUNCTION ROLES FOR PROTECTION OF THESE PRIFD SITES. I HAVE A LARGE NUMBER OF COLLABORATORS AND OF COURSE DR. GOTTESMAN TO ACKNOWLEDGE FOR THE WORK I'VE BEEN TALKING ABOUT. THANKS FOR YOUR TIME AND ATTENTION. MICHAEL AND I WILL BE HAPPY TO ANSWER QUESTIONS IF ANYONE HAS THEM FROM OTHER PARTS OF THE PRESENTATION. >> WE WERE UNUSUALLY CLEAR. WE'LL BE HERE FOR A FEW MINUTES SO IF YOU'RE SHY, WE'LL BE HAPPY TO TALK TO YOU.