Factors Influencing Drug Delivery to the Brain: Multiple Mechanisms at Multiple Barriers. Pharmacodynamics. Pharmacodynamics

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1 Factors Influencing Drug Delivery to the Brain: Multiple Mechanisms at Multiple Barriers Target biology Pharmacodynamics April 13, 2011 NJDMDG Somerset, New Jersey Pharmacokinetics William F. Elmquist Pharmaceutics CNS drug development : Must keep in mind the big questions! Why Does a Drug Work?? Why Doesn t t a Drug Work?? Why does this one work, and that one doesn t t?? CNS Pharmacodynamics (effects at the target) Pharmacokinetics (conc-time at the target) Connect - Disconnect of the PK-PD PD Relationship Balance Information flow?? Blood Pharmacokinetics (conc-time in blood) 1

2 CNS Pharmacokinetics and Pharmacodynamics (Drug Delivery) in the Era of Systems Biology Dose and dosing regimen Traditional PK/PD Black box models Mechanisms of delivery and action the Big Picture Molecular pharmacokinetics Systems model System structure function quantitative qualitative Nature Biotechnology 23, (2005) Connect - Disconnect of the PK-PD PD Relationship Understanding Sources of Variability in Drug Response Variability Cycle Genetic Factors - drug targets - drug transporters - drug metabolizing enzymes Environmental Factors -induction - inhibition Physiological Factors -age, disease, etc. 2

3 Locations of Variability in Drug Response Mechanisms that influence the fraction of the drug in the systemic circulation that is available for distribution to target tissue and the exposure of the tissue to the drug Oral- distribution Intestinal of blood flow Tissue Target site dosage - ratio of metabolism total clearance to a distributional distribution clearance formdistributional clearance - membrane permeability, competing carrier-mediated transport Systemic (influx or efflux), protein-binding, intracellular metabolism, tissue circulation transit time, capillary structure Total clearance will affect the availability of the drug in the blood to distribute to the tissue Drug action Intestinal absorption Liver metabolism Systemic clearance Cellular delivery Presystemic bioavailability questions ( traditional bioavailability) Site-specific bioavailability questions (drug targeting) Targeted Bioavailability Locations of Variability in Drug Response Intestinal metabolism Tissue distribution BTSC Target site Oral dosage form Systemic circulation Drug action Intestinal absorption Liver metabolism BBB Cellular delivery EGFR mtor BRaf Presystemic bioavailability questions ( traditional bioavailability) Site-specific bioavailability questions (drug targeting) Targeted Bioavailability 3

4 Examine a location considering the interplay between external factors and mechanism Physiology / Pathology Patient External factors Drug Metabolism Membrane Permeability Drug Transport Physicochemical Properties Drug Protein Binding Mechanisms Receptor Affinity Gene Regulation Targeted Protein Expression Bioavailability External factors Dosage Regimen Dosage form/regimen Pharmacological / Toxicological Response One Location : Blood-brain Barrier Importance of Transporters in the CNS Disposition of Drugs From Lee and Gottesmann. Journal of Clinical Investigation, 1998 illustration by Naba Bora, Medical College of Georgia. 4

5 GLUT1 brain Clin brain P-gp (p-glycoprotein) blood Co-localization GLUT1 - P-gp brain Clout Loscher,, Aug Nature Rev. Neurosci. BCRP P-gp Decisions limited by available data at specific sites input output exchange bolus IC, CED IV plasma brain capillaries extracellular fluid neuronal and glial cell membranes intracellular fluid ICV, IT choroid plexus arachnoid membrane cerebrospinal fluid ependyma pia mater Compartmental model for solute exchange in the brain 5

6 Modeling limited by available mechanistic data at specific barriers brain capillaries neuronal and glial cell plasma choroid plexus arachnoid ECF production Surface area Permeability Transporters Metabolism Regional variability extracellular fluid Convection Diffusion Transporters Permeability Metabolism Receptors Regional variability intracellular fluid CSF flow Surface area Permeability Transporters Metabolism Regional variability ependyma pia mater cerebrospinal fluid Convection, Diffusion Permeability, Regional variability Simplified Quantitative Analysis of Drug Transfer In CNS Drug Binding Plasma and Brain Plasma Cu,plasma Cplasma BBB PS CL eff CL in BCSFB [CLin, CLeff,, PS] Brain Cbrain Cu,brain CL bulk Ccsf CSF CL meta 6

7 Simplified Quantitative Analysis of Drug Transfer In CNS Extent - partitioning into brain parenchyma Tight-junction opening Substrate for Influx Transporter K p, free PS CL PS CL efflux CL uptake metabolism CL bulk Tight-junction opening Inhibition of Efflux transporter Drug Targeting Index (a measure of delivery) DTI AUC target,intervention AUC target,control AUC AUC blood,intervention blood,control A neutral intervention would lead to a targeting index of unity, where positive effect would lead to a DTI greater than one, and a negative effect would result in a DTI less than one. The critical issue in the quantitative assessment of drug targeting is the need to measure drug concentrations at the target site 7

8 Complexity of the Transporter Problem at Various Barriers basolateral intracellular apical Brain PSin Bidirectional PSout Like multiple enzymes forming the same metabolite and the fraction metabolized varies with location! Carrier in Carrier out Carrier in P-gp PSout Bcrp Blood PSin (TIGHT JUNCTIONS) (transmembrane permeability) Many processes can be occurring simultaneously! Complex System Complexity of the Transporter Problem at Various Barriers basolateral Brain P-gp Bcrp apical Blood Tools: Genetic knockouts Mdr1a/b (-/-)( Bcrp1 (-/-)( Mdr1/Bcrp (-/-)( Inhibitors Many processes can be occurring simultaneously! LY Ko143 GF

9 Case Study : Tyrosine Kinase Inhibitors for Glioblastoma Multiforme (Glioma) Molecularly-Targeted Agents. Can they find the target? Numerous clinical trials with targeted tyrosine kinase inhibitors for glioma have failed. Is there a PK-PD PD disconnect for these drugs in glioma? consilience : to give a purpose to understanding the details Targets Mol Cancer Ther 2007;6(7) Drugs 9

10 Target Locations Like Fighting a Forest Fire Tumor Initiation Intact BBB Where is the drug needed? Factors enhancing infiltration of tumor Broken BBB: MRI contrast 10

11 Locations Invasive Cell Migration Localized?? Normal Brain Invading cells necrosis Tumor core Glioma cell Glioma Invasive Stem Cell Molecularly-Targeted Agents. Can they find the target in the brain? In Vivo Methods Mouse models: Wild type: FVB Blood Brain Transgenic: Abcb1a/b-Abcg2 (-/-) 11

12 Quantitative Analysis of Drug Targeting Other Tissue 3 CL 31 Multiple transporters CL 13 CL out CL in Brain - glioma Target Site 2 BBB transport Blood 1 Targeting Intervention CL 10 Dasatinib Brain/Plasma ratios in triple knockouts (Abcb1a/b-Abcg2 (-/-)) Brain/Plasma Ratio * WT Tri-KO * * * * min 20 min 60 min 120 min 180 min Time The brain-to-plasma ratio of dasatinib in wild-type and triple-knockout FVB Mice (* p<0.05, n=4) Chen Y. et al., JPET September 2009 vol. 330 no. 3 pgs

13 Dasatinib genetic deletion of transporter genes Brian Conc (ng/g) WT P-gp-KO Bcrp1-KO Tri-KO B 0 20min 120min Brain concentration of dasatinib in WT mice with and without genetic deletion of efflux transport. Chen Y. et al., JPET September 2009 vol. 330 no. 3 pgs Dasatinib pharmacologic inhibition of transport Brain Conc (ng/g) WT WT+LY WT+Ko143 WT+GF A 0 20min 120min Brain concentration of dasatinib in WT mice with and without pharmacologic inhibition of efflux transport. Chen Y. et al., JPET September 2009 vol. 330 no. 3 pgs

14 7 Gefitinib Brain-to-Plasma Ratios at 90 min Postdose Brain-to-Plasma ratios WT P-gp KO BCRP KO TKO ** ** 0 WT P-gp BCRP TKO Agarwal S., Sane R., Gallardo J., Ohlfest J., Elmquist W.F., Brain Distribution of Gefitinib is Limited by P-glycoprotein P (ABCB1) and Breast Cancer Resistance Protein (ABCG2) Mediated Active Efflux, J Pharmacol Exp Ther Steady-State Brain/Plasma Ratios Lapatinib Brain Distribution WT 0.3 mg/hr/kg 3.0 mg/hr/kg ~ 40-fold ~ 40-fold P-gp BCRP TKO WT P-gp BCRP TKO WT P-gp BCRP P-gp/BCRP data from : Polli et al., An Unexpected Synergist Role of P-Glycoprotein and Breast Cancer Resistance Protein... DMD 37: ,

15 Steady-State Brain to Plasma Ratios Sorafenib - Influence of Multiple Transporters on Brain Distribution WT P-gp KO Bcrp KO TKO ** ** WT Pgp Bcrp TKO Steady-State Brain/Plasma Ratio Bcrp / P-gp and Erlotinib Brain Penetration Wild-type TKO 42-fold increase WT TKO Alzet pump IP, 24 hour infusion, n = 3 15

16 Erlotinib Brain-to-Plasma Concentration Ratio at Different Locations in Rat Brain Tumor Brain Tissue-to-Plasma Concentration Ratio (Mean ± SE) Tumor Core Brain Around Tumor Normal Brain ** ** Vehicle Control * - p < Compared to Control **- p < Compared to Core * * GF Relative Increase in Brain-to-Plasma Ratio Influence of Elacridar on Erlotinib Brain Penetration in Different Brain Regions Tumor Core Tumor Rim Normal Brain DTI Targeted bioavailability no effect 16

17 Differences in Brain Distribution Enhancement between Genetic Knockouts and Pharmacological Inhibition Generally knockouts show greater enhancement 1) accessibility of inhibitors? (dose, potency, parenchymal concentrations) 2) locations of transporters? (BBB vs parenchyma) 11 C-elacridar (GF120918) WT P-gp BCRP P-gp Dorner,, 2009 BCRP WT 17

18 P-gp/Bcrp wild-type P-gp KO P-gp WT Bcrp BCRP KO P-gp/BCRP KO K 1 Cb Cpdt ED 50 = mg/kg 18

19 In Vitro- In Vivo Correlations, What about BCRP vs P-gp? Kusuhara and Sugiyama Is BCRP Important in the BBB? - Does BCRP KO tell the story? Kp? Kp ratio brain BcrpKO WT Kp (brain) Ratio (Bcrp KO/WT)ss vs CFR for BCRP and MDR1 genistein dantrolene BCRP MDR Corrected Flux Ratio Flux Ratio (transfected) Flux Ratio (wild-type) Data from Enokizono et al., DMD 2008 In vitro prazosin 19

20 Erlotinib CFR Pgp = 4.6 CFR Bcrp = 1.4 Flavopiridol CFR Pgp = 4.7 CFR Bcrp = 2.0 Dantrolene CFR Pgp = 1.0 CFR Bcrp = 1.9 Quinidine CFR Pgp = 6.2 CFR Bcrp = 1.0 Relative Distributional Clearances Simple Kinetic Model to Explain Non-Additive Increases in Brain Distribution of Dual P-gpP / BCRP Substrates Kusuhara and Sugiyama 20

21 Kushuhara and Sugiyama, Drug Metab. Pharmacokint.. 24 (1):37-52, 2009 Relative Increase in Brain-to-Plasma Ratio Influence of Relative Distributional Clearances on the Brain-to-Plasma Ratios of Dual Substrates Dual - Pgp - dominant PS 2,u = 0.5 PS p-gp = 5 PS BCRP = 2 PS BCRP 0 Wild type P-gp BCRP e.g., dasatinib, erlotinib,, gefitinib, lapatinib TKO (affinity and capacity) 21

22 25 Influence of Relative Distributional Clearances on the Brain-to-Plasma Ratios of Dual Substrates Dual - BCRP - dominant Relative Increase in Brain-to-Plasma Ratio PS 2,u = 0.5 PS p-gp = 2 PS BCRP = 8 PS BCRP 0 Wild type P-gp BCRP TKO e.g., sorafenib Influence of Relative Distributional Clearances on the Brain-to-Plasma Ratios of Selective Substrates 12 Relative Increase in Brain-to-Plasma Ratio PS 2,u = 0.5 PS p-gp = 0 PS BCRP = 5 PS BCRP 0 Wild type P-gp BCRP TKO Selective - BCRP e.g., dantrolene 22

23 Influence of Relative Distributional Clearances on the Brain-to-Plasma Ratios of Selective Substrates 12 Relative Increase in Brain-to-Plasma Ratio PS 2,u = 0.5 PS p-gp = 5 PS BCRP = 0 PS BCRP 0 Wild type P-gp BCRP TKO Selective - Pgp e.g., quinidine 16 Influence of Relative Distributional Clearances on the Brain-to-Plasma Ratios of Dual Substrates Relative Increase in Brain-to-Plasma Ratio PS 2,u = 10 PS p-gp = 5 PS BCRP = 2 PS BCRP Increased Passive Permeability! 2 0 Wild type P-gp BCRP TKO 23

24 Integrity of Tight Junctions in WT vs Triple Knockout Mice 10 Brain Space % (Percent of Total Brain Volume) (Mean ± SE) Wild-type Control Mdr1a/1b (-/-) Bcrp1 (-/-) 0 [14C] Sucrose [14C] Inulin 10 min post bolus n = 4, each time point Brain Space Amount in brain Plasma Conc Total Brain Volume 100 De Novo Induction of Genetically Engineered Brain Tumors in Mice Using Plasmid DNA John Ohlfest, Brain Tumor Program, U of MN 24

25 Characterization of the NRAS/shP53/EGFRvIII model Large tumor in the right hemishphere invading the left hemisphere 20x increase magnification showing infiltrating tumor in normal brain John Ohlfest, Brain Tumor Program, U of MN Genetically-Engineered Mouse model of glioma Induce tumor txt? FVB Wild-type txt w/inh?? Induce tumor txt FVB Mdr1a/b (-/-)( Bcrp1 (-/-)( txt w/inh? 25

26 Experimental Design WT or TKO mice p53shrna PDGF 15 mg/kg Dasatinib Twice daily, 7 days GFP-Guided Guided Dissection Strategy Rim Normal Brain Core 26

27 Dasatinib Regional Brain Distribution Dasatinib Concentrations (ng/gm) Wild-type Triple Knockout 0 Normal Brain Brain Around Tumor Tumor NB rim core Regional Effect on Signaling Normal Brain Rim Core WT TKO WT TKO WT TKO AKT pakt SrC psrc Actin 27

28 Less Efflux = Superior Efficacy 14 vs. 33 days; p= mg / kg b.i.d For 7 days Conclusions: 1) multiple mechanisms at multiple barriers may limit glioma treatment to invasive brain tumor stem cells (barrier 1 (BBB); barrier 2 (BTSC)) 2) several molecularly-targeted drugs are substrates of critical transport systems that are in both barriers 3) molecularly-targeted drugs may be effective in glioma if delivery issues are overcome and allow personalized therapy depending on individual tumor 4) need dirty drug (s), sharp needle 28

29 Overview Major Challenges (Opportunities) in Describing the Kinetics of Drug Distribution in the CNS (systems( systems biology to do list) 1) limited knowledge of biochemical, anatomical, and physiological variables that influence drug transport and delivery in the CNS (to do: integrate locations and mechanisms) 2) develop methods to measure time and space dependent changes in drug concentration in and around the target site (to do: use complementary existing technologies and strive to develop new measurement techniques) Overview Major Challenges (Opportunities) in Describing the Kinetics of Drug Distribution in the CNS (to( do list) continued 3) design appropriate experimental and mathematical models that incorporate critical transport or transformation mechanisms, allowing predictions of concentration-time time and space profiles leading to the site of action (to do: make correlations between animal models and human application) 4) incorporate pharmacokinetic and pharmacodynamic information to help design and implement more effect treatments for CNS diseases (to do: : educate the many disciplines involved in the discovery, development and eventual use of new treatments) 29

30 Acknowledgements Graduate Students: Sagar Agarwal,, Ying Chen, Haiqing Dai, Tianli Wang, Li Li,, Ramola Sane Collaborators: John Ohlfest and group (Jose, Belle; Pediatrics and Neurosurgery, UMN) Mike Volgelbaum,, Cleveland Clinic Funding: NIH-NCI, NCI, Leukemia Research Fund, BMS, Novartis, Childrens Cancer Research Fund-Minneapolis, MN, Brain Tumor Program-UMn UMn,, Masonic Cancer Center- UMn,, Academic Health Center-UMn Consilience: Beware of the simplified boxes. Empiric Black-Box Pharmacokinetics Systems Biology Make things as simple as possible, but not simpler. Albert Einstein Mechanistic Molecular Pharmacokinetics 30