The primary purposes of this dissertation project were as follows: 1) to explore kinetic model(s) of efflux to better understand efflux kinetics with emphasis on risk assessment of clinical P-gp inhibition at the BBB, 2) to devise methodology to better assess the extent of CNS distribution of drugs, and 3) to use pharmacokinetic-pharmacodynamic modeling with opioids to assess the influence of P-gp-mediated efflux on opioid brain penetration and antinociception, and to determine the best surrogate of in vivo intrinsic potency for CNS active drugs.
Unexpected and complex experimental observations related to efflux transport have been reported in the literature. Current models and understanding of efflux kinetics do not adequately predict or explain these observations. Furthermore, the clinical risk of efflux
inhibition at the BBB is not well-defined. A better understanding of the relationship between efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of efflux transport, and predicting the clinical significance of efflux inhibition. The first goal of this dissertation project was to develop an alternative mathematical model for expressing and assessing efflux transport kinetics. The results of this model were applied to recent unexpected and complex experimental observations concerning efflux kinetics (Chapter 2) and towards the assessment of clinical risk of P-gp inhibition at the BBB (Chapter 3).
A second goal of this project was to evaluate the steady-state unbound plasma-to-unbound brain concentration ratio ([plasma],u/[brain],u) as a method for assessing the extent of CNS
distribution of drugs (Chapter 4). The [plasma],u/[brain],u ratio is expected to be equal to the
in vivo P-gp efflux if P-gp-mediated efflux is the only active process affecting brain disposition. Using this principle, studies were conducted to compare the degree of CNS distributional impairment expressed as the [plasma],u/[brain],u ratio to the P-gp efflux ratio
for 34 marketed drugs. Opioids, triptans, protease inhibitors, and antihistamines (n = 24 total) were included in the study because these classes of agents are known to include P-gp substrates, and the extent to which these compounds distribute into the CNS may have important implications regarding safety and efficacy. In addition, 10 marketed drugs from various drug classes with either poor CNS distribution or BBB efflux also were included as part of the analysis.
The third goal of this project was to define the impact of BBB efflux on opioid pharmacokinetics/pharmacodynamics (Chapter 5, 6, and 8) and to evaluate the applicability of in vivo brain extracellular fluid concentrations, obtained via brain-homogenate equilibrium
dialysis to estimate CNS biophase concentrations (Chapter 7). Pharmacokinetic/ pharmacodynamic studies were conducted with seven opioids to estimate ED50, serum EC50,
and brain EC50; relevant in vitro and clinical parameters were used to construct in vitro-to-
preclinical and preclinical-to-clinical comparisons of opioid potency (Chapter 7).
In summary, the overall goal of this research project was to evaluate a brain equilibrium dialysis method for predicting in vivo brain extracellular fluid concentrations in order to assess CNS penetration, as well as to provide a context for concentration-effect relationships for CNS-active drugs. Furthermore, a considerable portion of this project was devoted to mathematically and experimentally assessing the impact of BBB efflux on CNS pharmacokinetics and pharmacodynamics.
REFERENCES
1. Adachi Y, Suzuki H and Sugiyama Y (2001) Comparative studies on in vitro methods for evaluating in vivo function of MDR1 P-glycoprotein. Pharm Res 18:1660-1668. 2. Aitken PG, Breese GR, Dudek FF, Edwards F, Espanol MT, Larkman PM, Lipton P,
Newman GC, Nowak TS, Jr. and Panizzon KL (1995a) Preparative methods for brain slices: a discussion. J Neurosci Methods 59:139-149.
3. Aitken PG, Breese GR, Dudek FF, Edwards F, Espanol MT, Larkman PM, Lipton P, Newman GC, Nowak TS, Jr., Panizzon KL and et al. (1995b) Preparative methods for brain slices: a discussion. J Neurosci Methods 59:139-149.
4. Anthonypillai C, Sanderson RN, Gibbs JE and Thomas SA (2004) The distribution of the HIV protease inhibitor, ritonavir, to the brain, cerebrospinal fluid, and choroid plexuses of the guinea pig. J Pharmacol Exp Ther 308:912-920.
5. Aronica E, Gorter JA, Redeker S, van Vliet EA, Ramkema M, Scheffer GL, Scheper RJ, van der Valk P, Leenstra S, Baayen JC, Spliet WG and Troost D (2005) Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain. Epilepsia 46:849-857.
6. Assem M, Schuetz EG, Leggas M, Sun D, Yasuda K, Reid G, Zelcer N, Adachi M, Strom S, Evans RM, Moore DD, Borst P and Schuetz JD (2004) Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. J Biol Chem 279:22250-22257.
7. Balakrishnam A and Polli JE (2005) Bias in estimation of transporter kinetic parameters: Interplay of transporter expression level and substrate affinity. J Clin Pharmacol
45:1087.
8. Balakrishnan A, Sussman DJ and Polli JE (2005) Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT). Pharm Res 22:1269-1280.
9. Banker MJ, Clark TH and Williams JA (2003) Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding. J Pharm Sci 92:967-974.
10. Becker S and Liu X (2006) Evaluation of the utility of brain slice methods to study brain penetration. Drug Metab Dispos 34:855-861.
11. Breedveld P, Pluim D, Cipriani G, Wielinga P, van Tellingen O, Schinkel AH and Schellens JHM (2005) The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and brain penetration of imatinib mesylate (gleevec): Implications for the use of breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain penetration of imatinib in patients. Cancer Research 65:2577-2582.
12. Chen C, Liu X and Smith BJ (2003) Utility of Mdr1-gene deficient mice in assessing the impact of P-glyco-protein on pharmacokinetics and pharmacodynamics in drug
discovery and development. Curr Drug Metab 4:272-291.
13. Chen C and Pollack GM (1998) Altered disposition and antinociception of [D- penicillamine(2,5)] enkephalin in mdr1a-gene-deficient mice. J Pharmacol Exp Ther 287:545-552.
14. Chen W, Yang JZ, Andersen R, Nielsen LH and Borchardt RT (2002) Evaluation of the permeation characteristics of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs across the blood-brain barrier using an in situ
perfused rat brain model. J Pharmacol Exp Ther 303:849-857.
15. Choo EF, Kurnik D, Muszkat M, Ohkubo T, Shay SD, Higginbotham JN, Glaeser H, Kim RB, Wood AJ and Wilkinson GR (2006) Differential in Vivo Sensitivity to Inhibition of P-Glycoprotein Located in Lymphocytes, Testes, and the Blood-Brain Barrier. J Pharmacol Exp Ther.
16. Cordon-Cardo C, O'Brien JP, Boccia J, Casals D, Bertino JR and Melamed MR (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38:1277-1287.
17. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR and Bertino JR (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A 86:695-698. 18. Cox EH, Kerbusch T, Van der Graaf PH and Danhof M (1998) Pharmacokinetic-
pharmacodynamic modeling of the electroencephalogram effect of synthetic opioids in the rat: correlation with the interaction at the mu-opioid receptor. J Pharmacol Exp Ther 284:1095-1103.
19. Dagenais C, Ducharme J and Pollack GM (2001a) Uptake and efflux of the peptidic delta-opioid receptor agonist. Neurosci Lett 301:155-158.
20. Dagenais C, Graff CL and Pollack GM (2004) Variable modulation of opioid brain uptake by P-glycoprotein in mice. Biochem Pharmacol 67:269-276.
21. Dagenais C, Zong J, Ducharme J and Pollack GM (2001b) Effect of mdr1a P- glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds. Pharm Res 18:957-963.
22. Daniel WA (2003) Mechanisms of cellular distribution of psychotropic drugs. Significance for drug action and interactions. Prog Neuropsychopharmacol Biol Psychiatry 27:65-73.
23. Daniel WA, Wojcikowski J and Palucha A (2001) Intracellular distribution of psychotropic drugs in the grey and white matter of the brain: the role of lysosomal trapping. Br J Pharmacol 134:807-814.
24. de Lange EC (2004) Potential role of ABC transporters as a detoxification system at the blood-CSF barrier. Adv Drug Deliv Rev 56:1793-1809.
25. de Lange EC, Danhof M, de Boer AG and Breimer DD (1997) Methodological considerations of intracerebral microdialysis in pharmacokinetic studies on drug transport across the blood-brain barrier. Brain Res Brain Res Rev 25:27-49.
26. Di L, Kerns EH, Fan K, McConnell OJ and Carter GT (2003) High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem 38:223-232. 27. Eisenblatter T and Galla HJ (2002) A new multidrug resistance protein at the blood-
brain barrier. Biochem Biophys Res Commun 293:1273-1278.
28. Elsinga PH, Hendrikse NH, Bart J, Vaalburg W and van Waarde A (2004) PET Studies on P-glycoprotein function in the blood-brain barrier: how it affects uptake and binding of drugs within the CNS. Curr Pharm Des 10:1493-1503.
29. Gao J, Murase O, Schowen RL, Aube J and Borchardt RT (2001) A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells. Pharm Res 18:171-176.
30. Garberg P, Ball M, Borg N, Cecchelli R, Fenart L, Hurst RD, Lindmark T, Mabondzo A, Nilsson JE, Raub TJ, Stanimirovic D, Terasaki T, Oberg JO and Osterberg T (2005) In vitro models for the blood-brain barrier. Toxicol In Vitro 19:299-334.
31. Gerk PM and Vore M (2002) Regulation of expression of the multidrug resistance- associated protein 2 (MRP2) and its role in drug disposition. J Pharmacol Exp Ther 302:407-415.
32. Gillette JR (1971) Factors affecting drug metabolism. Ann NY Acad Sci 179. 33. Goldstein GW and Betz AL (1986) The blood-brain barrier. Sci Am 255:74-83. 34. Graff CL and Pollack GM (2004) Drug transport at the blood-brain barrier and the
choroid plexus. Curr Drug Metab 5:95-108.
35. Gros P, Ben Neriah YB, Croop JM and Housman DE (1986) Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 323:728-731.
36. Habgood MD, Begley DJ and Abbott NJ (2000) Determinants of passive drug entry into the central nervous system. Cell Mol Neurobiol 20:231-253.
37. Heinzen EL and Pollack GM (2004) Pharmacodynamics of morphine-induced neuronal nitric oxide production and antinociceptive tolerance development. Brain Res 1023:175- 184.
38. Ho RH and Kim RB (2005) Transporters and drug therapy: implications for drug disposition and disease. Clin Pharmacol Ther 78:260-277.
39. Hoffmann K, Gastens AM, Volk HA and Loscher W (2006) Expression of the multidrug transporter MRP2 in the blood-brain barrier after pilocarpine-induced seizures in rats. Epilepsy Res 69:1-14.
40. Hsiao P, Sasongko L, Link JM, Mankoff DA, Muzi M, Collier AC and Unadkat JD (2006) Verapamil P-glycoprotein transport across the rat blood-brain barrier:
cyclosporine A concentration-inhibition analysis and comparison with human data. J Pharmacol Exp Ther.
41. Hutchinson PJ, O'Connell MT, Kirkpatrick PJ and Pickard JD (2002) How can we measure substrate, metabolite and neurotransmitter concentrations in the human brain? Physiol Meas 23:R75-109.
42. Johnson BM, Zhang P, Schuetz JD and Brouwer KL (2006) Characterization of
transport protein expression in multidrug resistance-associated protein (mrp) 2-deficient rats. Drug Metab Dispos 34:556-562.
43. Juliano R (1976) Drug-resistant mutants of Chinese hamster ovary cells possess an altered cell surface carbohydrate component. J Supramol Struct 4:521-526.
44. Kakee A, Terasaki T and Sugiyama Y (1996) Brain efflux index as a novel method of analyzing efflux transport at the blood-brain barrier. J Pharmacol Exp Ther 277:1550- 1559.
45. Kakee A, Terasaki T and Sugiyama Y (1997) Selective brain to blood efflux transport of para-aminohippuric acid across the blood-brain barrier: in vivo evidence by use of the brain efflux index method. J Pharmacol Exp Ther 283:1018-1025.
46. Kalvass JC, Graff CL and Pollack GM (2004) Use of loperamide as a phenotypic probe of mdr1a status in CF-1 mice. Pharm Res 21:1867-1870.
47. Kalvass JC and Maurer TS (2002) Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 23:327- 338.
48. Kansy M, Senner F and Gubernator K (1998) Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J Med Chem 41:1007-1010.
49. Kurz A, Ikeda T, Sessler DI, Larson MD, Bjorksten AR, Dechert M and Christensen R (1997) Meperidine decreases the shivering threshold twice as much as the
vasoconstriction threshold. Anesthesiology 86:1046-1054.
50. Kusuhara H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y and Endou H (1999) Molecular cloning and characterization of a new
51. Kusuhara H and Sugiyama Y (2001) Efflux transport systems for drugs at the blood- brain barrier and blood-cerebrospinal fluid barrier (Part 1). Drug Discov Today 6:150- 156.
52. Kusuhara H, Suzuki H, Terasaki T, Kakee A, Lemaire M and Sugiyama Y (1997) P- Glycoprotein mediates the efflux of quinidine across the blood-brain barrier. J Pharmacol Exp Ther 283:574-580.
53. Lee YJ, Maeda J, Kusuhara H, Okauchi T, Inaji M, Nagai Y, Obayashi S, Nakao R, Suzuki K, Sugiyama Y and Suhara T (2005) In vivo evaluation of P-glycoprotein function at the blood-brain barrier in nonhuman primates using [11C]verapamil. J Pharmacol Exp Ther.
54. Letrent SP, Pollack GM, Brouwer KR and Brouwer KL (1999a) Effects of a potent and specific P-glycoprotein inhibitor on the blood-brain barrier distribution and
antinociceptive effect of morphine in the rat. Drug Metab Dispos 27:827-834. 55. Letrent SP, Polli JW, Humphreys JE, Pollack GM, Brouwer KR and Brouwer KL
(1999b) P-glycoprotein-mediated transport of morphine in brain capillary endothelial cells. Biochem Pharmacol 58:951-957.
56. Leysen JE, Gommeren W and Niemegeers CJ (1983) [3H]Sufentanil, a superior ligand for mu-opiate receptors: binding properties and regional distribution in rat brain and spinal cord. Eur J Pharmacol 87:209-225.
57. Lipton P, Aitken PG, Dudek FE, Eskessen K, Espanol MT, Ferchmin PA, Kelly JB, Kreisman NR, Landfield PW and Larkman PM (1995) Making the best of brain slices: comparing preparative methods. J Neurosci Methods 59:151-156.
58. Liu X and Chen C (2005) Strategies to optimize brain penetration in drug discovery. Curr Opin Drug Discov Devel 8:505-512.
59. Liu X, Smith BJ, Chen C, Callegari E, Becker SL, Chen X, Cianfrogna J, Doran AC, Doran SD, Gibbs JP, Hosea N, Liu J, Nelson FR, Szewc MA and Van Deusen J (2005) Use of a physiologically based pharmacokinetic model to study the time to reach brain equilibrium: an experimental analysis of the role of blood-brain barrier permeability, plasma protein binding, and brain tissue binding. J Pharmacol Exp Ther 313:1254-1262. 60. Lotsch J (2005) Pharmacokinetic-pharmacodynamic modeling of opioids. J Pain
61. Mahar Doan KM, Humphreys JE, Webster LO, Wring SA, Shampine LJ, Serabjit-Singh CJ, Adkison KK and Polli JW (2002) Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs. J Pharmacol Exp Ther 303:1029-1037.
62. Mandula H, Parepally JM, Feng R and Smith QR (2006) Role of site-specific binding to plasma albumin in drug availability to brain. J Pharmacol Exp Ther 317:667-675. 63. Matheny CJ, Lamb MW, Brouwer KR and Pollack GM (2001) Pharmacokinetic and
pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 21:778-796.
64. Maurer TS, Debartolo DB, Tess DA and Scott DO (2004) Relationship between Exposure and Nonspecific Binding of Thirty-Three Central Nervous System Drugs in Mice. Drug Metab Dispos.
65. Miksys S and Tyndale RF (2004) The unique regulation of brain cytochrome P450 2 (CYP2) family enzymes by drugs and genetics. Drug Metab Rev 36:313-333.
66. Miksys SL and Tyndale RF (2002) Drug-metabolizing cytochrome P450s in the brain. J Psychiatry Neurosci 27:406-415.
67. Niemegeers CJ, Lenaerts FM and Janssen PA (1974a) Loperamide (R 18 553), a novel type of antidiarrheal agent. Part 1: in vivo oral pharmacology and acute toxicity. Comparison with morphine, codeine, diphenoxylate and difenoxine.
Arzneimittelforschung 24:1633-1636.
68. Niemegeers CJ, Lenaerts FM and Janssen PA (1974b) Loperamide (R 18 553), a novel type of antidiarrheal agent. Part 2: in vivo parenteral pharmacology and acute toxicity in mice. Comparison with morphine, codeine and diphenoxylate. Arzneimittelforschung 24:1636-1641.
69. Niemegeers CJ, McGuire JL, Heykants JJ and Janssen PA (1979) Dissociation between opiate-like and antidiarrheal activities of antidiarrheal drugs. J Pharmacol Exp Ther 210:327-333.
70. Nies AT, Jedlitschky G, Konig J, Herold-Mende C, Steiner HH, Schmitt HP and Keppler D (2004) Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain. Neuroscience 129:349-360.
71. Okura T, Saito M, Nakanishi M, Komiyama N, Fujii A, Yamada S and Kimura R (2003) Different distribution of morphine and morphine-6 beta-glucuronide after
intracerebroventricular injection in rats. Br J Pharmacol 140:211-217.
72. Oldendorf WH (1970) Measurement of brain uptake of radiolabeled substances using a tritiated water internal standard. Brain Res 24:372-376.
73. Ooie T, Terasaki T, Suzuki H and Sugiyama Y (1997a) Kinetic evidence for active efflux transport across the blood-brain barrier of quinolone antibiotics. J Pharmacol Exp Ther 283:293-304.
74. Ooie T, Terasaki T, Suzuki H and Sugiyama Y (1997b) Quantitative brain microdialysis study on the mechanism of quinolones distribution in the central nervous system. Drug Metab Dispos 25:784-789.
75. Parepally JM, Mandula H and Smith QR (2006) Brain Uptake of Nonsteroidal Anti- Inflammatory Drugs: Ibuprofen, Flurbiprofen, and Indomethacin. Pharm Res. 76. Potschka H, Fedrowitz M and Loscher W (2003a) Brain access and anticonvulsant
efficacy of carbamazepine, lamotrigine, and felbamate in ABCC2/MRP2-deficient TR- rats. Epilepsia 44:1479-1486.
77. Potschka H, Fedrowitz M and Loscher W (2003b) Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity. J Pharmacol Exp Ther 306:124-131.
78. Rodriguez M, Ortega I, Soengas I, Suarez E, Lukas JC and Calvo R (2004) Effect of P- glycoprotein inhibition on methadone analgesia and brain distribution in the rat. J Pharm Pharmacol 56:367-374.
79. Sasongko L, Link JM, Muzi M, Mankoff DA, Yang X, Collier AC, Shoner SC and Unadkat JD (2005) Imaging P-glycoprotein transport activity at the human blood-brain barrier with positron emission tomography. Clin Pharmacol Ther 77:503-514.
80. Schinkel AH, Smit JJ, van Tellingen O, Beijnen JH, Wagenaar E, van Deemter L, Mol CA, van der Valk MA, Robanus-Maandag EC, te Riele HP and et al. (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491-502.
81. Schinkel AH, Wagenaar E, Mol CA and van Deemter L (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 97:2517-2524.
82. Shen DD, Artru AA and Adkison KK (2004) Principles and applicability of CSF sampling for the assessment of CNS drug delivery and pharmacodynamics. Adv Drug Deliv Rev 56:1825-1857.
83. Smith QR (1996) Brain perfusion systems for studies of drug uptake and metabolism in the central nervous system. Pharm Biotechnol 8:285-307.
84. Sugiyama D, Kusuhara H, Lee YJ and Sugiyama Y (2003) Involvement of multidrug resistance associated protein 1 (Mrp1) in the efflux transport of 17beta estradiol-D- 17beta-glucuronide (E217betaG) across the blood-brain barrier. Pharm Res 20:1394- 1400.
85. Sun H, Bungay PM and Elmquist WF (2001) Effect of capillary efflux transport inhibition on the determination of probe recovery during in vivo microdialysis in the brain. J Pharmacol Exp Ther 297:991-1000.
86. Takasato Y, Rapoport SI and Smith QR (1984) An in situ brain perfusion technique to study cerebrovascular transport in the rat. Am J Physiol 247:H484-493.
87. Takasawa K, Terasaki T, Suzuki H, Ooie T and Sugiyama Y (1997) Distributed model analysis of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine distribution in brain tissue and cerebrospinal fluid. J Pharmacol Exp Ther 282:1509-1517.