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Nuclear Imaging of
Multidrug Resistance (MDR)
Hee-Seung Bomcnuh.com
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Mechanisms of MDR
1. P-glycoprotein (Pgp)2. MDR-associated protein (MRP)3. DNA topoisomerase II4. Tripeptide GSH5. Lung resistant related protein (LRP)
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P-glycoprotein
ATP-dependent170-180 kDaencoded by the MDR1 gene
located on chromosome 7 (7q21.1)
Substrates:anthracyclines (doxorubicin, daunorubicin)vinca alkaloids (vincristine)epipodophyllotoxins (etoposide)taxanes (paclitaxel)
Modulators:calcium channel blockerscyclosporin A, PSC 833
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"flippase" model "vacuum cleaner"
"aqueous pore"-transporter
Hypothetical model for P-glycoprotein
Muller: J Hepatol, Volume 28(2).February 1998.344-354
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Hopfner K.P., Karcher A., Shin D.S., Craig L., Arthur L.M., Carney J.P. and Tainer J.A.
Structural biology of Rad50-ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
Cell, (2000) 101:789-800.
Molecular Mechanisms for the ABC-ATPase Superfamily Members
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Normal distribution of Pgp
adrenal cortexintestinal mucosal cellsbiliary hepatocytesrenal proximal tubule epitheliumpancreatic ductulespregnant uterusgastrointestinal epitheliumblood capillaries of the brain and testesCD34+ bone marrow stem cells
Function? protection by extruding toxins out of organs protect critical organs such as brain and testes
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Choroid plexus epithelial expression of MDR1 P-glycoproteinand MRP contribute to the blood-cerebrospinal-fluiddrug-permeability barrier
Proc Natl Acad Sci, USA 1999; 96:3900-5
GF120918(inhibitor of Pgp)
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Pgp is especially expressed in
Renal cell cahepatomapheochromocytomacolon ca
Many Pgp modulators in trials; ineffectivity >> side effects
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Techniques to measure MDR1 gene or MRP expression
DNA PCRSouthern blotting
mRNA RT-PCRNorthern blotting
protein immunohistochemistryWestern blotting
MDR1 mRNA =/= Pgp efflux pump
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F OExpression Function
100%
50
0
F: fresh mediumO: old medium
Prolongation of medium exchange is associated with a decrease in function but not expression of the P-glycoprotein pump in leukaemic cells. European Journal of Haematology. 1996; 56:12-22
DNR accumulation
F O
PCR
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expression functionmyeloblastic cell line
mature high lowimmature low high
Lack of correlation between expression and function of P-glycotprotein in acute myeloid cell lines. Leukemia 1995; 9:799-807
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Functional Imaging of Pgp
SPECTTc-99m sestamibiTc-99m tetrofosminTc-99m Q complex (Tc-99m Q12: furifosmin)
PET [C-11] daunorubicin[C-11] colchicine[C-11] verapamil[Ga-68] gallium(III) complexes[Co-64] bis(diphosphine) complexes
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Tc-99m sestamibi vs. Tc-99m tetrofosmin
Verapamil indexsimilar Wolf et al. EJNM 1996; 23:1095sestamibi Nakamura et al. EJNM 1996; 23:1142
MRP reversalsimilar Utsunomiya et al. EJNM 2000;27:1786
MIBI, TF vs. furifosmin
MDR(-) soft tissue sarcoma cell lines EJNM 2000; 27:1839
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Lessons from investigations using nuclear imaging on MDR
1. Tc-99m complexes (MIBI, TF, FF; Tc-C) are substrates of Pgp. Tl-201 is not.
2. Accumulation (esp. of MIBI) in resistant tumors is lower thanin sensitive tumors.
MDR(-) cell line
MDR(+) cell line
JNM 2001; 42:646-54
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3. Pgp expression is inversely proportional to accumulation of Tc-Cin tumor cells.
4. Intensity of Tc-C efflux out of tumor cells is good measure of MDR.
JNM 2001; 42:1476-83
accumulation efflux
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5. MDR modulators increase accumulation of Tc-C.(e.g. PSC 833, GW 0918, KR 30035)
10
20% Uptake of Tc-99m MIBI
L1210 Adr Cell Vcr Cell0
Basal
Cyclosporin 10 uM
KJNM 1999; 33:152-62
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Lesson from clinical studies using Tc-99m MIBI
1. Increased efflux rate from tumors with elevated Pgp expression,Increased uptake correlates with angiogenesis.
2. Rapid clearance out of tumor correlates with lack of response to chemotherapy.
3. Inverse correlation between T/N ratio and Pgp expression.Positive correlation between retention and chemosensitivity.
5.0
4.0
3.0
2.0
1.0
0
Tc-99m MIBI uptake (T/N 1h)
CR PR NR
p=0.006
JNM 1998; 39:91-4Response to chemotherapy in SCLC
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10min 180min10min 180min
Clin Nucl Med. 1999 ; 24:314-8.
Tc-99m MIBI Scintimammography
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0 10 20 30 40 50 60 70
Angiogenesis (CD34)
1.0
2.0
3.0
4.0 Tc-99m Sestamibi uptakeat 10 min (T/N10)
r = 0.47r = 0.47p= 0.008p= 0.008n = 31n = 31
Clin Nucl Med. 1999 ; 24:314-8.
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O I II III IV
40
30
20
10
0
-10 Pgp expression
Washout Index (%)= (TN10-TN180)/TN10 X 100
Clin Nucl Med. 1999 ; 24:314-8.
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4. In malignant lymphoma, MIBI uptake correlates withthe response to chemotherapy.
5. SCLC: Pgp expression correlates with T/N ratio but not with washout. (debate)
6. Bone and soft tissue tumor: Pgp expression correlates withwashout but not with uptake.
7. AML, ALL: Inverse correlation between BM/B ratioand Pgp expression.
10 min 180 min
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F-18 FDG PET
MDR(-) tumor
MDR(+) tumor
JNM 2001; 42:646-54
• GLUT-1 levels are diminished progressively with elevated Pgp levels. Cancer Lett. 1997;115:221-7
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Multidrug resistance-associated protein (MRP)
190 kDaMRP1; encoded by MRP1 gene
located on chromosome 16 (16p13.1)ATP-binding cassette (ABC) superfamily
present in almost all cells of the human bodycytoplasm: endoplasmic reticulum or Golgi apparatus
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MRP: Mechanism of action
Glutathione S-conjugate efflux pump (GS-X pump)GSH dependent
MRP1 ~ MRP5
Substrates of MRP: Leukotriens (LT)LTC4, LTD4, LTE4
Modulator: BSO (buthionine sulfoximine)
PET: N-[C-11]acetyl-LTE4SPECT: Tc-99m sestamibi
Tc-99m tetrofosmin
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1. Tc-C is substrate of MRP1 as well as Pgp.2. Lowering cellular glutathione increase Tc-C uptake in MRP1-
positive cells, not in Pgp-positive cells.
MRP(-) MRP(+)
Tc-99m TF uptake
GSH depletionby BSO
Basal1.58
3.16
Biochem Phramacol 2000; 60:413-26
buthionine sulfoximine
Lessons from investigations using nuclear imaging on MRP
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Canalicular transport systems
Basolateral transport systems
Trauner: J Hepatol, Volume 31(1).July 1999.165-178
HEPATOCYTE
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Mutations in MRP2 in patients with Dubin-Johnson syndrome
THOMPSON: Semin Liver Dis, Volume 20(3).August 2000.365-372
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DNA topoisomerase II enzyme
Target for many drugs: anthracyclines, epipodophyllotoxins
Decreased topoisomerase II = less DNA damage
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faciliate drug metabolism to less active compoundsdetoxification of drug-induced free radicals
Tripeptide GSH
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Lung resistant related protein (LRP)
110 kDaMajor vault proteinNucleo-cytoplasmic transport
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JNM 2001; 42:1476-83
Pgp vs. MRP vs. LRP
Tc-99m MIBIaccumulation
Tc-99m MIBIwashout
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Problems
• Various mechanisms for MDRPgp, MRP, topoisomerase II, LRP, etc.
• No specific markers for each genes or proteins
• Inefficient modulators with side effects