Nuclear Imaging of

Multidrug Resistance (MDR)

Hee-Seung Bomcnuh.com

Mechanisms of MDR

1. P-glycoprotein (Pgp)2. MDR-associated protein (MRP)3. DNA topoisomerase II4. Tripeptide GSH5. Lung resistant related protein (LRP)

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

"flippase" model "vacuum cleaner"

"aqueous pore"-transporter

Hypothetical model for P-glycoprotein

Muller: J Hepatol, Volume 28(2).February 1998.344-354

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

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

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)

Pgp is especially expressed in

Renal cell cahepatomapheochromocytomacolon ca

Many Pgp modulators in trials; ineffectivity >> side effects

Techniques to measure MDR1 gene or MRP expression

DNA PCRSouthern blotting

mRNA RT-PCRNorthern blotting

protein immunohistochemistryWestern blotting

MDR1 mRNA =/= Pgp efflux pump

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

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

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

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

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

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

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

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

10min 180min10min 180min

Clin Nucl Med. 1999 ; 24:314-8.

Tc-99m MIBI Scintimammography

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.

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.

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

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

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

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

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

Canalicular transport systems

Basolateral transport systems

Trauner: J Hepatol, Volume 31(1).July 1999.165-178

HEPATOCYTE

Mutations in MRP2 in patients with Dubin-Johnson syndrome

THOMPSON: Semin Liver Dis, Volume 20(3).August 2000.365-372

DNA topoisomerase II enzyme

Target for many drugs: anthracyclines, epipodophyllotoxins

Decreased topoisomerase II = less DNA damage

faciliate drug metabolism to less active compoundsdetoxification of drug-induced free radicals

Tripeptide GSH

Lung resistant related protein (LRP)

110 kDaMajor vault proteinNucleo-cytoplasmic transport

JNM 2001; 42:1476-83

Pgp vs. MRP vs. LRP

Tc-99m MIBIaccumulation

Tc-99m MIBIwashout

Problems

• Various mechanisms for MDRPgp, MRP, topoisomerase II, LRP, etc.

• No specific markers for each genes or proteins

• Inefficient modulators with side effects