1. Apatinib (YN968D1) reverses multidrug resistance by inhibiting the function of multiple ATP-binding cassette transporters
Yanjun Mi, Liwu Fu
State Key Laboratory of oncology in southern China,
Cancer Center, Sun Yat-sen University, Guangzhou, , China

2. Background
A successful cancer chemotherapy is limited by multidrug resistance (MDR).
The major inducers of MDR are the ABC transporters, such as ABCB1 (anthracyclins, vinca alkaloids, taxanes, and epipodophyllotoxins), ABCG2 (mitoxantrone, anthracyclins, topoisomerase I inhbitors, methotrexate).
Combination of modulator with conventional chemotherapeutic drugs to restore the sensitivity of MDR cells is a main strategy for overcoming MDR.

3. Background
Tyrosine kinase inhibitors (TKIs) belong to a new class of anticancer drugs.
TKIs including lapatinib, erlotinib and sunitinib, have been shown to overcome ABC transporters-mediated MDR in cancer cells.
Apatinib (YN968D1) is an oral small-molecule TKI that targets VEGFR-2, RET, c-Kit and c-Src tyrosine kinases.
It is conceivable that apatinib may inhibit functions of ABC transporters by binding to their ATP-binding sites.

4. Cytotoxicity of apatinib alone in all experimental cell lines
ABCB1
ABCG2
More than 85% of cells were viable at concentrations of apatinib up to 3.0 μM in all experimental cells.

5. Apatinib reversed MDR in vitro
Compounds
IC50 ± SD (μM) (fold-reversal) KB
KBv200 (ABCB1)
Doxorubicin
0.029 ± 0.002
(1.00)
2.277 ± 0.134
μM Apatinib
1.259 ± 0.099**
(1.81)
+ 1.5 μM Apatinib
0.030 ± 0.001
(0.98)
0.964 ± 0.070**
(2.36)
+ 3.0 μM Apatinib
0.028 ± 0.003
(1.04)
0.337 ± 0.016**
(6.81)
+ 10 μM Verapamil
0.029 ± 0.003
0.106 ± 0.006**
(21.4)
Paclitaxel ±
0.296 ± 0.027 ±
0.142 ± 0.013**
(2.08) ±
(0.95)
0.045 ± 0.004**
(6.58)
0.009 ± 0.001**
(32.9)
0.007 ± 0.001**
(42.3)
Cispatin
0.726 ± 0.055
1.284 ± 0.141
0.714 ± 0.057
(1.01)
1.292 ± 0.125
(0.99)
MCF-7
MCF-7/adr (ABCB1)
0.344 ± 0.037
(1.00 )
± 1.186
0.349 ± 0.011
3.021 ± 0.196**
(3.81)
0.331 ± 0.019
2.177 ± 0.273**
(5.28)
0.350 ± 0.036
(0.98 )
0.854 ± 0.056**
(13.5)
0.340 ± 0.038
0.540 ± 0.076**
(21.3)
5.811 ± 0.533
4.622 ± 0.371
5.624 ± 0.211
(1.03 )
4.531 ± 0.352
(1.02)

6. Apatinib reversed MDR in vitro
Compounds
IC50 ± SD (μM) (fold-reversal) S1
S1-M1-80 (ABCG2)
Mitoxantrone
0.194 ± 0.027
(1.00)
± 0.922
μM Apatinib
0.196 ± 0.041
(0.98)
6.434 ± 0.478**
(2.12)
+ 1.5 μM Apatinib
0.185 ± 0.058
(1.05)
2.070 ± 0.621**
(6.59)
+ 3.0 μM Apatinib
0.136 ± 0.067
(1.42)
1.188 ± **
(11.5)
+ 2.5 μM FTC
0.188 ± 0.011
(1.03)
0.892 ± 0.056**
(15.3)
Topotecan
0.262 ± 0.042
± 0.455
0.264 ± 0.022
(0.99)
4.089 ± 0.026**
(2.51)
0.247 ± 0.017
2.037 ± 0.083**
(5.04)
0.196 ± 0.055
(1.33)
1.188 ± 0.055**
(8.65)
0.254 ± 0.016
(1.02)
0.745 ± 0.068**
(13.8)
Cisplatin
± 1.181
± 1.322
± 1.990
(1.04)
± 1.452
HL60
HL60/ADR(ABCC1)
Doxorubicin
0.036 ± 0.002
5.704 ± 0.378
0.035 ± 0.002
5.493 ± 0.289
0.037 ± 0.002
(0.97)
5.528 ± 0.515
0.033 ± 0.004
(1.09)
5.719 ± 0.595

7. Apatinib reversed MDR in vitro
Compounds
IC50 ± SD (μM) (fold-reversal)
HEK293/pcDNA3.1
HEK293/ABCG2-R2
HEK293/ABCB1
Mitoxantrone ±
(1.00) ± ±
+ 3 μM Apatinib ±
(1.63)
± **
(20.55)
± **
(2.24)
+ 3 μM FTC ±
(0.98)
± **
(19.59) -
+ 3 μM PSC833 ±
(1.04)
± **
(2.01)
SN-38 ± ± ±
(1.62)
± **
(19.37) ±
(1.46)
± **
(18.00)
Vincristine ±
(1.00 ± ±
(1.30)
± **
(3.57)
+ 10 μM Verapamil ±
(0.97)
± **
(12.46)
Doxorubicin ± ± ±
(1.41)
± **
(3.42) ±
(0.77)
± **
(11.23)
Cisplatin ± ± ± ±
(1.19) ±
(1.16) ±
(0.90)

8. Potentiation of antitumor effects of paclitaxel by apatinib in a xenograft model of KBv200 cells in nude mice
Control: saline
Apatinib: 70 mg/Kg, P.O., q3dX4
Paclitaxel:18 mg/kg, i.p., q3dx4
Combination:
70 mg/Kg, P.O., q3dx4+18 mg/kg, i.p., q3dx4
The combination of Apatinib and paclitaxel produced a significantly inhibitory effect on the growth of the xenografts but did not result in the increased toxicity
52%

9. Effect of apatinib on the accumulation of doxorubicin

10. Effect of apatinib on the accumulation of rhodamine 123

11. Apatinib ABCB1 and ABCG2 ATPase activity Expression of ABCB1 and ABCG2
Intracellular accumulation of drugs in the MDR cells
Reverse ABCB1- and ABCG2-mediated MDR

12. Effect of apatinib on the expression of ABCB1 and ABCG2

13. Effect of apatinib on the expression of ABCB1 and ABCG2

14. Effect of apatinib on ABCB1 and ABCG2 ATPase activity
950nmol/L
10-12nmol/L

15. Effect of apatinib on photoaffinity labeling of ABCB1 and ABCG2 with [125I]-IAAP
IC50 = 2.9 ± 0.40 (μmol/L)
IC50 = 11 ± 4 (nmol/L)

16. Effect of apatinib on the phosphorylation of AKT and ERK1/2

17. Apatinib MDR ABCB1 and ABCG2 ATPase activity
Expression of ABCB1 and ABCG2
Intracellular accumulation of drugs in the MDR cells
Blockade of AKT and ERK1/2 activation
Reverse
MDR

18. Conclusion
Apatinib reverses ABCB1- and ABCG2-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 and ABCG2 function, resulting in elevated intracellular concentrations of substrate chemotherapeutic drugs.
The reversal of MDR is not associated with the blockade of tyrosine kinases. These findings may be useful for cancer combinational therapy of chemotherapeutic drug with apatinib in the clinic.

19. Acknowledgments
We like to thank Professor Suresh V. Ambudkar (Center for Cancer Research, NCI, NIH, Bethesda) and Zhe-Sheng Chen (College of Pharmacy and Allied Health Professions, St. John’s University ) for collaborating to finish this work.

20. Thank you