Table S1. Representative list of kinases with structural features disfavoring off-target inhibition
by PF-114
Active site-bound water molecule (analogous to B-Raf
active site water molecule)
Main chain carbonyl oxygen interacting with 1,2,4triazolo-moiety of PF-114
BMX/ETK
BRAF
BRAF
c-Src
EPHA3
ERBB4/HER4
EPHA7
FGFR1
FGFR1
FGFR2
FGFR2
KDR/VEGFR2
FLT1/VEGFR1
LOK/STK10
HCK
LYN
P38a/MAPK14
TIE2/TEK
P38b/MAPK11
TRKB
RET
TRKC
TIE2/TEK
TRKB
TRKC
Table S2. Kinase inhibition profile of PF-114, ponatinib, dasatinib and nilotinib at 100 nM of
inhibitor.
Residual activity:
<10%
10%<x<25%
25%<x<50%
50%<x<75%
>75%
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
ABL1
1,91
5,20
8,34
9,84
ABL2/ARG
2,33
0,29
4,51
8,45
ACK1
96,58
37,24
-0,74
95,39
AKT1
95,99
98,44
96,94
101,04
AKT2
100,38
94,10
99,06
97,31
AKT3
102,37
93,66
101,11
99,91
ALK
99,48
101,77
99,99
98,13
ALK1/ACVRL1
99,83
90,36
29,96
95,91
ALK2/ACVR1
91,82
90,16
135,32
98,10
ALK3/BMPR1A
91,92
100,01
108,78
97,21
ALK4/ACVR1B
102,07
97,56
121,34
101,81
ALK5/TGFBR1
97,73
102,52
102,77
100,91
ALK6/BMPR1B
105,78
90,83
118,92
88,89
ARAF
76,97
21,26
47,31
92,37
ARK5/NUAK1
100,94
106,98
99,59
103,48
ASK1/MAP3K5
89,69
108,34
104,61
95,10
Aurora A
104,66
103,54
103,32
82,20
Aurora B
102,52
83,87
98,90
100,07
Aurora C
89,92
102,48
98,66
102,48
AXL
62,79
77,35
70,95
76,35
BLK
13,01
0,64
1,41
96,43
BMPR2
70,19
79,67
99,59
95,03
BMX/ETK
83,09
10,95
0,26
96,61
BRAF
92,48
6,47
85,42
34,84
BRK
90,59
44,64
3,20
78,43
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
BRSK1
104,21
101,70
99,95
100,27
BRSK2
92,71
98,12
105,64
97,06
BTK
99,44
85,78
-1,35
100,13
c-Kit
61,11
32,16
14,47
65,48
c-MER
101,98
82,84
92,45
93,06
c-MET
89,14
112,75
95,45
95,10
c-Src
51,85
1,40
-0,53
93,62
CAMK1a
84,17
99,45
100,44
92,40
CAMK1b
93,68
94,78
99,29
93,54
CAMK1d
93,18
102,87
100,49
102,34
CAMK1g
79,32
95,73
92,74
99,90
CAMK2a
92,52
96,56
102,36
100,01
CAMK2b
100,04
92,95
101,96
92,18
CAMK2d
97,94
104,04
106,64
105,09
CAMK2g
96,24
103,58
106,41
105,30
CAMK4
103,95
105,40
100,30
105,75
CAMKK1
100,21
61,48
98,43
104,97
CAMKK2
97,10
55,47
93,65
99,69
CDC7/DBF4
95,91
103,91
104,11
101,57
CDK1/cyclin A
109,94
94,98
95,58
107,77
CDK1/cyclin B
94,63
91,53
88,94
84,81
CDK1/cyclin E
97,88
75,83
98,70
97,61
CDK2/cyclin A
92,39
95,29
89,29
87,47
CDK2/Cyclin A1
82,70
98,90
96,47
103,29
CDK2/cyclin E
84,70
96,74
92,36
94,12
CDK3/cyclin E
107,09
92,18
91,42
92,35
CDK4/cyclin D1
98,17
98,36
96,13
94,95
CDK4/cyclin D3
96,65
93,13
91,27
91,79
CDK5/p25
91,21
91,20
90,16
89,32
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
CDK5/p35
105,80
88,28
96,65
95,62
CDK6/cyclin D1
97,37
95,79
102,96
103,96
CDK6/cyclin D3
97,73
100,91
102,82
101,82
CDK7/cyclin H
96,03
98,47
101,39
102,33
CDK9/cyclin K
100,49
95,99
97,69
95,90
CDK9/cyclin T1
96,02
102,71
98,39
100,94
CHK1
103,45
111,37
104,02
110,89
CHK2
97,87
39,86
97,97
99,47
CK1a1
93,09
107,75
104,23
105,09
CK1d
104,34
91,87
88,32
85,24
CK1epsilon
88,95
94,25
102,19
150,00
CK1g1
88,67
104,66
104,38
109,15
CK1g2
89,85
101,69
98,72
98,28
CK1g3
103,54
99,67
93,83
92,73
CK2a
87,51
100,33
102,50
98,62
CK2a2
85,31
100,95
99,01
98,03
CLK1
90,70
69,48
104,38
96,64
CLK2
101,40
99,20
99,10
100,07
CLK3
99,44
110,72
107,81
110,83
CLK4
69,50
69,52
91,15
59,83
COT1/MAP3K8
107,76
92,35
91,15
88,43
CSK
67,40
4,87
2,48
53,42
CTK/MATK
104,13
99,31
99,90
100,16
DAPK1
103,05
70,00
85,13
77,48
DAPK2
100,62
108,63
105,02
102,75
DCAMKL1
91,23
92,24
105,35
98,02
DCAMKL2
106,34
98,84
106,72
100,76
DDR1
0,40
1,29
1,31
2,19
DDR2
3,34
2,31
2,28
2,95
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
DLK/MAP3K12
100,13
81,96
88,01
92,75
DMPK
103,79
112,14
110,01
110,66
DMPK2
94,33
89,77
96,53
95,38
DRAK1/STK17A
97,78
95,33
93,98
95,41
DYRK1/DYRK1A
89,95
92,36
99,01
95,92
DYRK1B
97,91
101,36
97,18
98,24
DYRK2
102,26
101,40
99,82
99,22
DYRK3
97,16
102,48
104,32
103,91
DYRK4
101,37
99,77
101,90
100,36
EGFR
96,23
83,72
39,38
100,17
EPHA1
96,53
38,34
4,80
86,62
EPHA2
17,65
1,83
2,49
24,50
EPHA3
60,02
2,16
1,33
70,10
EPHA4
61,44
3,46
1,80
48,98
EPHA5
53,46
0,86
0,82
49,85
EPHA6
54,82
0,37
80,08
79,32
EPHA7
50,28
6,12
96,37
94,86
EPHA8
29,00
2,41
2,34
39,39
EPHB1
50,00
4,50
3,70
33,57
EPHB2
40,63
2,66
0,74
14,26
EPHB3
69,02
3,73
2,53
31,09
EPHB4
77,46
4,91
1,47
21,18
ERBB2/HER2
85,94
93,44
97,94
101,05
ERBB4/HER4
86,72
33,04
16,76
95,74
ERK1
96,47
97,68
101,47
99,94
ERK2/MAPK1
98,34
96,70
95,13
93,75
ERK5/MAPK7
102,76
101,13
105,93
113,04
ERK7/MAPK15
97,41
61,52
102,47
91,54
FAK/PTK2
104,73
100,12
103,18
98,94
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
FER
100,92
88,36
101,25
101,16
FES/FPS
90,31
84,96
92,37
92,16
FGFR1
89,10
7,35
91,04
99,65
FGFR2
89,56
17,67
81,33
96,13
FGFR3
98,98
15,47
98,55
99,66
FGFR4
99,36
18,59
102,79
104,86
FGR
17,64
0,96
-0,38
70,32
FLT1/VEGFR1
56,71
5,72
91,74
90,05
FLT3
75,15
1,43
93,56
86,68
FLT4/VEGFR3
30,20
1,85
86,17
89,65
FMS
3,60
1,43
1,65
47,73
FRK/PTK5
5,58
1,90
1,28
23,07
FYN
24,38
-0,35
-0,99
97,70
GCK/MAP4K2
89,96
6,90
90,05
95,41
GRK1
96,67
95,34
84,49
91,59
GRK2
97,77
104,85
101,21
104,44
GRK3
95,59
95,45
102,23
97,20
GRK4
103,84
96,49
102,49
98,65
GRK5
103,25
92,88
98,84
98,85
GRK6
99,07
98,01
98,95
98,63
GRK7
95,40
103,22
99,18
105,34
GSK3a
101,56
103,69
104,76
107,10
GSK3b
98,40
107,13
104,54
108,47
Haspin
94,37
94,94
93,18
96,72
HCK
18,09
1,01
0,97
73,69
HGK/MAP4K4
92,49
46,52
83,61
101,91
HIPK1
100,72
94,17
90,18
89,11
HIPK2
92,11
94,11
100,15
103,34
HIPK3
110,04
104,49
103,35
101,20
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
HIPK4
109,35
92,56
100,82
97,48
HPK1/MAP4K1
93,97
15,53
95,10
97,08
IGF1R
97,48
96,65
95,66
93,88
IKKa/CHUK
91,01
87,07
101,11
97,45
IKKb/IKBKB
97,47
91,04
98,61
102,15
IKKe/IKBKE
98,68
98,87
90,55
94,93
IR
97,59
86,75
96,56
90,69
IRAK1
100,39
35,34
113,72
113,88
IRAK4
90,25
95,02
99,85
107,84
IRR/INSRR
104,49
94,59
99,42
101,48
ITK
96,05
91,43
97,84
101,44
JAK1
80,80
19,54
105,33
103,64
JAK2
103,24
73,80
94,87
99,73
JAK3
96,31
42,49
101,28
98,22
JNK1
97,48
104,64
106,99
101,45
JNK2
102,37
93,82
102,82
100,89
JNK3
91,22
92,34
90,77
93,07
KDR/VEGFR2
16,26
1,12
106,60
104,70
KHS/MAP4K5
92,44
21,51
5,43
98,77
LATS1
101,77
95,36
99,04
99,37
LATS2
96,11
73,82
99,53
98,88
LCK
1,51
1,21
1,29
57,29
LCK2/ICK
96,94
98,91
104,63
101,08
LIMK1
102,96
102,09
53,41
104,66
LKB1
99,41
95,23
100,38
100,81
LOK/STK10
50,68
7,45
80,32
98,67
LRRK2
87,75
38,14
90,80
94,37
LYN
1,30
0,94
0,99
62,41
LYN B
2,01
2,67
1,07
74,91
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
MAPKAPK2
136,06
94,58
104,11
97,83
MAPKAPK3
98,56
101,06
94,54
94,88
MAPKAPK5/PRAK
98,73
111,22
107,39
102,73
MARK1
103,11
90,25
95,44
110,34
MARK2/PAR-1Ba
95,33
95,00
101,20
100,11
MARK3
98,11
100,07
96,09
104,50
MARK4
107,58
97,19
91,12
101,04
MEK1
101,33
94,01
94,78
99,34
MEK2
92,26
87,38
84,32
93,52
MEK3
102,96
102,67
107,12
107,34
MEKK1
98,55
101,36
103,97
109,25
MEKK2
75,83
46,48
98,40
102,63
MEKK3
100,13
24,84
96,79
100,47
MELK
84,76
96,51
105,80
102,17
MINK/MINK1
108,50
139,52
91,65
104,24
MKK4
92,89
101,55
99,66
96,08
MKK6
99,22
100,62
101,07
104,56
MLCK/MYLK
94,10
105,95
107,03
111,21
MLCK2/MYLK2
89,89
99,34
102,47
106,16
MLK1/MAP3K9
99,48
92,40
94,29
100,45
MLK2/MAP3K10
95,52
85,66
87,13
89,86
MLK3/MAP3K11
101,39
85,43
87,86
93,34
MNK1
92,42
93,59
99,99
101,85
MNK2
100,36
61,20
103,78
101,67
MRCKa/CDC42BPA
99,11
106,51
101,36
107,59
MRCKb/CDC42BPB
101,76
106,70
102,20
104,68
MSK1/RPS6KA5
98,23
77,72
100,41
100,31
MSK2/RPS6KA4
98,00
95,61
101,85
105,20
MSSK1/STK23
96,73
95,53
101,18
97,56
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
MST1/STK4
107,94
100,73
102,03
103,87
MST2/STK3
93,71
99,35
100,72
100,68
MST3/STK24
101,98
159,80
100,66
99,72
MST4
97,40
115,54
84,83
97,95
MUSK
78,32
98,00
95,25
99,06
MYLK3
103,17
102,86
103,43
101,43
MYO3b
101,07
99,87
101,43
99,43
NEK1
77,74
71,37
95,52
101,08
NEK11
89,18
110,80
73,53
105,86
NEK2
90,49
98,67
101,16
99,19
NEK3
99,45
99,38
84,51
90,50
NEK4
75,93
23,68
93,62
98,25
NEK6
95,05
100,37
91,41
99,97
NEK7
94,24
94,07
94,12
92,64
NEK9
87,59
103,38
85,67
100,24
NLK
96,72
105,29
48,86
111,08
OSR1/OXSR1
86,94
94,76
98,97
95,88
P38a/MAPK14
29,11
11,06
74,23
62,40
P38b/MAPK11
93,81
28,89
94,94
31,08
P38d/MAPK13
99,24
87,28
114,57
105,93
P38g
94,91
67,48
100,02
101,65
p70S6K/RPS6KB1
98,28
44,38
94,20
94,45
p70S6Kb/RPS6KB2
103,31
92,89
94,17
95,94
PAK1
94,47
102,87
98,47
100,13
PAK2
96,60
103,07
103,44
101,00
PAK3
98,13
101,02
106,27
102,24
PAK4
100,76
104,30
105,91
108,96
PAK5
97,78
99,34
100,60
97,76
PAK6
96,02
91,64
104,48
98,83
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
PASK
91,29
92,81
107,60
107,31
PBK/TOPK
104,98
103,50
105,01
101,97
PDGFRa
-0,62
1,45
27,89
45,57
PDGFRb
10,98
4,80
7,84
57,85
PDK1/PDPK1
105,44
98,66
100,60
96,58
PHKg1
88,20
97,67
103,95
104,81
PHKg2
96,59
97,29
96,38
100,63
PIM1
90,86
91,49
93,55
97,13
PIM2
93,03
102,85
98,99
101,23
PIM3
93,35
98,79
95,04
97,77
PKA
99,69
92,99
85,61
101,70
PKAcb
99,97
110,93
113,57
117,66
PKAcg
91,37
44,11
78,90
83,19
PKCa
97,84
94,11
86,58
90,57
PKCb1
93,62
93,64
87,86
93,10
PKCb2
105,32
101,14
95,36
104,22
PKCd
90,08
92,19
94,74
93,88
PKCepsilon
101,62
99,60
94,04
105,23
PKCeta
101,27
103,86
102,87
102,63
PKCg
102,32
93,46
94,14
91,24
PKCiota
100,10
112,15
97,04
91,59
PKCmu/PRKD1
100,15
95,54
96,71
95,61
PKCnu/PRKD3
91,66
93,66
93,74
93,39
PKCtheta
79,26
83,25
81,83
85,15
PKCzeta
95,03
96,60
96,52
93,26
PKD2/PRKD2
95,03
105,78
101,98
109,20
PKG1a
97,18
91,63
91,91
97,50
PKG1b
91,80
92,99
95,87
92,95
PKG2/PRKG2
89,39
106,47
111,53
99,56
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
PKN1/PRK1
93,70
94,15
85,71
95,13
PKN2/PRK2
94,76
89,90
94,94
96,00
PKN3/PRK3
103,88
100,08
94,99
95,29
PLK1
97,97
99,38
97,97
98,59
PLK2
101,02
94,43
89,20
92,98
PLK3
93,78
97,21
99,56
98,20
PLK4/SAK
91,91
98,22
103,03
101,97
PRKX
104,20
99,13
108,09
103,08
PYK2
94,50
32,34
101,03
99,92
RAF1
23,38
3,66
69,59
28,51
RET
4,95
-0,75
87,46
94,13
RIPK2
78,13
36,49
43,31
98,32
RIPK3
24,85
5,46
82,92
138,29
RIPK5
88,38
95,25
92,63
90,67
ROCK1
98,80
98,58
100,77
99,34
ROCK2
96,32
92,03
95,83
97,61
RON/MST1R
93,09
105,63
101,90
99,77
ROS/ROS1
104,80
88,16
96,11
91,65
RSK1
101,91
112,44
112,72
110,47
RSK2
101,15
96,51
98,77
103,35
RSK3
78,97
96,96
97,96
104,13
RSK4
90,00
97,75
99,84
98,39
SGK1
102,89
109,80
108,14
108,72
SGK2
90,49
94,58
107,89
102,42
SGK3/SGKL
97,25
98,91
99,59
94,39
SIK1
87,47
25,30
-1,48
97,33
SIK2
99,83
86,16
1,78
99,77
SLK/STK2
85,58
40,00
86,74
95,09
SNARK/NUAK2
106,02
114,09
109,50
110,66
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
SRMS
108,53
9,15
27,04
105,28
SRPK1
89,98
103,40
94,21
83,81
SRPK2
100,41
98,81
92,91
97,46
SSTK/TSSK6
99,31
96,84
98,42
101,17
STK16
100,72
101,94
100,46
100,38
STK22D/TSSK1
90,90
103,61
100,34
97,00
STK25/YSK1
95,18
108,15
72,37
77,71
STK32B/YANK2
100,59
98,85
115,18
104,86
STK32C/YANK3
101,92
101,31
102,26
98,33
STK33
96,66
89,14
106,01
100,27
STK38/NDR1
98,15
101,16
106,58
111,84
STK38L/NDR2
102,01
80,49
101,64
96,29
STK39/STLK3
95,80
104,71
103,89
104,72
SYK
95,58
95,57
91,71
97,99
TAK1
102,80
7,29
98,85
105,01
TAOK1
82,18
79,31
104,15
96,03
TAOK2/TAO1
94,39
68,33
104,37
99,12
TAOK3/JIK
94,10
70,71
107,20
99,66
TBK1
100,76
94,33
96,33
95,85
TEC
101,19
101,30
2,61
104,29
TESK1
105,20
95,99
31,49
94,29
TGFBR2
96,88
66,40
100,39
99,31
TIE2/TEK
97,12
9,12
107,64
109,32
TLK1
105,95
103,41
109,14
107,09
TLK2
103,33
107,10
105,30
108,86
TNIK
97,75
38,11
53,52
90,14
TNK1
67,51
1,54
101,94
98,48
TRKA
92,43
5,71
98,13
93,25
TRKB
94,85
8,09
94,35
95,58
Kinase
PF-114
Ponatinib
Dasatinib
Nilotinib
TRKC
90,76
10,31
90,98
90,55
TSSK2
100,47
98,01
103,46
104,99
TSSK3/STK22C
112,00
106,40
110,45
109,86
TTBK1
90,87
105,36
103,89
103,79
TTBK2
103,50
105,87
98,46
105,64
TXK
90,57
12,83
0,88
87,99
TYK1/LTK
90,30
100,62
100,22
105,47
TYK2
95,59
65,41
103,22
106,33
TYRO3/SKY
74,08
98,38
99,79
102,79
ULK1
108,31
99,74
105,37
100,32
ULK2
95,63
105,27
103,46
114,01
ULK3
98,07
34,71
89,96
97,18
VRK1
100,21
93,97
87,34
92,75
VRK2
107,07
100,24
102,57
94,79
WEE1
95,20
99,47
102,55
98,54
WNK1
102,44
101,91
103,96
98,12
WNK2
98,58
107,81
95,02
103,86
WNK3
100,17
102,30
105,50
107,44
YES/YES1
61,75
4,68
3,19
91,36
ZAK/MLTK
27,06
2,32
25,99
35,80
ZAP70
106,79
101,82
99,82
99,87
ZIPK/DAPK3
95,06
100,06
99,32
100,46
Toxicology studies for PF-114
Single dose toxicology studies for PF-114 in mice, rats and dogs
In a single dose toxicology study mice received PF-114 in a dose range between 250 mg/kg and 1450
mg/kg. 2/12 animals receiving 850 mg/kg died at day 4 and 8, respectively; 2/12 animals receiving
1000 mg/kg died at day 2 and 4, repsectively; 2/12 animals receiving 1150 mg/kg both died on day 2;
7/12 animals receiving 1450 mg/kg died between day 2 and 8. Based on these observations, LD 10 and
LD50 were determined at 800±100 and 1400±100 mg/kg, respectively. Clinical signs of toxicity
included dyspnea, decreased activity, rough hair coat, stomach bloating, focal alopecia.
In a single dose toxicology study rats received PF-114 in a dose range between 350 mg/kg and 3000
mg/kg. 2/5 animals receiving 3000 mg/kg died at days 4 and 10. Based on these findings, LD10 and
LD50 were determined at 2000 and 3400±700 mg/kg, respectively. In a single dose toxicology study
Beagle dogs received PF-114 in a dose range between 5 mg/kg and 135 mg/kg. No mortality was
observed. Clinical signs of toxicity included aqueous diarrhea and vomiting. Based on these
observations MTD was 45 mg/kg.
Repeated dose toxicity studies for PF-114 in rats and dogs
In a 28-day repeated dose toxicology study were performed in rats and Beagle dogs. Rats received PF114 in a range of doses starting from 9 mg/kg up to 73 mg/kg. No test item-related mortality was
documented in all study groups. Clinical signs of toxicity included piloerection, erythema, focal
alopecia. Basing on these observations the dose of 9 mg/kg of PF-114 per rat was considered as NOEL
(Supplementary table S3).
Beagle dogs received PF-114 in a dose range between 2 mg/kg and 22 mg/kg. No mortality was seen.
Clinical signs of toxicity included watery and mush feces and sporadic vomiting. Based on these
observations the dose of 7 mg/kg of PF-114 per dog was considered as NOEL (Supplementary table
S3).
Table S3. Repeated dose toxicity findings in 28-day studies of PF-114 in rats and dogs.
Species
N/sex/dos
e
Route
Duration
Doses,
mg/kg
9
Rat
N=5
26
Gavage
28-day
Rat
N=8
73
Significant findings
No clinical signs. All hematological and biochemical
parameters within normal ranges.
Only sporadic findings: piloerection and mild erythema in one
♀ for only 7-8 day of the study.
With the exception of one ♂ and one ♀ all animals presented
piloerection, mild erythema, and slight focal alopecia strating
from the day 6 of the study. These symptoms gradually
disappeared. In ♂s food consumption and body weight
decreased as compared to control animals.
2
7
Dog
N=2
Dog
N=4
Gavage
28-day
No clinical signs.
No clinical signs.
Sporadic vomiting in one ♂ and one ♀. Watery or mush feces
strating from day 9 of treatment in all animals but one ♀.
An increase of the mean LDH and ALT activities was
determined in both sexes during the administration period as
compared to starting levels (in males slightly above the
reference ranges).
The mean AST and CK serum activities were increased in ♂ (~
25% above reference range) and normal in ♀.
22
Table S4. Mortality in the 28-Day Rat study of ponatinib a
Dose, mg/kg
1,5
3
6
Mortality
1/46
6/46
11/46
a
U.S. Food and Drug Administration, Center for Drug Evaluation and Research. Iclusig NDA 203469 approval
letter, November 19, 2012. Retrieved March 14, 2013, from
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/203469Orig1s000PharmR.pdf. Page 35
Pharmacokinetics of PF-114 in comparison to ponatinib
Table S5. Pharmacokinetics of PF-114 in comparison to ponatinib (p.o. suspension in 0,5%
aqueous methyl cellulose)
species
Tmax, hr
Cmax,ng/ml
T1/2, hr
AUC0-24,
hr*ng/ml
a
PF-114
40 mg/kg
p.o.а
mice
2
764
2,1
6632
Ponatinib
25 mg/kg
p.o.а
mice
2
968
4,4
7485
PF-114
10 mg/kg
p.o.b
rat
3,7
240
3,8
4385
Ponatinib
3 mg/kg p.o.c
Ponatinib
6 mg/kg p.o.c
rat
8
72
NEe
1050
rat
8
140
NEe
2215
Ponatini
b
45 mgd
Human
4,8
77
22,0
1300
Study was performed by Shanghai Chempartner Co., Ltd.
Study was performed by PharmInnTech LLC
c
U.S. Food and Drug Administration, Center for Drug Evaluation and Research. Iclusig NDA 203469 approval
letter, November 19, 2012. Retrieved March 14, 2013, from
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/203469Orig1s000PharmR.pdf. Page 44.
d
Cortes, et al.;Ponatinib in refractory Philadelphia chromosome-positive leukemias;N Engl J
Med;2012;367;22;2075-88
e
Not estimated due to insufficient characterization of the terminal phase of the mean concentration-time curve
b
Supplementary Materials and Methods
Colony assay on primary murine hematopoietic stem and progenitor cells (HSPCs)
Sca1+/lin- HSPCs were isolated from 8- to 12-week-old female C57BL/6 N mice (Janvier, St.
Berthevin, France) after euthanization by CO2 asphyxiation. Bone marrow (BM) was
harvested from the femur and tibia by flushing the bones with a syringe and 26-gauge
needle. Sca1+ cells were purified by immunomagnetic beads using MACS cell separation
columns according to the manufacturer’s instructions (Miltenyi, Bergisch-Gladbach,
Germany). The cells were ‘‘lineage depleted’’ by labeling the cells with biotin-conjugated
lineage panel antibodies against B220, CD3e, Gr-1, Mac-1 and Ter-119 (BD/Pharmingen,
San Diego, CA). Labeled cells were removed using streptavidin loaded ‘‘MACS’’ cell
separation columns. The purified cells were pre-stimulated for 2 days in DMEM
supplemented with 10% FCS (Hyclone/Perbio Science, Bonn Germany), 1% L-Glutamine,
1% Penicillin/Streptomycin, mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL)
(Cell Concepts, Umkirch, Germany). The cells were plated at 5x103cells/mL in methyl-cellulose
supplemented with mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/ml). On day 10 after
plating, the number of colonies was determined.