Discovery and pharmacological characterization of the second generation of Btk inhibitors with improved target selectivity and enhanced in vivo efficacy Jiayin Zhang1, Dong Liu1, Ru Shen1, Yinfa Yan1, Liuqing Yang1, Minsheng Zhang1, Jun Feng2, BeiBei Fu2, Jerry Hu2, Biao Lu2, Hong Wan2, Lei Zhang2, Weikang Tao2, Lianshan Zhang2, and Jingsong Cao1 1Eternity Bioscience Inc. 2005 EastPark Blvd, Cranbury, NJ 08512, USA 2Shanghai Hengrui Pharmaceutical Co. LTD. 279 Wenjing Rd, Minhang Hi-tech Zone, Shanghai, China 200245
RESULTS Table 1. Potency of BTK inhibitors in kinase and cell-based assays IC50/GI50 (nM) BTKi
BTK Kinase
TMD-8 (Btk-dependent)
SU-DHL-1 (Btk-independent)
p-BTK (TMD-8)
EBI-1266
1.4
0.5
>5,000
0.9
EBI-1367
4.7
0.5
>5,000
0.7
EBI-cpdX
42.0
1.3
>5,000
0.04
Ibrutinib
3.3
0.6
>5,000
0.9
40%
***
36%
***
66%
Fig. 1. BTK signaling pathways • •
Targeting BTK with small molecule inhibitors has been clinically proved to be an effective treatment option for B-cell lymphomas. We have discovered multiple structurally distinct, pharmacologically potent and selective BTK inhibitors.
2000
200
20
2
0.2
0.02
(nM)
2000
200
20
2
0.2
0.02
##
36%
pERK
60% tERK
*** Vehicle
2000
200
20
2
0.2
0.02
(nM)
2000
200
20
2
92%
***
0
24
0
*** 0.2
0.02
(nM)
Compounds
***, P < 0.001 vs. Vehicle; ##, P < 0.01
tERK
Kinases
19% 43%
56%
*
72%
**
EBI1367
EBIcpdX
Ibrutinib
1.4
4.7
42.5
3.3
BTK
468 – R P I F I I T E Y M A N G C L L N Y L R E M R H R F Q T Q Q
294.6
0.3
7.9
0.2
BLK
306 - E P I Y I V T E Y M A R G C L L D F L K T D E G S R L S L P
22.5
1.7
2.9
2.5
BMK
483 – Y P I Y I V T E Y I S N G C L L N Y L R S H G K G L E P S Q
651
321
3345
96
ITK
429 - A P I C L V F E F M E H G C L S D Y L R T Q R G L F A A E T
42.1
4.2
11.0
2.9
TEC
436 - K P I Y I V T E F M E R G C L L N F L R Q R Q G H F S R D V
144.9
5.1
48.2
3.5
TXK
337 - K P L Y I V T E F M E N G C L L N Y L R E N K G K L R K E M
1643
48.9
3411
119.1
EGFR
784 - S T V Q L I T Q L M P F G C L L D Y V R E H K D N I G S Q Y
9191
226.1
7474
342.4
ERBB2
792 - S T V Q L V T Q LMPY G C L L D H V R E N R G R L G S Q D
187.4
1.1
195.7
1.4
ERBB4
790 – P T I Q L V T Q L M P HG C L L E Y V H E H K D N I G S Q L
>5000
60.2
9547
79
JAK3
896 - Q S L R L V M E Y LPS G C L R D F L Q R H R A R L D A S R
**
Fig. 5. BTK inhibitors had no effects on body weight gain
0 6
12 Time (h)
18
24
100 0 0
6
12 18 Time (h)
24
Tmax (h)
0.25 ± 0.0
Cmax (ng/ml)
266±133
AUCall (h*ng/ml)
279 ± 94
AUCinf (h*ng/ml)
290 ± 91
EBI-1266 0.25 ± 0.0 31.4 ± 9.5 134 ± 57 142 ± 53
EBI-1367 0.33 ± 0.14 1279 ± 214 6837 ± 2341 7969 ± 2264
EBI-cpdX 0.42 ± 0.14 914 ± 278 2290 ± 859
i.v.
1500
i.g.
1000 500 0 0.0
3.0 6.0 9.0 Time (h)
12.0
t1/2 (h)
EBI-cpdX
900
CL (ml/min/kg)
i.v. i.g.
600 300
Vz/F (ml/kg)
0 0
6
12 18 Time (h)
24
Bioavailability (%)
2.99 ± 0.83
2.32 ± 1.0
4.09 ± 1.25
i.v.-0.5 mg/kg
50
i.g.-2.0 mg/kg
25 0 0.0
1.0
4.62 ± 1.63
654 ± 276
74801 ± 2545
145903 ±
24.5
11 ± 3.1
3827 ± 1264
121515 14.7
51.9
4.0
EBI-1367
1000
i.v.-0.5 mg/kg
150
i.g.-2.0 mg/kg
100 50 0 0.0
0.5
1.0 1.5 Time (h)
2.0
39.2 ± 12.1 15421 ± 7458 58.1
i.g.-2.0 mg/kg
600
EBI-cpdX
500
i.v.-0.2 mg/kg
800
305 ± 88
2.0 3.0 Time (h)
EBI-1266
200
75
Table 4. The PK parameters of BTK inhibitors in dogs after an oral dose at 5 mg/kg (mean ± SD)
Parameters
Ibrutinib
EBI-1266
EBI-1367
EBI-cpdX
Tmax (h)
0.75±0.29
0.50 ± 0.35
0.88 ± 0.25
1.25 ± 0.50
Cmax (ng/ml)
8.01±4.11
6.46± 2.24
656 ± 165
315 ± 80
AUCall (h*ng/ml)
9.33±3.68
5.95 ± 2.36
1094 ± 181
1290 ± 238
t1/2 (h)
-
-
4.09 ± 1.25
1.78 ± 0.15
CL (ml/min/kg)
-
-
11 ± 3.1
26.3 ± 5.4
Vz/F (ml/kg)
-
-
3827 ± 1264
4010 ± 628
3.61
2.04
114
61.6
2304 ± 862
1200
EBI-1367
Ibrutinib Concentration (ng/ml)
100
i.g. i.v.
200
Fig.3. The time course of BTK inhibitor plasma concentration over 24 hrs following an i.v (0.5 mg/kg) or oral (2 mg/kg) administration in dogs
Concentration (ng/ml)
200
300
Ibrutinib
0
6
12 Time (h)
18
24
day3
day5
day7
EBI-1266
EBI-1367
EBI-cpdX
EBI-1367 20、60、200 mg/kg, p.o, Q.D
Death
None
None
None
Clinical Observation
Nothing significant to report
Nothing significant to report
Nothing significant to report
Body weight
Slight ↓ at 200 mg/kg
Slight ↓ at 200 mg/kg
Slight ↓ at 200 mg/kg
Food intake
Slight ↓ at 200 mg/kg
Slight ↓ at 200 mg/kg
Normal
Hematology
Slight ↓ in RET at 200 mg/kg
Slight ↓ HDW, RDW-CV, and CHCM at 200 mg/kg
Slight ↑ in GRAN and WBCP at 200 mg/kg
Blood Biochemical
Slight ↓ in CK at 200 mg/kg
ALT and ALB slight alteration at 200 mg/kg
Slight alteration in ALP and BUN at 200 mg/kg
Urine Biochemical
Normal
Normal
Normal
Pathology
Normal
Enlarged spleen and abnormal thymus at 200 mg/kg
Normal
Organ weight
Normal
Normal
Normal
Conclusion
NOAEL - 60 mg/kg，MTD＞200 mg/kg
NOAEL - 60 mg/kg，MTD＞200 mg/kg
NOAEL - 60 mg/kg，MTD＞200 mg/kg
• EBI-1367 has a remarkable improvement in in vivo exposure in multiple preclinical species, translating to a superior in vivo efficacy • EBI-1266 has a cleaner kinase selectivity profile • EBI-cpdX showed improvement in both in vivo exposure and kinase selectivity
Concentration (ng/ml)
i.g. i.g. i.v. i.v.
Table 3. The PK parameters of BTK inhibitors in rats after an oral dose at 5 mg/kg (mean ± SD)
Parameters
0
• As compared with the first-in-class BTK inhibitor Ibrutinib, each of the three compounds shows unique and favorable pharmacological features –
100
EBI-1266
24
• We have discovered multiple series of novel BTK inhibitors (represented by EBI-1266, EBI-1367 and EBI-cpdX) with potent in vitro and in vivo anti-tumor efficacies in BTK-driven models
*, P < 0.05, **, P< 0.01 vs. Vehicle.
Concentration (ng/ml)
300
0
Concentration (ng/ml)
400
PCI-32765 Ibrutinib
18
CONCLUSIONS
PK Analysis of BTK inhibitors in rats and dogs (marked improvement in in vivo exposure of EBI-1367 and EBI-cpdX)
400
12 Time (h)
Model 1367-10mg/kg
66%
Sequence Alignment
EBI1266
Fig.2. The time course of BTK inhibitor plasma concentration over 24 hrs following an i.v (0.5 mg/kg) or oral (5 mg/kg) Administration in rats
6
500
Table 5. Summary of safety evaluation of BTK inhibitors in two-week toxicity studies in rats
tBTK
BTK and ERK phosphorylation was measured by standard western blot analysis. Xenograft model: Antitumor activity of BTK inhibitors were evaluated in NOD-SCID mice bearing TMD8 cells. Tumor-bearing mice were orally administrated with BTK inhibitors for 18 days. Tumor volume and body weight were measured twice a week.
2000
Dose regimen
2000
•
4000
1000
71%
pBTK (Y223)
Concentration (ng/ml)
•
Proliferation assay: CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega).
18
day1
***
EBI-cpdX
EBI-1367 Vehicle
12 Time (h)
10 9 8 7 6 5 4 3 2 1 0
tBTK
Concentration (ng/ml)
•
Enzymatic assay : LanthaScreen™ TR-FRET (Invitrogen) was used to detect BTK kinase activities.
6
(nM)
pBTK (Y223)
Concentration (ng/ml)
•
Compounds – all testing compounds were synthesized by Eternity Bioscience or Shanghai Hengrui Pharmaceutical Co. LTD
0
EBI-1266 Vehicle
MATERIALS AND METHODS •
200
6000
EBI-1459 (3mg/kg) EBI-1459 (10mg/kg) EBI-1459 (30mg/kg) EBI-1459 (60mg/kg)
Toe clinical score
Ibrutinib Vehicle
BTK inhibitors
B cell activation and proliferation Malignant B-cell survival, growth, and division
400
***, P<0.001 vs. Vehicle
IC50 (nM)
MAPK
EBI-1266(30 mg/kg)
8000
1500
Fig. 7. BTK inhibition by EBI-1367 significantly improved the arthritic score in CIA model
Table 2. Selectivity profiles of BTK inhibitors in human kinases containing a Cystein in catalytic domain (Cleaner selectivity of EBI1266 and EBI-cpdX)
PI3K
EBI-1266(10 mg/kg)
600
EBI-1367(3 mg/kg) EBI-1367(10 mg/kg) EBI-1367(30 mg/kg)
Note: Cmax for Ibrutinib at 10 mg/kg was 19.5 ng/ml.
Estimated EC50 is 10 mg/kg
• BTK is an essential component of the B-cell receptor (BCR) signaling pathways regulating survival, activation, proliferation, and differentiation of B lymphocytes. (Fig. 1)
NF-kB
800
***
pERK
Lyn
EBI-1266(3 mg/kg)
Fig. 1. Suppression of BTK and ERK Phosphorylation by BTK inhibitors in TMD-8 cells
BACKGROUND
BTK
10000
1000
0
Estimated EC50 less than 3 mg/kg
Syk
Fig. 6. BTK inhibitors had a dose-dependent in vivo exposure in the TMD8 xenograft mice model Concentration (ng/ml)
Fig. 4. Antitumor activity of BTK inhibitors in the TMD8 xenograft mice model
Concentration (ng/ml)
Bruton’s tyrosine kinase (BTK) is an essential component of the B-cell receptor (BCR) signaling pathways regulating survival, activation, proliferation, and differentiation of B lymphocytes. The first Btk inhibitor, Ibrutinib, has demonstrated a significant clinical efficacy in a variety of B-cell malignancies. We have discovered multiple series of novel and potent BTK inhibitors, represented by EBI-1266, EBI-1367, and EBI-cpdX, respectively. All three compounds showed high potency in inhibiting BTK kinase activity and growth of an aggressive lymphoma cell line driven by aberrant BCR signaling through BTK, with IC50 value within low nanomolar range. Mechanistic studies examining BCR signaling pathway in related B cell lymphoma cell lines revealed that all three compounds potently inhibited BTK phosphorylation and downstream Erk phosphorylation. Furthermore, each of these three compounds showed unique and favorable pharmacological properties when compared with Ibrutinib: EBI-1266 had a cleaner selectivity profile against a panel of kinases with a cysteine residue in the conserved catalytic domain; EBI-1367 exhibited >10-fold higher in vivo exposure in multiple preclinical species; and EBI-cpdX showed significant improvement in both selectivity and in vivo exposure. In a tumor xenograft mouse model where tumor growth is dependent on Btk activity, all three compounds showed significant oral anti-tumor activities, as evidenced by dose-dependent inhibition of tumor growth. In addition, we have observed a good correlation between anti-tumor activity and compound exposure level in circulation and tumor tissues. Our data also showed that BTK inhibition by EBI1367 led to a marked inhibition of collagen-induced arthritis in mice. No evidence of overt toxicities was observed in rodents with prolonged oral administration for two weeks with doses at least 3-fold above a highly efficacious dose. In summary, we have discovered multiple series of novel BTK inhibitors with high potency and significant improvement in target selectivity and in vivo exposure. These results suggest that our compounds have a great potential to become the next generation of BTK inhibitors, offering advantages in pharmaceutical development, enhanced in vivo efficacy and reduced toxicities. We are currently performing IND-enabling studies aiming to initiate phase I clinical trials for these BTK inhibitors.
Concentration (ng/ml)
SUMMARY
400
i.v.-0.5 mg/kg
300
i.g.-2.0 mg/kg
200
400
Bioavailability (%)
100
200 0 0.0
2.0
4.0 6.0 Time (h)
8.0
0 0.0
3.0
6.0 9.0 Time (h)
12.0
• The initial 14-day toxicity studies in rats revealed excellent therapeutic margin for all three compounds • These findings present our BTK inhibitors as meaningfully differentiated molecular entities with best-in-class potential • IND-enabling studies ongoing, looking for collaborators for clinical development in ex-China markets
