SUPPLEMENTAL MATERIAL
METHODS
Body Weight Changes after Drug Treatment The body weight of individual animals was measured twice weekly until the study end. The figures were plotted as percentage change as compared to the first day of treatment.
Hematological Toxicity Analysis Whole blood was collected three days after treatment by cardiac puncture. Hematological analysis was performed using an automatic hematological analyzer (HEMAVET 950, Drew Scientific Corp. Dallas, TX). Hematological parameters measured included total count of white blood cells (WBC) and differential count of lymphocytes (LY), neutrophils (NE) and monocytes (MO). Statistical significance between the treated groups monotherapy versus control group or versus combotherapy was evaluated by Dunnett's Multiple Comparison Test, one-way ANOVA using Prism GraphPad software.
RESULTS
Effect of TH-302 in combination with doxorubicin or gemcitabine on hematologic changes.
We examined whether the addition of TH-302 will increase the reduction of white counts caused by MTD dose of gemcitabine and doxorubicin in different combination schedules (Supplemental Figure 1). The results showed that TH-302 monotherapy reduced the counts of WBC, NE, LY and MO by 29%, 45%, 23% and 19%, whereas doxorubicin monotherapy reduced them by 47%, 59%, 41% and 42%, respectively (Supplemental Figure 1 A-D). The TH-302 and doxorubicin combination groups did not show more inhibition on WBC, LY and MO counts when compared to the effects of doxorubicin alone. However, some additional suppression was observed on NE counts in the combination therapy group compared to doxorubicin alone. TH-302 that was administered 0hr, 2hr, 4hr and 8hr apart from doxorubicin inhibited NE by 76%, 69%, 67% and 68% compared to 59% inhibition induced by doxorubicin alone. The simultaneous dosing showed lowest counts as compared with other time intervals, though the difference didn’t reach statistical significance.
The separate study showed that TH-302 monotherapy reduced the counts of WBC, NE, LY and MO by 25%, 23%, 25% and 28%, whereas gemcitabine monotherapy reduced them by 37%, 47%, 31% and 56% respectively (Supplemental Figure 1 E-H). The combination groups of all the time intervals showed more inhibition on the cell counts than gemcitabine alone. On average they reduced the counts of WBC, NE, LY and MO by 51%, 65%, 41% and 65%. Same as the other study of TH-302 in combination with doxorubicin, the effect of TH-302 in combination with gemcitabine on neutrophils are more dramatic than their effects on other cell counts. This observation is consistent with clinic investigation where neutropenia is the dose limiting toxicity.
In summary, TH-302’s toxicity on hematology is mild. It added 10% and 18% more inhibition on neutrophils when combined with doxorubicin and gemcitabine respectively. It added no or 10% more inhibition on WBC, LY and MO cell counts when combined with doxorubicin and gemcitabine respectively.
Supplemental Figure 1. Effect of TH-302 alone and in combination with gemcitabine (Gem) or doxorubicin (Dox) on hematologic changes in the CD1 mice. The time in the legends indicate the delay interval between TH-302 and chemotherapeutics administration. TH-302 was administered prior to the chemotherapeutics. The blood was collected three days after drug treatment. The means and standard errors from 6 mice per group are presented. (A-D) The effect of TH-302 and doxorubicin on white blood cell, neutrophil, lymphocyte and monocyte counts. TH-302 was given at 100mg/kg, ip, doxorubin was given at 6mg/kg iv. (E-H) The effect of TH-302 and gemcitabine on white blood cell, neutrophil, lymphocyte and monocyte counts. TH-302 was given at 75mg/kg, ip, gemcitabine was given at 300mg/kg ip. *, P < 0.05 or **, P < 0.01 as compared to Vehicle; #, P < 0.05, ##, P < 0.01 as compared to doxorubicin or gemcitabine alone.
Supplemental Figure 2. Model for complementary cytotoxic activity of companion chemotherapy and TH-302 selectively targeting two compartments (normoxic and hypoxic) in the tumor microenvironment [2].
Reference
Name / Dose (mg/kg) / Regimen and administration route
H460 / NSCLC, large-cell carcinoma / 4.7 / Docetaxel / 10 / Q7Dx2, iv / [9]
Pemetrexed / 100 / QDx3/wk x 2wks, ip / [4]
Cisplatin / 6 / Q7Dx2, iv / [1]
Irinotecan / 20 / QDx5/wk x 2wks, ip / [3]
Calu-6 / NSCLC, large-cell carcinoma / 11.0 / Doxorubicin / 4 / Q7Dx2, iv / [5]
HT29 / colon adenocarcinoma (primary tumor) / 7.7 / Cisplatin / 6 / Q7Dx2, iv / [1]
PC3 / prostate adenocarcinoma (bone metastasis ) / 8.9 / Docetaxel / 10 / Q7Dx2, iv / [9]
HT1080 / fibrosarcoma / 5.2 / Doxorubicin / 4 / Q7Dx2, iv / [5]
Stew2 / melanoma / 13.8 / Temozolomide / 25 / QDx5/wk x 2wks, ip / [7]
A375 / melanoma / 7.3 / Temozolomide / 50 / QDx5/wk x 2wks, po / [7]
Hs766t / pancreatic adenocarcinoma (lymph node metastasis) / 4.5 / Gemcitabine / 60 / Q3Dx5, ip / [8]
SU.86.86 / pancreatic adenocarcinoma (liver metastasis) / 7.5 / Gemcitabine / 60 / Q3Dx5, ip / [6]
MIA PaCa-2 / pancreatic adenocarcinoma / 7.3 / Gemcitabine / 60 / Q3Dx5, ip / [6]
BxPC-3 / pancreatic adenocarcinoma / 7.7 / Gemcitabine / 60 / Q3Dx5, ip / [8]
Supplemental Table 1. Characteristics of human tumor xenografts and regimens for dosing of companion chemotherapy compounds.
Supplemental Table 2. Median and range of days of individual animals to reach a tumor size of 500mm3.
Tumor Model / Chemo-therapeutic Drugs / Monotherapy of Chemotherapeutic Drugs / Monotherapy of TH302 / DSg / Timeh (hr) / Combination Therapy / Vehicle Median Days to 500mm3 (Range)Median Days to 500mm3 (Range) / Median Days to 500mm3 (Range) / Median Days to 500mm3 (Range)
Supplemental Data of
Table 1 / H460 NSCLC / Cisplatina / TC / 2 / 31 (11-32+) / 14.5 (9-32+)
TC / 0 / 31.5 (22-32+)
CT / 2 / 31.5 (24-32+)
Docetaxele,f / 17.5 (12-40+) / 23.5 (16-40+) / TC / 48 / 24.5 (14-40+) / 12.5 (10-19)
TC / 24 / 26 (18-40+)
TC / 4 / 24.5 (20-40+)
TC / 1 / 24.5 (18-40+)
CT / 4 / 28 (18-35)
CT / 24 / 29 (12-40+)
CT / 48 / 20 (16-40+)
Gemcitabineb / 31 (24-38) / 21 (13-44) / TC / 24 / 19 (20-46) / 14 (8-30)
TC / 8 / 22 (18-45)
TC / 4 / 21 (18-46)
TC / 2 / 17 (24-38)
TC / 0 / 19 (20-46)
HT1080 Fibrosarcoma / Doxorubicinc / 27 (20-78+) / 25 (13-30) / TC / 24 / 27 (18-78+) / 17 (6-19)
TC / 8 / 35 (18-45)
TC / 4 / 36 (26-60)
TC / 2 / 33 (29-78+)
CT / 0 / 40 (22-78+)
CT / 2 / 37 (18-63)
PC3 Prostate Carcinoma / Docetaxele,f / 36 (22-36+) / 28 (17-28+) / TC / 24 / 36 (24-36+) / 17 (9-28)
TC / 4 / 42 (28-42+)
TC / 2 / 36 (22-36+)
TC / 0 / 12 (7-36+)
CT / 2 / 31 (7-36+)
CT / 4 / 38 (26-39+)
CT / 24 / 34.5 (4-36+)
Supplemental Data of
Figure 1E / H460 NSCLC / Docetaxel / 23 (11-29+) / 33 (26-53+) / 34 (30-45) / 14 (10-24)
30.5 (26-39) / 44 (15-53+)
32.5 (24-39+) / 30 (16-39+)
Supplemental Data of
Table 2 / H460 NSCLC / docetaxela / 24 (10-37) / 24.5 (20-51) / 36.5 (22-56) / 11.5 (9-19)
pemetrexeda / 12.5 (5-29) / 19.5 (6-42) / 29.5 (9-42) / 10 (4-17)
cisplatina / 23.5 (16-47+) / 23 (16-35) / 40 (14-47+) / 15 (12-47+)
irinotecana / 29 (14-67+) / 26 (18-38) / 39 (26-52) / 14 (5-26)
Calu6 NSCLC / doxorubicina / 26 (22-38) / 25 (8-36+) / 33 (24-46+) / 18 (10-27)
HT29 Colon Carcinoma / cisplatinb / 23.5 (16-47+) / 23 (16-35) / 40 (14-47+) / 15 (12-47+)
HT1080 Fibrosarcoma / doxorubicina / 29 (20-29+) / 18.5 (15-29+) / 34.5 (20-53+) / 14 (8-20)
doxorubicinc / 27 (20-78+) / 25 (13-30) / 36 (26-60) / 17 (6-19)
PC3 Prostate Carcinoma / docetaxela / 60 (37-60+) / 31 (21-60+) / 59.5 (45-60+) / 17.5 (12-32)
MiaPaCa-2 Pancreatic Carcinoma / gemcitabined / 44 (27-52) / 38 (28-45) / 62 (43-73) / 35 (28-40)
Hs766t Pancreatic Carcinoma / gemcitabined / 7 (4-24) / 42.5 (5-100) / 58.5 (6-70) / 6.5 (3-13)
SU.86.86 Pancreatic Carcinoma / gemcitabined / 73.5 (15-120+) / 12 (5-56) / 62.5 (45-120+) / 12 (5-23)
BxPC-3 Pancreatic Carcinoma / gemcitabined / 7 (3-14) / 6 (4-16) / 11.5 (4-23) / 6 (2-10)
Stew2 Melanoma / temozolomidea / 29 (20-29+) / 18.5 (15-29+) / 34.5 (20-53+) / 14 (8-20)
A375 Melanoma / temozolomidea / 16 (8-60+) / 17 (10-36) / 20 (10-31) / 15 (2-60+)
a TH-302 was administered at 50 mg/kg ip QDx5 per week for two weeks
b TH-302 was administered at 100 mg/kg ip Q3Dx5
c TH-302 was administered at 100 mg/kg ip Q7Dx2
d TH-302 was administered at 75 mg/kg ip Q3Dx5
e Docetaxel was administered at 10mg/kg iv once
f TH-302 was administered at 150mg/kg ip once
g DS is abbreviation for dosing sequence
h time refers to time interval between chemotherapeutic drugs and TH-302
Supplemental Table 3. Comparison of Docetaxel efficacy and toxicity at MTD and half-MTD dose on H460 xenograft model.
(%) / TGD500
(d) / MBL
(%)
Vehicle / 0.1
Docetaxel 10mg/kg, Q7Dx3, iv / 58 / 8 / 4.1
Docetaxel 20mg/kg, Q7Dx3, iv / 58 / 8 / 8.4
TH-302 50mg/kg, QDx5/wkx2wks, ip / 79 / 15 / 3.7
TH-302 + Docetaxel (10mg/kg) / 91 / 24 / 6
TH-302 + Docetaxel (20mg/kg) / 95 / 28 / 11.6
No significant difference was observed between TGIs of docetaxel 10mg/kg and 20mg/kg in combination with TH-302 groups (P=0.45)
Supplemental Figure 3. Body weight changes of TH-302 alone and in combination with different chemotherapeutic drugs in the ectopic H460 human lung carcinoma xenograft model. TH-302 was administered at 50mg/kg ip, QDx5 per week for two weeks. (A) Body weight changes of TH-302 and docetaxel alone and in combination. (B) Body weight changes of TH-302 and pemetrexed alone and in combination. (C) Body weight changes of TH-302 and cisplatin alone and in combination. (D) Body weight changes of TH-302 and irinotecan alone and in combination.
Supplemental Figure 4. Body weight changes of TH-302 in combination with different chemotherapeutic drugs on various ectopic xenograft models. (A) Body weight changes of TH-302 alone and in combination with cisplatin on HT29 colon carcinoma xenograft. TH-302 was administered ip at 100mg/kg Q3Dx5. Cisplatin was administered iv at 6mg/kg Q7Dx2. (B) Body weight changes of TH-302 alone and in combination with doxorubicin on HT1080 fibrosarcoma xenograft. TH-302 was administered ip at 100mg/kg Q7Dx2. Doxorubicin was administered iv at 4mg/kg Q7Dx2. (C) Body weight changes of TH-302 alone and in combination with doxorubicin on Calu6 NSCLC xenograft. TH-302 was administered ip at 50mg/kg daily for five days per week for two week. Doxorubicin was administered iv at 4mg/kg Q7Dx2. (D) Body weight changes of TH-302 alone and in combination with docetaxel efficacy on PC3 prostate cancer xenograft. TH-302 was administered ip at 50mg/kg daily for five days per week for two week. Docetaxel was administered iv at 10mg/kg Q7Dx2. (E-H) Body weight changes of TH-302 alone and in combination with gemcitabine on Hs766t, SU.86.86, MIA PaCa-2 and BxPC-3 pancreatic xenografts. TH-302 and gemcitabine was administered ip at 75mg/kg and 60mg/kg Q3Dx5 respectively. (I-J) Body weight changes of TH-302 alone and in combination with temozolomide on Stew2 and A375 melanoma xenograft. TH-302 was administered ip at 50mg/kg daily for five days per week for two weeks. Temozolomide was administered ip at 25mg/kg and po at 50mg/kg daily for five days per week for two weeks on Stew2 and A375 melanoma xenografts respectively.
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