Supplemental Material
HPLC methods for Cytarabine and Daunorubicin
For cytarabine analysis, 10 µL of plasma samples were mixed with 50 µL of
methanol by vortexing, and added to 440 µL of HPLC grade water. The mixture was
vortexed and centrifuged at 3200g for 10 minutes and 20 μL of supernatant was injected
onto the HPLC for quantitation. For daunorubicin analysis, 10 µL of plasma samples
were mixed with 490 µL of acidified methanol (acetic acid:methanol = 1:5, v/v) by
vortexing. The mixture was centrifuged at 3200g for 10 minutes and 20 μL of supernatant
was injected onto the HPLC for quantitation.
Cytarabine was quantified using a
Phenomenex Luna C18(2) reverse phase analytical column with a UV detector set to
273.7 nm.
The mobile phase was 1 mL/min 25 mM ammonium acetate (pH 4.8).
Daunorubicin was evaluated using a Phenomenex Luna C18(2) reverse phase analytical
column with a multi  fluorescence detector set at excitation/emission wavelengths of
480/560 nm. The mobile phase was 1 mL/min 25 mM ammonium acetate:acetonitrile
(67.5:32.5, v/v at pH 4.8). For free drug analysis, cytarabine and daunorubicin were
administered at the free cocktail MTD of 600 mg/kg and 9 mg/kg respectively. Samples
were injected i.v. and blood was collected at 5, 15 and 30 minutes, 1 hour, 2 hour and 8
hours and plasma isolated as outlined previously. Free cytarabine was stabilised by
addition of tetrahydrouridine then samples were frozen at -20ºC until analysis. The HPLC
method used in this manuscript was suitable for separating daunorubcin from the active
metabolite daunorubicinol. This metabolite was detected at very low levels in the plasma
of mice treated with free drug cocktail but was absent in CPX-351 treated mice. At 2
hours after intravenous administration of the free drug cocktail, neither daunorubicin or
its metabolite were detectable.
Estimation of bone marrow volume
Femurs were obtained from age-matched CD-1 nude mice. The femurs were
fixed in formalin, decalcified using hydrochloric acid/formic acid, processed for
histology and stained with hematoxylin and eosin (WaxIt Inc., Vancouver, BC). Major
and minor dimensions from internal cross sections were determined using an inverted
microscope fitted with a stage micrometer. The average cross sectional area (A) was
determined using the formula for an ellipse where A = π (a) (b) in which a = ½ major
dimension and b = ½ minor dimension. The average femur length post bone marrow
collection was determined using digitally enlarged photographs. The area multiplied by
the length was used to calculate the volume. The average area of the ovoid bone marrow
cross sections was 0.76 mm2 and the average length of the femurs post bone marrow
collection was 11.7 mm resulting in an average volume of 8.9 L/femur.
Analysis of CPX-351 Therapeutic Activity for In Vivo Synergy
In order to elucidate the degree to which CPX-351 increased the therapeutic
activity of cytarabine:daunorubicin treatment, and assess the contribution of the two
agents to the overall therapeutic effect of CPX-351, we utilized a more stringent format
of the P388 leukemia tumor model whereby initiation of the Q3Dx3 i.v. treatment was
delayed until day 4 post tumor inoculation rather than on day 1 which is typically
employed for this model. Given the rapid doubling time of P388 tumor cells (16 hours),
this delay increased the tumor burden by approximately 50-fold at the time of treatment.
When
CPX-351
was
administered
at
10:4
mg/kg
(0.8
MTD)
cytarabine:daunorubicin in the high stringency P388 model, a median survival time of 38
days (increase in life span, ILS, of 443%) was obtained, with a long-term (day 55)
survival rate of 17% (1/6 mice, Figure 4A). In comparison, matched doses of liposomal
cytarabine (10 mg/kg) and liposomal daunorubicin (4 mg/kg) administered individually
provided significantly reduced antitumor activity as reflected by ILS values of 186% and
43%, respectively. The degree of antitumor activity obtained after the final treatment was
estimated using the following calculation: Log cell kill (LCK) = [T-C]/3.32(Td) where TC is the difference in median survival time between treatment and control groups and Td
is the tumor doubling time in days, which for P388 is 0.66 [1]. Liposomal cytarabine (10
mg/kg) and liposomal daunorubicin (4 mg/kg) each individually provided LCK values of
5.9 and 1.4, respectively.
In comparison, CPX-351 dosed at 10:4.4 mg/kg
cytarabine:daunorubicin provided a LCK value of > 10, the theoretical maximum value
obtainable.
If the enhanced antitumor activity observed with CPX-351 over the
individual liposomal drugs was additive, a LCK value for CPX-351 of approximately 7.3
would be predicted as the sum of the individual values. The fact that the LCK for CXP351 was approximately three logs higher than that predicted for additivity is consistent
with strong in vivo synergy.
Since the log cell kill estimations described above may be complicated by the
multiple day treatment regimen used here, we also compared the antitumor activity of
CPX-351 with individual liposomal drugs based on the number of tumor cells remaining
after final treatment. This could be determined by correlating treatment-induced survival
extension with the median survival times for P388 tumor cell inoculums titrated between
10 cells and 108 cells in the absence of treatment [2]. For individual liposomal cytarabine
(10 mg/kg) and liposomal daunorubicin (4 mg/kg) treatment, the residual tumor burden
post treatment was estimated to be 1.6 x 104 and 5.0 x 108 tumor cells, respectively. In
comparison, the median survival time of 38 days for CPX-351 is beyond that predicted
without curing mice and consequently the average residual tumor burden after final
treatment was estimated to be between 1-10 cells per mouse which is consistent with the
occurrence of 1/6 long term survivors in this treatment group (Figure 4A). This analysis
also revealed approximately a three log increase in antitumor activity for CPX-351 over
that predicted for additive activity of the individual encapsulated cytarabine and
daunorubicin components.
Weight Loss Data for Efficacy Studies
All studies were conducted within MTD doses as defined survival in the absence of
significant tumor burden with < 15% body weight loss nadir lasting < 2 days. Druginduced weight loss associated with MTD doses was sometimes obscured by the
progressive increase in ascitic fluid, as occurred in the P388, L1210, WEHI models; prior
MTD studies were relied upon for accurate equitoxic dosing of the efficacy studies.
Drug-related weight loss nadirs generally occurred between 1 and 3 days post final
injection except in the L1210 tumor model where the nadir weight loss occurred 6 days
post final injection for the high-dose liposomal daunorubicin. Weight loss occurring at
later times was noted in the CCRF-CEM and the HL-60 tumor models and was associated
with localized and/or disseminated tumor burden that resulted in mortality.
Figure 3 Weight loss data
20
% Weight loss
10
0
-10
-20
0
10
20
30
40
50
60
70
Day of Experiment
Saline Control
Cytarabine:Daunorubicin 15:3 mg/kg (12:1)
Cytarabine:Daunorubicin 10:4 mg/kg (5:1)
Cytarabine:Daunorubicin 7.7:10 mg/kg (3:1)
Cytarabine:Daunorubicin 12.5:5.4 mg/kg (1:1)
Figure 4A Weight loss data
Figure 4B Weight loss data
30
20
20
% Weight loss
% Weight loss
10
0
-10
10
0
-10
-20
-20
0
10
20
30
40
Day of Experiment
Saline Control
CPX-351 (10:4.4 mg/kg)
Liposomal Cytarabine (10 mg/kg)
Liposomal Daunorubicin (4.4 mg/kg)
50
0
10
20
30
40
Day of Experiment
Saline Control
CPX-351 (12.5:5 mg/kg)
Liposomal Daunorubicin (10 mg/kg)
Liposomal Cytarabine (15 mg/kg)
Free Drug Cocktail (30:12 mg/kg)
Free Drug Cocktail (600:9 mg/kg)
50
Figure 5B Weight loss data
20
20
10
% Weight loss
30
10
0
0
-10
-10
-20
-20
-30
0
10
20
30
40
50
60
70
80
90
20
30
40
Day of Experiment
50
60
70
Day of Experiment
Saline Control
CPX-351 (12:5.3 mg/kg)
Ratio Matched Cocktail (12:5.3 mg/kg)
Dose Pushed Cocktail (300:4.5 mg/kg)
Saline Control
CPX-351 (10:4.4 mg/kg)
CPX-351 (6.3:2.5 mg/kg)
Dose Pushed Cocktail (300:4.5 mg/kg)
Table 1 Weight loss data
10
0
% Weight loss
% Weight loss
Figure 5A Weight loss data
-10
-20
10
20
30
40
Day of Experiment
Saline Control
CPX-351 (5:2 mg/kg)
CPX-351 (2.5:1 mg/kg)
Dose Pushed Cocktail (200:3 mg/kg)
Ratio Matched Cocktail (10:4 mg/kg)
References
1) Corbett T, Polin L, LoRusso P, Valeriote F, Panchapor C, Pugh S et al. In vivo
methods for screening and preclinical testing: Use of rodent solid tumors for drug
discovery. In: Teicher BA, editor. Anticancer drug development guide: preclinical
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screening, clinical trials, and approval. Totowa, New Jersey: Humana Press; 1997. p.
99-124.
2) Waud WR. Murine L1210 and P388 leukemias. In: Teicher BA, editor. Anticancer
Drug Development Guide. New Jersey: Humana Press;1997.p.59-74.