Anti proliferative activity of ELACYT™ (CP-4055) in combination with cloretazine (VNP40101M), idarubicin, gemcitabine, irinotecan and topotecan in human leukemia and lymphoma cells

Figures & data

Figure 1. Structures of drugs under study.

Figure 2. Composite dose-response curves in human leukemia cells. Data from replicate dose-response experiments for HL-60 (open circles) and U937 (filled circles) leukemia cell lines were combined and the mean values plotted for the various drugs under study. Line: data fit to the Hill Equation (a form of the median-effect equation); bar: intra-assay standard error.

Table I.  Potency of CP-4055 and combination partners in human leukemia cells

Drug	HL-60		U937
	IC50 (nM)	N	IC50 (nM)	N
CP-4055	13,387 ± 1428	24	710 ± 78	14
Cloretazine	136,150 ± 11,146	7	59,996 ± 13,852	7
Idarubicin	12.1 ± 0.6	8	30 ± 5	7
Gemcitabine	9.4 ± 0.7	18	23 ± 2	10
Irinotecan	4742 ± 253	8	3823 ± 518	7
Topotecan	20.2 ± 1.0	7	34 ± 2	7
Cytarabine	2989 ± 322	5	167 ± 29	4

Figure 3. Combination index plots of Chou-Talalay and impact of drug sequence in HL-60 cells. Composite dose-response curves were constructed for drugs alone and in combination as described in Methods. The respective fraction affected (F_a) was interpolated at 10 percent Intervals and the values used to compute the combination index for simultaneous administration of CP-4055 (open circles), for CP-4055 given 24 h prior to (open triangles) or 24 h after (open squares) the second drug in the combination listed in each panel. For cytarabine, only simultaneous combinations are shown combined with CP-4055 (closed circles), and with gemcitabine (closed triangles).

Figure 4. Combination index plots of Chou-Talalay and impact of drug sequence in U937 cells. Drug interaction was assessed by the combination index method as described in Figure 3 in U937 cells for the drug combination partners listed in each panel.

Table II.  Combination index at the IC50 in human leukemia cells

	Molar ratio	N	CI
HL-60 Combination
Cytarabine + Gemcitabine	1:0.01	3	0.39 ± 0.01
CP-4055 + Gemcitabine	1:0.0025	11	0.41 ± 0.01
CP-4055 + Topotecan	1:0.00125	6	0.78 ± 0.01
CP-4055 + Irinotecan	1:0.125	6	0.81 ± 0.07
CP-4055 + Idarubicin	1:0.0025	6	0.88 ± 0.09
CP-4055 + Cloretazine	1:10	6	0.91 ± 0.08
Cloretazine + Gemcitabine	1:0.00025	3	1.10 ± 0.11
Cloretazine + Idarubicin	1:0.00025	3	1.20 ± 0.03
Idarubicin + Gemcitabine	1:1	3	1.51 ± 0.04
CP-4055 + Cloretazine  + Gemcitabine	1:10:0.001	2	0.36 ± 0.07
CP-4055 + Idarubicin + Gemcitabine	1:0.001:0.001	2	0.30 ± 0.17
CP-4055 + Cloretazine  + Idarubicin	1:10:0.001	2	0.92 ± 0.15
U937 combination
CP-4055 + Gemcitabine	1:0.02	8	0.82 ± 0.04
CP-4055 + Cloretazine	1:40	6	0.94 ± 0.10
Cytarabine + Gemcitabine	1:0.2	3	1.00 ± 0.05
CP-4055 + Irinotecan	1:2	4	1.11 ± 0.02
CP-4055 + Topotecan	1:0.02	4	1.11 ± 0.06
CP-4055 + Idarubicin	1:0.01	6	1.16 ± 0.11

Table III.  Dose reduction index for CP-4055 combined with gemcitabine in HL-60 cells

Fraction affected	Dose reduction index^1
	CP-4055	Gemcitabine
0.1	2.2	1.4
0.2	3.3	1.6
0.3	4.9	1.9
0.4	6.9	2.4
0.5	9.8	3.0
0.6	14.2	3.8
0.7	21.5	4.7
0.8	35.7	5.8
0.9	71.8	6.6

¹Combined in a molar ratio of 400:1 (CP-4055:Gemcitabine).

Figure 5. Effect of ratio on drug interaction in HL-60 cells. 3-Dimensional drug interaction analysis was performed for binary combinations of CP-4055 in HL-60 cells as described in Methods. The combination effect surface is presented as a color-coded contour map where green represents additivity, red antagonism and blue synergy. For clarity, the color spectrum has been constrained to the range −6 to +6. Values beyond those extremes were observed, but not discriminated (i.e. the blue corresponding to CE = 6 actually represents CE ≥ 6).

Figure 6. Effect of ratio on drug interaction in U937 cells. 3-Dimensional analysis was performed as in Figure 5 for the same drug combinations but in the U937 cell line.

Figure 7. Effect of exposure time on the interaction of CP-4055 plus gemcitabine. Panel A: IC50 versus exposure time in HL-60 cells Is shown for CP-4055 (diamonds) and gemcitablne (triangles) as single agents. Panel B: HL-60 (circles) or U937 (squares) cells were exposed simultaneously to CP-4055 in combination with gemcitabine at a fixed ratio of 1:0.0025 or 1:0.02, respectively. The combination index at the IC50 level of effect was then determined at each indicated time point. Bars: intra-assay standard error; lines: fit of data to the pharmacodynamic model Cⁿ × T = k.

Table IV.  Pharmacodynamic model parameters for the simultaneous combination of CP-4055 plus gemcitabine.*

Drug	HL-60			U937
	n	Log(k)	R^2	n	Log(k)	R^2
CP-4055	1.78	9.11	0.86	1.09	4.47	0.84
Gemcitabine	1.23	1.59	0.99	1.06	2.89	0.87

*Based on log(T) = n log(C) + log(k), where C is the IC50 concentration, n is the concentration coefficient, k is the exposure constant and R² is the Pearson linear correlation coefficient.

Table V.  Reported potency of CP-4055 versus cytarabine

Cell line	IC50 (nM)		Ratio^1	Assay	N	Reference
	Cytarabine	CP-4055
CCRF-CEM	50	ND^4		SRB^2	119	NCI^3
CCRF-CEM	40	70	1.75	Cell Count	3 – 4	Breistol (ref. 10)
HL-60(TB)	200	ND^4		SRB	119	NCI
K-562	15849	1140	0.07	SRB	119	NCI
RPMI-8226	316228	172330	0.54	SRB	119	NCI
SR	251	973	3.87	SRB	119	NCI
MOLT-4	50	2	0.04	SRB	119	NCI
MOLT-4	0.1	0.9	9	Cell Count	3 – 4	Breistol (ref. 10)
U937	167	710	4.25	ATP	14	Current study
HL-60	2989	13387	4.48	ATP	24	Current study

¹Ratio of the IC50 for CP-4055 to that of cytarabine.

²Sulforhodamine B stain for total protein.

³National Cancer Institute 60 cell line screen (http:dtp.nci.nih.gov; cytarabine is NSC 63878).

⁴Not determined.