Table 1. Results of curve shift analysis.

Experiments were conducted at two levels of folic acid. Combination ratio indicates the trimetrexate-to-AG2034 ratio in their actual concentrations (μM). Initial concentrations are the starting concentrations, which were subsequently diluted at fixed concentration ratios. IC_{50, combination} is the combination concentration in IC₅₀ equivalents, as calculated by Eq. 4. SE is the corresponding standard error. The critical value for the inverse cumulative T-distribution (type I error rate = 0.025, two sides, degrees of freedom = 53) is equal to 2.01. IC_{50, combination} +2.01 SE <1, IC_{50, combination} −2.01 SE <1< IC_{50, combination} +2.01 SE, and IC_{50, combination} −2.01 SE >1 indicate synergy, additivity, and antagonism, respectively. Parameter n is the curve shape parameter describing the steepness of the concentration-effect relationship. IC₅₀ values for pure agents are as follows. AG2034 in 2.3 μM folic acid: 0.0063±0.0004 μM, in 78μM folic acid: 0.56±0.08 μM (universal response surface estimates: 0.0035 and 0.414μM, respectively). Trimetrexate in 2.3 μM folic acid: 0.0014±0.0009 μM, in 78 μM folic acid: 0.013±0.001 μM (universal response surface estimates: 0.0015 and 0.013 μM, respectively).

PteGlu, μM	Combination Ratio	Initial concentration before dilution		IC50, combination, IC50 equivalents	n	Evaluation at 50% Effect Level
	Trimetrexate:AG2034	Trimetrexate, μM	AG2034, μM	Mean±SE	Mean±SE
2.3	AG2034 alone	0.00	2.78	1.00±0.06	3.12±0.53
	1:0.1	0.55	0.05	0.96±0.06	2.29±0.28	Additive
	1:0.2	0.54	0.11	0.91±0.08	2.26±0.42	Additive
	1:0.5	0.51	0.25	0.90±0.10	2.82±0.77	Additive
	1:1.25	0.45	0.56	0.89±0.03	3.81±0.31	Synergy
	1:2.5	0.37	0.93	0.89±0.06	5.00*	Additive
	1:5	0.28	1.39	0.63±0.02	5.00*	Synergy
	1:5 repeat	0.28	1.39	0.58±0.02	5.00*	Synergy
	1:10	0.19	1.85	0.56±0.01	4.92±0.43	Synergy
	1:20	0.11	2.22	0.53±0.03	5.00*	Synergy
	1:50	0.05	2.53	0.26±0.01	5.00*	Synergy
	1:125	0.02	2.67	0.99±0.07	3.19±0.59	Additive
	1:250	0.01	2.73	0.60±0.02	3.87±0.57	Synergy
	Trimetrexate alone	0.56	0.00	1.00±0.64	2.32±0.33
78	AG2034 alone	0.00	27.78	1.00±0.14	1.45±0.23
	1:1	0.55	0.54	0.72±0.05	2.80±0.43	Synergy
	1:2	0.54	1.07	0.37±0.02	3.53±0.49	Synergy
	1:5	0.51	2.53	0.21±0.01	3.12±0.52	Synergy
	1:12.5	0.45	5.56	0.14±0.01	2.54±0.35	Synergy
	1:25	0.37	9.26	0.13±0.01	3.55±0.74	Synergy
	1:50	0.28	13.89	0.10±0.01	2.00±0.10	Synergy
	1:50 repeat	0.28	13.89	0.12±0.01	2.59±0.42	Synergy
	1:100	0.19	18.52	0.12±0.01	2.96±0.34	Synergy
	1:200	0.11	22.22	0.15±0.01	3.21±0.54	Synergy
	1:500	0.05	25.25	0.18±0.02	3.16±0.82	Synergy
	1:1250	0.02	26.71	0.27±0.02	3.03±0.69	Synergy
	1:2500	0.01	27.24	0.36±0.04	2.55±0.61	Synergy
	Trimetrexate alone	0.56	0.00	1.00±0.08	3.73±0.62

^*The fitted value of n is limited to 5.00. At this value, effect declines over the effect range (e.g. from 90% to 3% effect) between adjacent data points at the employed $\sqrt{10}$ or 3.16-fold sequential dilution, and higher values cannot be accurately estimated.