potentiation of 5-fluorouracil-leucovorin activity by a2a ... · interfaced with an ibm/ps2...

Vol. 1, 33-40, January 1995 Clinical Cancer Research 33

Potentiation of 5-Fluorouracil-Leucovorin Activity by

a2a-Interferon in Colon Adenocarcinoma Xenografts1

Janet A Houghton,2 Pamela J. Cheshire,

Christopher L. Morton, and Clinton F. StewartDepartment of Molecular Pharmacology [J. A. H., P. J. C., C. L. M.]

and Pharmaceutical Department [C. F. S.], St. Jude Children’s

Research Hospital, Memphis, Tennessee 38101

ABSTRACT

Previous studies using cultured colon adenocarcinoma

cells demonstrated that a mixture of the diastereoiosomers ofthe biologically active (6S) and inactive (6R) forms of (6RS)

leucovorin or 5-formyl-H4PteGlu (LV) and recombinant hu-man a2a-interferon (rLFN-a2a) in combination significantly

increased the cytotoxicity of 5-fluorouradil (FUra) (by 10-14-

fold) whereas FUra combined with single modulators was less

potentiated (3-fold). Maximum cytotoxicity was achieved with48-h drug exposures when drugs were applied continuously,and modulatory rIFN-a2a concentrations were obtained at�50 International units (IU)/ml. We therefore examined

whether such interactions could occur in vivo using HXGCI

clTKc3 colon adenocarcinoma xenografts, deficient in thy-

midine salvage. Potentiation of FUra activity was significantly

greater when FUra was combined with both LV and rIFN-a2a

in comparison to the use of single modulators using a 5-dayschedule for 3 courses. In mice receiving LV, the maximum

level of potentiation of FUra-induced growth inhibition was

independent of the rIFN-a2a dose between 25,000 and 600,000

lu examined in contrast to rIFN-a2a used as a single modu-lator. After administration of 25,000 IU rWN-a2a, plasmarIFN-ot2a concentrations �50 lU/mI were maintained for 6-8h, comparable to exposure times achievable clinically. Dataindicate that intennittent rWN-a2a exposure potentiatesFUra-LV activity in vivo. The efficacy of FUra combined with

dual versus single modulators will thus be of importance to

evaluate in randomized phase III clinical trials in patients withcolorectal cancer.

INTRODUCTIONWe have previously shown significant potentiation (10-

14-fold) of the cytotoxic activity of FUra3 against human colon

adenocarcinoma cell lines using dual modulation with LV and

rIFN-a2a (1). Cytotoxic effects were considerably greater than

with the use of single modulators, where a 3-fold enhancement

of FUra efficacy was obtained. Potentiation of FUra-LV cyto-

toxicity was influenced by the concentration of rIFN-a2a, and

modulation was obtained at clinically achievable rIFN-a2a con-

centrations (�50 IU/ml). Maximum interaction among FUra,

Received 6/24/94; accepted 7/26/94.

t Supported by National Cancer Institute Awards CA-23099 and CA-

21765 (Cancer Center Support Grant) and by the American Lebanese

Syrian Associated Charities.

LV, and nIFN-a2a required a minimum period of 48 h of

continuous drug exposure (2).

The mechanism of FUra-LV interaction is well understood

in the human colon adenocarcinoma cell lines and xenografts

used in this laboratory (3-6). Under conditions of exposure to

physiological folate, LV enhances FUra cytotoxicity by target-

ing FUra to the TS locus. Leucovonin has induced elevation of

critical intracellular pools of 5,10-methylenetetrahydrofolate

that enhance binding of the FUra metabolite FdUMP to TS

within a covalent ternary complex. Enhanced complex stability

mediated by elevated reduced folate pools further contribute to

potentiation of the degree and duration of TS inhibition and to

depleted dTFP pools, while drug resistance has been associated,

in pant, to inadequate pools of 5,10-methylenetetrahydrofolate

(3).

Mechanistically, the locus of the interaction among FUra,

LV, and rIFN-a2a has been identified in the GC3/cl colon

adenocarcinoma cell line (2). FUra cytotoxicity in the presence

of modulating agents was reversible by dThd. However, TS was

determined not to be the primary drug interaction site since (a)

direct assays of TS indicated no significant difference in the

level, activity, or accumulation among FUra ± LV ± rIFN-a2a

treatment groups; (b) nIFN-a2a did not potentiate the cytotoxic

activity of 10-pnopargyl 5,8-dideazafolate, a TS inhibitory

quinazoline; and (c) in TS-deficient cells rIFN-a2a cytotoxicity

was not influenced by the concentration of dThd. Data thus

indicated the requirement for the nucleotide analogue (i.e.,

5-fluoropyrimidine) in the interaction, and this suggested that

potentiation of FUra by rIFN-a2a may be a consequence of

increased incorporation of fraudulent nucleotide into DNA. The

level of DNA single-strand and double-strand breaks obtained

with FUra that were enhanced when combined with single

modulators and further increased in the presence of dual mod-

ulators paralleled the cytotoxic activity of FUra-LV-rIFN-a2a

combinations. Thus, rIFN-a2a may exert its effects by enhance-

ment of FUra base excision or incorporation into DNA, events

that subsequently become influenced by TS inhibition and

dThd-less stress and are further potentiated by LV (2).

Clinically, FUra-LV combinations have demonstrated sig-

nificantly increased response rates (33-48%) in comparison to

2 To whom requests for reprints should be addressed, at Department of

Molecular Pharmacology, St. Jude Children’s Research Hospital, P.O.Box 318, 322 North Lauderdale, Memphis, TN 38101.3 The abbreviations used are: FUra, 5-fluorouracil; LV, a mixture of the

diastereoiosomers of the biologically active (65) and inactive (6R) forms

of (6R5) leucovorin or 5-formyl-H4PteGlu; rIFN-a2a, recombinant hu-

man cr2a-interferon; IFN, interferon; FdUMP, 5-fluoro-2’ -deoxyunidy-

late; TS, thymidylate synthase; TK, thymidine kinase; dThd, deoxythy-

midine; AUC, area under curve; CR, complete response; PR, partial

response of �50% reduction in tumor volume that also includes CRs; C,

no regrowth during the observation period; CLs, clearance; IU, Inter-

national units.

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34 FUra-LV Activity in Colon Adenocarcinoma Xenografts

the use of FUra administered alone (7-15%) when evaluated in

phase III randomized clinical trials conducted in patients pre-

senting with colon cancer (7-10). In addition, significant in-

creases in time to disease progression (8-10) and in patient

survival (8, 9) have been indicated. Thus, by building on this

combination with additional therapeutic approaches, response

rates and survivorship may be improved. rIFN-a2a has been

evaluated for this purpose, commencing in single institutional

trials. Initially, FUra and rIFN-a2a combinations were evalu-

ated in previously untreated colon carcinoma patients and re-

sponse rates (26-63%) greater than those anticipated for FUra

treatment alone, and equal to those obtained with FUra-LV

combinations, were reported (1 1-13). In a subsequent phase II

multiinstitutional trial, Wadler et al. (14) confirmed a high

response rate of 42%. The potential for the interaction of FUra

and rIFN-a2a at a locus distinct from that of FUra and LV,

along with their nonoverlapping toxicities, suggested that rIFN-

a2a would be of importance to evaluate in combination with

FUra-LV. Subsequently, Grem et al. (15) evaluated the triple

combination in previously untreated colon carcinoma patients

and determined a response rate of 45%.

As demonstrated from both preclinical and clinical studies,

the use of rIFN-a2a in combination with FUra-LV clearly has

potential for improving therapeutic outcome in patients with

colon carcinoma. However, several key issues remain to be

addressed that will determine the utility of rIFN-a2a as a mod-

ulator of FUra-LV action. The minimum dose of rIFN-a2a that

can be used to achieve modulatory plasma concentrations must

be determined. In colorectal carcinoma patients, the rIFN-a2a

dose may be limited by fatigue and neurological toxicities (16,

1 7) and also by elevation in the plasma concentrations of FUra

(15). The duration of rlFN-a2a exposure must be sufficient to

enhance FUra-LV action, and it is evident from preclinical

studies that modulation has not been achieved with single, short

(2-h) exposures (2). The interdependence of these two parame-

tens on FUra-LV modulation at clinically achievable plasma

concentrations of rIFN-a2a is of importance for clinical therapy.

Finally, whether the effect of dual modulation versus the effect

of single modulators significantly enhances the therapeutic ac-

tivity of FUra will impact on the application of rIFN-a2a in

clinical trials.

Previously, we have extensively characterized the thera-

peutic activity of FUra-LV combinations in human colon ade-

nocarcinoma xenografts maintained in immune-deprived mice

(18). We have therefore used these models to address some

of the key issues outlined above regarding the influence of

rIFN-a2a action on the in vito activity of FUra or FUra-LV

combinations. In the studies presented, we have examined the

therapeutic utility of FUra, FUra-LV, FUra-rIFN-a2a, and

FUra-LV-rIFN-a2a combinations. The xenognaft models used

were the parental HxGC3/cl colon adenocarcinoma that sal-

vages dThd and HxGC3/clTKc3, a subline selected for its

inability to salvage dThd because of a deficiency in TK

HxGC3/clTKc3 tumors may be more representative of the

situation in humans, where reduced dThd salvage capability due

to considerably lower plasma dThd concentrations has been

demonstrated (18, 19). Specifically, we have examined (a) the

influence of dose of rIFN-a2a on FUra or FUra-LV-induced

antitumor activity in mice; (b) pharmacokinetics of nIFN-a2a

given to mice; and (c) duration of systemic exposure to rIFN-

a2a at modulatory dose levels.

MATERIALS AND METHODS

Immune Deprivation of Mice. Female 4-week-oldCBA/CaJ mice (The Jackson Laboratory, Bar Harbor, ME) were

immune deprived by thymectomy and subjected 3 weeks later to

whole-body irradiation (950 cGy) using a ‘37Cs source. Mice

received 3 X 106 nucleated bone marrow cells within 6-8 h of

irradiation (18).

Tumor Lines. HxGC�/c1 was originally derived from a

poorly differentiated adenocarcinoma of the colon that was

established as a xenograft in immune-deprived mice (HxGC3).

Subsequently, the line was cultured and cloned (GC3/cl; Refs.

20-22) and reestablished as a xenograft in mice (HxGC3/cl).

HxGC3/clTKc3, a TK-deficient variant of HxGC�/cl was

initially selected from GC3/cl in tissue culture (21, 22), and

inoculated s.c. into immune-deprived mice to produce the xc-

nografted line. All tumors were maintained as xenografts by

passage in the s.c. space of female CBA/CaJ immune-deprived

mice.

Growth Inhibition Studies. Mice bearing bilateral s.c.

tumors were randomized and received drugs when the tumors

had reached a diameter of “0.5 cm. Tumor response was

determined at 7-day intervals using digital calipers (Maxcal)

interfaced with an IBM/PS2 microcomputer. Two perpendicular

diameters were used to compute volumes (23). Growth inhibi-

tion was calculated from the difference in days required for

treated tumors to grow to four times their volume at the start of

treatment as compared with untreated controls. Each treatment

group comprised six tumor-bearing mice. Relative tumor vol-

umes were calculated using the formula RTV = V,JV�, where V�

represents the tumor volume on day X and V(, indicates the

tumor volume at the initiation of treatment. Volume regressions

were also determined and expressed as CR, PR or C, and

maximum percentage of treated/control ratio.

Formulation and Administration of Drugs. FUra (50

mg/ml), obtained from Solo-Pak Laboratories (Franklin Park,

IL) as a pharmaceutical preparation, was diluted to a concen-

tration of 15 mg/mI in sterile 0.9% saline. It was given to mice

by iv. bolus injection weekly via the tail vein for three courses

(days 1, 8, and 15) after randomization of the animals. The dose

used was 75 mg/kg (“�225 mg/m2) given in a volume of 0.1

ml/20 g body weight and constituted the maximum tolerated

dose on that schedule as described previously (18).

Leucovonin, purchased from Sigma Chemical Co. (St.

Louis, MO), was given to mice by i.v. bolus injection daily at a

dose of 500 mg/m2 on days 1-5, 8-12, and 15-19 immediately

before FUra commencing on the first day of FUra injection as

published previously (18). Leucovorin was dissolved in 0.9%

sterile saline at a concentration of 23.6 mg/ml, and after addi-

tional filtration was injected at volumes of 0.17-0.27 ml/20 g

body w as described previously (18). The dose relationship

between mg/kg and mg/m2 body-surface area was determined

for individual mice as reported previously (5, 6, 24).

rIFN-a2a was obtained, in part, as a generous gift from

Hoffmann La Roche Inc., Nutley, NJ. The lyophilized powder

representing 18 X 10� IU rIFN-a2a was initially dissolved in 1



Clinical Cancer Research 35

ml sterile water. Alternatively the pharmaceutical formulation

(Roferon A; 6 X 106 IU/ml) was purchased. Further dilutions

were made in sterile 0.9% saline, and the agent was given i.p. to

mice on days 1-5, 8-12, and 15-19 in volumes of 0.1 ml!

10 g body weight at dose levels ranging from 25,000 to 600,000

lU/mouse. A 25,000-lU dose of nIFN-a2a in mice was equiva-

lent to 3.3 X 106 IU/m2 or 5.7 X 106 IU in humans. a2a-

Interferon was given immediately before leucovonin. Because of

the lack of interaction between high concentrations of rIFN-a2a

and LV in cultured cells in the absence of FUra (1, 2) and the

lack of antitumor activity or host toxicity of the combination in

initial studies in mice, the agents were examined in combination

in vivo as a control group.

Statistical Analyses of Antitumor Activity. The results

of studies were evaluated and compared by one-way analysis of

variance using the number of days required to reach 4 times the

original tumor volume as the dependent variable followed by the

Newman-Keuls range test for differences between means (18,

25). Only tumors from mice that survived the entire study were

included in the analyses, and any tumor that failed to reach 4

times its original volume was assigned a default value of 140

days for the maximum duration of the study. The percentage of

tumors showing partial and/or complete regressions, along with

the extent of regression, were also analyzed. Data are presented

as the mean and SE.

Pharmacokinetic Analyses. Plasma concentrations of

nIFN-a2a were determined following i.p. injection of non-tu-

mon-beaning immune-deprived mice at dose levels of 25,000 and

200,000 lU/dose. Time points, using 4 mice at each point,

ranged from 0.25 to 6 h following nIFN-a2a administration.

Additionally, groups of mice receiving 25,000 IU nIFN-a2a on

day 1 were injected with the same dose level of rIFN-cz2a at

24-h intervals over a period of 5 days. On day 5, samples were

obtained between time 0 (just before injection) and 6 h (4

mice/point) after rIFN-a2a administration. Blood was drawn by

cardiac puncture into hepaninized syringes and plasma prepared

(18).

Plasma concentrations of rIFN-a2a were measured by an

adaptation of an enzyme immunoradiometnic assay (Celltech

Limited, Berkshire, United Kingdom; Ref. 26). Calibrators were

prepared using pure rIFN-a2a (Hoffman La Roche Inc.) in

pooled mouse plasma over a concentration range of 25-2500

IU,’ml. The lower limit of sensitivity of the assay was 15 IU!ml.

A one-compartment model was fit to the nIFN-ot2a plasma

concentration-time data by the use of maximum likelihood

estimation (27). Parameters estimated included volume of the

central compartment (Vd�) and elimination rate constant (Ke).

Calculated parameters included t112, AUC versus time curve,

maximum drug concentration, systemic CLs, and length of time

plasma concentrations of nIFN-cz2a exceeded 50 IU/ml.

For purposes of estimating systemic exposure to rIFN-a2a

in humans from existing clinical dosing schedules, a simulation

analysis was performed. A one-compartment model was fit to

concentration versus time data from a previously published

study in humans (28). The parameters of Vd� and Ke were used

to simulate cumulative nIFN-a2a systemic exposure for two

clinically relevant dosage schedules [i.e., 5 X 106 IU!m2 daily

for S consecutive days (15) and 9 X 106 IU on alternate days for

three doses; (Ref 17)].

RESULTSPotentiation of FUra-LV Activity by rIFN-a2a in Vivo.

Previous reports in the literature indicated that a standard dose

of 200,000 IU IFN-a in mice induced growth inhibition of

xenognafted tumors (29, 30) or was modulatory to the action of

other agents (30, 31). Consequently, this dose level of rIFN-a2a

was initially evaluated for its modulatory effects on FUra or

FUra-LV activity in mice beaning HxGC3/clTKc3 xenografts,

deficient in dThd salvage (Fig. 1, left). In mice receiving no

treatment or LV-rIFN-a2a only, tumors grew at similar rates

and reached four times their pretreatment size at similar times

(35 and 38 days, respectively), indicating no direct effect of

LV-rIFN-a2a on tumor growth. When FUra was given once

weekly for 3 weeks, volume regressions were obtained and

growth was delayed by 33.5 days relative to controls. When

FUra was combined with rIFN-a2a or LV as single modulators,

volume regressions increased and further increases in growth

delay were obtained. However, when FUra was combined with

both modulators, additional and significant decreases in tumor

volume and further enhancement of growth inhibition (P <

0.001) was evident, effects considerably greater than those

achieved with single modulators (Fig. 1).

Since data obtained with cultured GC/cl cells had mdi-

cated that FUra-LV-rIFN-a2a cytotoxicity was reversible by

dThd (1, 2), modulation of FUra activity was subsequently

determined in HxGC�Jc1 xenografts capable of dThd salvage

(Fig. 1, right). In this experiment, the dose level of rIFN-a2a

was increased by 3-fold to 600,000 IU, although drug adminis-

tration schedules remained the same. In contrast to the HxGC/

clTKc3 xenognafts, no volume regressions or growth inhibi-

tion were induced in HxGC3!cl tumors when FUra was

combined with dual modulators, confirming results obtained

with cultured cells.

Influence of rIFN-a2a Dose on FUra ± LV Modulation.Since significant potentiation of the in vivo activity of FUra had

been obtained in HxGC3!clTKc3 xenografts by dual modula-

tion with rIFN-a2a administered at a dose of 200,000 IU, the

influence of nIFN-a2a dose (systemic exposure) on therapeutic

activity was examined. In these experiments, the rIFN-a2a dose

was increased to 600,000 IU to examine whether FUra or

FUra-LV activity could be potentiated further. In addition, the

dose level of rIFN-a2a was reduced to 25,000 IU (considered to

be a low dose level in mice; Ref. 29) to determine the minimum

dose exhibiting a modulatory effect. Data for rIFN-ct2a com-

bined with FUra alone as a single modulator, or combined with

LV in dual modulation studies, are shown in Tables 1 and 2 and

Fig. 2.

Tumor Growth Inhibition. The time required in days

for tumors to reach four times the volume at the initiation of

treatment has been compared for control and all drug-treated

groups (Table 1). The differences between untreated (42.1 days)

and FUra-treated (69.6 days) tumors, or between FUra-treated

and FUra-LV-treated (88.6 days) groups were significantly dif-

fenent (P < 0.05). When the efficacy of FUra treatment was

compared with on without rIFN-a2a, the time required to reach

the value at four times the volume ranged from 77.1 to 118.8

days, significantly different at rIFN-a2a doses of 25,000 and

600,000 IU (P < 0.001). Modulation of FUra activity by rIFN-



HxGC3 /clTKc3

ci,E:�

0

E

ci,>

ccici)

a:

Group

ControlFUra

FUra-nIFN-a2a

Dose of rIFN-ct2a (lU) Days to 4X ± SE

P

<0.001

<0.05


0.1

0 2 4 6 8101214161820 0123456789

Weeks Weeks

Fig. I Modulation by rIFN-a2a of the activity of FUra or FUra-LV against HxGC3/cITK c3 (left) or HxGC3/cl (rig/it) xenografts maintained in

immune-deprived mice. 0, control; #{149},LV-rlFN-a2a; V. FUra; V, FUra-rIFN-a2a; 0, FUra-LV; #{149},FUra-LV-rIFN-a2a. Six mice bearing bilateral

tumors were randomized to each group, and results represent the mean ± SD for tumor volumes determined at each time point. Drug treatments were

conducted as follows: FUra, 75 mg/kg iv. days 1,8, and 15; LV, SOt) mg/rn2 iv. daily on days 1-5, 8-12, and 15-19: rIFN-#{128}s2a, 2(X),0(X) lU

(HxGC�/TK c3) or 600,0()() IU (HxGC�/cl) i.p. daily on days 1-5, 8-12, and 15-19. Tumor volumes were determined at weekly intervals.

Table I Delay in tumor growth induced by FUra-LV-rlFN-x2a combinations in HxGC�/clTK c3 xenografts”

Days to 4X” � ri � Group Days_to 4X ± SE N

42.1 ± 3.5 50 FUra 69.6 ± 3.2 29

69.6 ± 3.2 29 FUra-LV 88.6 ± 4.1 52

FUra-LV-rIFN-a2a

�1 Days to 4X ± SE n P

25,0(R) 96.5 �: 5.4 35 1 15.0 ± 4.7 35 <0.05

20(),0()() 77.1 � 5.4 10 1 1 1.2 ± 8.3 8 <0.001

600,0(X) 118.8 ± I 1.5 10 124.2 ± 7.9 12 >0.05

(‘ FUra vs. FUra-rlFN-u2a, P < 0.001 at 25,0(X)-lU and 60(),0()0-IU doses; FUra-LV i’s. FUra-rlFN-a2a, P < (1.05 at 600,000-lU dose; FUra-LV

vs. FUra-LV-rlFN-a2a (all doses), P < 0.05.

I, Days to 4X, time required to reach 4 times the pretreatment volume.

� P, comparison of FUra-rlFN-a2a with FUra-LV-rIFN-a2a at equivalent rIFN-cs2a dose levels.

cs2a alone appeared to be dependent on dose and was greatest at

the highest dose level of rIFN-a2a used. Data for FUra-LV

activity compared with FUra-rlFN-a2a responses were similar

(P > 0.05) at the 25,000- and 200,000-lU dose levels of rIFN-

a2a (P > 0.05), but were also greater at the high rIFN-a2a dose

level (P < 0.05). When FUra was combined with both modu-

lators however, days required to reach four times the volume

increased (range, I I I .2-1 24.2 days) and was independent of the

rIFN-a2a dose. Comparison of FUra-LV-rIFN-a2a responses

with the response to FUra-LV indicated significant differences

over the entire dose range of rIFN-a2a examined (P < 0.05).

Similarly, a comparison of FUra-LV-rIFN-a2a responses with

the responses to FUra-rIFN-a2a at equivalent rIFN-a2a dose

levels indicated increased FUra activity in the presence of dual

modulators at doses of rIFN-a2a � 200,000 IU (P < 0.05), but

not at the 600,000-lU rIFN-a2a dose.

Volume Regressions. The maximum percentage of TIC

for all treatment groups is shown in Fig. 2, and the percentage

of PR and CR responses for tumors that did not regrow during

C are presented in Table 2. FUra treatment alone induced 62%

PR that was increased significantly (P < 0.01) to 83% PR, 12%

CR, and 10% C in the presence of LV. When FUra was corn-

bined with rIFN-a2a, volume regressions were dependent on the

rIFN-a2a dose, being significantly greater at the 600,000-lU

dose of rIFN-a2a only (P < 0.001) in comparison to FUra or

FUra-LV treatment groups. Additionally, FUra-LV-rIFN-a2a-

treated tumors were smaller and demonstrated less rIFN-a2a

dose dependency, but were significantly smaller than in the

FUra-LV-treated group, only at the highest dose level of rIFN-

a2a (P < 0.01). However, when FUra-rIFN-a2a activity was

compared either in the absence or in the presence of LV at

equivalent rIFN-a2a dose levels, tumor volume regressions



00

501

40

IC.)

I 20

1�

10

0 nFUrs

RhI � 200.000

200.000 25.000 000.000

I IL IFUrs + rlPN-a2a (flJ) FUrs + LV + riFN-u2a Intl

Fig. 2 Maximum percentage of treated/control (TIC) for tumors in

mice from all drug treatment groups. Data are the mean ± SE of 8-52

tumors/treatment group.


were significantly greater in groups receiving LV at doses of

rIFN-ct2a � 200,000 IU (P < 0.05), but not at the 600,000-lU

rIFN-a2a dose.

Taken together, these data suggested that (a) rIFN-a2a

significantly modulated the activity of FUra-LV; (b) modulation

of FUra-LV-induced growth inhibition was independent of the

dose of rIFN-a2a over the dose range examined; and (c) the use

of dual modulation had greater effects on FUra-induced tumor

growth inhibition and volume regression than the use of single

modulators. Hence maximum potentiation of FUra by rIFN-a2a

alone was achievable only at the suprapharmacological rIFN-

a2a dose level of 600,000 IU.

Toxicity of FUra-Modulator Combinations. It has pre-

viously been shown that FUra (75 mg/kg) given i.v. to mice

once weekly for 3 weeks was the maximum tolerated dose.

Toxicity, as determined from deaths occurring within the first 3

weeks after completion of therapy, was not increased in the

presence of LV (500 mg/rn2; Ref. 18). From the data presented

in Table 3, addition of rlFN-a2a did not enhance the toxicity of

FUna-LV in tumor-bearing mice.

rIFN-a2a Pharmacokinetics. Concentrations of rIFN-

a2a achieved in the plasma of mice following i.p. administration

of the agent at dose levels of 25,000 IU on days I and 5, and at

200,000 IU on day 1, are shown in Fig. 3. These are dosages

equivalent to 3.3 x l0� and 26.4 X 106 IU/m2, respectively, in

humans. Peak plasma levels occurred by 1 h after rIFN-a2a

administration. Model estimated peak plasma levels of rIFN-

a2a achieved were 3400 and 4500 IU/ml (25,000-lU dose) on

days 1 and 5, respectively.

Pharmacokinetic analyses of these data are shown in Table

4. The AUC and systemic CLs on days 1 and S were similar for

the 25,000-lU rIFN-ct2a dose. The elimination half-life was

lower (0.87-1.1 h) for the 25,000-lU dose level in comparison

to the 200,000-lU dose (t11, = 16.4 h). The elimination half-life

for the 25,000-lU dose was 26% greater on day S compared with

day I . The AUC for 200,000 IU rIFN-a2a was considerably

greater and CLs lower than predicted from data obtained at the

lower rIFN-a2a dose, suggesting saturation of elimination pro-

cesses.

The duration of maintenance of rIFN-a2a concentrations

above 50 lU/ml, a minimal modulatory concentration of

FUra-LV cytotoxicity determined for 72-h continuous expo-

sures in cultured cells (1) is shown in Table 4. For the 25,000-lU

dose level of rIFN-a2a, plasma concentrations of 50 lUImI were

maintained for 6 h on day 1 and 8 h on day 5, and for the

200,000-lU dose for >24 h.

Pharmacokinetic modeling oftwo clinical dosing schedules

used in humans is shown in Fig. 4. Cumulative systemic expo-

sure (AUC) following five daily doses of nIFN-a2a (5 X l0�

IU/m2) was 5270 lU!h/ml, and following three doses adminis-

tered on alternate days, 3310 lU/h/mI. Although in mice at the

200,000-lU rIFN-a2a dose level accumulation was suggested,

simulations in humans were more consistent with the lower

dosage studied in the mice where no accumulation was apparent

between days 1 and 5 as evidenced by no changes in CLs or

AUC. The simulation study also indicated that plasma concen-

trations of rIFN-a2a �50 IU/ml would be maintained for pen-

ods of 8 h following nIFN-ct2a administration, consistent with

the systemic exposure observed for the lowest rIFN-a2a dose in

the xenograft study.

DISCUSSION

In this study we extend the relationship between rIFN-a2a

concentrations that modulate FUra-LV cytotoxicity determined

in vitro to the level of rIFN-a2a systemic exposure associated

with dosages modulating the cytotoxicity of FUra and FUna-LV

in mice bearing human colon adenocancinoma xenografts. Fun-

then we show that the length of time above an in vitro modula-

tory concentration is similar between the mouse xenognaft

model and pharmacokinetic simulations of dosing schedules

used clinically.

rIFN-a2a has been shown to modulate the in vitro cyto-

toxicity of FUna and FUna-LV against two human colon adeno-

carcinoma cell lines, GC/cl and VRC5/cl (I). Previously we

had demonstrated, using cultured colon adenocancinoma cells,

that 500 lU/mI and 5,000 lU/mI nIFN-a2a produced similar and

significant modulation of FUra-LV cytotoxicity (10-14-fold;

>95% reduction in clonogenic survival) when cells were cx-

posed continuously for 72 h (1). Reduction in clonogenic sun-

vival by 60% was achievable when FUra-LV were combined

with as low as 50 IU/ml rIFN-a2a for 72 h (1). In addition, in

the presence of LV lower modulatory concentrations of rIFN-

a2a could be combined with a noncytotoxic FUra concentration

to achieve a cytotoxic response. In this in vitro system maximal

cytotoxicity required a minimum of 48 h of continuous drug

exposure (2).

With the knowledge that rlFN-a2a modulated the activity

of FUra in vitro, we evaluated three rIFN-a2a doses (25,000 IU,

200,000 IU, and 600,000 IU) against human colon adenocanci-

noma xenografts in combination with FUra and FUna-LV to

investigate the potential for enhancing in vivo thenapeutic activ-

ity. Using HxGC3/clTKc3 xenografts deficient in dThd sal-

vage, significant potentiation of FUna-LV activity was obtained

in the presence of nIFN-a2a, determined by enhanced volume



10�

10�

10�I10

0 1 2 S 4 5 0

Tim. �4

38 FUra-LV Activity in Colon Adenocarcinoma Xenognafts

Table 2 Volume regressions induced by FUra-LV-nIFN-a2a combinations in HxGC�/c1TKc3 xenognafts’�

Group %PR %CR %C n

Control 0 0 0 50

FUra 62 0 0 29

FUra-LV 83 12 10 52

FUna-nIFN-a2a FUna-LV-nIFN-a2a

Dose of nIFN-a2a (IU) % PR % CR % C n % PR % CR % C n

25,000 31 9 3 35 94 23 20 35 <.05

200,000 67 0 0 10 100 17 8 8 <.05600,0(X) 100 60 60 10 100 67 67 12 >05

a FUra-LV > FUra, P < 0.01; FUra-nIFN-a2a > FUna, P < 0.001 at 600,000 lU only; FUna-nIFN-a2a > FUna-LV, P < 0.05 at 600,000 IU

only; FUna-LV-nlFN-a2a > FUna-LV, P < 0.01 at 600,000 IU only. The PR group contains all tumors that achieved at least 50% reduction in tumorvolume and hence also includes CRs.

S p comparison of FUna-nIFN-a2a with FUra-LV-rIFN-ct2a at equivalent nIFN-cs2a dose levels.

Table 3 Toxicity of FUra-LV-nIFN-cc2a combinations in tumor-

bearing mice

Group % Survival” n

Control 100 30FUra 97 30

FUra-LV 100 30

FUna-LV-FUna-nIFN-a2a rIFN-a2a

Dose of rlFN-a2a (lU) % Survival n % Survival n

25,000 100 18 100 18

200,000 83 6 100 6

600,()00 100 6 100 6

“Survival was determined within the first 3 weeks after completion

of therapy.

regressions and inhibition of tumor growth. Responses were

considerably greaten than those achieved with FUra combined

with either LV on nIFN-a2a as single modulators. The drug

interaction among FUra, LV, and nIFN-a2a was dependent on

reduced ability to salvage dThd, thereby confirming previous

data obtained using cultured GC3/cl cells (1, 2).

We then evaluated the influence of the rIFN-a2a dose or

systemic exposure on FUra activity. When FUra was combined

with rIFN-a2a as a single modulator, the greatest effect was

seen at the 600,000-lU dose level. However, this would consti-

tute a suprapharmacological exposure not achievable for clinical

studies. In contrast to results with FUra-rIFN-a2a, potentiation

of FUna-LV-induced growth inhibition was independent of the

nIFN-a2a dose oven the dose range examined, as was the extent

of volume regressions at the 25,000- and 200,000-lU dose

levels. The lowest nIFN-a2a dose (25,000 IU) yielded signifi-

cant potentiation of FUna-LV-induced growth inhibition, as high

as achieved with the 600,000-lU rIFN-a2a combined with FUra

± LV. Thus, in combination with LV, lower pharmacologically

relevant doses of nIFN-a2a were effective in modulating FUra.

As recently reviewed by Galpin and Evans (32), many

studies have shown systemic exposure to anticancer drugs to be

nelated to both toxicity and efficacy. Different approaches have

been used to estimate systemic exposure, including AUC, peak

plasma concentration, and time above a minimum effective

Fig. 3 Plasma concentrations of nIFN-cx2a on day 1 of administrationon on day 5 following daily i.p. injections. Dose levels of nIFN-a2awere: 0, 25,000 IU and A, 200,000 IU on day 1, and #{149},25,000 IU onday 5. Data represent the mean ± SD of 4 determinations at each time

point. Solid line, ‘ ‘best-fit’ ‘ curve generated from maximum likelihoodestimates of pharmacokinetic parameters; dashed line, limit of assay

sensitivity (15 IU/ml).

concentration. Studies of the antiviral properties of nIFN-a2a

showed that 2’,5’-oligoadenylate synthetase activity was related

to dose and route of nIFN-a2a administration (33). At similar

dosages, 2’,5’-oligoadenylate synthetase activity in peripheral

blood mononuclear cells was greaten when nIFN-a2a was given

i.m. compared with i.v. administration, paralleling prolonged

systemic exposure at low concentration from the i.m. route and

short exposure with an initial high peak level from the i.v. route.

This would suggest antiviral activity is associated with prolong-

ing rIFN-a2a exposure above a minimum effective concentra-

tion for some period of time rather than association with a high

peak concentration. Additionally, Wills et al. (28), studying the

pharmacokinetics of rIFN-a2a in volunteers, observed that ad-

verse effects from rIFN-a2a were related to exceeding and

maintaining serum concentrations above a threshold level.

For comparison with nIFN-a2a systemic exposure deter-

mined in mice, we simulated the rIFN-a2a plasma concentra-



100

10

.�

:a

Co

CCo0C0

0

0 24 48 72 96 120 144


Table 4 Phanmaco kinetics of nIFN-a2a in mice

Apparent DurationDose Vd� CLs AUC0 . . above

(IU) �t/2 (h) (ml/m2) (mi/him2) (IU-mlih) 50 lUimI (h)

25,000, 0.87 501 399 8,536 6

day 1

25,000, 1.1 712 448 7,533 8

day 5

200,000, 16.4 410 164 88,762 >24.0day I

tions achievable in humans following s.c. administration at the

nIFN-a2a dose levels used in clinical tnials of FUra-rIFN-a2a

(1 1-14) on FUna-LV-nIFN-a2a (15). Simulations were con-

structed for 5 X l0� IU/m2 nIFN-cx2a administered daily for S

days (15) or 9 X 10� IU administered on alternate days for 3

doses (1 1-14). The total AUC measured in mice following

25,000 IU rIFN-a2a administered daily for 5 consecutive days

was 41 and 66% of that determined from phanmacokinetic

simulations of the two clinical dosing schedules. Similarly,

estimated peak plasma concentrations of rIFN-a2a were lower

in both clinical studies (-90 lU/mi) than determined in mice

(�‘4000 lU/mi). The reduced effect of nIFN-ct2a dose on tumor

volume regressions and the lack of a dose-response relationship

for nIFN-a2a when combined with FUra and LV in inducing

growth inhibition in the xenograft studies suggested that time

above a minimum effective concentration may be more impon-

tant than the peak plasma concentration in inducing a modula-

tory response to FUna-LV activity.

Simulations of dosing schedules used clinically indicated

plasma rIFN-a2a concentrations would be maintained above SO

lU/mi each day for a period of 8 h, and with the alternate day

schedule, for 8 h three times per week. Of interest from this

study was that the 25,000-lU dose level in mice maintained a

nIFN-a2a plasma concentration of �50 lU/mI for 6-8 h. This

concentration of rIFN-cs2a for the durations obtained provided

in vito modulation of FUra-LV activity in mice. Thus, when

combined with LV it may be possible that intermittent and

repeated exposure to lower dosages (i.e. , concentrations) of

rIFN-cr2a is sufficient to modulate and potentiate the activity of

FUna.

Clearly, intermittent exposure to rIFN-a2a has been mod-

ulatony in the xenograft studies, and maintenance of critical

rIFN-a2a levels for 6- to 8-h periods daily appears to be suffi-

cient to modulate the activity of FUna in the presence of LV.

Since the window of intermittent nIFN-a2a exposure is very

similar between mice and humans, it is conceivable from the

simulations presented that the rIFN-a2a doses used clinically

will modulate FUra-LV activity. It is also evident from previous

studies with cultured cells (1, 2) and from the xenograft studies

that lower rIFN-cx2a doses may be used to modulate FUra

activity in the presence of LV. However, the optimal schedule

remains to be elucidated.

In patients, an additional effect of intenfenons has been to

increase FUna systemic exposure, thereby incneasing FUra-as-

sociated toxicity. Most of these investigations have been con-

ducted with an alternative IFN-a subtype (IFN-a2b; Ref. 34),

although effects appear to be similar with rIFN-a2a (15). In

0.1

Time (hr)

Fig. 4 Plasma concentration versus time plot for pharmacokinetic sim-

ulation of two clinical dosing schedules for rlFN-a2a as described in

‘ ‘ Materials and Methods.”

these studies pharmacokinetic analyses indicated that FUra CLs

decreased by 20-35% (34). Grem et al. (15) determined that

reduction in FUna CLs was related to the dose of nIFN-a2a,

resulting in an increase of FUna AUC of 30 and 50% in patients

receiving S x 10� and 10 X 10� lU/rn2, respectively, of nIFN-

a2a. However, the effects of intenferons are species specific, and

for the xenognaft studies, a human IFN-a subtype was used in a

munine model system bearing human tumors. Thus, as antici-

pated, no increase in FUna-associated toxicity was observed in

these studies. It is unlikely therefore that suitable data on the

influence of rIFN-a2a on FUna toxicity will be forthcoming

from studies conducted in mice. In this regard, it is noteworthy

that even at the highest rIFN-a2a dose, the toxicity of FUra on

FUna-LV was not enhanced. Consequently, this type of infon-

mation will be more readily gained from clinical studies.

Modulation of the activity of FUra by rIFN-a2a has been

possible in an in vito model system, and has been considerably

greaten at a more clinically relevant lower dose level of nIFN-

ct2a in the presence of LV. Plasma levels of nIFN-a2a achieved

in mice have been maintained above critical concentrations for

periods of time achievable clinically. It is feasible that dual

modulation may enhance the clinical activity of FUna in patients

with colorectal cancer, although whether responses and survi-

vonship are increased significantly oven the use of single mod-

ulatons will await results from randomized phase III clinical

trials in this population.

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1995;1:33-40. Clin Cancer Res J A Houghton, P J Cheshire, C L Morton, et al. alpha2a-interferon in colon adenocarcinoma xenografts.Potentiation of 5-fluorouracil-leucovorin activity by

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