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Experimental Therapeutics, Preclinical Pharmacology |
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i.v. x 1 >> daily x 5. In contrast, the rank order of toxicity was daily x 5 >> weekly x 3 i.v. x 1. ZD1694 at the MTD produced 20% complete tumor regression and 20% partial tumor regression (PR) with i.v. x 1 and weekly x 3 schedules and 12-day tumor growth delay with daily x 5 schedule against FaDu xenografts. No complete tumor regression was achieved with ZD1694 with any schedule against A253; a 20% PR, 40% PR, and 10-day tumor growth delay were observed with i.v. x 1, weekly x 3, and daily x 5 schedules, respectively. The data indicate that ZD1694 was slightly more effective against FaDu than against A253. Of interest and potential clinical importance was the observation that ZD1694 was still active at doses lower than the MTD (
MTD), which showed a high therapeutic index and wide safety margin. Study of ZD1694 compared with 5-fluorouracil and 5-fluoro-2'-deoxyuridine at the MTD revealed that the antitumor activity of ZD1694 was comparable with or superior to 5-fluorouracil and 5-fluoro-2'-deoxyuridine against both A253 and FaDu xenografts, with less toxicity.
High plasma thymidine in mouse relative to human (
1.3 µM and <0.1 µM, respectively) may complicate the study of antitumor activity and toxicity of TS inhibitors with human tumor xenografts grown in the mouse. To test this hypothesis, we preadministered methoxypolyethyleneglycol-conjugated thymidine phosphorylase (MPEG-TPase; 2500 units/kg/dose) to reduce mouse plasma thymidine, then treated with various doses of ZD1694 using the daily x 5 or i.v. x 1 schedules in the A253 tumor model. MPEG-TPase significantly increased the toxicity of ZD1694; the MTD of ZD1694 plus MPEG-TPase was reduced 3- and 10-fold compared with ZD1694 alone for i.v x 1 and daily x 5 schedules, respectively. However, preadministration of MPEG-TPase did not potentiate the antitumor activity of ZD1694 with either schedule. The data indicate that the study of TS inhibitors in rodent models may not be suitable for predicting a safe dose for clinical study. However, rodent models, particularly human tumor xenografts, are still useful models for evaluation of antitumor activity and schedule selection for TS inhibitors.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ZD1694 is a quinazoline antifolate that is a highly specific TS inhibitor (7) . Cellular uptake of ZD1694 is via the reduced-folate carrier and it subsequently undergoes rapid and extensive intracellular polyglutamylation (8) . Polyglutamylation increases drug potency and intracellular retention, which allows a convenient infrequent dosing schedule (8) . Polyglutamylation also occurs in various tissues, resulting in a high tissue:plasma drug ratio (8) .
In vitro studies showed ZD1694 to be a potent inhibitor of the growth of both mouse and human tumor cell lines, with an IC50 in the 1–10 nM range for continuous exposure (8) . This is equivalent in potency to methotrexate, but 94- and 56-fold more potent than FUra alone and FUra plus 10 µM LV, respectively (8) . In vivo studies with ZD1694 showed it was curative in mice bearing L5178Y TK -/- lymphoma and L1210:ICR ascitic tumors (8 , 9) . It was also active against human colon, gastric, lung, and ovarian cancers and was superior to FUra and methotrexate against these xenografts (10) .
Clinical trials with ZD1694 demonstrated that it is active in a number of solid tumors, including colorectal (11, 12, 13, 14, 15) , breast (11 , 16) , head and neck (17) , ovarian (11) , non-small-cell lung (11) , and pancreatic (11) cancers. ZD1694 is currently being investigated in Phase I and II studies in patients with hormone-resistant prostate cancer, soft tissue sarcoma, and pediatric and adult leukemias (15) . ZD1694 had similar efficacy to FUra/LV therapy, with a 25–30% response rate in patients with advanced colorectal cancer, but induced less mucositis and leucopenia. In addition, patients who received ZD1694 spent a substantially shorter time in the hospital for drug administration than those who received FUra/LV (13, 14, 15) . ZD1694 treatment also offered more frequent palliative benefits and a more convenient administration schedule (13, 14, 15) . ZD1694 is now available in more than 10 countries for first-line treatment of advanced colorectal cancer (15) . In a Phase II study of advanced breast cancer, ZD1694 achieved a 26% overall response rate and a 44% response rate in patients with measurable liver lesions (16) .
Cytotoxicity of ZD1694 is completely reversed by coincubation with dThd or LV in vitro (9
, 18
, 19)
. The toxicities and antitumor activity of ZD1694 can be abrogated by coadministration of dThd or LV in vivo (9
, 20)
. These results demonstrate that TS is the locus of action of ZD1694 for both antitumor effect and toxicity. High circulating levels of dThd in rodents relative to humans (1.3 and <0.1 µM, respectively) may complicate the study of toxicity and antitumor activity of TS inhibitors (7
, 21)
. Wilson et al. (22)
reported that administration of 2500 units/kg of MPEG-TPase could reduce mouse plasma dThd levels from 1–2 µM to undetectable levels (<0.2 µM); coadministration of MPEG-TPase with 1843U89, a benzoquinazoline TS inhibitor, potentiated the in vivo activity of 1843U89 in a tumor-specific manner by reduction of dThd.
The principal objectives of this study were to determine: (a) the antitumor efficacy of ZD1694 against human HNSCC established in nude mice; (b) the role of the dosing schedule of ZD1694; and (c) the effect of plasma dThd on the antitumor activity and toxicity of ZD1694 in nude mice. The human HNSCC A253 and FaDu models in athymic nude mice were used to test the antitumor efficacy and determine the role of the dosing schedule of ZD1694 with three clinically relevant schedules [single i.v. push (i.v. x 1) versus i.v. push once a week for 3 weeks (weekly x 3) versus i.v. push once a day for 5 days (daily x 5)] and to compare the effect of ZD1694 with FUra and FdUrd on the weekly schedule. We also investigated the antitumor activity and toxicity of ZD1694 after preadministration of MPEG-TPase in the A253 model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice.
Female athymic nude mice (nu/nu, body weight 20–25 g), 8–12 weeks of age, were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were housed five mice/cage under specific pathogen-free conditions with water and food ad libitum, according to an institutionally approved protocol.
Tumors.
The HNSCC cell lines A253 and FaDu were purchased from American Type Culture Collection (Manassas, VA) and maintained as a monolayer in RPMI 1640 supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA). Xenografts were initially established by implanting s.c. 106 cultured A253 or FaDu cells and passed several generations by transplanting 50 mg nonnecrotic tumor tissues before treatment.
Preparation of MPEG-TPase.
MPEG-TPase was prepared essentially as described (22
, 23)
. Briefly, TPase (Sigma T-3678; 12,500 units, 370 mg total protein), supplied in 0.5 M K-phosphate (pH 7.0), 2 mM uracil, 0.02% NaN3, and 2.5% (w/v) BSA was dialyzed for 24 h at 4° against 150 mM Na-phosphate (pH 7.0), containing 2.5 mM thymine, with three changes of 1 liter each. To the dialyzed TPase, solution (30.5 ml) was added slowly with stirring, 3.5 g of solid MPEG-succinimidyl succinate (Sigma M3152; 75% pure, molecular weight (MW) 5000). The mixture was allowed to stir at 4° for 3.5 h, and the reaction was quenched by the addition of 0.4 ml of 1 M glycine-NaOH (pH 7.0) and further stirring for 30 min. The MPEG-TPase was dialyzed for 24 h at 4° against 10 mM K-phosphate (pH 7.0), with three changes of 1 liter each, and concentrated by nitrogen pressure over an Amicon PM-30 membrane to 20.5 ml. This material was flash-frozen in dry-ice acetone and stored below -80°, where it was stable for months. Overall recovery of activity was about 40%, and no unmodified protein was observed on SDS-PAGE analysis (data not shown).
TPase activity was measured spectrophotometrically (24) ; one unit of activity hydrolyzes 1 µM TdR/min. Protein concentrations were determined colorimetrically using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), according to manufacturer’s instructions, using BSA as the standard.
Drug Solutions.
ZD1694 was dissolved in sterile saline, and the solution was adjusted to pH 7.4 with NaOH immediately before use.
Drug Doses and Schedules.
ZD1694 was administered by i.v. injection via the tail vein of animals using three schedules: (a) i.v. x 1 (day 0); (b) weekly x 3 (days 0, 7, and 14); and (c) daily x 5 (days 0–4). Two schedules were used for experiments with ZD1964 plus MPEG-TPase: (a) with daily x 5 of ZD1694, MPEG-TPase (2500 units/kg) was administered by i.p. injection 1 h before the first, third, and fifth doses of ZD1694 (three doses in total); and (b) with the single dose of ZD1694, MPEG-TPase (2500 units/kg) was administered by a single i. p. injection 1 h before ZD1694.
MTD and Toxicity Evaluation.
The MTD was defined as the maximum dose that caused no drug-related lethality and produced <20% loss of initial animal weight. Drug-induced toxicities, including body weight loss and lethality, were determined daily for a minimum of 3 weeks after treatment.
Tumor Measurement.
Two axes of the tumor (L, longest axis; W, shortest axis) were measured with a Vernier caliper. Tumor weight (mg) was calculated as: 
(L x W2) (mm).
Relative tumor volume (%) was calculated by actual tumor weight (ATW) over initial tumor weight (ITW, day 0) as following: ATW ÷ ITW x 100%.
Measurements were taken once a day during the first 10 days and two to three times a week thereafter.
Antitumor Activity.
Drug treatments were initiated 7–8 days after tumor transplantation when tumor weight was 
200–250 mg, as described previously (25)
. Antitumor activity was assessed by TGI, which is mean tumor weight (MTW) of the treated group (TG) relative to the untreated control group (CG) on day 12, as calculated as: (MTWTG –MTWCG) ÷ MTWCG x 100%. The TDT was defined as the mean time for the tumor to reach twice its initial weight (at treatment beginning, day 0). Tumor response was defined as PR, when tumor weight was temporarily reduced by at least 50%, and CR, when tumor was undetectable by palpation for 90 days after treatment, at which time the mouse was sacrificed. The response rate was expressed as a percentage of animals in the group. In general, tumor in mice with PRs regrew within 2 weeks after therapy. However, tumors rarely (<5%) regrew after CRs occurred (cure). As a general policy, animals were sacrificed when the tumor weight exceeded 2000 mg. Each experimental group had five mice, and each experiment was repeated at least once.
Statistical Analysis.
Differences between the mean values were analyzed for significance using the unpaired two-tailed Student’s t test for independent samples; P 
0.05 was considered to be statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We also compared i.v. to i.p. injection; the MTD of i.p. ZD1694 was the same as for i.v. injection with single and weekly x 3 schedules, but the MTD for i.p. ZD1694 was lower (20 mg/kg/day) with the daily x 5 schedule. In general, lethality was observed in a significant number of animals when body weight loss was >20% of their original weight.
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summarizes the antitumor activity and toxicity of ZD1694 with three different schedules at various doses ( MTD). ZD1694 at the MTD produced a 20% CR and a 20% PR with i.v. x 1 or weekly x 3 schedules, and 12-day tumor growth delay (Fig. 2)
with the daily x 5 schedule against FaDu xenografts. ZD1694 did not achieve CR against A253; however, 20% PR, 40% PR, and a 10-day tumor growth delay (Fig. 2)
were observed with i.v. x 1, weekly x 3, and daily x 5, respectively. Of note, ZD1694 was still active at doses lower than the MTD against both tumors (Table 1)
, demonstrating its significant chemotherapeutic index and safety margin. The tumor growth inhibitory curves and the animals’ body weight loss produced by ZD1694 at the MTD with three different schedules are illustrated in Fig. 2
. The data clearly demonstrated that single and weekly schedules were more active and less toxic than the daily schedule (Fig. 2)
. ZD1694 administered i.p. also showed a similar pattern of antitumor activity and toxicity with the three schedules (data not shown).
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showed that the antitumor activity of ZD1694 was comparable with FUra, but superior to FdUrd, against A253 xenografts and was more active than both FUra and FdUrd, in terms of tumor regression against FaDu xenografts. The animals experienced less toxicity (as weight loss) when treated with ZD1694 than with FUra or FdUrd at the MTD (Table 2)
. However, ZD1694 showed wider deviation of tumor response in individual animals than did FUra and FdUrd in both models (Fig. 3)
.
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. However, MPEG-TPase did not alter the pattern of antitumor activity or toxicity of ZD1694; TGI, TDT, and tumor response and weight loss at the MTD of ZD1694, with or without MPEG-TPase, were comparable (P > 0.05;Fig. 5
and Table 3
).
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and Table 4
shows no significant difference in TGI, TDT, and tumor response between ZD1694 plus MPEG-TPase and ZD1694 alone (P > 0.05; previous experiments with ZD1694 alone were excluded). The data indicate MPEG-TPase did not augment the antitumor activity of ZD1694 at an equitoxic dose, but a 3-fold dose reduction was evidenced.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ZD1694 is a direct and specific TS inhibitor that lacks the nonspecific effects on RNA and protein synthesis of FUra; it was hypothesized that ZD1694 would have similar antitumor efficacy, but fewer side effects than FUra (28, 29, 30, 31)
. Indeed, in clinical Phase III trials of patients with advanced colorectal cancer, ZD1694 showed similar efficacy compared with FUra/LV therapy, but induced less toxicities and improved palliative benefits (13, 14, 15)
. Our data herein also demonstrate that ZD1694 had a better therapeutic index than FUra and FdUrd in human HNSCC models (Table 2)
.
Earlier preclinical studies demonstrated that ZD1694 is active against a range of human solid tumors including colon, ovarian, lung, gastric, bladder, and breast xenografts (8)
. Preliminary data from Phase I trials showed ZD1696 was moderately active in patients with head and neck cancer; two of five patients achieved minor responses (15)
. ZD1694 as monotherapy with or without radiotherapy in patients with head and neck cancer has entered a Phase II trial in Europe and the United States (32
, 33) . To our knowledge, no preclinical in vivo data with ZD1694 against head and neck tumor has been reported to date, although it was active against a wide panel of murine and human tumors (8, 9, 10)
. Our data indicate that ZD1694 is moderately active against human HNSCC FaDu and A253 xenografts in a schedule-dependent manner (Table 1
and Fig. 2
). Of interest and potential clinical importance was the finding that ZD1694, unlike FUra with which optimal antitumor activity is achieved only with doses at or near the MTD (34)
, was active at doses lower than the MTD, particularly with weekly and single schedules (Table 1)
. The data indicate that ZD1694 had a large chemotherapeutic index and safety margin and may not need to be given at the MTD in the clinic. The comparative study of ZD1694 with FUra and FdUrd showed the antitumor activity of ZD1694 was similar to FUra, but superior to FdUrd, against A253 and was more active than both FUra and FdUrd, in terms of CR against FaDu with less toxicity (Table 2)
. However, ZD1694 showed wider deviation of tumor response in an individual animal than FUra and FdUrd in both models (Fig. 3)
; this may relate to different individual plasma dThd levels. The in vivo data also indicate that A253 is slightly less sensitive than FaDu to ZD1694 (Table 1
and Fig. 2
); this is consistent with early findings with the same tumor cell lines in vitro (18)
. Because ZD1694 is not highly active in these models, to achieve higher response rate, it may need to be combined with other chemotherapeutic agents with different mechanisms of action, such as a topoisomerase I inhibitors (CPT-11), platinum compounds (cisplatin, carboplatin), and/or radiotherapy. The study of antitumor efficacy and role of sequence of ZD1694 in combination with CPT-11 or cisplatin is under way in this laboratory.
The data presented herein demonstrated that the antitumor activity and toxicity of ZD1694 was highly schedule-dependent. Single-dose and weekly schedules were more active and less toxic than the daily schedule (Table 1
and Fig. 2
). Higher toxicity was observed with the daily x 5 schedule, probably from rapid drug accumulation and retention in mouse tissues due to extensive polyglutamylation, resulting in high drug levels (50–100-fold higher than plasma) associated with prolonged TS inhibition in tissues (8)
. This confirms the argument that frequent dosing schedules with ZD1694 may produce unacceptable toxicity (8)
. Unlike drugs with different mechanisms of action, such as CPT-11, with which higher antitumor activity is usually associated with prolonged drug administration (35)
, ZD1694 showed higher antitumor efficacy with an infrequent dosing schedule (single or weekly). A possible explanation is that ZD1694 was rapidly and efficiently polyglutamylated with resultant high intracellular drug levels and more potent, prolonged inhibition of TS (36)
. However, other mechanisms must be involved because the dose difference in MTD is small between the weekly and the daily schedule (60 versus 30 mg/kg). More importantly, with the weekly schedule, lower drug doses (20–40 mg/kg) produced higher antitumor activity than the daily schedule at the MTD (Table 1)
. We hypothesize that host immune function may be decreased with the daily schedule because of high toxicity. The mechanism of action of ZD1694 with different schedules should be further investigated. Nevertheless, these data demonstrate that current clinical trials with infrequent administration of ZD1694 are appropriate.
It was believed that the high level of plasma dThd in mouse relative to human might affect the toxicity and antitumor activity of TS inhibitors (8
, 21)
. Indirect supporting evidence was that the cytotoxicity of ZD1694 was completely reversed by coincubation with dThd in vitro (9
, 18
, 19)
and the toxicity and antitumor activity of ZD1694 can be abrogated by coadministration of dThd in vivo (9
, 20)
. We preadministered MPEG-TPase to reduce plasma dThd in the A253 xenograft model, then treated with various doses of ZD1694. The results showed that MPEG-TPase significantly increased the toxicity of ZD1694, produced a 3–10-fold dose reduction in MTD with the two tested schedules (Fig. 4
; Tables 3
and 4
). However, MPEG-TPase did not potentiate the antitumor activity of ZD1694 (Tables 3
and 4
; Figs. 5
and 6
). The data indicate that low plasma dThd would profoundly affect toxicity, but has little effect on the antitumor activity of ZD1694. We agree that the MTD of a TS inhibitor obtained from rodent models should not to be used to predict a safe dose for clinical Phase I study. It is better to choose other models with low plasma dThd level similar to humans, such as dogs, to determine the clinical starting dose. However, we believe that rodent models, particularly human tumor xenografts, are still useful models for predicting the outcome of antitumor activity of TS inhibitors and for schedule selection for clinical trails.
In conclusion, these studies with human HNSCC models show ZD1694 was moderately active against human HNSCC xenografts. Antitumor efficacy of ZD1694 is comparable with or superior to FUra and FdUrd, with a better chemotherapeutic index and safety margin in these model systems. The antitumor efficacy and toxicity of ZD1694 is schedule-dependent. It is less active and more toxic with frequent daily dosing. The data provide direct evidence for the rationale of current clinical studies with ZD1694. Animal models with high plasma dThd are not the most suitable models for assessment of the toxicity of and for selecting a safe dose for clinical trial of TS inhibitors. However, these models are useful for in vivo study of antitumor activity and schedule selection with TS inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants CA65761, CA65755, and Institute Core Grant CA16056 from the National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-1638; Fax: (716) 845-8857; E-mail: scao{at}sc3101.med.buffalo.edu
3 The abbreviations used are: FUra, 5-fluorouracil; HNSCC, head and neck squamous cell carcinoma; TS, thymidylate synthase; ZD1694, tomudex (raltitrexed); FdUrd, 5-fluoro-2'-deoxyuridine; dThd, thymidine; MPEG-TPase, methoxypolyethyleneglycol-conjugated thymidine phosphorylase; CR, complete tumor regression; PR, partial tumor regression; TGI, tumor growth inhibition; TDT, tumor doubling time; MTD, maximum tolerated dose; MWL, maximum weight loss; LV, leucovorin.
4 S. Cao and Y. M. Rustum, unpublished data.
Received 3/11/99; revised 4/14/99; accepted 4/16/99.
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-methylene-10-deazaaminopterin is not essential for antitumor activity. Clin. Cancer Res., 2: 707-712, 1996.[Abstract]
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, i.v. x 1 (day 0); 
, weekly x 3 (days 0, 7, and 14); •, daily x 5 (days 0–4). Animals were observed for up to 3 months after treatment. Each group represents 10–30 animals of two to six independent experiments.
, control; •, 300 mg/kg ZD1694, i.v. x 1 (day 0); 
, ZD1694 + MPEG-TPase. MPEG-TPase was administered at 2500 units/kg i.p. 1 h before the first, third, and fifth doses of ZD1694 in a total of three doses. Each group represents 10–20 animals of two to four independent experiments.
, 30 mg/kg ZD1694 (MTD); 