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Cancer Letters, 49 (1990) 133- 137 Elsevier Scientific Publishers Ireland Ltd.

Antitumor activity of brequinar sodium (Dup-785) against human head and neck squamous cell carcinoma xenografts B.J.M. Braakhuisa, G.B. Snowa “Departments Boelelaan

G.A.M.S.

of Otolayngology

11 I 7, 1081

van Dongen”,

and Head

HV Amsterdam

G.J.

Petersb, M. van Walsum” and

and Neck Surgery and bMedical Oncology,

Free University Hospital,

de

(The Netherlands)

(Received 31 July 1989) (Accepted 14 September 1989)

Summary

Introduction

The effect of Brequinar sodium on the growth of xenografts established from head and neck squamous cell carcinomas (HNSCC) was assessed. Brequinar sodium is a novel drug, known to inhibit dihydroorotic acid dehydrogenase (DHO-DH), resulting in a

Most patients with advanced squamous cell carcinoma of the head and neck (HNSCC) cannot be cured by surgery and/or radiotherapy. The rationale to include chemotherapy in the combined modality treatment of these patients is clear. Although responses to chemotherapy are often seen, as with the cisplatin/5-fluorouracil schedule [9], randomized clinical trials do not indicate any consistent improvement in survival for patients receiving chemotherapy in addition to local treatment as compared with patients receiving local treatment alone [15,16]. The need for new active drugs for HNSCC remains. The recent failure of new drugs in phase II HNSCC clinical trials made us decide to use an in vivo model as a preclinical screening system. Nude mice bearing human tumor xenografts represent a system with high clinical predictability [18]. Generally, there is a good correlation of drug effectiveness in the nude mouse with clinical results in the donating patient [7,8]. So new drugs, already selected in the in vitro primary screening, can be evaluated in this model in order to decide which drug and/or schedule to designate for phase II HNSCC trials. A drug selected in this approach was Brequinar

decrease of the pyrimidine de novo synthesis. The drug was administered i.p. to tumor-bearing nude mice, once a day, during 5 days at a

maximum tolerated dose of 50 mg/kg/day. Statistically significant growth delaying effects were observed in 4 out of 5 lines tested. In 3 of these lines the effect was moderate and short lasting, whereas in one line (HNX-LP) tumor growth rate was totally inhibited for a 17-day period. In this line, Brequinar sodium was superior to 5 drugs known to be active in HNSCC patients. In two tumor lines DHO-DH activity could be measured and the results are in agreement with the concept that there is a relation between Brequinar sodium sensitivity and enzyme activity.

Keywords: brequinar sodium; head and neck cancer; pyrimidine de novo; squamous cell carcinoma; xenografts. 0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland

Ireland I_td

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sodium (or Dup-785) that was undergoing a phase I evaluation at the time of start of the experiments [ 141. Brequinar sodium is an inhibitor of the de novo ,pyrimidine synthesis by inhibition of the enzyme dihydroorotic acid dehydrogenase (DHO-DH) [4,10]. In murine and xenograft models Brequinar sodium has shown good activity [6]. Because of the urgent need for new active agents for HNSCC, we have decided to test Brequinar sodium in this xenograft model. Another rationale to study this drug is that - to our knowledge - little is known about the activity of de novo pyrimidine synthesis inhibitors in HNSCC, neither from clinical nor from preclinical studies. We decided to use a schedule with daily injections during 5 days, because in vitro data indicate that prolonged exposure to the drug is necessary to produce long lasting depletion of pyrimidine nucleotide pools [13]. Also in vivo data show an increase in therapeutic activity when the drug is given more frequently [6]. Materials and methods

Animals and tumors Female nude mice (NMRI, 8-10 weeks old) were obtained from Harlan Olac CPB, Zeist, the Netherlands. The conditions under which the mice are kept were reported elsewhere [Z]. Characteristics of tumor lines are shown in Table 1. The tumor lines HNX-LP and PI were established from tumor material

obtained from previously untreated patients referred to our hospital. HNX-HEp-2, 14C and 14A were derived from established cell lines growing as monolayer cultures. The lines designated HNX-14C and 14A were initiated from the UM-SCC-14C and 14A cell lines, which were kindly provided by Dr. T. Carey, Ann Arbor, U.S.A., and characterized by Roa et al. [12]. The HEp-2 cell line was obtained from Flow Laboratories, Herts, United Kingdom. Serial transplantation of tumor lines was performed by inserting slices measuring approximately 3 x 1 x 1 mm S.C. in the lateral thoracic region on both sides of the animal. Tumor volume in mm3 was measured biweekly using vernier calipers and calculated according to the formula: length x width X height x 0.5. Chemotherapy

Treatment was started when the tumor reached a volume between 50 and 150 mm3. The tumors were randomly divided into treatment and control groups (5-8 tumors/ group). All drugs were injected at a maximum tolerated dose level, corresponding to a weight loss between 5 and 15%. An antitumor effect was expressed in terms of a growth delay factor (GDF). GDF is defined as the difference between the mean values of the time required by tumors of treated and control animals to double their volume, divided by the mean value of the time required by the tumors of control mice to double their volume. Differ-

Tablel. Characteristics of HNSCC tumor lines used for chemotherapy studies. Designation

Histology

Site

HNX-LP HNX-PI

Mod. diff. see Undiff. ca.

HNX-HEp-2 HNX-14A HNX-14C

Poorly diff. see Poorly diff. see Poorly diff. see

Oral cavity Metastasis, unknown primary Larynx Oral cavity Metastasis, Oral cavity

Mod. diff. see,moderately differentiated squamous cell carcinoma; Ca, carcinoma.

Doubling time (days)

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ences in growth rate between tumors from treated and control animals were analyzed for a statistical difference with the Student’s t-test. DHO-DH assay Enzyme activity was determined as described by Peters et al. [ll]. Tumors from untreated animals were homogenized by using a Potter-Elvehjem apparatus and mitochondria were isolated by differential centrifugation. Enzyme activity was determined at 37 OC in the presence of 158 FM L-DHO. Inhibition of DHO-DH activity was measured by performing the reaction in the presence of 1.3 PM Brequinar sodium. Drugs Bleomycin was obtained from Lundbeck, Amsterdam, the Netherlands, cisplatin from Bristol Myers, Weesp, the Netherlands, methotrexate from Lederle, Etten-Leur, the Netherlands, 5-fluorouracil from HoffmanLa-Roche, Hoofddorp, The Netherlands and cyclophosphamide from Farmitalia, Bournonville-pharma, Almere, the Netherlands. Brequinar sodium was donated by Dr. M. Barbu, Du Pont De Nemours, Geneva, Switzerland. For all drugs, formulations prepared for patients were used. The drugs were dissolved and stored as indicated by the manufacturers. Results and discussion Nude mice bearing HNSCC xenografts were treated intraperitoneally with Brequinar sodium at a dose of 50 mg/kg/day during five days. Treatment with this schedule did not cause toxicity related deaths and led to a maximal weight loss of 7% on the seventh day after the start of treatment (Fig. 1). Brequinar sodium was significantly active in 4 out of 5 HNSCC tumor lines (Student’s t-test, P < 0.05, Table 2), with GDF-values varying from 0.6 to 2.3. In the HNX-LP line with the highest GDF-value of 2.3, growth was inhibited for a period of 17 days (Fig. 1). The activity of Brequinar sodium was com-

Relative tumor volume

Animal weight (%I

Days after start therapy

M

Control

M

Brequinar sodium

Effect of Brequinar sodium treatment on tumor Fig. 1. growth and animal weight. Tumor volumes are plotted semilogarithmically and related to the initial volume (mean (n = 6) + S.E.). A single arrow indicates an i.p. injection with 50 mg/kg Brequinar sodium. The weight curves constructed are based on data derived from treated and untreated animals from all experiments reported here. Error bars are omitted because they were less than 5%.

pared with drugs, known to be clinically active against HNSCC. Mice were treated with equitoxic doses of methotrexate, bleomycin, cisplatin, 5-fluorouracil and cyclophosphamide. When a GDF-value exceeding 1 was taken as criterion [17], response rates between 50 and 80% were found for almost all drugs, Brequinar sodium included. Additionally, cisplatin and cyclophosphamide had the capacity to

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Table 2.

Efficacy of anticancer

Drug

Bleomycin Brequinar Sodium Cisplatin Cyclophosphamide Methotrexate 5-Fluorouracil

drugs in

HNSCCxenograft

lines.

GDF no. in tumor lines

HNX-LP

HNX-PI

HNX-HEpZ

HNX-14A

HNX- 14C

1.4 2.3 0.4 0.6 0.3 0.7 -

1.7 1.2 CR” CR*** 0.0 -1.7

-0.9 0.6 0.3 1.2 0.4 -1.0

1.3 0.2 1.2 n.d. 0.3 0.5

-2.0 1.0 CR’ 0.4 0.1 -1.2

Underlined values mean significant difference (Student’s t-test, P < 0.05) in tumor volume doubling times between tumors from treated and control animals. Schedules: Bleomycin 15 mg/kg, day O-4; cisplatin 5 mg/kg day 0, 7, 14; cyclophosphamide 100 mg/kg, day 0, 7; methotrexate 125 mg/kg day 0, 7; 5fluorouracil50 mg/kg, day 0, 7; Brequinar sodium 50 mg/kg day O-4. All drugs were injected i.p. with the exception of cisplatin, which was given i.v. CR’, 2/7 tumors showed regression without regrowth; CR’ * , 3/6 tumors showed regression without regrowth; CR’ ’ , 6/7 tumors showed regression without regrowth; GDF no., growth delay factor = TDt - TDc/TDc (TD = tumor volume doubling time, t = treated, c = control). N.D., not determined. l

cause complete tumour regressions. Methotrexate was hardly active in HNSCC tumor lines, a result that was discussed in earlier reports [ 1,3]. For Brequinar sodium we observed an interesting sensitivity profile in the line HNX-LP. In this line, Brequinar sodium showed a superior activity in comparison to all other drugs tested. However, from the same panel of drugs tested on the HNX-PI tumour line, Brequinar sodium was the least active one. In an attempt to elucidate the background of the differences in intrinsic sensitivity to Brequinar sodium, we were able to measure DHODH activity in xenografts derived from the HNX-LP and -14C lines, grown in control animals from treatment experiments. Baseline activity was lowest in the most sensitive (HNXLP) line: 84 f 27 nmol/h/mg protein (mean f SD., n = 4). For HNX-14C this was 123 f 41 (n = 3). In the presence of 1.3 FM Brequinar sodium enzyme activity was 11 f 4.8 and 20 + 13.0 nmol/h/mg protein, respectively. So, the line with the lower baseline and residual DHO-DH activity was more sensitive to Brequinar sodium. Although it is hard to draw generalized conclusion from these data,

they are in line- with previous reported in vitro data [5] and also with in vivo data, obtained in murine colon tumors, where the Brequinar sodium sensitive tumor was as responsive as the HNX-LP tumor, reported here [ 111. These findings support the concept that there is a relation between cytotoxicity and activity of DHO-DH, the target enzyme known to be inhibited by the drug [4, lo]. At this moment a phase II trial with Brequinar sodium in HNSCC is ongoing, activated by the Early Clinical Trial Group of the EOBTC. The drug is administered i.v. weekly at a dose of 2000 mg/m2. It is not yet known what the results from this trial will be. It has to be considered that clinical trials with schedules employing more frequent administrations can also be successful. Our results indicate that frequent administrations can lead to responses, confirming the results with colon xenografts [6]. Moreover, in vitro data indicate that prolonged exposure to the drug is necessary to produce long lasting depletion of pyrimidine nucleotide pools [ 131. In conclusion, Brequinar sodium, administered daily for 5 days, is a drug with activity in HNSCC xenografts.

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References Braakhuis, B.J.M, Schoevers, E.J. Heinerman, Sneeuwloper G. and Snow G.B. (1983) Chemotherapy of human head and neck cancer xenografts with three clinically active drugs: cis-platinum, bleomycin and methotrexate. Br. J.

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and Loehr, G.W. (1984) Development and characterization of 51 tumour models for large bowel, stomach and esophageal cancers. Dig. Surg. 1.225-235. Mattern, J. and Volm, M. (1988) Human tumor xenografts as model for drug testing. Cancer Metast. Rev., 7, 263281. Mercier, R.J., Neal G.D., Mattox, D.E., Gates, G.A. Pomeroy, T.C. and Vonhoff, D.D. (1987) Cisplatin and 5-

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flurouracil chemotherapy in advanced or recurrent squamous cell carcinoma of the head and neck. Cancer, 60, 2609-2612. Peters, G.J., Sharma, E., Laurensse, E. and Pinedo, H.M. (1987) Inhibition of pyrimidine de novo synthesis by DUP 785 (NSC 368390). Invest. New Drugs, 5, 235244. Peters, G.J., Nadal, J.C., Laurensse, E., de Kant E. and Pinedo, H.M. (1989) Retention of in vivo antipyrimidine effects of Brequinar sodium (DUP-785, NSC 363390) in murine liver, bone marrow and colon cancer. Biochem. Pharmacol., in press. Roa, A., Carey, T.E., Passamani, P.P, Greenwood, J.H., Hsu, S., Ridings, O., Schwartz, D.R., Wolf, G.T. and Hudson, J.L. (1985) DNA content of human squamous cell carcinoma cell lines. Arch. Otolaryngol. 111, 565575. Schwartsmann G., Peters, G.J., Laurensse, E., de Waal, F.C., Loonen, A.H., Leyva, A. and Pinedo, H.M. (1988) DUP 785 (NSC 368390): schedule-dependency of growth-inhibitory and antipyrimidine effects. Biochem. Pharmacol., 37,3257-3266. Schwartsmann G., Van der Vijgh, W. J.F., Klein, I., Van Groeningen, C.J., Vermorken, J.B. and Pinedo, H.M. (1988) Pharmocokinetics of Brequinar sodium (NSC 368390; DUP 785) in cerebrospinal fluid. Eur. J. Cancer Clin. Oncol. 24,1903-1904. Snow, G.B. and Vermorken J.B. (1989) Neo-adjuvant chemotherapy in head and neck cancer, state of the art 1988. Clin. Otolaryngol., in press. Tannock, I.F. and Browman, G. (1986) Lack of evidence for a role of chemotherapy in the routine management of locally advanced head and neck cancer. J. Clin. Oncol. 4: 1121-1126. Van Dongen, G.A.M.S., Braakhuis, B.J.M., Leyva, A., Hendriks, H.R., Kipp, B.B.A., Bagnay, M. and Snow, G.B. (1989) Anti-tumor and differentiation-inducing activity of N,N-dimethylformamide (DMF) in head-and-neck cancer xenografts. Int. J. Cancer 43.285-292. Winograd, B., Boven, E., Lobbezoo, M.W. and Pinedo, H.M. (1987) Tumor xenografts in the nude mice and their value as test models in anticancer drug development. In Vivo, 1, l-14.

Antitumor activity of brequinar sodium (Dup-785) against human head and neck squamous cell carcinoma xenografts.

The effect of Brequinar sodium on the growth of xenografts established from head and neck squamous cell carcinomas (HNSCC) was assessed. Brequinar sod...
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