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Leukemia cell lines resistant to cytosine arabinoside, cyclophosphamide or daunomycin are equally sensitive to the drug acetyldinaline as the parental cell line

HM El-Beltagi, ACM Martens & A Hagenbeek

University Medical Center Utrecht, Jordan Laboratory/Hemato Oncology, Utrecht, The Netherlands

Correspondence to: A Hagenbeek, UMCU, Jordan Laboratory/Hemato Oncology G03-647, PO Box 85500, 3508 GA Utrecht, The Netherlands; Fax: 31-30-251-1893

Acetyldinaline (ACD) (CI-994, GOë 5549, 4-acetylamine-N-(2'-aminophenyl)-benzamide) is the active and predominant metabolite of the parent compound dinaline (GOë 1734, 4-amino-N (2'-aminophenyl)-benzamide). Both compounds revealed a marked cytostatic and immuno-suppressive capacity in various animal tumor models that are highly refractory to commonly used anticancer agents.¹ In addition, ACD showed a high anti-leukemia effect in the BNML rat acute myelocytic leukemia model,² while at the same time ACD has only minor toxicity to normal pluripotent hemopoietic stem cells (HSC) as we have previously shown.³ ACD is currently being evaluated in phase 1 clinical trials.

Despite successful remission induction and consolidation treatment of acute leukemia with cytostatic agents, the majority of AML cases eventually develop a leukemia relapse. This implies that the leukemic cells have either developed drug resistance or that they already had a natural resistance and were selected out by the treatment.⁴

Here, we report on the absence of cross-resistance between ACD and daunomycin (Dauno), cytosine arabinoside (Ara-C) or cyclophosphamide (CP) using resistant sublines for each of these drugs in the BNML leukemia model in the rat.^5,6,7 Normal BN/HsdRij rats (SPF quality) were injected on day 0 with 1  10⁷ cells i.v. from either the BNML parental cell line BNML/P or with the BNML/Dauno-R, BNML/CP-R or BNML/Ara-C-R sublines. ACD treatment was started on day 7 after leukemia transfer for the BNML/CPR, BNML/Ara-C-R and the BNML/P groups and on day 5 for the BNML/Ara-C-R group because this line showed slightly more rapid growth kinetics. Thus, the leukemic cell load was comparable for each of the drug-resistant BNML variants. ACD was administered intragastrically, as a suspension in methyl cellulose, in a dose of 11.85 mg/kg/day for 5 consecutive days. For each resistant BNML subtype an untreated group of rats carrying the same cell line served as control. All experimental groups consisted of eight rats. To calculate the anti-leukemic effect of ACD the increase in survival time (IST) of treated animals vs that of the controls was determined. The ISTs for each of the treatment groups was compared with that of the corresponding non-treated controls group. Based on the known relationship between cell load reduction and increase in survival time for each BNML subline the LCK induced by drug treatment can be deduced. Finally, the effect of ACD on the resistant cell line was compared in terms of LCK, with the efficacy of ACD in the parental BNML line. The survival curves for ACD treatment of the three BNML-resistant sublines and that of the parental BNML/P cell line are shown in Figure 1. ACD treatment of rats carrying the BNML/P cell line resulted in an IST with 18 days (median survival time, MdST 45 days) compared to the non-treated control rats (MdST 27 days). ACD treatment group of rats injected with the BNML/Dauno-R subline resulted in an IST of 14 days (MdST 25 days) compared with the corresponding non-treated control group (MdST 11 days). ACD treatment of rats injected with the BNML/Ara-C-R subline resulted in an IST of 15 days (MdST 28 days) compared with the corresponding non-treated control group (MdST 13 days). ACD treatment of rats carrying the BNML/CP-R cell line resulted in an IST of 12 days (MdST 28 days) compared with the corresponding non-treated control group (MdST 16 days). In all groups all rats died from leukemia relapse. In Table 1 the leukemic cell load reducing effects of a single course of ACD treatment for each of the resistant sublines and for the BNML/P cell line are listed. For the BNML/P the 18 days increase in the survival time reflects a LCK of 4.5; for the BNML/Dauno-R the calculated LCK is 4.7; for the BNML-Ara-C-R 5.0 and for the BNML-CP-R line 4.0, respectively.

These results show that the sublines of the BNML rat leukemia model that acquired resistance for either Dauno, Ara-C or CP during repeated in vivo administration of the respective drugs retained the sensitivity for ACD. During the treatment of AML the leukemic cells often acquire resistance to the frequently used drugs Dauno, Ara-C or CP. The exact mode of action of ACD is not yet known although there are reports that it affects intracellular amino acid transport thus interfering with protein metabolism, DNA synthesis and cell proliferation.⁸ On the basis of the preclinical study reported here it is concluded that the mechanisms involved in the resistance to Dauno, Ara-C and CP, do not effect the susceptibility of the resistant cells for ACD. Clinical evaluation of ACD in acute leukemia, especially in leukemia patients resistant to those cytostatic drugs, is warranted.

²  El-Beltagi HM, Martens ACM, Dahab GM, Hagenbeek A Efficacy of acetyldinaline for treatment of minimal residual disease (MRD) – preclinical studies in the BNML rat model for human acute myelocytic leukemia Leukemia 1993 7: 1795–1800 MEDLINE

³  El-Beltagi HM, Martens AC, Lelieveld P, Haroun EA, Hagenbeek A Acetyldinaline: a new oral cytostatic drug with impressive differential activity against leukemic cells and normal stem cells. Preclinical studies in a relevant rat model for human acute myelocytic leukemia Cancer Res 1993 53: 3008–3014 MEDLINE

⁴  Skipper HE Some thoughts on intrinsic versus acquired drug resistance in cancers that are classified as responsive, refractory or very refractory to chemotherapy. Booklet 10 Southern Research Institute: Birmingham, AL 1986

⁵  Martens ACM, Van Bekkum DW, Hagenbeek A The BN acute myelocytic leukemia (BNML) (a rat model for studying human acute myelocytic leukemia (AML)) (review) Leukemia 1990 4: 241–257 MEDLINE

⁶  Hagenbeek A, Martens AC, Colly LP In vivo development of cytosine arabinoside resistance in the BN acute myelocytic leukemia Semin Oncol 1987 14 (2 Suppl. 1): 202–206

⁷  Martens AC, de Groot CJ, Hagenbeek A Development and characterisation of a cyclophosphamide resistant variant of the BNML rat model for acute myelocytic leukaemia Eur J Cancer 1991 27: 161–166 MEDLINE

⁸  Schaider H, Haberkorn U, Stohr M, Berger MR Dinaline inhibits amino acid transport and proliferation of colon carcinoma cells in vitro Anticancer Res 1995 15: 2501–2509 MEDLINE

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