In viva antitumour activity of Ilmofosine

Dieter B. J. Herrmann,* Wulf Pahlke, Hans-Georg Opitz and Uwe Bicker

Boehringer Mannheim GmbH, Department of Immunopharmacology, Sandhofer Strasse 116, 6800 Mannheim 31, F.R.G.

Introduction

We have for some time been interested in the antitumour activity of alkyllysophospholipids (ALPS). They are synthetic derivatives of the cell membrane component lysophospha- tidylcholine and represent a new class of antitumour drugs. We are particularly inter- ested in thioether analogues such as Ilmofbsine. This compound has recently been tested in clinical Phase I studies in adult patients with refractory cancers (11, 13) and is now under evaluation in clinical Phase II trials.

The mode of action of ALPs in viva is still unclear, but direct cytostatic/cytotoxic effects on neoplastic cells (10, 18) and/or immunomodulating mechanisms are possibilites ( 1, 3, 18).

The present investigations were designed to clarify this question for Ilmofosine by studying its in vivo antitumour activity, alone and in combination with clinically established cytotoxics, in the MethA fibrosarcoma and 3Lewis-lung carcinoma systems.

Materials and methods

Drugs

Ilmofosine was synthesized by Boehringer Mannheim GmbH (Mannheim, F.R.G.). Details of the synthesis and chemistry are published (6). Cyclophosphamide was obtained from Asta Pharma AG, Bielefeld, and cis-DDP from Farmitalia/Carlo Erba GmbH, Freiburg, F.R.G. The drugs were dissolved in PBS and diluted correspondingly to the final treatment dosages.

In vivo tumour systems

MethAJibrosarcoma Ascites forms of MethA fibrosarcoma cells were propagated i.p. in female CBGF, mice (Iffa Credo, L’Arbresle, France). For chemotherapy experiments, 1 x lo” cells/mouse were injected S.C. into female CBGF, mice. Tumour growth in controls and in drug-treated animals was determined by measuring two diameters of the growing neoplasms at weekly intervals as previously described (14).

Dedicated to Dr D. G. Waldecker on the occasion of his 65th birthday. * ‘I.0 whom correspondcnw should bc addressed.

0305-7372/90/2&3247 f06 $03.00/O

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0 1990 Academic Press Limited

248 D. B. J. HERRAMANN ET AZ.

n Control El 0.625mg/kg q 1.25mglkg

7 14 21 28 Doys after tumour cell inoculation

Figure 1. Dose- and time-dependent effect of Ilmofosine on the MethA-fibrosarcoma growth. Female CBGF, mice (n = 20) were treated with the appropriate daily p.o. doses of Ilmofosine or Cyclophosphamide from day 1-21. Tumour volumes are expressed as % of placebo (PBS)-treated controls. Means+SEM, *Jo < 0.01,

**p < 0.001, Mann-Whitney test.

3Lewis-lung carcinoma The 3Lewis-lung carcinoma was maintained by serial passage in CB57B1/6 mice (Ifla Credo). For studying the therapeutic effect of test compounds, 1 x 10’ cells/mouse were injected into the right hind foot-pad of female C57B1/6 mice. Details on tumour transplantation, determination of primary tumour growth and surgical removal of tumour bearing hind legs have been published ( 15). E xamination of pulmonary meta- stases was performed using the Wexler method (23).

Results and discussion

Ilmofosine was introduced into in vivo test programs because ofits unique pharmacological in vitro profile which is characterized by a differential cytostatic/cytotoxic activity (11, 22)) lack of platelet activating factor properties (4, 1 l), lack of bone marrow toxicity (8) and lack of mutagenicity at clinically relevant concentrations (11).

The therapeutic effect of Ilmofosine on the MethA fibrosarcoma growth in vivo was assayed with doses ranging from 0.625 to 40 mg/kg, daily administered p.o. over 3 weeks starting on day 1 through day 21 after S.C. inoculation of 1 x 10’ tumour cells. Tumour volumes and numbers of tumour-free animals were monitored on days 7, 14, 21 and 28. Tumour weights were determined on day 28.

Figure 1 illustrates the dose- and time-dependent inhibition of MethA sarcoma growth induced by Ilmofosine. A highly significant tumour suppression can already be found after a continuous 7- to 14-day treatment period, even with doses as low as 0.625 and 1.25 mg/kg/day. Furthermore, an inhibitory effect of Ilmofosine can still be observed on day 28, i.e. 1 week after termination of the oral treatment on day 21. Cyclophosphamide (2.5 mg/kg/day) was always less effective compared to the corresponding dose of Ilmofosine.

In accordance with this tumour parameter, the mean tumour weights on day 28 decreased dose-dependently. Moreover, the number of tumour-free animals increased dose- and time-dependently in the same experiment (data not shown).

The 3Lewis-lung carcinoma system is of particular relevance for testing new anticancer

ILMOFOSINE 249

80.

i 6oi 2 5 I 40, cl7

20 (

r Treotment

0 IO 20 30 40 50 60 Days after tumour cell inoculation

Figure 2. Dose-dependent effect of Ilmofosine on survival time of 3Lewis-lung carcinoma-bearing mice. Female C57Bl/6 mice (n = 20) were inoculated with 1 x lo6 turnour cells/mouse on day 0. Animals were treated orally

from day l--9.

drugs because some of its biological features are very similar to some human solid tumours, e.g. metastases formation and resistance to the majority of chemotherapeutics (7, 9).

In the first series of experiments in this system, groups of 20 mice were inoculated S.C. with 1 x lo6 3Lewis-lung tumour cells into the foot-pad of the right hind leg. Animals were then treated with doses ranging from 0.625 to 40 mg/kg daily p.o., either from day 1 through day 9 or from day 11 through day 28. Cyclophosphamide (10 mg/kg/day) served as positive control. The tumour parameters measured included (a) primary tumour growth, (b) survival time, and (c) cumulative numbers of metastases-free animals.

Figure 2 shows the dose-related increase in survival time under Ilmofosine treatment from day 1 through day 9. In agreement with this result a dose-dependent inhibition of primary tumour growth and a dose-dependent increase in the numbers of metastases-free animals was observed in the same experiment (data not shown). Furthermore, the next set of experiments which was aimed at evaluating the therapeutic efficacy of Ilmofosine in this model by starting treatment 1 day after removal of the primary tumour (amputation of the tumour-bearing hind leg) on day 11, revealed similar findings.

Taken together the almost linear, dose- and time-dependent dose-response relationship in the MethA-fibrosarcoma and “Lewis-lung carcinoma models for all tumour parameters studied indicate that direct cytostatic/cytotoxic effects rather than the postulated immune- modulating mechanisms of ALPS (3, 18) ‘g ‘fi sr m cantly contribute to the antitumour activity of Ilmofosine in uzvo. These results are in agreement with a dose-dependent antitumour effect of Ilmofosine in the MNU-induced mammary carcinoma model (5) and with the dose-dependent increase in plasma and tumour concentrations of Ilmofosine in MethA sarcoma bearing mice (12). Similar results were also described for the structurally related compound ET-18-OCH, in the MethA-fibrosarcoma (21), MNU-induced mammary carcinoma (5), myeloma MPC-11 (18) and L 1210 leukemia (19). On the other hand, no convincing evidence for immunomodulatory properties of ET-18-OCH3 was found in uivo

120). The completely different mode of action of ALPS (IO, 17) compared to clinically

established cytostatics make these compounds interesting candidates for combination chemotherapy. This is particularly true if one keeps in mind that ALPS are characterized by a lack of mutagenicity at clinically relevant concentrations (2, 11).

250 D. B. J. HERRMANN ET /IL

Ilmofosim pa./ Ilmofosine p.o./ Cyclophosphemidc po. cis-DPP ix

'IO ‘IO

1 0,

~ C

l/IO hL 3/10

WI0

0 Cir IL Cis+ I

0 cy IL cy+ 2.5 2.5 IL 0.5 2.5 IL

Figure 3. Combination chemotherapy of MethA-fibrosarcomas. Female CBGF, mice (TI = 10) were treated with 2.5 mg/kg daily Ilmofosine p.o. immediately followed by 2.5 mg/kg Cyclophosphamide p.o. from day I-14. Cis- DDP (0.5 mg/kg) was administered i.v. three times a week. Columns represent turnour volumes on day 14.

Figures above the columns give the numbers of tumour-free mice out of 10 animals per group on day 28.

Therefore, Ilmofosine has been tested in combination with a variety of different cyto- statics in vitro (16) and in uivo. Figure 3 illustrates, for example, the combined therapeutic effect of Ilmofosine together with either cyclophosphamide p.o. or &-DDP iv. in the MethA-fibrosarcoma model. There was a clear synergistic effect for both combinations, particularly with regard to the clinically most relevant parameter, ‘numbers of tumour- free animals’.

In conclusion, the data presented here indicate that the therapeutic activity of Ilmofosine in vivo is related to its direct cytostatic/cytotoxic effect against tumour cells. Moreover, the antimetastatic activity and the combined therapeutic effects with other cytostatics make Ilmofosine an interesting new compound for adjuvant and combination cancer chemo- therapy, respectively.

Summary

Ilmofosine is a cytostatic/cytotoxic thioether phospholipid derivative. The in uiuo anti- tumour activity of this compound was investigated in a methylcholanthrene (MethA)- induced fibrosarcoma and in the “Lewis-lung carcinoma systems, respectively. Ilmofosine showed antineoplastic and antimetastatic properties at oral doses ranging from 0.625 to 40 mg/kg/day. Combination of Ilmofosine (p.0.) together with either cyclophosphamide (p.o.) or cis-DDP (i.v.) resulted in synergistic effects in viva. These results demonstrate the in uiuo antitumour activity of Ilmofosine in two tumour systems. The data indicate that direct cytostatic/cytotoxic effects of Ilmofosine are mainly responsible for its antitumour activity in uivo and which are increased by other cytotoxics.

ILMOPOSINE 251

References

1. Andrerscn, R., Osterholz, J., Luckenbach, A., Costabel, U., Schulz, A., Speth, V., Munder, P. G. & Lohr, C. W. (1984) Tumor cytotoxicity of human macrophages after incubation with synthetic analogues of 2- lysophosphatidylcholine. ,J. N.C.I. 72: 53-59.

2. Bauchingrr, M., Drcsp, J., Schmid, E. & Berdcl, W. E. (1983) Cytogenetic effect ofan alkyl-lysophospholipid derivative in human peripheral lymphocytes exposed in oitru and in viva. Mutalion Res. 121: 225-231.

3. Berdcl, W. E. & Munder, P. C. Metastatic growth of ‘Lewis-lung carcinoma in mice treated with alkyl- lysophospholipids and lysophospholipid-induced peritoneal macrophages. ~lnticancer Res. 1: 3977402.

4. Berdcl, W. E., Korth, R., Rcirhert, A., Houlihan, W. J., Bicker, U., Nomura, H., Vogler, W. R., Benvenistc,

J. & Rastetter, J. 11987) Lack ofcorrclation between cytotoxirity of agonists and antagonists of platclet- activating factor (PAP-accther) in neoplastic cells and modulation of [‘HI-PAF-accthrr binding to platrlrts from humans in &ro. rlnricancer Rej. 7: 1181-l 188.

5. Bcrgcr, M. R. & Schmahl, D. (19871 Modulation of chrmical carcinogcnesis in rats by alkyl-

lysophospholipids. Lipids 22: 9355942. 6. B&es, E., Hrrrmann, D. B. J,, Bicker, U., Gall, Ra. hi Pahlkc, W. (1987) Synthrsis of thiocther phospho-

choline analogues. I,$&& 22: 947 951. 7. Budzynski. L\‘. (1982) Lewis lung carcinoma in mice as an experimental therapy model. I. Thr growth

kinrtics and the rffcct of tumor on host. Arch. Immund. Thu. Lxp. 30: 363-372. 8. Fromm. M., Berdrl, W. E., Schick, H. D., Fink, U., Pahlke, W., Bicker, U., Reichert, A. & Rastctter, J,

(198ii Antinroplastic activity of the thioether lysophospholipid derivative BM 41.440 in vitro. Lipids 22: 916-918.

9. Goldin. A., Vrnditti, J. M., MacDonald. J. S., Muggia, F. M., Hcnney, J. E. & DeVita, V. 7‘. (1981) Current results of the screening program at the Division of Cancer ‘Treatment, National Cancer Institute. Eur. J. cancel- 17: 129-142.

IO. Herrmann, D. B. J. (1985) Changes in cellular lipid synthesis of normal and ncoplastir cells during cytolysis induced by alkyllysophospholipid analogues. ,J.N.C.I. 75: 423-430.

I I. Hcrrmann, D. B. J. & Bicker. U. 119881 Ilmofosinr fBhl 41.4401, a nrw cytotoxir ctllcrphosph(,liI,id. fjru,qs f;rr/rm 13: 543-554.

12. Hcrrmann, 1). B..J., H&k, J. P., Pahlke, W., Besmfeldrr, E.. Bosies, E:., Ncubert. l’., bndele, R., Bicker, U. & hlundrr, P. C. (1989) Antitumor activity of Ilmofosinr (BM 41.440) and its analogs in L&J and in

Go. Eur. J. Clin. I’harmacol. 36 (Suppl.): A67.

13. Herrmann, D. B. J., Neumann, H. A., Hcim, M. E., Bcrdcl, W. E., Fromm, M., Andrccsrn, R., QuciBcr, iv., Bocrncr. D., Sterz, R., Bcsrnfclder, F,. & Bicker, U. (1989) Short- and long-trrm tolerability study of the thiorthcr phospholipid derivative Ilmofosine (BM 41.440) in cancer patients. In: Eckhardt, S., Holzner,

J. H. & Nagcl. G. A., cds., Contribu/ions lo Oncolqy: Nez I1ry.t in Oncalo~y, Vol. 37, pp. 236-247. Bawl: Kargcr.

14. Herrmann, D. B. J., Pahlke, W., Bicker, U. & Mundcr, P. G. (1990) Antitumor activity of Ilmofosine (BM 41.440) in a methylcholanthrene-induced fibrosarcoma model in vitro and in I;& (in press).

1.5. Herrmann, 1). B. J., Pahlke, W., Munder, P. G. & Bicker, C. (1990) ilntitumor activity of Ilmofosine (BM 41.4401 in the ‘lxwis-lung carcinoma model (in prrss!.

16. Hofmann, J., tibcrall, F., Posch. I,., Maly, K., Herrmann, D. B. J. & Crunickc, H. (1989) Synergistic cnhancemcnt of thr antiproliferative activity of c&Diamminedichloroplatinum I II i by the ether lipid analogue BM 41.440, an inhibitor of protein kinase C. I,i$ds 24: 3 12 3 17.

17. hlodolcll, hl., Andrrcscn, R., Pahlkc, W., Brugger, C. & Mundcr, I’. C. (1979) Disturbances ofphospholipid mrtabolism during srlcctive destruction of tumor cells induced by alkyllysophospholipids. Gzncer Res. 39: 4681-4686.

18. Xlundcr, 1’. G., Modolell, M., Bausert, W., Oettgen, H. F. & Wcstphal, 0. (1976) Alkyllysophospholipids in cancer thrrapy. In: Hersh, E. hl., Chirigos, M. A. and Mastrangclo, M, J., rds, Augnen/in~q &wls in Gncer Thera/g~, pp. 441-447. New York: Raven Press.

19. Mundcr. P. G., iv4odolel1, M., Andrccsrn, R., Weltzicn, H. U. & Wcstphal, 0. (1979) Lyso- pl~(~sphatidylchofinc (lysolccithin) and its synthrtic analogucs. Immunr-modulating and other biologic ctlccts. Springer Seminars Immunopathol. 2: 187-203.

20. Talmadgc, J. E., Schneider, M., Ixnz, B., Phillips, H. 8i Loug, C. (1987) lmmunomodulatory and thcrapcutic proprrties ofalkyllysophospholipids in micr. Lifd 22: 871-877.

21. Tarnowski, C. S., Mountain, J., Stock, C. C., Munder, P. G., LVeltzicn, H. U. & Ft’rstphal, 0. (1978)

Eli&t oflysolrrithin and analogs in mouse ascites tumor. Cancer Re.r. 38: 339~ 344.

252 D. B. J, HERRMANN ET AL.

22. Vogler, W. R., Olson, A. C., Okamoto, S., Shoji, M., Kuo, J. F., Berdel, W. E. & Haydu, J. (1989) Structure-function relations in the selective toxicity of ether lipids (EL). 3rd International Confcrenw on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, 8-12 May, 1989,

p. 56. 23. We&r, H. (1966) Accurate identification ofexperimental pulmonary metastases. J.N.C.I. 36: 641~645.