Brtish Joumal of Cancer (1998) 78(3). 336-343c 1998 Cancer Research Campaign

Interaction of thalidomide, phthalimide analogues ofthalidomide and pentoxifylline with the anti-tumouragent 5,6-dimethylxanthenone-4-acetic acid:concomitant reduction of serum tumour necrosisfactor-alpha and enhancement of anti-tumour activity

L-M Ching, WL Browne, R Tchernegovski, T Gregory, BC Baguley and BD Palmer

Auckland Cancer Society Research Centre. University of Auckland School of Medicine. Auckland. New Zealand

Summary DMXAA (5,6-dimethylxanthenone-4-acetic acid), a novel anti-tumour agent currently undergoing clinical evaluation. appears tomediate its anti-tumour effects through immune modulation and the production of the cytokine tumour necrosis factor-a (TNF). Our previousstudies have shown that thalidomide, a potent inhibitor of TNF biosynthesis that has numerous biological effects, including inhibition of tumourangiogenesis. unexpectedly augments the anti-tumour response in mice to DMXAA. We show here that thalidomide (100 mg kg-) has noeffect when administered with inactive doses of DMXAA, and that it must be given simultaneously with an active dose of DMXAA to have itsmaximum potentiating effect on the growth of the munne Colon 38 adenocarcinoma. To address the issue of whether inhibition of serum TNFproduction is important for potentiation of anti-tumour activity, we have tested three potent analogues of thalidomide. All three analogues.when co-administered with DMXAA to mice at doses lower than those used with thalidomide, inhibited TNF production and were effective in

potentiating the anti-tumour activity of DMXAA against transplanted Colon 38 tumours. One of the analogues, N-phenethyltetrafluorophthalimide.was 1000-fold more potent than thalidomide and at a dose of 0.1 mg kg-' in combination with DMXAA (30 mg kg-') cured 1000 of mice.compared with 670o for the group treated with DMXAA alone. We also tested pentoxifylline and found it to suppress TNF production inresponse to DMXAA and to potentiate the anti-tumour effect of DMXAA. The results are compatible with the hypothesis that pharmacologicalreduction of serum TNF levels might benefit the anti-tumour effects of DMXAA and suggest new strategies for therapy using this agent.

Keywords: 5,6-dimethylxanthenone-4-acetic acid; thalidomide: phthalimide: pentoxifylline: tumour necrosis factor: anti-tumour activity:Colon 38

We hax e dex eloped a series of xanthenone analogues of thedrug flax one acetic acid (FAA that are highly actix e againsttransplantable murine tumours w-ith an established Xasculature(Rex-castle et al. 1989. 1991). The most potent of these. 5.6-dimethN-lxanthenone4-acetic acid (DMXAA). is now- in phase Iclinical trials in New Zealand and the UK. In mice. DNIXAA is12-fold more potent than FAA and induces a higher percentage ofcures acainst the Colon 38 carcinoma (Rexwcastle et al. 1991).DNIXAA and FAA share a mechanism of action that is differentfrom that of conventional direct cvtotoxic anti-cancer drugs. Bothappear to actix-ate. through host and tumour cell components. acomplex series of responses involving shutdow n of tumour bloodfloxw (Z"-i et al. 1989. 1994). stimulation of immune responses(Ching and Bagulev. 1987: Ching et al. 1991) and elimination ofthe tumour.

NManv of the biolo2ical actix-ities of DMNXAA and FAA havebeen attributed to their abilitv to induce cxtokines. in particularTNF and the interferons (Mace et al. 1990: Futami et al. 1992:

Received 9 September 1997Revised 21 January 1998Accepted 28 January 1998

Correspondence to. L-M Ching. Auckland Cancer Society Research Centre.University of Auckland School of Medicine. Private Bag 92019. Auckland.New Zealand

Philpott et al. 1995). w-hich have peak serum concentrations 2-3 hafter DMNIXAA administration. The early vascular effects appear tobe mediated by tumour necrosis factor-a (TNT) as antibodies toT-NF ablate FAA-induced tumour x-ascular collapse (Mahadex an etal. 1990). DMXAA induces hiaher lexels of serum TNF than FAA.and the anti-tumour response correlates w ell w-ith TNF productionw ithin a series of DMXAA analoaues (Philpott et al. 1995).As an approach to inx estigating the role of TNF induction in the

anti-tumour action of DNMXAA. wxe co-administered thalidomidewxith DMXAA to mice x ith subcutaneous Colon 38 tumours(Ching, et al. 1995). Thalidomide. best knowxn for its sedatixe andteratogenic effects (Fabro et al. 1967). has receixed attention inrecent -ears as a selectixe inhibitor of TNT production Sampaioet al. 1991 ). apparently acting, by increasing the rate of degradationof TNT m_RNA (Moreira et al. 1993). We haxe shoxwn that thalido-mide inhibits DMXAA-induced serum TNF lexels. but. unexpect-edly. potentiates the anti-tumour response (Ching et al. 1995).While these results appear to argue against a role for TNF in theanti-tumour action of D.MXA. they indicate a noxvel approach toaugmenting the anti-tumour action of DMXAA.

Recently. sexeral phthalimide-derixved analogues of thalidomidehax-e been described that are more potent than thalidomide inmodulating TNF production in cells stimulated with a phorbolester (Sasaki et al. 1995). The druc pentoxif-lline is also knoxxnto suppress TNF production in response to administration of

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DMXAA and TNF inhibition 337

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0 OH3

CH3CO(CH2)4 N,, N

ICH3

Pentoxifylline

Figure 1 Structures of thalidomide. PP. PEP. PEFP. pentoxifyiline and DMXAA

lipopolx-sacchande (Noel et al. 1990). In this report. w-e havefurther investioated the interaction of thalidomide and DMXAAand has-e investigated a series of three phthalimide derivatives. as

ell as pentoxif lline. to determine whether they also suppress

TNF production in response to DMXAA and w hether thevpotentiate DMXAA-induced anti-tumour effects.

MATERIALS AND METHODS

Materials

DMXAA. svnthesized in this laboratorv by Dr GA Rew-castle(Rewcastle et al. 1991). w-as dissolved in 5%z7c sodium bicarbonateand xwas injected intraperitoneallv (i.p.) in a volume of 0.01 ml g-'

bodv weight. (->-Thalidomide. N-phenylphthalimide (PP).N-phenethylphthalimide (PEP). and N-phenethyltetrafluorophthal-imide (PEFP) (chemical structures in Figure 1) w ere synthesizedaccording to published methods (Casini and Ferappi. 1964: Sasakiet al. 1995). dissolved in dimethyl sulphoxide and injected at2.5 I1 g-' body wei5ht. Clinical formulations of pentoxifylline(Trental. Hoeschst. Frankfurt. Germany) were diluted in saline to

required concentrations for i.p. injections.

Measurement of anti-tumour activity

All experiments were carried out in 8- to 12-vveek-old C57B1/6 x

DBAI2 F (BDF, mice bred in the laboratorx animal facilitv andtreated according to institutional auidelines. Fragments of Colon38 tumour (1 mm¼ ) were implanted subcutaneously in the flank ofanaesthetized (sodium pentobarbital. 90mr kg-' i.p.) animals.Experiments were carried out on tumours approximately 4-5 mmin diameter. grenerally 10 days after implantation. Tumour-bearing,mice (at least five per group) were injected w-ith drugs and thetumours measured with callipers two or three times per weekthereafter. Tumour volumes were measured as 0.52 a: x b. wherea and b are the minor and major axes of the tumour. The arithmeticmean and standard error of the means A-ere calculated for each

time point. including animals having zero measured tumour

volume. and expressed as a fraction of the pretreatment volume.Growth delav was determined as the difference in the number ofdays required for the control and treated tumours to reach fourtimes the pretreatment xolume. Statistical tests w-ere carried out

using SigmaStat (Jandel Scientific. San Rafael. CA. USA). Mice

cured of tumours were kept for at least 3 months to ensure thattumours did not regrow.

Histological examination of tumour sections

Mice w-ith Colon 38 tumours w-ere treated with DMXAA (singledose. 24 mg kg-'). either alone or w-ith thalidomide (100 mg kg-').Tumours were excised 1-10 dayvs after treatment. fixed overnightin 10% formalin. Fixed tumours were then embedded in paraffinwax and sections were stained w-ith haematoxvlin and eosin. Thesection across the major diameter of the tumour w-as examined on

a grid marked at 0.4-mm intervals and scored for the percentage

area of viable tissue as previously described (Bagulev et al. 1989).

Determination of serum TNF

Mice were anaesthetized using halothane and wA-ere bled from theocular sinus at indicated times after treatment. Blood w as allowedto clot ovemirht on ice and the serum collected by centrifu2ation(2000 g. 30 min) and stored at -20 C until it was assayed for TNFactivity. using the standard L929 cvtotoxicitx assay as described(Philpott et al. 1995). L929 cells (3 x 10-) were allowed to adhereox erniht to the bottom of flat-bottomed 96- ell plates.Actinomycin D (final concentration 8 ,gr ml- ') was then added tothe wells followed by serial dilutions of the serum to be assayed.Killinc of the L929 cells was assessed after 24 h by a colorimetricassay using 3-(4.5-dimethvl-2-thiazoly l)-2.5-dipheny-l-2H-tetra-zolium bromide. One unit of TNF A as defined as that reducing cellstaining in this assay by 50%. and corresponded to the activitxobtained with 10-" of punrfied murine TNF protein.

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Figure 2 Potentiation of DMXAA-induced tumour growth inhibition by thalidomide. Growth of Colon 38 tumours in mice with no treatment (-); with DMXAA15 mg kg-' (A): with 20 mg kg-' DMXAA (B): with 25 mg kg-' DMXAA (C): with 30 mg kg-' DMXAA (D) either alone (open symbols) or together with thalidomideat 100 mg kg-' (closed symbols). Veriical bars indicate s.e.m. In some cases. s.e.m. values are smaller than the size of the symbol

RESULTS

Potentiation of anti-tumour activity of DMXAA bythalidomide

Thalidomide potentiates the anti-tumour response of DMXAA at

its optimal therapeutic dose of 30 mg kg-I when administeredsimultaneously (Ching et al. 1995). To investigate whether thalido-mide potentiated suboptimal doses of DMXAA. mice with Colon38 tumours were treated with thalidomide (100 mg kg-') and a

range of doses of DMXAA from 15 mg, kg-I to the maximal-tolerated dose (MTD) of 30 mg kg-'. Tumour growth delays andcure rates appeared to be increased by thalidomide after DMXAAdoses of 25 and 20 mc kg-' but not after a dose of 15 mg kg-I(Figure 2 and Table 1). How-ever. the differences between groups

were not statistically significant. analysis beina complicated by thepercentage of cures in each group. We also examined the effect ofchanging the timinc of administration. using DMXAA at its MTD.W'hile ggrowth delays were potentiated by thalidomide given simul-taneously wvith DMXAA. in agreement w-ith previous results

(Ching et al. 1995). they were not significantly increased whenthalidomide w-as given 1 day before. or 1. 2 or 3 davs afterDMXAA (Figure 3 and Table 1).

Histology of tumour regrowth after treatment withDMXAA and thalidomide

The histology of tumours treated with DMXAA either alone or in

combination with thalidomide (100 mr kg-') was examined bymeasuring the proportion of viable tissue in tumour sections taken at

different times after treatment. A suboptimal dose of DMXAA w as

used (24 mg kg-' ) to permit a significant amount of viable tissue tobe measured. Extensive haemorrhagic necrosis w-as evident in bothtreatment groups when measured after 24 h. However. pockets ofviable tissue that were visible in tumours treated with DMXAAwere less evident than those in tumours treated w-ith the combina-tion. and the amount of viable tissue in tumours treated with thecombination w-as lower than that in tumours treated with DMXAAalone (Figure 4. While the proportion of regenerating viable tumour

British Joumal of Cancer (1998) 78(3). 336-343

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Table 1 Cure rates and growthldelays of tumours treated with DMXAA and thalidomide time dependence (data from Figures 2 and 3)

Dose Time between DMXAA Growth delay (days) Percentag cures(mg kg-') and thalidomide (h)

DMXAA DMXAA+ DMXAA DMXAA+thalidomide thalidomide

15 0 6 6 0 2820 0 10 15 36 4825 0 15 25 60 8030 0 20 - 67 10030 -24 17 8330 +24 9 7530 +48 16 4030 +72 23 56

c t100,

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Figure 3 Effect of time of administration of thalidomide in relation to that ofDMXAA. Growth of Colon 38 tumours in mice with no treatment (0), with30 mg kg- DMXAA (_ ), with thalidomide (100 mg kg-') given at the sametime as DMXAA (hexagons), one day before (*), one day after (=). 2 days

)and 3 days afterC)

tissue increased steadilv w-ith time in the animals treated withDMXAA only. the proportion in tumours from mice treated with thecombination increased only for 3 days after treatment (Figure 4).

10 12

Figure 4 Measured percentages of viable Colon 38 tumour in tissuesecfions taken at various times after administration of DMXAA (24 mg kg-')either alone (0) or together with thalidomide (100 mg kg-) (- )

no effect was found on the take-rate or growth of the Colon 38tumour. Rather. the rate of growth was slightly accelerated bvthalidomide treatment. and the tumours became palpable sooner in

the treated amnmals (Figure 5). Thus there wias no evidence forinhibition of neoxascularization. and thus of tumour growth anddevelopment. usinc thalidomide alone at doses of 100 mg kg-

Effect of thalidomide alone on tumour developmentand growth

Thalidomide is known to inhibit anciooenesis (DlAmato et al.1994). and angiogenesis antagonists can increase the anti-tumour

response to radiation and chemotherapy through inhibition oftumour neovascularization (Teicher et al. 1993: D'Amato et al.1994). To test the hypothesis that thalidomide. administered alone.was affecting the growth of Colon 38 tumours. mice w-ere treatedeither w-ith a single dose or ,iith multiple doses of thalidomide(100 mg kg- per injection). No inhibition of tumour growth wasobserved in mice treated w-ith a single injection of thalidomide(100 mg kg-'). or ith injections three times A eekly for the dura-tion of the experiment (1100 mg kg- per injection). Furthermore.Awhen thalidomide alone A as gixven at the time of tumour implanta-tion and three times A eeklv thereafter (100 mg kg-' per injection).

Ability of analogues of thalidomide to inhibit serumTNF production and potentiate the anti-tumour actionof DMXAA

A series of simple phthalimide derivatives that are more potent

than thalidomide in modulating serum TNF production has beenreported (Sasaki et al. 1995). We synthesized three of these more

dose-potent derivatives (PP. PEP and PEFP) and compared theirabilitxv ith thalidomide to suppress DMLXAA-induced serum TNFproduction and to potentiate anti-tumour action. PEFP w-as themost toxic of the three compounds. inducing deaths at 3 days at

doses above 50 mg kg-'. whereas the other two derivatives wereA-ell tolerated at 100 mg kg-1. In comparison. thalidomide was

well tolerated up to 250 mg kg-I in mice (Ching et al. 1995).Comparisons of inhibition of serum TNF production w-ere

carried out using a dose ofDMXAA (50 mg kg-') that Awas optimal

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Figure 5 Effect of thalidomide on tumour establishment. Mice (12 pergroup) were implanted with Colon 38 fragments and either untreated (openbars) or treated with thalidomide (100 mg kg-') at the time of implantation andthrice weekly for the duration of the experiment (shaded bars). Mice werechecked for palpable tumours (greater than 2 x 2 mm2) after implantation

for the TNF response 2 h after treatment but that caused no toxiceffects at the time of the assay (Philpott et al. 1995). Each of thephthalimide derixatixves inhibited DMXAA-induced serum TNFproduction (Figure 6). PEP. PP and PEFP %vere more dose-potentthan thalidomide and. "-hen administered at a dose of 0.1 mg kg-.inhibited TNF lexels to 58. 18 and 8.5%k. respectively. of DMXVAAcontrols (Figure 6). They xx-ere thus more potent than thalidomide."-hich was actix-e at a dose of 0.3 mg kg-. In contrast to thalido-mide. none of the phthalimide derivatixves caused sedation (resultsnot sho"-n).We next compared the ability of the phthalimide analogues at a

dose (10 mg kg-') that suppressed serum TNF production (Ficure6) to potentiate the anti-tumour response of DMXAA (30mgrkg- ). Combination of DMXAA A-ith PEFP and PP produced curesagainst the Colon 38 tumour in 100% of the mice (Ficure 7). A-hilecombination with PEP caxve cures in 75%7e of the mice and extendedthe grow th delay to oxer 60 days. At this dose. thalidomide "-as theleast actix-e of the four agents. Thus. all three phthalimide derixa-tixes appeared to be more potent than thalidomide in potentiatingthe anti-tumour actix itv of DMXAA.As PEFP "-as the most actix-e in reducing serum TNF lexels

Ficure 6). A e tested this compound at low er doses for potentiationof the anti-tumour action. PEFP A.1 mg kg- in combination "-ithDNIX.AA (30 mc kg-1) induced cures in 100%' of the animals(Figrure 8) and thus >-as at least 1000-fold more potent thanthalidomide. A hich required a dose of 100 mc ko' in combinationwvith DNMXAA for a 100% cure rate (Table 1). PEFP had no anti-tumour effects on its own at its maximal-tolerated dose (Figure 8).and this >-as also the case for PEP and PP (100 mg k-': datanot shox-n).

Inhibition of serum TNF production and potentiation ofDMXAA anti-tumour action by pentoxifylline

The studies w-ith the phthalimide derixatixes extended ourprexious demonstration that inhibition of serum DMXAA-induced TNF production xxas concomitant x-ith the potentiation

of DMXAA anti-tumour action (Figures 6 and 7). We thereforeexamined pentoxifylline. a structurallv unrelated inhibitor(Figyure 1). w-hich. like thalidomide. inhibits TNF production inresponse to lipopolx-saccharide (Noel et al. 1990). We found thatco-administration of pentoxifylline at doses of 12.5-100 mg kg-1reduced DMXAA-induced serum T'NF bv 50-805 (data notshown). Co-administration of pentoxifxlline (50 mg kgY-increased the growth delav induced by DNIXAA (30 mg kg- )from 19 to more than 40 dayvs (Figure 9A) and increased the curerate from 64A' to 82%. but these differences were not statisticallysi2nificant. Co-administration of DNIXAA w-ith pentoxifylline(100 mg kgx- ) induced complete tumour regressions in 100% ofthe mice (Figure 9B

DISCUSSION

W'e have previously demonstrated that thalidomide. w hilereducina DNIXAA-induced increases in serum TNF. potentiatesthe anti-tumour response of DNIXAA (Ching et al. 1995 ). We haveshow n similar effects to thalidomide firstly for three derivatives ofphthalimide structurally related to thalidomide (Sasaki et al. 1995)and secondly for pentoxifA-lline. w-hich differs from thalidomide inboth structure and mechanism of TNT inhibition (Han et al. 1990:Sampaio et al. 1991). The augmentation of anti-tumour activitv bvthese drugs (Figures 7-9) is difficult to assess statisticallv becauseDMXAA alone induces a percentage of cures. and a large numberof animals are therefore required to achiex-e statisticall1 sienificantdifferences in cure rates. How-ever. taken together. the results areconsistent w-ith the conclusion that the potentiation of the anti-tumour action of DMNXAA is associated w ith the common propertyof pentoxifylline. thalidomide and the phthalimides to decreaseserum TNF production. Co-administration of agents low erincyserum TNF may thus represent an innoxvative strateav forincreasing the anti-tumour efficacy of DMIX.AA. a drug that iscurrently in phase I clinical trial.

Thalidomide has a number of pharmacological actions.includinc the inhibition of angiogenesis (DAmato et al. 1994).raisin2 the question of w hether potentiation of DMXAA actix ity ismediated by its anti-angiogenic properties. Administered alone.thalidomide had no inhibitorx effect on the row-th of the Colon 38tumour (Ching et al. 1995). and repeated dosing did not affect thedevelopment and growxth of the Colon 38 tumour (Fioure 5. Asingle dose. administered at the same time as DMXAA (Fiaure 3).was required for the synergistic effect. and thalidomide did notaffect tumour growth when administered with an ineffective doseof DMXAA (Figure 2). Inhibition of tumour angiogenesis requiresrepeated or continuous application of the angiogenesis antaconist(D'Amato et al. 1994). and it is likely that higher doses than thatused in these experiments are necessarx for inhibition of neox ascu-larization (RJ d'Amato. personal communication). W'hile we havenot ruled out completely that thalidomide may be potentiating theanti-tumour activitv of DMXAA through inhibition of anaioeen-esis. it appears more likely that it is acting in some other fashion.Histoloaical studies indicate that the augmented tumour growthinhibition of the combined therapy stems from an acute effectresulting in a greater reduction in tumour cell survival at 24 h andfrom a subsequent slower rate of regeneration (Figure 4. Theseobserx ations suggest that thalidomide minht in some w-ax increasethe actions of the induced cvtokines.The mechanism bx which thalidomide. the phthalimide derixa-

tixes and pentoxifx lline inhibit serum TNF production is not vet

British Joumal of Cancer (1998) 78(3). 336-343 0 Cancer Research Campaign 1998

DMXAA and TNF inhibition 341

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Figure 6 Suppression of DMXAA-induced serum TNF actvity by thalidomide and its analogues. Individual mice were treated with DMXAA(50 mg kg-') alone or together with thalidomide, PP, PEP or PEFP at the indicated doses, or with drug aloe. Sera were collected 2 h later and assayed for TNFactty. The control actvity for PP and PEP was 3393 units and that for PEFP and thalidomide was 2440 units

understood. In cultured cells. thalidomide can either inhibit or

enhance TNF production. depending on the conditions and the celltype. TNF production by HL-60 cells in response to phorbol esters

as increased by co-incubation with thalidomide, as well as by thephthalimide analogues used in this study (Nishimura et al. 1994).Thalidomide also enhanced TNF svnthesis (in response to

lipopolysaccharide) in THP-1 cells and in human mononuclearcells enriched for adherent cells. but inhibited TNF svnthesis in

cultures of unfractionated peripheral blood cells (Shannon andSandoval. 1996). We do not yet know whether these mechanismswill apply in vivo or whether other mechanisms are insols ed. Weare currently investigating whether these drugs affect the in vi--o

pharmacokinetics of DMXAA.

The observation that lowering of serum TNF levels is associatedwith improving the anti-tumour activity of DMXAA appears to

contradict previous studies that show that antibodies to TNFinhibit tumour vascular collapse and ameliorate FAA-inducedanti-tumour action (Mahadevan et al. 1990: Pratesi et al. 1990). Inanother tumour model. we have shown that antibodies to TNFpartly inhibit the action of DMXAA. consistent with the aboveobservations (WL Browne et al. manuscript in preparation). Inrecent studies. we have shown that TNF is produced in response to

DMXAA in tumour tissue and that co-administration of thalido-mide. while reducing serum TNF. does not reduce tumour-associ-ated TNF (WL Browne et al. manuscript in preparation). Furtherstudies are now in progress to determine the mechanism by which

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Figure 7 Effect of thakdomide and its analogues on DMXAA-inducedtumour growth inhibition. Colon 38 tumours were measured either untreated(_) or after treatment with DMXAA (30 mg kg-') (0), DMXAA (30 mg kg-')plus thalidornkle (10 mg kg-') (), DMXAA plus PEP (10 mg kg-') (=),DMXAA plus PEFP (10 mg kg-') (A) or DMXAA plus PP (10 mg kg-')(hexagons)

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Figure 8 Effect of PEFP alone or in combination with DMXAA on tumourgrowth. Colon 38 tumours were measured either untreated (0) or aftertreatment with PEFP alone (50 mg kg-') (0), DMXAA alone (U), DMXAA(30 mg kg-') plus PEFP (0.1 mg kg-') (A) or DMXAA (30 mg kg-') plus PEFP(10 mg kg-') (hexagons)

TNF is produced in tumours and to identify the source of the TNTin serum. How-ever. we can conclude from the data obtained so farthat the inhibition of serum TNF bv thalidomide and the otherdrugs investigated here does not contradict the hypothesis thatTNF mediates the effect of DMXAA in tumours.

In conclusion. we have shown here that pharmacologicalmodulators of TNT production can be applied to reduce serumlevels of TNF while increasing the anti-tumour effects ofDMXAA. The phthalimide derivatives are more activ-e and potent

A100

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Figure 9 Potentiation of DMXAA-induced tumour growth inhibition bypentoxify1line. (A) Growth of Colon 38 tumours in mice with no treatment (0).with pentoxifyfline alone (U), DMXAA (30 mg kg-') (A) or DMXAA togetherwith pentoxifylline (50 mg kg-') (*). (B) A similar experiment usingpentoxifylline at a dose of 100 mg kg-

than thalidomide and lack the sedatory effects. presumablythrough the elimination of the Olutarimide substituent of the mole-cule and interaction with the glutamate receptors in the brain. Theco-administration of pharmacological TNF modulators such asthese may lead to improved strategies to exploit the novel biolog-ical properties ofDMXAA in a clinical situation.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the excellent technical assis-tance of Ms Belinda H Gummer in the measurement of anti-tumour responses and of Mr Zhi-Feng Xu in the pentoxifyllinestudies. This work was supported by the Auckland DivisionCancer Societv of New Zealand and by the Health ResearchCouncil of New Zealand.

REFERENCES

Baguley BC. Calveley SB. Crowe KKI Fray L.M. O Rourke SA and Smith GP1989) Comparison of the effects of flavone acetic acidL fostnecin.

homoharrincatonine and tumour necrosis factor alpha on Colon 38 tumors inmice. Eur J Cancer Clin Oncol 25: 263-269

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DMXAA and TNF inhibiton 343

Casini G and Ferappi M ( 1964) Prepartion of one optical antipode of2-phthalimidoglutarimide. Farmaco 19 563-565

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0 Cancer Research Campaign 1998 Britsh Jourmal of Cancer (1998) 78(3), 336-343