Patent Application: US-89681104-A

Abstract:
this invention relates to a compound or group of compounds present in an active principle derived from plants of species euphorbia peplus , euphorbia hirta and euphorbia drummondii , and to pharmaceutical compositions comprising these compounds . extracts from these plants have been found to show selective cytotoxicity against several different cancer cell lines . the compounds are useful in effective treatment of cancers , particularly malignant melanomas and squamous cell carcinomas . in a preferred embodiment of the invention , the compound is selected from the group consisting of jatrophanes , pepluanes , paralianes and ingenanes , and pharmaceutically - acceptable salts or esters thereof , and more particularly jatrophanes of conformation ii .

Description:
the invention will be described in detail by reference only to the following non - limiting examples and to the figures . the ability of sap of three euphorbia species , euphorbia peplus , euphorbia hirta and euphorbia drummondii to inhibit the growth of three different human tumour cell lines was tested . the activity against normal skin fibroblasts was tested as a control : the cell lines were maintained in rpmi medium containing 5 % foetal calf serum ( fcs ), and assays were performed in the same medium . sap was collected from plants growing randomly on cultivated soil on a farm at palmwoods , in the sunshine coast hinterland , south - east queensland . the plant stem surface was briefly washed with 70 % ethanol , and scissors washed in ethanol were used to cut the stem and release the milky latex sap . the sap was collected into 10 ml sterile plastic centrifuge tubes , transported at 4 ° c . to brisbane and stored frozen at − 20 ° c . prior to use , the sac was serially diluted five - fold up to 1 in 3125 into sterile 1 . 5 ml eppendorf tubes using sterile milliq water . 10 μl aliquots of each dilution were added to each two of microtitre plate wells containing 100 μl of the cell lines . assays were performed in duplicate . after 5 days , cells were examined blind , for inhibition of growth compared to control untreated cell samples . the results are summarized in tables 3 to 6 , in which the cell lines tested were nff normal skin fibroblasts mm96l malignant melanoma , brain metastasis hela cervical cancer hacat spontaneously - transformed human keratinocytes and the scale is 0 = no effect to 5 = complete cell death the dilution in the table heading refers to the dilution of the sample before addition to the culture . thus , the dilution in the final culture is approximately 10 - fold greater . a ) e . peplus was active against hela cells , and to a lesser extent against mm96l cells . b ) e . hirta was active against mm96l cells and very strongly active against hela cells . c ) e . drummondii had a lesser effect against . mm96l than the other two samples , and inhibited hela cells only at the highest concentration tested . d ) nff normal fibroblasts were severely affected at the ⅕ dilution , but only mildly affected at the other dilutions . for example , at a dilution of { fraction ( 1 / 25 )}, there was mild inhibition of nff cells ( rating 2 ), but severe inhibition of mm96l cells ( rating 4 ). at a dilution of { fraction ( 1 / 125 )}, no effect was observed against nff cells -( rating 0 ), but severe inhibition of mm96l cells ( rating 4 ) was observed for one sample , and milder inhibition ( rating 1 ) with the duplicate sample ). hacat cells , which could be considered as representative of normal keratinocvtes , were only inhibited at the highest concentration . at high concentrations of e . peplus sap , it appeared that there was direct killing of ms196 cells . however , at lower concentrations ( down to a dilution of { fraction ( 1 / 625 )}), although no growth inhibition was observed , the surviving cells were dendritic , and had the appearance or normal melanocytes . without wishing to be limited to any proposed mechanism , it appears that e . peplus sap may contain at least one agent which promotes differentiation , rather than directly cytotoxic agents which damage dna . effect of heat or acetone trea bent on activity of euphorbia sap the experiment described in example 1 was repeated for e . peplus and e . hirta by a different person , using different cell line preparations , different plant samples and a different rating scale . the samples were either prepared as described in example 1 , or were subjected to treatment with heat or acetone . undiluted extracts of plant sap were heated at 95 ° c . for 15 minutes . for the acetone treatment , 40 μl extract was suspended in 400 μl acetone , and the tube shaken on a vortex mixer . contents were centrifuged at 10 , 000 g for 3 minutes and the supernatant ( acetone - soluble fraction ) removed to a separate tube . both the pellet and supernatant were left in open tubes at room temperature in the fume hood overnight with exhaust fan operating to evaporate the residual acetone . the results are shown in tables 7 to 9 , in which +++ indicates no effect , and − indicates 100 % cell death . “ c ” indicates that the culture was contaminated . using this rating scale the results were even more striking than in example 1 , with strong inhibitory activity being observed up to a dilution of 1 : 3125 . however , some growth inhibition of nff cells was seen in this experiment . neither heat nor acetone affected the anti - tumour activity significantly . with acetone treatment , most activity was found in the pellet , particularly in the case of e . hirta , though some activity was also present in the soluble fraction . this suggests that the compounds responsible are not protein in nature , and that at least one component may be a lipid . since e . peplus is the most abundant of the three plants tested in these studies , further experiments utilised extracts from this species . this is not to be taken to imply that activity is not present in the other two species . example 2 was repeated , using mm229 and mm220 human malignant melanoma cells and b16 mouse malignant melanoma cell lines , in addition to nff and mm96l cells . assays were performed in duplicate , using addition of an equivalent amount of water as a control , and dilutions of the pellet and supernatant fractions after acetone treatment from { fraction ( 1 / 20 )} to { fraction ( 1 / 12500 )}. the results are summarised in table 10 . the results confirm those obtained in example 2 . at a dilution of { fraction ( 1 / 100 )} to { fraction ( 1 / 50 )} there was no effect on nff cells , but significant inhibition of mm96l cells was observed . the melanoma cells surviving at these dilutions had the appearance of normal melanocytes . inhibition of the other two human melanoma cell lines and of the mouse melanoma cell line was also observed . similar results were obtained using merkel cell carcinoma ( mcc 16 ) or squamous cell carcinoma ( colo 6 ) calls . the results are shown in table 11 . dendritic cell morphology was displayed by squamous cell carcinoma , even at 1 in 500 , 000 dilution . this extreme potency of the crude extract was also evident for merkel cell inhibition , which was also still evident at 1 in 500 , 000 dilution . a fresh preparation of sap from e . peplus was subjected to extraction with 95 % aqueous ethanol . ethanol was removed from the soluble fraction after extraction by vacuum centrifugation , and the fraction was reconstituted to its original volume in tissue culture medium ( rpmi1640 ) containing 5 % foetal calf serum and antibiotics . the pellet remaining after ethanol extraction was dried by vacuum centrifugation and reconstituted to its original volume in tissue culture medium as described above . the crude sap ( c ), the soluble fraction ( s ) and the pellet ( p ) were tested as described above against nff cells , the melanoma cell lines mm96l , m1537 , mm229 and mm2058 , and also against the colon cancer cell line lim4215 and the lung cancer cell line a549 . assays were performed in triplicate , and were assessed after four days culture following addition of the sample . the results are shown in table 12 , in which + indicates normal appearance of cells , ++ indicates a possible increase in cell numbers , and − indicates cell death . the results obtained were consistent with those of the previous experiments . again at low doses the mm96l cells had a dendritic appearance . all of the tumour cell lines as well as the normal fibroblast cell line nff were killed by the crude sap and by the soluble fraction obtained by ethanol extraction at a dilution of { fraction ( 1 / 20 )}. it appeared that the majority of the activity partitioned to the ethanol - soluble fraction . the lung cancer cell line a459 appeared to be particularly susceptible , being affected at a dilution of up to { fraction ( 1 / 2500 )} by both the crude sap and by the soluble fraction . reporter assay for gene expression in transfected mm96l malignant melanoma cell line e . peplus sap in phosphate - buffered saline diluent was added to wells containing mm96l cells or the breast cancer cell line mcf7 transfected with a construct consisting of the sheep metallothionein promoter , upstream of a β - galactosidase reporter gene which had been substituted for the metallothionein gene . the assay thus becomes a measure of gene expression and in particular , of potential transcription , translation and expression of the metallothionein gene . cells were treated with 4 extract in microtitre plates for 20 hr , 100 μm znso 4 was added and the plates incubated for a further 5 hr , and the medium was removed . β - galactosidase activity was then measured by incubation of the cells for 1 - 2 h at 37 ° c . with a chromogenic substrate . this assay is used as a sensitive test for transcriptional activation of genes . this shows that there was a marked stimulation of metallothionein gene activation , as measured by increased β - galactosidase reporter gene expression , which surprisingly became more evident as the sample further was diluted . the mechanism by which e . peplus sap mediates this effect is not understood . whereas known drugs specific for inhibition of histone deacetylase activity demonstrate increasing expression of the reporer gene with increasing concentration of drug , e . peplus exhibits an inverse dose response . however , the results indicate that this assay can be used to monitor purification of the active agents ( s ) in e . peplus sap or the plant itself . the metallothionein protein has antioxidant activity , and is implicated in a protective role against heavy metal - induced cancers . activation of the metallothionein promoter occurred at concentrations of e . peplus sap too low to effect direct cell killing , except for the extremely sensitive breast cancer cell line mcf7 ( fig2 ). the change in appearance of mm96l melanoma cells to the dendritic morphology of normal melanocytes at these dilutions possibly implicates the metallothionein gene in these effects . the soluble fraction obtained by extraction with 95 % ethanol , performed as in example 4 , was subjected to isocratic reverse - phase high - performance liquid chromatography ( rp - hplc ). 100 μl of crude extract was dissolved in 1 ml 95 % ethanol and periodically shaken at 4 ° c . overnight . the extract was centrifuged at 10 , 000 × g for 4 minutes , and the supernatant was removed and dried by vacuum centrifugation . the solids were reconstituted in 100 μl running buffer centrifuged briefly , and the soluble material applied to a brownlee aquapore rp - 300 column ( c8 ), 220 × 4 mm , with a 30 × 4 mm rp - 300 guard column . the running buffer was acetonitrile : water 50 : 50 ( v / v ), and the flow rate was 0 . 75 ml / mn . fractions were collected at 0 . 5 min intervals , and the absorbance profile at 195 nm was monitored . the absorbance profile is shown in fig3 . fractions were dried by vacuum centrifugation , reconstituted in 500 μl pbs , and assayed against mm96l cells and in the metallothionein reporter assay as described above . fractions 13 to 28 all induced complete reversion of km96l cells to a dendritic appearance , but cell death was not observed . the effect was much more striking in the reporter assay , in which activity was still observed at a dilution of { fraction ( 1 / 10 , 000 )} ( ie . at a final concentration in the culture of { fraction ( 1 / 100 , 000 )}) in addition to the foregoing results , the inventor has found that following ultracentrifugation , activity against mm96l cells is found both in the supernatant and in the pellet , and that activity cannot be removed by passing a sample through a molecular weight cut - off membrane . in addition to the cell lines tested above , proliferation of cells of the mcf7 breast cancer cell line was inhibited by e . peplus sap at a final dilution of up to { fraction ( 1 / 100 , 000 )}. cell numbers were assessed using the bicinchoninic acid reagent ( pierce ). results are shown in fig2 . further solvent fractionation of the crude latex of e . peplus was effected by a series of solvents of increasing polarity . to 1 ml crude latex was added 20 ml diethyl ether in a centrifuge tube . the tube was shaken and centrifuged at 5000 g for 5 minutes to partition the layers . the diethyl ether upper layer was removed and the procedure repeated twice . the ether fractions were combined , concentrated to dryness on a rotary evaporator and reconstituted in 1 ml dme for bioassay . in a similar manner , the residue was extracted with ethyl acetate , followed by methylene chloride . the initial ether extract obtained the majority of the activity as measured by decrease in cell numbers of mcf7 breast cancer cells and reversion to a dendritic appearance . however , activity was also demonstrated from the fractions derived from the ethyl acetate and methylene chloride layers . no activity was seen in the final water — soluble ( aqueous ) fraction . the results are summarised in table 13 . cmv promoter activity was assayed in hela cells infected with a replication - deficient adenovirus construct , in which the e1a gene had been replaced by the cmv promoter driving β - galactosidase . the results , shout in table 14 , are expressed as a percentage of the control values of infected , untreated cells . the results obtained are qualitatively similar to those seen with other differentiation - inducing agents , such as histone deacetylase inhibitors or butyrate , albeit with more potent activity than seen with these agents . the lower promoter activity observed with the crude and the diethylether extracts at higher concentrations probably reflects cell killing effects against hela cells seen at those concentrations . in a further solvent fractionation experiment , the crude sap was partitioned between methanol : water ( 17 : 3 ) and n - hexane , a solvent partition expected on the basis of previous reports to separate diterpenes ( polar phase ) from the triterpenes ( heptane phase ) ( evans and kinghorn 1977 ). unexpectedly , however , activity was detected in both phases , suggesting that the active principles behave anomalously in this system . another solvent fractionation approach was suggested by the need to clarify samples prior to hplc analysis . the crude latex was mixed with ethanol to 70 - 95 % and shaken overnight at 4 ° c . the mixture was centrifuged at 1 , 000 g for 10 min and the supernatant was removed and concentrated to approx one third the original volume of crude sap . to the concentrate was added 100 % acetonitrile to 30 - 60 %. the resulting white precipitate was removed by centrifugation at 12 , 000 g for 10 minutes . the supernatant was enriched in macrocyclic diterpenes ( jatrophanes and pepluane ), as determined by tlc and mass spectroscopy . this observation points the way to a suitable large scale process for enrichment of the active principles fractions 14 and 15 from the hplc subfractionation described in example 7 and fig3 were further purified by repeated chromatography , selecting the dominant symmetrical peak with constant diode array sectra ( eg . fractions 14 and 15 ; results for fraction 14 are shown in fig4 and 5 ). activity of the purified fractions in causing reversion of mm96l to the dendritic appearance was confirmed by cell assay . the features of the change to m96l cells after the addition of fraction 15 are shown in fig6 . cells were visualised as photomicrographs , using an antibody coupling procedure . the first antibody , a mouse monoclonal directed towards tyrosinase - related protein 1 ( trp - 1 ), was detected with a second antibody , sheep anti - mouse — alkaline phosphatase conjugate , using bromo - chloro - indolyl phosphate and nitroblue tetrazolium ( bcip / nst ) as developing substrates . after four days of incubation ( fig6 a and 6b ) there was a marked reduction in the number of melanoma cells and a pronounced change in their morphology . the cells had reverted to a long , spindly ( dendritic ) appearance , characteristic of normal mature melanocytes . all cells in the field appeared to have adopted this altered morphology , which is surprising given the heterogeneous nature of the m96l cell population . after 21 days of incubation , the treated cells were seen to align somewhat parallel to one another in clusters , as shown in fig6 c and 6d , a characteristic of normal , mature melanocytes . similar features have been observed with all dendritic cell - inducing fractions from e . peplus , including the crude sap . electrospray mass spectroscopic analyses for fractions 14 and 15 indicated the presence of 2 , 5 , 7 , 14 - tetraacetoxy - 3 - benzoyloxy - 8 , 15 - dihydroxy - 9 - nicotinoyloxyjatropha - 6 ( 17 ), 11e - diene ( jatrophane 5 , jakupovic et al , 1998a ) with an m / z of 780 ( calculated 779 . 315 ). nuclear magnetic resonance ( nmr ) analysis , using 1d nmr , on fraction 14 gave down - field signals between 7 and 9 . 4 ppm which are consistent with a pyridine - like moiety , as is present in the nicotinoate group at ring position 9 . also , a trans double bond was evidenced by the large coupling constant at 5 - 6 ppm , in agreement with the 11 , 12 internal double bond in the jatrophane ring structure . also identified in fraction 14 by electrosorav in the negative ion mode was 2 , 5 , 7 , 9 , 14 - pentaacetocy - 3 - benzoyloxy - 8 , 15 - dihydroxy - jatropha - 6 ( 17 ), 11e - diene ( jatrophane 6 , jakupovic et al , 1998a ), with m / z 716 ( calculated 716 . 304 ), 673 ( m - ketene ), 656 ( m - acoh ). fraction 15 contained 2 , 3 , 5 , 7 , 15 - pentaacetoxy - 9 - nicotinoyloxy - 14 - oxojatropha - 6 ( 17 ), 11e - diene ( jatrophane 1 , jakupovic et al , 1998a ) with m / z 597 ( m - ketene - acoh ). thus , by spectroscopic analysis , the early - eluting fractions at 7 - 7 . 5 minutes on hplc with cell killing and dendritic activity contained a mixture of jatrophanes 5 , 6 , and 1 . this result is consistent with the behaviour of hplc fractions 14 and 15 when chromatographed on hptlc , using toluene : acetone 9 : 1 as the developing solvent . uv - positive spots did not move from the origin , r f 0 . 0 ( approx ), in contrast to later - eluting fractions ( eg fractions 20 - 22 , r f 0 . 3 - 0 . 5 ). this indicates the relatively polar behaviour of jatrophanes 5 , 6 , and 1 , in comparison to jatrophanes 3 , 2 and 4 , as demonstrated by chromatography on hptlc , using either toluene : acetone 9 : 1 or hexane : ethyl acetate 4 : 1 as developing solvents . these results are similar to those obtained by jakupovic et al , 1998a , using petrol : methyl - tert - butyl ether ( 1 : 1 ) as the developing solvent , eg : jatrophane 5 : r f 0 . 04 , jatrophane 6 : r f 0 . 10 ( 3 ×), and jatrophane 1 : r f 0 . 11 . there was no evidence in the mass spectroscopic data from the early hplc fractions of the presence of ingenane derivatives ( see later ), or other components reported from the literature and presented in table 1 , in e . peplus crude extracts . biological activity - guided purification of crude and ether - soluble extracts on thin layer chromatography ( tlc ) and high performance thin layer chromatography ( hptlc ) ( a ) the ether - soluble fraction , prepared as in example 7 , was reconstituted in ethylene glycol dimethyl ether ( dme ) and chromatographed on 20 × 20 cm silica gel plates , using chloroform : ethyl acetate ( 82 : 18 ) as the developing solvent ( fig7 ). the plate was viewed on a ( tv light box and the tv positive bands were identified , excised from the gel , eluted with dme , and tested for inhibitory activity and morphology reversal against mm96l melanoma cell line . by slicing the whole gel into uv and non - uv absorbing fractions , it was demonstrated in preliminary experiments that activity was associated with the uv - absorbing bands . staining the side strips of the gel with 0 . 1 % iodine in chloroform revealed other iodine strongly positive bands . however , these were found to possess negligible activity . uv - absorbing bands at rf 0 . 0 ( a ), rf 0 . 16 - 0 . 18 ( b1 ), rf 0 . 22 - 0 . 24 ( b2 ), rf 0 . 73 - 0 . 80 ( c ), rf 0 . 80 - 0 . 96 ( d ) were biologically active , with observable decrease in cell numbers and complete reversion to dendritic cell appearance at { fraction ( 1 / 5 , 000 )} dilution . zones b 1 , c and d were further purified by chromatography on silica gel 60 plates , using a two - dimensional solvent system with hexane : ethyl acetate ( 1 : 1 ) in the first dimension and toluene : acetone ( 9 : 1 ) in the second dimension ( fig8 a to 8 c respectively ). uv - absorbing spots with inhibitory activity towards mm96l of greater than 30 % of cell numbers and with complete reversion to dendritic cell appearance at { fraction ( 1 / 500 )} dilution are indicated on the figures . the strongly uv - absorbing spots 22 and 23 derived from zone d ( see fig8 c ) were excised from the gel , eluted with dme and dried by vacuum centrifugation . mass spectroscopic analysis of fractions 22 and 23 revealed the presence of 5 , 8 , 9 , 10 , 14 - pentaacetoxy - 3 - benzoyloxy - 15 - hydroxypepluane , m / z 639 . 5 [ m - acoh ] − , ie pepluane . ( b ) whole crude sap was chromatographed on 10 × 10 cm hptlc silica gel 60 plates with concentrating zones ( merck cat no . 013748 . 1000 ), using toluene : acetone ( 9 : 1 ) as the developing solvent , as shown in fig9 . the uv - positive zones ( 1 , r f 0 . 14 ; 2 , r f 0 . 23 ; 3 , r f 0 . 49 ; 4 , r f 0 . 54 ; 5 , r f 0 . 57 ; 6 , r f 0 . 63 ; and 7 , r f 0 . 73 ) were excised from the gel and eluted with dme / diethyl ether . the fractions were tested against mm96l as described above , and fractions 1 , 3 , 4 , 5 and 6 were demonstrated to possess cell inhibitory activity and cell reversion activity . these fractions were separately chromatographed on hptlc plates using hexane : ethyl acetate ( 4 : 1 ) as the developing solvent , yielding uv positive bands a , r f 0 . 17 ; b , r f 0 . 24 ; c , r f 0 . 42 ; d , r f 0 . 48 ; e , r f 0 . 52 ; f , r t 0 . 58 ; g , r f 0 . 62 ( fig1 ) and h , r 0 . 02 ( fig1 ). all fractions except g ( iodine positive , see fig1 ) were active against mm96l cells , in terms of cell growth inhibition and reversion to complete dendritic morphology , at 1 in 5000 dilution . mass spectroscopic analyses of fractions a - f ( b missing ) and h are shown in table 15 , with a tentative assignment of compounds from the known molecular mass ions of the published constituents of e . peplus : table 16 1 h chemical shift * data for fraction h h ppm multiplicity indicative jatrophane ring backbone signals 1α 2 . 816 brd 1β 2 . 056 d 3 5 . 918 d 4 3 . 731 brd 5 5 . 730 brd 7 5 . 390 d 8 ( oh ) 2 . 948 d 9 4 . 971 s 11 6 . 145 d 12 5 . 640 dd 13 2 . 840 cm 14 5 . 110 s 15 ( oh ) 3 . 645 s 16 1 . 489 s 17 4 . 438 d 17 ′ 4 . 788 d 18 1 . 052 s 19 1 . 152 s 20 1 . 353 d ester substituent signals onic 9 . 290 brd 8 . 340 ddd 8 . 805 brdd 7 . 390 brd onic 9 . 079 brd 8 . 202 ddd 8 . 767 brdd 7 . 327 brd obz 8 . 040 aa ′ 7 . 403 bb ′ 7 . 541 c oibu 1 . 972 qq 0 . 912 d 0 . 449 d 0 . 450 d * chemical shifts are measured at 295 k relative to chloroform at 7 . 24 ppm . these assignments indicated the presence of a jatrophane ring structure as determined from dqf - cosy , noesy and tocsy two - dimensional spectra . the spectrum or fraction h was consistent with the presence of jatrophane 3 in two diastereomeric conformations ( considered most likely ), a mixture of two or more similarly substituted jatrophanes , or a new jatrophane with two nicotinate , one benzoate , and an iso - butyrate moiety . the likely ring confirmation was ii , as per jakupovic et al ( 1998a ), with a j4 , 5 of approximately 6 hz and strong noe &# 39 ; s between 5 and 8 , and 4 and 7 ; with j7 , 8 and j8 , 9 practically zero — as evidenced by total lack of cross peaks in the dqf cosy spectrum . there were no signals consistent with the presence of any ingenol structure . the sample was retrieved from the magnet , and an aliquot demonstrated potent activity against mm96l , evidenced by complete cell death at 20 μg / ml , and complete reversion to a dendritic appearance at less than 20 pg / ml . fraction a was further purified by chromatography on hptlc using hexane : ethyl acetate ( 4 : 1 ) as the developing solvent . as an adjunct to absorbance on a uv light box , a side strip was stained by spraying the gel with 70 % phosphoric acid in methanol , and development by heating the gel with a hair drier revealed an intense blue band under uv light , separable from the major uv absorbing band . the unstained region equivalent to this band was excised , eluted with ether and dried by vacuum centrifugation . approx . 1 mg of this material was accumulated from 4 ml latex . the material was subjected to nmr analysis , and subsequently bioassayed and demonstrated to be active in terms of reversion to complete dendritic morphology at 1 in 5 × 10 6 dilution , representing a 1 ng / ml final concentration . this material was identified by nmr as c 27 h 36 o 7 , 20 - acetyl - ingenol - 3 - angelate as shown in table 17 . this finding is consistent with the mass spectroscopic evidence presented in table 15 . however , the absence of 20 - acetyl - ingenol - 3 - angelate from the mass soectra of the activity - guided purifications by hplc , and in other tlc fractions apart from fraction a , indicates that this is not the only active fraction . rather , jatrophanes 1 - 6 and pepluane are also implicated by deduction from the nmr and mass spectroscopic data . this is particularly true of fractions h as prepared by tlc ( jatrophane 3 na + m / z 830 ; see also 1d nmr results in table 16 ) and fractions 13 and 14 as prepared by hplc ( jatrophane 5 , m / z 779 and 1d dlr ; jatrophane 6 , m / z 716 ; jatrophane 1 or jatrophane 6 derivative , m / z 597 . jakupovic et al ( 1998a ) have proposed that the paraliane class of compounds are intermediates in the pathway between jatrophanes and pepluane . since anti - cancer cell activity and dendritic cell reversal by both jatrophanes and pepluane have been demonstrated in this invention , it seems reasonable to conclude that the paralianes will also exhibit these properties . preparation of material for the mouse experiments by preparative thin layer chromatography 15 ml crude sap in 70 % ethanol was extracted with diethyl ether as described in example 6 . the extract was concentrated by vacuum centrifugation and resuspended in approx 5 ml dme . the dme extract was chromatographed on preparative tlc plates ( merck plc , silica gel 60 , cat no . 005745 . 1000 ) using hexane : ethyl acetate ( 4 : 1 ) as the developing solvent . zones corresponding to regions “ h ” and “ a - f ” as shown in fig1 were excised and combined , eluted with ether / dme , and dried by vacuum centrifugation . the extract was enriched in jatrophanes 2 , 3 and 4 , pepluane , and the ingenane acetate . the pellet was suspended in 95 % ethanol and centrifuged at 10 , 000 g for 10 minutes . the supernatant ( 6 . 0 ml , 10 mg / ml ) was distributed into 0 . 2 ml aliquots and stored at − 20 ° c . this extract was assayed against mm96l melanoma cell line , and showed high potency , with dendritic cell morphology still evident at 1 in 5 × 10 6 dilution ; this replicated the potency of the crude sap . the extract so prepared was enriched in jatrophanes 2 , 3 and 4 , pepluane , and the ingenane acetate . just prior to injection , 20 μl was diluted to 1 ml with rpmi 1640 tissue culture medium containing 5 % foetal calf serum for injection of 0 . 1 - 0 . 2 ml . the ethanol solution ( 10 mg / ml ) was absorbed on a cotton bud ( 0 . 2 - 0 . 4 ml ) and used for topical application in mice . ( a ) five 4 week old nude mice were injected s . c . at 4 different sites with 0 . 1 ml of tissue culture medium containing 2 × 10 6 mm96l human melanoma cells . the three treated mice were injected on days 1 , 2 , 3 , 5 , 6 , 7 , and 8 with 0 . 1 ml rpmi medium containing 5 % foetal calf serum and 20 μg ethanol extract . in addition , the treated mice received up to four topical applications of approx 5 - 10 μl of 10 mg / ml ethanol extract or crude undiluted sap . two separate sites on each treated mouse received topical treatment with either ethanol extract or crude sap . one mouse received topical treatment on days 12 , 13 and 14 , and the other two treated mice received topical treatment on days 15 , 19 , 20 and 22 . tumour volume was measured on day 32 . prior to the topical applications , injection of extract had no apparent effect on tumour volume . following topical application of ethanol extract there was an overnight change in tumour appearance . the tumours became greyish - black in colour , then developed a hard , lumpy black appearance followed by scab formation . tumours treated with crude sap showed similar changes a day later . with time , the overall effects of ethanol extract and crude sap were similar , so measurements for the topically treated lesions have been combined . on the mice given injection plus topical treatment , tumour volume was reduced by 76 % ( p & lt ; 0 . 2 ). one tumour which had been treated with the ethanol extract had completely disappeared , as shown in fig1 , and eight others were reduced to flat black scabs . the other three treated tumours initially showed similar colour changes and tumour regression , but had regrown following cessation of topical treatment 10 days before the measurements were taken . ( b ) six 4 week old c57 black ( c57b1 ) mice were injected with 0 . 1 ml of tissue culture medium containing 10 5 b16 melanoma cancer cells at two sites on the underbelly . the tumours were allowed to develop for 4 days , and then were subjected to a regimen of three injections ( 20 μg ethanol extract in 0 . 1 ml rpmi medium containing 5 % foetal calf serum ( days 1 , 2 and 4 ) and 1 topical treatment ( 5 - 10 μl of 10 mg / ml ethanol extract on day 4 ). 8 days after the first injection the areas of the tumours were measured using a ruler . treatment reduced the size of the b16 melanoma tumours by 64 % ( p & lt ; 0 . 05 ) on the three treated mice by comparison with the size of tumours on the three control mice . human melanoma cells of the mm 96 l cell line , cultured in 150 cm 2 plates in rpmi 1640 medium containing 10 % foetal calf serum , were incubated with purified extract for 4 hr at 37 ° c . in 5 % co 2 / air . cells were washed with phosphate buffered saline ( pbs ), scraped in pbs , pelleted , resuspended in 1 ml pbs , pelleted and taken up in 300 μl np - 40 lysis buffer , left on ice for 15 min , pelleted and the supernatant treated with proteinase k and sds at 370c for 15 min , extracted with phenol chloroform and the total rna precipitated by ammonium acetate / ethanol at − 20 ° c . overnight . the promega mrna isolation kit was used to isolate mrna , which was then reverse transcribed in the presence of 33 p - labelled dctp to generate cdna . the latter was hybridised on a genome systems human gene discovery array 1 . 2 ( gda ) according to the manufacturer &# 39 ; s instructions . the array was quantitated with a molecular dynamics phosphorimager , and analysed with imageqant and excel software . the ratio of duplicate spot volumes from treated and untreated cells was calculated , and used to define the level of gene activation ( ratio & gt ; 1 ) or inhibition (& lt ; 1 ). backgrounds were typically 500 - 1000 counts , but were not subtracted ; thus the stated ratios will tend to be underestimates of the actual changes . the array contained cdna spots from over 18 , 000 unique sequences , so - called expressed sequence tags ( ests ), of which approximately 3000 were from identifiable expressed genes of human cells . many est sequences in the human melanoma cells tested were either up - or down - regulated by the extract treatment . only changes based on duplicates which had standard deviations & lt ; 30 % of the ratio were considered to be biologically significant at this stage . it should also be noted that a relatively short treatment time of 4 hr was used in order to identify the earliest and most critical targets for the agent . it is likely that further , major changes in gene expression , dependent upon the primary response , will occur after this time . results from the changes in level of the transcripts of some relevant known genes , considered to be beneficial either directly or indirectly for the control of cancer cells , are summarized in table 19 . the changes in cell morphology observed in the examples can be expected to result from the major down - regulation of a number of proteins that bind to actin , a major cytoskeletal protein . an increase in the retinol binding protein may also be involved here , as well as in induction of the differentiated phenotype through increasing the intracellular level of retinoids . repair of current and future dna damage induced by solar uv irradiation may be enhanced by the observed induction of xp repair proteins . in addition , the decrease in gadd45 and ionising radiation - resistance protein ( dap3 ) may be useful in sensitising tumour tissue to radiotherapy . the latter change is also notable because it is strongly upregulated in mm96l cells by uvb , the cause of skin cancer and melanoma . a number of molecules relevant to enhancing the immune response were induced , particularly g - csf . some of these , such as proteins of the major histocompatability complex ( mhc ), are considered to be useful attributes for immunotherapy , enhancing killer t - cell activity . the changes most significant for control of cell growth relate to the detected alterations of the g - protein and pkc pathways , and enhancement of proteosome activity . intracellular signalling is critical for many cell processes , including proliferation and alterations in the normal equilibrium of pathways and pathway interactions , such as those mediated by ras signalling are likely to have adverse consequences for the cell . the level of induction of the proteosome component limp7 - e1 was among the highest found for any gene in the experiment , and would be expected to greatly alter the processing of many proteins via the ubiquitin pathway . on the basis of the gene expression array data , the compounds of this invention are expected to have activity : 1 . in modulating gene expression in the g - protein , pkc and ras signalling pathways , in a manner that leads to anticancer activity in vivo . 2 . in ameliorating damage from solar uv and like agents , by enhancing dna repair and the immune response , either in the target or effector cells . 3 . as an adjuvant to radiotherapy or to therapy with other dna - damaging agents , on the basis of down - regulation of protective proteins ( gadd45 and dap3 ). ethics committee approval was obtained from the queensland institute of medical research for a clinician supervised trial of use of crude sap of e . peplus for treatment of a facial solar keratosis in a human subject . crude extract obtained from australian - grown plants and stored in 50 % glycerol for 2 weeks at − 20 ° c . was applied with a cotton bud applicator to the surface of a clinically diagnosed solar keratosis , approximately 5 mm in diameter , on the left temple of the face of a male human volunteer . approximately 50 μl was delivered to the surface . one day later , a second application was made to the same site . after the first application no reaction was noted for 4 - 5 h , whereafter an inflammation reaction occurred at the site and extended to an area of 80 - 100 mm in diameter . one day later , there was localised swelling , and blister formation at the site of application and on localised patches distal to the area of application , as if new premalignant sites were also targeted . after four days following the first treatment , the swelling subsided and scab formation was evident at the affected sites . after fourteen days , the scabs had sloughed off , leaving new skin underneath . after six weeks , the treated areas still had a pinkish tinge , but there was no sign of the original solar keratosis . as a control , a 1 cm 2 patch of normal skin on the forearm of the same volunteer was similarly treated . there was localised mild inflammation , which disappeared 7 - 10 days after treatment . the strong inflammatory reaction associated with treatment of the solar keratosis could reflect recruitment and proliferation of killer - t cells , as suggested by the results for immune response obtained from the gene array screen in example 13 , and the observation of in vitro proliferation of t - cells by e . peplus crude sap in example 15 below . enhancement of killer - t cell activity is considered to be a key step in destruction of cancer cells by the immune system and may help to explain the recognition and attack of premalignant lesions distal to the site of original treatment . effect of crude sap and purified fractions “ a ” and “ h ” from tlc on normal melanocyte cell numbers 12 - o - tetradecanoylphorbol - 13 - acetate ( tpa ) is essential for the culture of normal melanocytes in vitro , since these cells grow very poorly without tpa . in a preliminary experiment , e . peplus fractions were added to medium without added tpa from the start of the experiment . e . peplus fractions were added to fresh medium , and the cell numbers scored compared to fresh media without e . peplus fractions or tpa . under this regimen , higher numbers of melanocytes were obtained than with the “ control ” cells grown in tpa - deficient medium . interestingly , the cells in the medium with e . peplus fractions looked healthier than those cells grown in so - called “ standard ” medium with tpa . thus e . peplus - derived compounds may provide a superior alternative to the use of tpa as a tool in cell culture . in a second experiment , normal melanocytes were plated at 5000 cells per well , in rpmi 1640 medium containing 10 % foetal calf serum , cholera toxin , antibiotics , and tpa . after 24 hours , the medium was removed from the cells by suction , and replaced with fresh medium without added tpa , but with the additions as specified . cells were scored after a further 10 days of incubation . the results are shown in table 20 . it is evident that even at a 1 in 5 , 000 , 000 dilution a cell proliferation effect was noted with crude and purified fractions , in contrast to cell inhibitory effects observed at these concentrations against cancer cell lines as shown in earlier examples . in a separate test , in vitro proliferation of t cells was also obtained following treatment of t cells with crude e . peplus sap . since both normal melanocytes and t - cells were induced to proliferate by fractions from e . peplus sap , this agent may have wide application as a cell proliferation agent for normal cells , either in vivo or in vitro , in any medical condition where regeneration of normal cells would be advantageous , including but not limited to a ) multiplication of skin cells ( keratinocytes ) for rapid wound healing in trauma cases and after surgery , and in recovery from burns . b ) multiplication of pancreatic islet cells for implantation c ) multiplication of t - cells and other cells of the immune system . it is interesting to note that the expansion of action past the point of application in the human volunteer trial on treatment of solar keratosis may be explained by a recruitment of natural killer - t cells to the region of application . d ) regeneration of aged or necrotic tissue from liver , kidney , colon , lung and eye . e ) multiplication of host tissue as an alternative to organ transplantation betaines of different types were solubilised in sterile milliq ™ water to a final concentration of 1 mg / ml , and diluted into 0 . 1 ml tissue culture medium containing 5000 mm96l cells as described previously . cells were scored after 4 days incubation . the results are shown in table 21 . whereas most betaines tested had no effect on cell numbers , β - alanine betaine hydrochloride ( homobetaine ) depressed cell numbers at a final concentration of 20 μg / ml , and the cells had a dendritic appearance . t - 4 hydroxy n , n - dimethyl proline also inhibited cell numbers at a final concentration of 20 μg / ml ; however , the cell morphology changed to that of polydendritic forms , the significance of which is unknown . it is envisaged that β - alanine betaine hydrochloride ( homobetaine ) may be a suitable formulation agent for e . peplus crude sap or its purified active principles , including ingenol , pepluane , and jatrophanes 1 - 6 , either separately or in combination . this could be used for topical application against premalignant skin lesions at low dilutions of e . peplus principle ( s ), or formulated as an anticancer - drug with higher concentrations of e . peplus principle ( s ). it has been suggested that betaines per se are useful as anti - cancer agents ; see for example u . s . pat . no . 5 , 545 , 667 by wiersema et al . because of their surfactant properties , betaines are widely used as formulation ingredients in cosmetics . due to their zwitterion properties , betaines could also assist transport of other ingredients into the deeper layers of the skin . a betaine to be used in a skin cosmetic preparation along with very dilute extracts of e . peplus sap or purified fractions derived therefrom , such as jatroohanes , pepluane , paraliane , or ingenane , separately or in combination , should desirably have complementary properties of all the betaines tested , including glycine betaine , only β - alanine betaine hydrochloride ( homobetaine ) had a phenotype reversal effect , albeit modest , as compared to e . peplus sap and fractions . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . beljanski m and crochet , s . int . j . oncol ., 1996 8 1143 - 1148 belkin , m . and fitzgerald , d . b . j . natl . cancer inst ., 1952 13 139 . bosch , r . r ., patel , a . m ., van emst - de vries , s . e ., smeets , r . l ., de pont , j . j ., willems , p . h ., pont , j . j . eur j pharmacol 1998 346 345 - 351 djafarzadeh , s . exp cell res . 1997 nov . 1 ; 236 ( 2 ) 1997 , 236 427 - 435 evans , f . j . and kinghorn , a . d . botanical journal of the linnean society , 1977 74 23 - 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( tokyo ), 1994 42 1803 - 1807 this is a continuation of copending application u . s . ser . no . 09 / 888 , 178 , filed on jun . 21 , 2001 , which is a continuation application of u . s . ser . no . 09 / 486 , 199 , now u . s . pat . no . 6 , 432 , 452 , filed on feb . 22 , 2000 , which was filed under 35 u . s . c . 371 based on pct / au98 / 00656 , filed on aug . 19 , 1998 , which claims benefit of priority to australia application no . po - 8640 , filed aug . 19 , 1997 . these application are explicitly incorporated herein by reference in their entirety and for all purposes .