British Journal of Cancer (1999) 79(11/12), 1633—1640
`© 1999 Cancer Research Campaign
`Article no. bjoc.1998.0261
`
`Review
`
`Making the most of rodent tumour systems in cancer
`drug discovery
`
`MC Bibby
`
`Clinical Oncology Unit, University of Bradford, Richmond Road, Bradford, West Yorkshire. BD7 1DP, UK
`
`It is clear that even after almost halfa century of intensive effort to
`develop effective treatments for common solid cancers there is
`still seine way to go before a major impact on survival of patients
`with these malignancies is achieved. Much of the paucity of
`success is blamed on the lack of appropriate models and there is a
`commonly held belief amongst cancer researchers that
`trans—
`plantable tumours in rodents are sensitive to drug therapy, are easy
`to cure and therefore not predictive of responses in humans. it is
`true that,
`in the past, when one considers the large number of
`compounds evaluated, murine tumour models have identified only
`a limited number of clinically useful agents and not a single
`cancer—specific drug has resulted from a murine tumour screen. It
`is easy with hindsight to blame this disappointing lack of really
`effective therapies on inappropriate test systems and screening
`strategies but, although drug discovery from rodent systems has
`been sparse, in reality, an enormous contribution to cancer therapy
`principles has emanated from such studies. This work really began
`in the mid 1950s when the National Cancer Institute (NCI) in the
`USA initiated a large—scale anti~cancer drug—screening programme
`using three murine
`tumour models:
`sarcoma
`180, L1210
`leukaemia and carcinoma 755, (Plowman et al. 1997). Of major
`significance in the early days was the work of Skipper and
`colleagues who, by the use of experimental
`leukaemia models,
`established the principle of
`survival/inoculum relationship
`(Skipper ct a1, 1957) and also the concept offractional cell kill, i.e.
`that the percentage ofthe tumour cell population killed by a given
`dose of drug was relatively constant (Skipper et a1, 1964). They
`also described a close relationship between dose level and
`percentage
`of
`leukaemic
`cell
`population
`killed,
`i.e.
`a
`doscmrcsponse relationship. Other useful
`therapeutic strategies
`were first established in rodent systems. The principle of optimum
`scheduling was first described by Goldin et a1 (1956) using the
`L1210 leukaemia. Combination chemotherapy and the concept of
`therapeutic synergism was first
`investigated in rodent models
`(Goldin and Mantel, 1957) as was combining the modalities of
`surgery and chemotherapy
`adjuvant chemotherapy (Martin,
`1981).
`studies utilized murine
`the early experimental
`Many of
`leukaemias which grew very rapidly, had a high growth fraction
`and proved to be sensitive to a number of agents that were subse—
`quently shown to have more activity against
`leukaemias and
`lymphomas than against solid carcinomas or sarcomas and to be
`toxic to the bone marrow (Muggia, 1987). A number ofinvestiga—
`
`Received 10 July 1998
`Revised 24 August 1998
`Accepted 16 September 1998
`
`Correspondence to: MC Bibby
`
`tors believed that the use of more appropriate, slower growing
`tumour models of solid cancers. and adoption ofclinically relevant
`end-points, would improve the usefulness of preclinical studies,
`particularly for agents with potential activity against the common
`solid cancers. Corbett et a1 (1987) pointed out that most of the
`agents that had entered the clinic at that time had poor, or no,
`activity against the majority of transplantable solid tumours in
`mice but they also suggested that, as negative findings are rarely
`reported, the casual reader of the drug discovery literature may
`have gained the inaccurate impression that transplantable tumours
`in mice are highly vulnerable to a large proportion of agents that
`make the clinic.
`
`THERAPEUTIC INDEX
`
`The importance of addressing the therapeutic index of investiga—
`tional agents has been stressed by many groups over the years,
`including our own (Double and Bibby, 1989) and we have advo—
`cated a more thorough preclinical evaluation of new cytotoxic
`entities which assesses efficacy and normal tissue toxicity in the
`same setting (Bibby ct a1, 1988). Taking mitozolomide (Stevens
`et a1. 1984) as an example compound that had recently entered
`clinical trial, we evaluated anti-tumour activity in a limited panel
`of refractory murine tumours but we also investigated normal
`tissue toxicity at the active doses. Mitozolomide showed a poor
`therapeutic ratio in this test system with modest anti—tumour
`effects being seen close to maximum tolerated dose (MTD) only.
`At this dose level there was very severe normal tissue toxicity in
`mice (Table l). Mitozolomide is an example of (at that time) a
`novel chemical that showed exciting pre—clinical activity in some
`experimental tumour systems with cures in L1210 and P388 at half
`the LDlO but went on to behave poorly in the clinic due to unman-
`ageable haematological toxicity. Although rodent studies do not
`
`Table 1 Bone marrow toxicity of mitozolomide assessed by spleen colony
`forming unit (CFU-S) assay in mice
`
`
`Donor
`Recipient
`Dose
`Radiation
`No. of bone marrow cells
`CFU-S
`
`(mg kg")
`(Gy)
`injected
`
`»~
`20
`30
`40
`
`11.7
`11.7
`11.7
`11.7
`
`1.2 x 10"
`1.0 x 105
`9.0 x 10“
`1.0 x 105
`
`4O
`4
`3
`0
`
`n = 6 mice per group. Data taken from Bibby et a1 1988.
`
`Genentech 2075
`Celltrion v. Genentech
`|PR2017—01122
`
`1633
`
`Genentech 2075
`Celltrion v. Genentech
`IPR2017-01122
`
`

`

`1634 MC Bibby
`
`necessarily predict human organ toxicity, they do appear to be
`helpful
`in predicting effects in some organs. e.g. bone marrow.
`Hindsight has shown that mitozolomide was selected for clinical
`trial on the basis of results from inappropriate pie—clinical models
`that did not reflect the resistance of clinical disease, but the group
`has gone
`on
`to develop
`a useful
`less
`toxic
`analogue,
`Temozolomide (Newlands et al, 1997).
`
`THERAPEUTIC END-POINTS
`
`In the early 19805, Martin and colleagues discussed the signifi—
`cance ofthe methodology used in recording response (Martin et a1,
`1984). They assessed the importance of selecting an appropriate
`end—point
`in preclinical studies by comparing response rates in
`breast cancer patients to six agents and then evaluating the same
`six agents in a murine model of spontaneous breast cancer. the
`CD8F1 (Stolfi et a1, 1988). The breast cancer patients responded
`only to melphalan, cyclophosphamide and 5—iluorouracil (S-FU).
`When the authors evaluated response in the murine system by
`percentage tumour inhibition, all six agents could be classified as
`active, whereas when they employed tumour regression as a
`measure of activity, as in the patients, only the three clinically
`active agents were effective (Table 2). Despite these observations
`cytotoxic agents have still progressed to clinical study on the basis
`of tumour growth delay in rodents at lVlTD and have often been
`shown to be ineffective in humans at doses that can be tolerated.
`Obviously there are certain ethical considerations that need to
`be addressed when selecting model systems and end-points for in
`vivo studies. As long as appropriate guidelines for animal welfare
`are followed (e.g. UKCCCR; Workman et al, 1998), subcutaneous
`models of neoplasia are relatively straightforward to use and
`potential animal suffering can be monitored and appropriate
`humane end-points, such as tumour size, utilized. However, the
`use of ascites tumour models and survival end—points to investi-
`gate the activity of intraperitoneally administered agents is diffi-
`cult to justify on scientific or ethical grounds. These should not be
`used for preclinical screening as they can result in undue suffering
`and provide little more information than well-designed in vitro
`assays. Lethality end-points should be avoided in general. even for
`models of systemic disease, and histological and/or metastatic
`colony counting techniques should be employed to assess anti—
`tumour effects. As modern therapeutic strategies develop, more
`
`effort is required to ensure suitable pharmaeodynamic end—points
`are in place to assess whether or not a designed therapy is
`achieving its goal in vivo.
`
`IN VITRO CELL LINE PANELS
`
`As a result of expanding knowledge, but also limited success, in
`the identification and development of drugs with useful activity in
`common solid cancers, drug—screening programmes in the NCI
`and elsewhere have
`evolved considerably over
`the years
`(Plowman et a1, 1997) until in 1985 the NCI began to assess the
`feasibility of using human tumour cell
`lines for
`large—scale
`compound screening (Boyd, 1989). It is beyond the scope of this
`article to extol the virtues or otherwise of this approach to drug
`discovery but it is clear that, as a result ofits inception, many inter-
`esting molecules are continually emerging and these will require
`appropriate in vivo evaluation. The NCI cell
`line panel has
`expanded since it was initially set up and there is ongoing develop-
`ment and characterization of potential new molecular targets.
`Although the initial strategy for the cell line screen was to develop
`a disease—orientated approach, the addition of the molecular char~
`acterization of the cell
`line panel opens up the possibility for
`target-orientated screening. The option to use genetically modified
`cell lines to address specific molecular targets at the in vitro stage
`is now available. This scientifically sound strategy oftarget-orien-
`tated drug development must be extended into in vivo evaluation
`protocols, so not only should in vivo studies be carried out
`in
`tumours derived from cell lines that were identified as sensitive in
`
`vitro, but it is imperative that adequate characterization of these
`models demonstrates that the molecular characteristics of the cell
`
`line are retained in the in vivo setting.
`
`PHARMACOKINETIC CONSIDERATIONS
`
`Going from cell—free screening strategies and cell lines to in vivo
`studies represents a major step in the drug—discovery pathway and
`one very important requirement is knowledge of the pharmaco—
`kinetics of investigational agents at an early stage. ideally, such
`studies should be carried out alongside efficacy investigations
`with a view to either selecting those agents with the best pharma-
`ceutical properties or, alternatively, formulating or even modifying
`the active component to optimize the therapeutic effect.
`
`
`
`Table 2 importance of clinically relevant endpoints in predictability of preclinical anti-tumour studies
`Drug
`Human
`CD8F1 spontaneous
`breast cancer
`breast tumours
`
` inhibited (%)a Assessment Regression Assessmentb
`
`
`
`
`
`
`
`Meiphalan
`Cycio
`FU
`
`+
`+
`+
`
`88
`80
`79
`
`+
`+
`+
`
`7/24
`5/25
`13/60
`
`+
`+
`+
`
`N-phosphon-acetylaspartate
`—
`63
`+
`2/47
`~
`Ara-c
`—
`33
`+
`1/37
`«
`— 52 + 0/456‘thioguanine —
`
`
`
`
`
`
`
`
`
`
`
`aStatistically significant; bat least 50% reduction. Data taken from Stoifi et a1 1988.
`
`British Journal of Cancer (1999) 79(11/12), 7633—1640
`
`© Cancer Research Campaign 1999
`
`

`

`HOLLOW-FIBRE ASSAYS
`
`One relatively new approach to in vivo drug testing has been
`developed at NC] utilizing human cell-lines growing in hollow
`fibres. It is intended as a method for prioritizing compounds for
`testing in xenografts. At present. 10 000 compounds are screened
`in vitro against the cell line panel each year by NCl and 840% are
`referred for in vivo testing (Plowman et al, 1997). For the hollow—
`fibre assay,
`tumour cells are inoculated into l—mm internal
`diameter hollow—fibres that are heat—sealed and cut
`into 2cm
`
`lengths. The fibres are maintained in in vitro culture for 24—48 h
`and then implanted intraperitoneally and subcutaneously into nude
`mice. Three different cell lines can be grown in two different sites
`in the same mouse. The effects of treatment are determined by
`MTT assay on removal of the fibres 6~~8 days post—implantation.
`On the face of it, this technique would seem an efficient way
`of identifying lead compounds of promise because it requires
`relatively small expenditure and a
`limited quantity of test
`compound. Of course, as mentioned earlier, it cannot be assumed
`that expression ofa particular target will be identical in vivo to that
`expressed when the same cells are grown in vitro or vice versa.
`
`TUMOUR CHARACTERIZATION
`
`For several years our laboratory has been involved in studies of
`potential bioreductive drugs but in particular we have developed
`an interest in the role of the dimeric flavoprotein DT—diaphorase
`(DTD, NAD(_P)H:Quinone acceptor reductase, E.C.1.6.99.2) in the
`activation of a number of quinone based anti-cancer drugs
`(Phillips, 1996). There is a wealth of literature indicating a good
`correlation between enzyme activity in cell
`lines and aerobic
`chemosensitivity to compounds like E09. 3‘hydroxy—S-aziridinyl—
`l—methyl—2( 1 H-indole-4,7-dione)-prop-B-en—0t—ol
`(Robertson et
`al, 1992; Smitskamp-Wilms et al, 1994: Collard et al, 1995;
`Fitzsimmons et al, 1996). However, when attempts were made to
`translate these in vitro observations into in vivo studies it became
`
`apparent that DTD activity in human tumour xenografts derived
`from cell lines did not usually mirror levels seen in vitro (Collard
`et al, 1995),
`indicating the necessity to further characterize
`xenografts for the appropriate target for which therapeutic mole-
`cules are being sought.
`In the case of DTD, antibodies are
`available (Segura—Aguilar et al, 1994) so it is possible to identify
`the precise location of the protein within tissue sections (Segura—
`Aguilar et al, 1994; Phillips et al, 1998), and similar immunolocal—
`ization can be carried out now with a whole host of target enzymes
`and other molecular targets. It is certain that the majority oftargcts
`currently being investigated for drug development strategies will
`be influenced by their local environment, so not only will there be
`differential target expression in different tumours but there will
`also be different expression in vivo from that seen in in vitro
`culture of the same tumour cells. Furthermore,
`it
`is clear that
`tumour site within the body can influence the expression of
`specific targets, cg. P—glycoprotein. Fidler and colleagues investi-
`gated response to doxorubicin (DOX) or S—FU in three tumour
`types growing in different anatomical sites (Dong et al, 1994;
`Fidler et al, 1994). Sensitivity to 5~FU did not alter with anatom-
`ical site but
`lung and tumour deposits were resistant to DOX
`whereas subcutaneous (s.c.) tumours were sensitive. The authors
`determined that
`the difference in response was not due to
`differences in DOX distribution or potency but resulted from
`
`Rodent tumour systems in cancer drug discovery 1635
`
`over expression of mdrl mRNA in the resistant sites, they made
`the valid point that human colon cancer xenografts growing sub—
`cutaneously in nude mice often respond to DOX, whereas human
`colon cancer does not. These observations reiterate the require-
`ment for continual target characterization in in vivo models. Since
`modern molecular biology allows for the dissection of signalling
`pathways instrumental in cell proliferation and death, the number
`of potential molecular targets for drug development is increasing.
`Preclinical systems must be selected or designed in such a way to
`ensure that the appropriate target is expressed, and the appropriate
`controls in which the target is down—regulated or missing should
`be used in order to establish proof of principle and to ensure the
`validity of the therapeutic mechanism.
`
`HOST EFFECTS
`
`Although a molecular target approach using high—tlu‘oughput,
`robotic cell—free in vitro screens or cell
`line panels followed by
`properly characterized in vivo models should increase the effi—
`ciency of drug—discovery programmes, novel therapeutic strategies
`for cancer are clearly not going to be identified only by in vitro
`screening. These screens do not take into consideration indirect
`mechanisms such as host metabolism to an active species or
`immunomodulation. Within the rapidly expanding field oftumour
`biology several new therapeutic avenues are being explored that
`address host/tumour relationships or exploit specific features of
`solid tumours,
`thus relying even more on clinically relevant
`preclinical in vivo models.
`
`TU MOUR BLOOD SUPPLY
`
`One area of solid tumour biology that has been receiving a great
`deal of attention over recent years is the tumour blood supply.
`Broadly speaking,
`to date there are four different strategies
`attempting to exploit the tumour blood supply for therapeutic gain
`and all rely not only on differences in the architecture and cellular.
`biochemical and molecular properties between normal and tumour
`vasculature but also on the use of a properly characterized model.
`The major thrust at present seems to be concerned with anti-angio—
`genesis. i.e. a strategy to prevent the development of new blood
`vessels and to restrict solid tumour growth. There has been an
`enormous leap in our understanding of the processes and molec-
`ular control of tumour angiogenesis in recent years and the anti-
`angiogenic approach has been the subject of seine excellent
`comment articles and reviews (Folkman, 1990, 1997; Baillie et al,
`1995; Pluda, 1997). Modulation of tumour growth by the use of
`monoclonal antibodies to vascular endothelial growth factor
`(VEGF) has proved successful in preclinical studies (Warren et a1,
`1995) but the rational design of drugs which will specifically inter—
`fere with steps in the angiogenic process in tumours is in its
`infancy as is the development of gene therapy approaches (Kong
`and Crystal, 1998). However,
`targets are being identified. so
`continual development, improvement and characterization of in
`vivo tumour models in which to test specific strategies is required.
`The other attempts to exploit tumour blood supply have concen-
`trated on the existing blood vessels within solid tumours. One
`approach relies on the lack of smooth muscle and innervation of
`the new blood vessels growing in solid tumours. Several experi-
`mental studies have demonstrated potentiation of the activity of
`both standard and investigational drugs by combination with a
`
`@ Cancer Research Campaign 7 999
`
`British Journal of Cancer (1999) 79(11/12), 1633—1640
`
`

`

`1636 MC Bibby
`
`Normal tissue
`
`*
`
`9!
`
`’k
`Lymphatic drainage
`69
`\G)
`
`6),!
`
`it
`
`*
`it
`*
`it
`{37*
`ff :miteddrainage
`im‘fififl *
`
`‘k”:;€*7fir* G
`H: :12? 1:
`r
`*
`
`*
`
`at
`
`Continuous endothelium
`
`Leaky endothelium
`
`Figure 1
`
`Diagramatic representation of Enhanced Permeability and Retention in tumours (Matsumura and Maeda, 1986)
`
`
`
`i
`
`Glycine
`
`r
`i
`$HCH2‘Q
`90
`W CH3
`
`CH2
`
`l
`Phenylalanine
`i
`1
`Leucine
`
`
`Glycine
`
`
`
`Figure 2 Chemical structure of PK1 (Duncan, 1992)
`
`variety of vasoactive agents. A large number of agents alter blood
`flow in tumour models (Hirst and Wood, 1989) but many experi—
`mental studies utilized the anti-hypertcnsive hydralazine which
`has been shown to decrease blood flow through transplantable
`tumours in rodents (Chan et a1, 1984; Jirtle, 1988). Hydralazine
`enhanced the effectiveness of bioreductive drugs, such as the
`nitroimidazole RSU 1069 (Chaplin and Acker, 1987), tirapaza-
`mine (Brown, 1987), 1309 (Bibby et a1, 1993) and mitomycin C
`(Cowen et a1, 1994) but potentiation was also seen with melphalan
`
`(Stratford et a1, 1988) and tauromustine (Quinn et a1, 1992).
`Although these and other similar studies indicated a potential ther—
`apeutic strategy they have not yet resulted in a clinical advantage.
`Rowell ct a1 (1990) showed that single-dose oral hydralazine
`caused the blood flow through human lung tumours to decrease
`rather than increase. A study by Field et a1 (1991) demonstrated a
`difference in the effects of hydralazine on vascular perfusion
`between a series of primary and subcutaneously transplanted
`malignancies. The transplanted tumours responded to hydralazine
`
`British Journal of Cancer (1999) 79(17/72), 1633—7640
`
`© Cancer Research Campaign 1999
`
`

`

`whereas the primary tumour from which they were derived did not.
`Only 4/11 primary tumours responded to hydralazine. All
`the
`tumours used in the preceding papers were transplanted subcuta—
`neously in mice so, although they may have established proof ofa
`particular hypothesis,
`the studies are unlikely to be clinically
`predictive. An investigation from this laboratory (Cowen et al,
`1995) demonstrated that hydralazine was more effective at shut—
`ting down blood supply to so murine colon tumours than to the
`same turnouts transplanted orthotopically, and the same dose of
`hydralazine was completely ineffective in liver metastasis in this
`model. These studies highlight
`the lack of prediction by s.c.
`tumours for this strategy and the importance of selecting clinically
`relevant tumour models in appropriate anatomical sites.
`Another approach to targeting solid tumours by means of their
`poorly formed blood vessels relies on the enhanced permeability
`and retention effect (EPR) coined by Matsumura and Maeda
`(1986) (Figure 1). Many experimental tumours appear to selec-
`tively concentrate macromolecules of around 20 kDa molecular
`mass. This phenomenon is not due to just the leakiness of the
`tumour vasculature but also lack of the organized lymphatic
`drainage that usually occurs in normal tissues. One strategy that
`seems to be clinically useful at present is the incorporation of cyto-
`toxic moieties into copolymers of hydroxypropylmethylacryl—
`amide (HPMA) (Duncan, 1992). One ofthese compounds, PKl, is
`a copolymer with pcptidyl side—chains terminating in DOX (Figure
`2). The stable side—chain is thought to ensure that there is no extra-
`cellular release of DOX so that normal tissue toxicity is reduced.
`The polyrnerwdrug conjugate was designed so that
`the intact
`rnacromolecule is taken up into cells by pinocytosis and trans~
`ferred to the lysosomal compartment where it
`is exposed to an
`array of enzymes resulting in release of DOX. PKl containing at
`least four times the normal clinical dose of DOX was delivered to
`
`patients in a phase I trial and two responses were seen (Connors
`and Finedo, 1997). This kind of targeting seems a worthwhile
`strategy to pursue, but
`it
`is imperative that appropriate in vivo
`tumour models are employed in order to ensure the correct mole-
`cules are selected rapidly for clinical study. Preclinical studies with
`PKl
`indicated that, not only was it active against vascularized
`solid tumours in mice, it was also active against L1210 leukaemia
`when delivered by the intraperitoneal route (Duncan et al, 1992).
`Recent studies in this laboratory have also demonstrated that
`tumours in which the blood vessels are not leaky are no more
`responsive to PKl
`than to DOX. This study also demonstrated
`activity against avascular murine colon cancer
`lung colonies
`(Loadman et al, 1998) suggesting that, although there is impelling
`evidence from numerous studies that EPR plays a major role in the
`activity of PKl,
`the absence of such a phenomenon from
`rnicrornetastases would not preclude activity. Appropriate studies
`are required to further investigate this interesting approach.
`There is evidence emerging that
`the endothelium of solid
`tumour vasculature itself may be a useful target for drug therapy. It
`has been known since the 19305 that colchicine caused anti-
`
`vascular effects in experimental tumours (Clearkin, 1937) and it is
`now clear that some clinically useful tubulin interactive agents
`possess a vascular component in their mechanism of action against
`murine solid cancer models (Hill et al, 1993). However,
`these
`effects are seen only at doses close to MTD; the therapeutic index
`is extremely small so the ratio of sensitivity of tumour endothe~
`lium to normal cells is also small. A number of stilbenes,
`the
`cornbretastatins, has been isolated from the South African bush
`
`Rodent tumour systems in cancer drug discovery 1637
`
`willow, Combrerum crqffrum, and based on them a series of
`synthetic analogues has been synthesized (Pettit et al, 1989, 1995).
`Cornbretastatin A4 has been shown to bind to tubulin at
`the
`colchicine—binding site (McGown and Fox, 1989) and cause
`tumour blood flow reduction (Chaplin ct al, 1996), and Dark et al
`(1997) have demonstrated vascular shutdown with combretastatin
`A4 phosphate prodrug in so. experimental tumours at less than
`one—tenth of the MTD. Fearing the possibility of transplantation
`artefacts in these anti—vascular effects we have examined the
`
`efficacy of both combretastatin A4 and its prodrug on orthotopi—
`cally transplanted rnurine colon tumours and ensuing metastatic
`
`’D1o
`
`3
`'2{as}
`
`
`
`Figure 3 Haemorrhagic necrosis in an orthotopically implanted colon
`tumour and its metastasis following treatment with the investigational agent
`combretastatin A4 (courtesy of K Grosios). (A) Control caecal implant;
`(B) treated caecal implant; (C) treated metastatic deposit
`
`© Cancer Research Campaign 1999
`
`British Journal of Cancer (1999) 79(11/12), 1633—1640
`
`

`

`1638 Me Bibby
`
`deposits (Grosios et a1, 1997). Both parent compound and prodrug
`caused major haemorrhagic necrosis in tumours at the orthotopic
`site and in metastatic deposits (Figure 3). Careful examination of
`morphological effects indicated that
`lung deposits that had not
`undergone neovascularization did not respond to either compound,
`indicating that vaseularization and not
`transplantation site
`seems to be the important component for the activity of these
`compounds. The cornbretastatin A4 prodrug is awaiting phase 1
`clinical trial and it remains to be seen whether the vascular effects
`
`predicted from the preclinical models will occur.
`Another compound selected for clinical
`trial on the basis of
`preclinical anti—vascular effects is 5,6-dimethyl xanthenone acetic
`acid (XAA). This is a more potent analogue of flavone acetic acid
`(FAA) (Atassi et a1. 1985) from a series developed by Baguley and
`colleagues in Auckland, New Zealand (Atwell et
`a1, 1989;
`Rewcastle et a1, 1989, 1991a, 1991b, 1991c). FAA is an interesting
`molecule that had impressive experimental activity but, disap—
`pointingly, failed to show any clinical activity (reviewed by Bibby,
`1991; Bibby and Double, 1993). XAA again showed impressive
`haemorrhagic necrosis in vascularized solid tumour models but
`this time at tenfold lower doses than FAA (Rewcastle et al, 1991a:
`Ching et a1, 1992; Laws et al, 1995a) and also caused widespread
`haemorrhagic necrosis in an orthotopic model of human colon
`cancer (Laws et al, 1995b). Phase 1 clinical trials are ongoing in
`Auckland and in Mt Vernon Hospital/Gray Laboratory and
`Bradford, UK. To date, tumour vascular effects have been noted in
`two patients (Rustin et a1, 1998) suggesting that the preclinical
`models may well have been predictive in this case but, more
`importantly, demonstrating proof of principle, i.e. that a vascular
`effect demonstrated in in vivo preclinical models can also occur in
`solid malignancies in man. These clinical effects suggest that this
`anti—vascular strategy may well be worth pursuing with other more
`potent chemical entities.
`A particularly interesting development that again relies on thor-
`ough characterization of in vivo models has come from the identi—
`fication that certain peptides home specifically to the vasculature
`ofspecific organs (Pasqualini and Ruoslahti, 1996) and to tumour
`vasculature (Arap et a1, 1998). In the latter study the authors have
`demonstrated that it may be possible to target chemotherapeutic
`drugs specifically to tumour vasculature on the basis of differential
`expression of receptors.
`
`ORTHOTOPIC TUMOUR MODELS
`
`It is now clear from large numbers of studies, some of which are
`cited here, that it is necessary to take account of not only the rele-
`vanee of the tumour type utilized for the in vivo evaluation of
`novel drugs and other therapeutic strategies but also tumour site.
`In order to do this it is necessary to use model systems that reflect
`the morphology and growth characteristics of clinical disease but,
`equally as importantly, must be technically feasible and ethically
`acceptable to carry out. For the last 20 years good studies have
`been reported in the literature indicating that such orthotopic
`systems exist (eg. Tan et a1, 1977; Goldrosen et a1, 1986; Bresalier
`et a1, 1987; Morikawa et a1, 1988; Fu et a1, 1991) and the worth of
`such systems has been extensively reviewed (Fidler et a1, 1990;
`Fidler, 1991; Gutman and Fidler, 1995; Hoffman, 1997).
`It
`is
`important to remember that, in the case of common solid cancers
`in humans, the primary tumour mass is usually excised by the
`surgeon and it
`is the metastases that require novel
`therapeutic
`
`strategies, so models should reflect these clinical metastases. It is
`still necessary to refine orthotopic techniques in order to improve
`the predictability of these models in the future, either by character—
`ization for specific molecular targets within these clinically rele—
`vant sites of metastasis, or to develop similar models with tumour
`cells appropriately transfected to express the target of interest. It
`must be remembered that, if one adopts an orthotopic approach
`with models for drug discovery, due attention is given to appro-
`priate end-points. Lethality should not be seen as an alternative to
`a specific pharmacodynamic end—point such as changes in blood
`flow in the case of anti-vascular strategies or production of
`cytokines by immunornodulators etc.
`
`CONCLUSIONS
`
`It is clear that murine tumour models have been very helpful in
`determining basic principles of cancer chemotherapy and to date
`have been instrumental
`in identifying and evaluating a limited
`number of clinically useful agents, Contrary to popular belief
`many murine tumours are not easy to cure with standard
`chemotherapeutic agents and, ifadequate attention is paid to clini-
`cally relevant end—points and therapeutic index, they can be good
`predictors of clinical activity. Of course today a whole host of
`approaches and assays is available to drug—discovery teams:
`
`-
`-
`
`'
`-
`
`high throughput robotic cell-free screens
`in vitro cell lines (sometimes genetically modified to express a
`specific target)
`hollow—fibre in vivo assays
`induced rodent tumour systems W usually intraperitoneal or
`subcutaneous
`
`spontaneous rodent tumours
`-
`- models ofmetastasis
`
`-
`-
`-
`
`orthotopic models
`human tumour xenografts
`transgenic systems.
`
`Modern drug discovery should be much more mechanistically
`based and target-driven, so the first two of these systems should
`continue to lead to identification of structures that have potential
`for development. Assays like the NCI hollow-fibre screen provide
`a rapid and relatively inexpensive method for assessing in vivo
`potential. The remaining systems should be carefully selected
`based on the particular therapeutic approach. The evidence from
`the literature is clear in that it indicates that rodent tumours should
`
`not be used for random screening. lt is necessary to fully charac-
`terize tumour models to ensure that
`the precise target for the
`strategy being evaluated is expressed in vivo. Since targets are
`known to vary with different anatomical sites, more effort should
`be placed on developing models in clinically relevant sites rather
`than the continuous use of subcutaneously transplanted tumours
`whilst ignoring their tissue of origin or likely site of metastasis in
`humans. Ultimately, regardless ofthe molecule being investigated
`and developed,
`it
`is necessary to produce a safe pharmaceutical
`preparation that can be delivered in an appropriate way to reach
`the target for which it has been designed, so to make real progress
`in drug development appropriate in vivo rodent models are still
`essential. However,
`in addition to scientific considerations, due
`ethical consideration must be given to ensure selection of the
`appropriate model for the task in hand and animal welfare guide—
`lines rnust be followed throughout.
`
`British Journal of Cancer (1999) 79(11/12), 1633—1640
`
`© Cancer Research Campaign 1999
`
`

`

`In drug—discovery programmes it is not financially viable, nor
`does it make scientific sense,
`to use large panels of murine’
`tumours or human tumour xenografts
`for extensive in vivo
`screening, particularly when these xenografts are often poorly
`characterized. The most efficient and ethical approach must be
`to design limited, but
`relevant, experiments to address key
`questions, cg:
`
`- Can effective drug concentrations based on previous in vitro
`data be achieved in vivo?
`
`'
`
`'
`
`Is the molecule reaching its designated clinically relevant
`target?
`Is there efficacy or, in other words, is there proof of principle?
`
`If these questions can be answered in the affirmative then the
`compound should be scheduled for clinical trial without the need
`for further extensive in vivo testing.
`In summary,
`there are certain key requirements for in vivo
`studies:
`
`‘ Models must have known clinically-relevant target status.
`- Appropriate bioavailability oftest molecules should be demon-
`strat