[CANCER RESEARCH 52, 5940-5947. November 1, 1992]
`
`Phase II Preclinical Drug Screening in Human Tumor Xenografts: A First
`European Multicenter Collaborative Study1
`
`Epie Boven,2 Benjamin Winograd,3 Dietmar P. Berger, M. Patrick Dumont,‘ Boudewijn J. M. Braakhuis,
`(bystein Fodstad, Simon Langdon, and Heiner H. Fiebig
`Department of Medical Oncology, Free University Hospital, Amsterdam,
`the Netherlands IE. 3.]; New Drug Development Office of the European Organi-
`zation for Research and Treatment of Cancer, Amsterdam, the Netherlands [8. WJ; Department of Internal Medicine, University of Freiburg, Freiburg, Germany
`/D. P. 8., H. H. E]; Institut Jules Bordet, Brussels, Belgium [M. P. D.]; Department of Otalaryngology, Free University Hospital, Amsterdam, the Netherlands
`[8. J. M. 8.]; Department of Tumor Biology, Norwegian Radium Hospital, Oslo, Norway [0. Ii},- and Imperial Cancer Research Fund, Medical Oncology Unit,
`Western General Hospital, Edinburgh, Scotland IS. L]
`
`ABSTRACT
`
`In a European joint project carried out in 6 laboratories a disease-
`oriented program was set up consisting of a panel of 7 tumor types, each
`represented by 4 to 8 different human tumor lines, for secondary screen-
`ing of promising anticancer drugs. Human tumor lines were selected on
`the basis of differences in histology, growth rate, and sensitivity to
`conventional cytostatic agents. Xenografts were grown s.c. in nude mice,
`and treatment was started when tumors reached a mean diameter of
`6 mm in groups of mice where at
`least 6 tumors were evaluable.
`Drugs were given at the maximum talented dose. For evaluation of
`drug efficacy, median tumor growth curves were drawn, and specific
`growth delay and treated/control x 100% were calculated. Doxorubicin
`(8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and
`specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal
`cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers,
`4 of6 small cell lung cancers, 0 of3 melanomas, and 3 of6 ovarian
`cancer lines. Amsacrine (8 mg/kg i.v. days 1 and 8) was not effective,
`while datelliptium (35 mg/kg i.p. days 1 and 8) was active against 2 of
`6 small cell lung cancer lines. Brequinar sodium (50 mg/kg i.p. days
`1-5) showed efficacy in 4 of 5 head and neck cancers, 5 of 8 non-small
`cell lung cancers, and 4 of 5 small cell lung cancer lines. The project has
`been shown to be a feasible approach. Clinical activity for doxorubicin
`and inactivity for amsacrine against solid tumor types was confirmed in
`the human tumor xenograft panel. Additional anticancer drugs will be
`studied in the European joint project to further define the reliability of
`this novel, promising screening approach.
`
`INTRODUCTION
`
`Since 1985 the screening program for new anticancer drugs
`used by the American National Cancer Institute has been the
`subject for discussions on improving drug discovery (1). Up to
`1985, the screening system had consisted of the in viva P388
`murine leukemia prescreen and a secondary screen formed by a
`panel of 5 or 6 murine tumor models and 3 human tumor
`xenografts. Compounds without minimal activity in P388 leu-
`kemia were usually rejected from further testing, unless they
`showed activity in other biological or biochemical test models
`(“bypass compounds”). In view of the clinical data for com-
`pounds selected by the system from 1975—1985, it was con-
`cluded that the screen had low predictive capacity and had only
`defined a few new anticancer drugs (2-4). Most of the clinically
`effective drugs appeared to be active against selected tumor
`
`Received 3/25/92; accepted 8/25/92.
`The costs of publication of this article were defrayed in part by the payment of
`page charges. This article must therefore be hereby marked advertisement in accord
`ance with 18 U.S.C. Section 1734 solely to indicate this fact.
`' Supported by Dupont de Nemours lntemational, Geneva, Switzerland, and
`Sanofi Recherche. Montpellier, France.
`3 To whom requests for reprints should be addressed, at the Free University
`Hospital, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands.
`3 Present address: Bristol Myers-Squibb. Brussels, Belgium.
`‘ Present address: Centre National de la Recherche Scientifique, Toulouse Ce-
`dex. France.
`
`types, mainly leukemias and lymphomas (5,6). Thus, the use of
`P388 leukemia alone as a prescreen would limit the potential to
`discover and develop different types of compounds with pref-
`erential activity against solid tumor types.
`Improvement of anticancer drug discovery may be accom-
`plished by modifying the tumor-oriented approach into a dis-
`ease-oriented program. At the National Cancer Institute, a ma-
`jor effort‘has been put into the development of large-scale in
`vitro assays based on relatively large panels of human cell lines
`representing various tumor types (1). Using these assays as an
`in vitro prescreen would be an effective way to identify com—
`pounds which could otherwise be lost in the P388 prescreen.
`Also, tumor types might be defined to be responsive to a par-
`ticular drug. The in vitro drug screening leads should require
`follow-up testing in vivo. In the stage of in vivo drug analysis, we
`consider the use of human tumor xenografts grown s.c. in nude
`mice to be an excellent means of generating supplementary data
`on the potential activity of a promising compound in specified
`human solid tumors (7).
`In Europe, several investigators have gained experience in the
`establishment of human tumor lines from tumor tissue obtained
`from patients, the characterization of these lines, and the de-
`termination of sensitivity to both conventional and investiga-
`tional anticancer drugs (8). Human tumor xenografts do not
`only retain the histological, biochemical, and antigenic charac-
`teristics but also the chemosensitivity of the tumor tissue of
`origin (9—13). Moreover, human tumor lines representing a
`particular tumor type have the capacity to identify compounds
`with clinical activity in that disease (14). In 1988, a European
`multicenter collaboration was created to assess the feasibility
`and the reliability of “preclinical” phase II studies in a large
`number of human tumor xenografts (15). We now report on the
`first results of this joint project in which doxorubicin, amsa-
`crine, brequinar sodium, and datelliptium were studied in a
`panel of human tumor lines representing 7 tumor types.
`
`MATERIALS AND METHODS
`
`Animals. Participants in the joint project used female nude mice
`of a strain available in their laboratories, NMRI nu/nu (E. 3., D. P. 8.,
`B. .1. M. 3., H. H. F.), BALB/c nu/nu (M. P. D., 0. F.), or nudes of a
`mixed background (S. L.). The animals were maintained in cages in
`isolation under sterile conditions and fed ad Iibitum. Animal handling
`was carried out under sterile conditions by a limited number of persons
`either in laminar flow hoods or within isolators.
`
`Tumor Lines. A selection was made from a large pool of human
`tumor lines suitable for chemotherapy studies. The criteria for selection
`were based on differences in histology, growth rate, and sensitivity to
`conventional cytostatic agents and will be described in a separate report.
`The panel was composed of a choice of 7 tumor types which were
`represented by 4 to 8 different human tumor lines (Table 1).
`
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`Hospira v. Genentech
`M... W WWJERZQJ7-00737
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`1
`
`

`

`PHASE ll SCREENING IN HUMAN TUMOR XENOGRAFTS
`
`Table 1 Characteristics of human tumor liner grown s.c. in nude mice
`Tumor line
`Histology
`TD“
`Breast
`MAXF 401
`MAXF 449
`MAXF 583
`MAXF 857
`Colorectal
`CXF 158
`CXF 233
`CXF 243
`CXF 280
`Head and neck
`HNX-HEp—Z
`HNX-LP
`HNX-14C
`HNX—Pl
`HNXe14A
`Lung-NSCLC
`EVXOA
`AHXOL
`LXFE 397
`LXFE 409
`LXFL 529
`LXFA 629
`NXEE 002
`CXEA 117
`
`Papillary adenocarcinoma
`Solid adenocarcinoma
`Adenocarcinoma
`Ductal carcinoma
`
`Adenocarcinoma
`Adenocarcinoma
`Adenocarcinorna
`Undifferentiated carcinoma
`
`Squamous poorly differentiated
`Squamous moderately differentiated
`Squamous poorly differentiated
`Undifferentiated
`Squamous poorly differentiated
`
`Adenocarcinoma
`Large cell carcinoma
`Squamous cell carcinoma
`Squamous cell carcinoma
`Large cell carcinoma
`Adenocarcinoma
`Squamous cell carcinoma
`Adenocarcinoma
`
`5—11
`7—1 l
`6—1 2
`5—10
`
`7—12
`6—8
`6-8
`3—5
`
`5—8
`8—11
`4-7
`6—9
`7-10
`
`6—1 1
`9-12
`2—4
`3-5
`3—5
`5—8
`4-10
`8—12
`
`Evaluation. Tumor volume was calculated according to the formula
`
`0.5 x length x width x width
`
`The increase in tumor volume from the start of treatment (V0) until the
`value at any given time (V,) was calculated for each tumor and day of
`measurement and expressed as the relative tumor volume (Vt/V0). The
`median of these values for all evaluable tumors in the control and the
`treated groups was used to calculate treatment efficacy. Complete re-
`missions were recorded separately and represent tumors that disap-
`peared for a period of at least 4 weeks. Tumors that had not reached
`4 mm in one diameter at the start of treatment were considered uneval-
`uable. Animal deaths occurring within 2 weeks after the final drug
`injection were considered toxic and were excluded from the evaluation.
`Three evaluation criteria were used in parallel to express treatment
`efficacy: (a) median tumor growth curves; (b) specific growth delay over
`one and two doubling times; (c) the optimal growth inhibition (T/C%)5
`at a particular day within 4 weeks after the last drug administration. To
`calculate SGD, the end point of evaluation was taken at a median
`relative tumor volume of twice and 4 times the treatment size from day
`0 for control and treated tumors according to the formula (15)
`
`Tdtmted — Tdeomzol
`Wm
`
`For the T/C% calculations, the lowest ratio was taken of the median
`relative volume of treated tumors over that of control tumors multiplied
`by 100% (15).
`Central data evaluation was performed under the responsibility of
`Drs. Berger and Fiebig, using software specifically designed for the
`evaluation of nude mouse—human tumor experiments. A growth curve
`was calculated on the basis of tumor volume experiments for each study
`group and tumor line. Data were processed on a Sperry Univac 1100/
`82, for which the software was designed with the use of Fortran 77.
`
`RESULTS
`
`The schedules of doxorubicin and amsacrine were derived
`from the treatment regimens in solid tumors in the clinic
`(16, 17). Clinical cycles of 3—4 weeks were converted to weekly
`cycles x 2. As daily administration for brequinar sodium was
`required,
`the drug was administered i.p. on days 1-5. For
`datelliptium the weekly schedule was selected. Because this
`drug caused acute toxicity at a dose of 30 mg/kg i.v., experi-
`ments were carried out using the i.p. approach. The maximum
`tolerated dose for all four drugs was determined in non-tumor-
`bearing mice first by Dr. Boven. This dose was based on a
`median weight loss of 10% in mice in the week following the
`first injection. In the ultimate experiments some variation was
`evident for the effect of a particular drug on weight loss for the
`various mouse strains in the six laboratories. This effect was
`
`most pronounced for doxorubicin. When this occurred, exces-
`sive weight loss or toxic deaths compromised the evaluability of
`the experiments. Delayed toxicity resulting in late deaths has
`not been observed with any of the drugs.
`The treatment results of the various experiments obtained
`with the four drugs in the human tumor lines are presented in
`Tables 2-5. In each of the tables the following variables are
`given per tumor line: (a) the number of evaluable tumors;
`(b) the maximum median decrease in body weight for evalu-
`able animals as compared to the body weight at the start of
`treatment (%); (c) the number of animals dead from toxicity
`
`Lung-SCLC
`LBXOS
`LXFS 538
`LXFS 605
`LXFS 638
`LXFS 650
`NXES 004
`WXES 322
`Melanoma
`THXO
`FEMXO
`MEXF 274
`MEXF 394
`MEXF 514
`
`Small cell lung carcinoma
`Small cell lung carcinoma
`Small cell lung carcinoma
`Small cell lung drcinoma
`Small cell lung carcinoma
`Small cell lung carcinoma
`Small cell lung carcinoma
`
`Amelanotic
`Melanotic
`Amelanotic
`Amelanotic
`Melanotic
`
`9—12
`7—13
`6—~10
`4
`5-10
`3—8
`4-9
`
`6—7
`12—15
`9—14
`5—8
`7—12
`
`Ovarian
`6—9
`Mucinous moderately differentiated
`Ov.Pe
`6—8
`Mucinous moderately differentiated
`Ov.He
`5—6
`Mucinous poorly differentiated
`FMa
`Mucinous moderately/poorly differentiated 8—14
`Ov.Gl
`Serous moderately differentiated
`9—13
`Ov.Ri
`Serous moderately differentiated
`8—13
`FKo
`‘ Tumor doubling time given as the range in days.
`
`Drugs. Doxorubicin (commercially available from Farmitalia) as a
`solution of 10 mg/ml in distilled water was injected i.v. at a dose of 8
`mg/kg on days 1 and 8. Amsacrine (provided by Warner Lambert,
`Hoofddorp, the Netherlands) in a 50 mg/ml solution was further di-
`luted in L—lactic acid to a concentration of 5 mg/ml. At the day of
`administration a l-mg/ml solution was made by adding 5% glucose, and
`injections were given i.v. at a dose of 8 mg/kg on days 1 and 8. Bre-
`quinar sodium (NSC 368390; provided by DuPont de Nemours Inter—
`national, Geneva, Switzerland) was dissolved in water at a concentra—
`tion of 10 mg/ml and further diluted in 0.9% NaCl
`to 2 mg/ml.
`Injections were given i.p. at a dose of 50 mg/kg on days 1—5. Datellip-
`tium hydrochloride (SR 9515613; provided by Sanofi Recherche, Mont-
`pellier, France) as a solution of 10 mg/ml in water was given i.p. at a
`dose of 30—35 mg/kg on days 1 and 8.
`Treatment. Animals were implanted s.c. in both flanks with 2—3-mm
`diameter
`fragments obtained from established human tumor xe-
`nografts. Tumor growth was assessed weekly by caliper measurements
`of the tumor in two dimensions. For tumor lines with a volume dou-
`
`bling time <5 days these measurements were carried out twice weekly.
`At each day of measurement animal weights were recorded. Treatment
`was started at the time tumors had reached a mean diameter of 6 mm.
`The number of mice per treatment per control group had to be sufficient
`to yield at least 6 evaluable tumors.
`
`5 The abbreviations used are: T/C, treated/control; SGD. specific growth delay;
`SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer.
`5941
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`

`

`Table 2 Activity of doxorubicin, 8 mg/kg i.v. on days I and 8, in human tumor lines
`Optimal T/C
`
`PHASE ll SCREENING IN HUMAN TUMOR XENOGRAFI‘S
`
`Tumor
`No. of
`Maximum
`No. of
`
`line
`evaluable tumors
`BWC" (-%)
`toxic deaths
`MAXF 401
`11
`3
`0/7
`MAXF 449
`3
`6
`4/6
`MAXF 857
`10
`0
`1/6
`
`CXF 158
`CXF 233
`CXF 243
`CXF 280
`
`HNX~HEp—2
`HNX—LP
`HNX-14C
`HNX-Pl
`HNX-I4A
`
`EVXOA
`AHXOL
`LXFE 397
`LXFE 409
`LXFL 529
`LXFA 629
`NXEE 002
`
`LBXOS
`LXFS 538
`LXFS 605
`LXFS 650
`NXES 004
`WXES 322
`
`THXO
`FEMXO
`MEXF 274
`MEXF 394
`
`7
`10
`10
`12
`
`6
`8
`6
`7
`6
`
`7
`13
`9
`0
`12
`7
`8
`
`7
`6
`8
`5
`9
`6
`
`10
`11
`5
`6
`
`10
`25
`14
`8
`
`11
`9
`4
`6
`4
`
`NA
`3
`13
`
`1
`14
`2
`
`3
`4
`23
`5
`0
`0
`
`NA
`NA
`8
`13
`
`1/5
`0/5
`0/5
`0/6
`
`0/4
`0/5
`0/5
`0/4
`2/6
`
`217
`0/7
`0/6
`4/4
`0/6
`0/5
`0/6
`
`0/7
`0/6
`0/6
`1/8
`0/6
`1/5
`
`1/6
`0/6
`2/5
`1/5
`
`96
`56
`34
`58
`
`72
`43
`57
`34
`
`23
`47
`54
`62
`31
`
`93
`49
`44
`
`8
`28
`59
`
`10
`22
`2
`10
`39
`36
`
`33
`54
`14
`54
`
`At day
`35
`28
`21
`
`28
`28
`13
`28
`
`24
`31
`10
`17
`14
`
`7
`38
`14
`
`28
`21
`11
`
`17
`35
`28
`22
`21
`28
`
`32
`35
`28
`14
`
`SGD
`
`1—2
`0.7
`NR
`0.4
`
`0
`0.4
`0.1
`0.9
`
`1.4
`0.7
`0
`0.5
`1.8
`
`0
`0.7
`1.4
`
`9.5
`4.2
`0.1
`
`2.8
`4.2
`7.5
`5.6
`0.4
`0.2
`
`0
`0.3
`2.1
`0
`
`1—4
`0.5
`NR
`0.5
`
`0.2
`0.6
`0.5
`2.2
`
`2.3
`0.6
`0.7
`NR
`0.9
`
`0
`0.7
`1.5
`
`4.1
`0.9
`0.5
`
`1.4
`1.5
`3.3
`2.7
`0.3
`1.0
`
`0.2
`0.3
`3.0
`0.2
`
`_
`.
`Evaluabthty?
`y/n
`y
`n
`y
`
`y
`n
`y
`y
`
`y
`y
`y
`y
`y
`
`y
`y
`y
`11
`y
`y
`y
`
`y
`y
`y
`y”
`y
`y
`
`y
`y
`n
`y
`
`y
`0.3
`0.5
`42
`56
`0/6
`3
`12
`Ov.Pe
`y
`NR
`1.7
`29
`38
`2/8
`10
`11
`Ov.He
`y
`1.1
`0
`31
`41
`0/7
`8
`14
`FMa
`y
`NR
`3.4
`41
`37
`0/6
`4
`12
`Ov.Gl
`y
`0.5
`0.2
`33
`62
`2/8
`10
`11
`Ov.Ri
`
`FKo y 12 2 0/6 89 7 0 0
`
`
`
`
`
`
`
`" BWC. body weight change; NA. not available; NR. volume 400% not reached.
`b Only five tumors evaluable; six others too small at the start of treatment.
`
`within 14 days after the last drug administration; ((1) the opti—
`mal treatment result expressed as median relative tumor vol-
`ume as compared to control and the day of measurement; (e)
`the SGD related to one (100—200 mm3) and two (100—400
`mm’) tumor volume doubling times; (f) the evaluability of each
`experiment.
`1n Table 6 a summary is given for the responding tumor lines
`of the seven tumor types to each of the four drugs tested. Based
`on the experience of the participating investigators, a T/C
`<50% or a SGD >1.0 were considered minimum values for
`antitumor activity. When the results were reviewed it was de~
`cided that combining both evaluation criteria would reflect the
`best drug efficacy.
`A total of 35 human tumor lines were tested for sensitivity to
`doxorubicin. Line LXFS 605 was included in the evaluation
`
`6 NSCLC lines doxorubicin was active as well as in l of 3 of the
`colorectal cancer lines and in 2 of 5 of the head and neck cancer
`lines. In melanoma, a tumor type not responsive to doxorubi-
`cin, none of the lines responded to the drug. A T/C (25% and
`a SGD >2.0 were obtained in the HNX—HEp—Z head and neck
`cancer line, the LXFL 529 NSCLC line, and the four SCLC
`lines LBXOS, LXFS 538, LXFS 605, and LXFS 650.
`Amsacrine was investigated in 33 human tumor lines, of
`which 30 were considered evaluable. Except for CXF 233 and
`LXFE 409, no major body weight change was observed. This
`might suggest that amsacrine was tested at a suboptimal dose.
`It should be noted that amsacrine was diluted for this study as
`indicated on the instruction leaflet for clinical use. This limited
`
`the maximum dose to 8 mg/kg (0.24 ml i.v. in a 30—g mouse).
`However, at a dose of 10 mg/kg i.p. in LXFS 605-bearing mice,
`because the excessive weight loss of 23% appeared to be partly
`3 of 6 animals did not survive day 17, and the antitumor effect
`induced by amsacrine was not improved. In view of the low level
`attributable to tumor response, and all animals survived beyond
`day 21. In tumor types known to be clinically responsive dox-
`of overall activity of this drug, it is improbable that significant
`orubicin showed activity in 0 of 2 breast cancer lines, 4 of 6
`activity would be reached with the optimal dose in other tumor
`lines.
`SCLC lines, and 3 of 6 ovarian cancer lines. In addition, in 3 of
`5942
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`

`PHASE ll SCREENING IN HUMAN TUMOR XENOGRAFTS
`
`Table 3 Activity ofamsacrine, 8 mg/kg i.v. on days I and 8, in human tumor liner
`Optimal T/C
`
`SGD
`
`Tumor
`No. of
`Maximum
`No. of
`Evaluability?
`
`line
`evaluable tumors
`BWC “ (—96)
`toxic deaths
`‘16
`At day
`1—2
`1—-4
`y/n
`MAXF 401
`10
`0
`0/7
`82
`42
`0
`0.2
`y
`MAXF 449
`9
`0
`1/6
`65
`21
`0.6
`0.3
`y
`MAXF 857
`9
`0
`1/6
`55
`21
`0.4
`0.4
`y
`
`CXF 158
`CXF 233
`CXF 243
`CXF 280
`
`HNX-HEp—Z
`HNx-LP
`HNX-NC
`HNX-Pl
`
`EVXOA
`AHXOL
`LXFE 397
`LXFE 409
`LXFL 529
`LXFA 629
`NXEE 002
`
`LBXOS
`LXFS 605
`LXFS 650
`NXES 004
`WXES 322
`
`THXO
`FEMXO
`M EXF 274
`MEXF 394
`
`10
`8
`9
`11
`
`6
`8
`4
`6
`
`5
`13
`11
`5
`12
`9
`6
`
`13
`6
`8
`9
`7
`
`10
`11
`7
`4
`
`0
`19
`0
`0
`
`0
`3
`’)
`4
`
`NA
`2
`0
`9
`0
`2
`0
`
`0
`4
`0
`0
`NA
`
`NA
`NA
`0
`0
`
`0/5
`0/5
`0/5
`0/6
`
`0/4
`1/6
`0/4
`0/5
`
`0/4
`0/7
`0/6
`1/4
`0/6
`0/5
`1/6
`
`0/8
`2/6
`0/7
`0/6
`0/5
`
`1/6
`0/7
`0/5
`2/5
`
`78
`71
`87
`74
`
`32
`98
`76
`70
`
`60
`65
`60
`31
`57
`100
`84
`
`61
`46
`80
`88
`55
`
`89
`77
`34
`37
`
`28
`42
`4|
`7
`
`28
`21
`IO
`12
`
`5
`38
`14
`35
`17
`
`9
`
`10
`28
`14
`24
`16
`
`27
`35
`42
`35
`
`0.2
`0
`0
`0.6
`
`0.5
`0
`0.3
`0
`
`o
`0.1
`1.8
`0
`0.3
`0
`0
`
`0.8
`0.5
`0.2
`0
`0.2
`
`0
`0.1
`0.5
`0.3
`
`O
`0.3
`0
`0.3
`
`0.4
`0
`0.4
`0.4
`
`0
`0.2
`0.7
`0
`0.7
`0
`0.1
`
`0.4
`0.5
`0
`0
`0.2
`
`0
`0.1
`0.4
`0.2
`
`0
`0.2
`28
`81
`0/6
`0
`12
`Ov.Pe
`0.3
`0.2
`36
`71
`0/6
`0
`12
`0v.He
`0.2
`0
`9
`84
`0/6
`1
`12
`FMa
`0.3
`0.5
`16
`70
`0/6
`0
`12
`Ov.Gl
`0.2
`0.4
`32
`68
`0/6
`0
`10
`Ov.Ri
`
`
`0
`0/6
`75
`8
`0.3
`0.2
`10
`“(0
`" BWC, body weight change; NA, not available.
`0 Only five tumors evaluable; three others too small at the start of treatment.
`‘ Only five tumors evaluable; one other measurement on day 0 only missing.
`
`y
`n
`y
`y
`
`y
`y
`n
`y
`
`y"
`y
`y
`y ‘
`y
`y
`y
`
`y
`y
`y
`y
`y
`
`y
`y
`y
`n
`
`y
`y
`y
`y
`y
`y
`
`DISCUSSION
`
`mainly observed between days 7 and 14 (Table 4), while the
`optimal T/C% for doxorubicin was usually reached after day
`14 (Table 2).
`
`Of 35 human tumor lines studied for sensitivity to brequinar
`sodium, 33 were considered evaluable. The drug appeared spe-
`cifically active in tumor lines of head and neck cancer, NSCLC,
`and SCLC with, respectively, 4 of 5 lines, 5 of 8 lines, and 4 of
`5 lines responding.
`The fourth compound studied, datelliptium, could be evalu-
`ated in 34 of 35 human tumor lines. In three lines the drug
`Since 1988, several discussions have taken place between the
`induced a body weight change > —15%. Since no animal death
`investigators of this European joint project on the design of
`occurred and the lines were not responsive to datelliptium, the
`phase II drug screening in human tumor xenografts. General
`experiments were considered evaluable. In only two SCLC
`consent was obtained on the tumor types to be studied, the
`lines, LBXOS and LXFS 650, the drug was able to induce
`selection of tumor lines for characteristics representing the tu-
`significant growth inhibition.
`mor type, and the number of lines per tumor type. Agreement
`In Fig. l, the effects of treatment on the growth curves of four
`was reached on the size and the number of xenografts at the
`human tumor lines are visualized. The squamous cell NSCLC
`start of treatment, the measurement of tumors, and the evalu-
`line LXFE 397 was calculated to be responsive to brequinar
`ability of the experiments (15). In the first step it was decided to
`sodium. 1n the LXFL 529 large cell NSCLC line efficacy was
`include four anticancer drugs, i.e., doxorubicin as a drug known
`observed for doxorubicin and brequinar sodium. Except for
`to be active in a variety of human solid tumors in the clinic,
`amsacrine, the LBXOS SCLC line was responsive to all drugs
`amsacrine as an inactive drug in these malignancies, the inves-
`studied. The SCLC line LXFS 605 was responsive to doxoru-
`tigational compound brequinar sodium that had just entered
`bicin and brequinar sodium. In those lines responding to bre-
`phase 1 clinical trials, and datelliptium, which was still in the
`quinar sodium significant growth inhibition (T/C <50%) was
`preclinical phase of drug development. Maximum tolerated
`5943
`
`1“,“. .wlnndnfll ‘LAM AAAAAAAAAAAAAAAAAAA I nnnnnn “AAAML‘AW O find—I
`
`{A 4nnr~1 AMAh—ir‘nv‘ A AAAAIAHA" {AV l" AAAAA
`
`4
`
`

`

`PHASE 11 SCREENING IN HUMAN TUMOR XENOGRAFI‘S
`
`Table 4 Activity of brequinar sodium, 50 rug/kg i.p. on days [—5, in human tumor line:
`SGD
`No. of
`Optimal T/C
`Maximum
`No. of —— ————————————
`evaluable
`
`BWC" (—%)
`toxic deaths
`96
`At day
`tumors
`1—2
`1-4
`12
`2
`0/7
`81
`7
`0
`0
`4
`19
`3/6
`58
`7
`1.2
`0.1
`8
`11
`2/6
`52
`7
`1.5
`0.6
`
`Evaluability?
`y/n
`y
`n
`y
`
`8
`10
`8
`9
`
`6
`6
`6
`6
`7
`
`9
`l3
`9
`8
`10
`6
`8
`6
`
`12
`6
`7
`8
`7
`
`11
`10
`8
`9
`
`15
`10
`7
`11
`
`4
`4
`6
`5
`5
`
`NA
`5
`14
`11
`12
`8
`7
`0
`
`2
`11
`4
`0
`4
`
`NA
`NA
`0
`0
`
`0/5
`0/5
`1/5
`0/6
`
`0/4
`0/3
`0/4
`0/4
`0/4
`
`0/7
`0/7
`1/6
`0/4
`1/6
`0/5
`0/6
`0/4
`
`1/8
`2/5
`0/6
`0/6
`0/5
`
`0/6
`0/6
`1/5
`0/5
`
`59
`63
`53
`57
`
`65
`12
`26
`28
`35
`
`60
`83
`14
`10
`38
`44
`38
`56
`
`49
`15
`50
`48
`30
`
`38
`84
`36
`53
`
`28
`7
`13
`7
`
`7
`14
`5
`12
`14
`
`7
`7
`7
`14
`10
`7
`11
`9
`
`31
`28
`10
`7
`9
`
`27
`35
`28
`14
`
`0.8
`0.4
`1.4
`1.2
`
`0.7
`1.7
`1.6
`2.1
`1.4
`
`0.3
`0
`6.0
`4.3
`1.8
`2.0
`1.1
`0.2
`
`1.5
`2.5
`0.7
`1.6
`2.6
`
`0.7
`0
`1.0
`0.5
`
`0.3
`0.5
`0.5
`0.6
`
`0.2
`0.6
`0.5
`0.6
`0.6
`
`0
`0
`1.9
`1.2
`1.2
`0.1
`0.8
`0.1
`
`1.0
`1.4
`0.1
`0.8
`1.1
`
`0.4
`0.1
`0.5
`0.4
`
`y
`n
`y
`y
`
`y
`y
`y
`y
`y
`
`y
`y
`y
`y
`y
`y
`y
`y
`
`y
`y
`y
`y
`y
`
`y
`y
`y
`y
`
`Tumor
`line
`MAXF 401
`MAXF 449
`MAXF 857
`
`CXF 158
`CXF 233
`CXF 243
`CXF 280
`
`HNX-HEp—Z
`HNX-LP
`HNX-14C
`HNX—Pl
`HNX-14A
`
`EVXOA
`AHXOL
`LXFE 397
`LXFE 409
`LXFL 529
`LXFA 629
`NXEE 002
`CXEA 117
`
`LBXOS
`LXFS 605
`LXFS 650
`NXES 004
`WXES 322
`
`THXO
`FEMXO
`MEXF 274
`MEXF 394
`
`y
`0.5
`0.9
`42
`45
`0/6
`2
`12
`Ov.Pe
`y
`0.5
`0.7
`15
`70
`0/6
`10
`12
`Ov.He
`yb
`0.2
`0.6
`7
`71
`0/8
`5
`15
`FM:
`y
`0.3
`0.1
`28
`66
`0/7
`6
`14
`Ov.Gl
`y
`0.1
`0.1
`7
`82
`0/7
`3
`14
`Ov.Ri
`
`4 y 0/7 80 28 0.3 0.1
`
`
`
`
`
`14
`FKo
`" BWC. body weight change; NA. not available.
`” Test done at 60 mg/kg Up. 1. 2, 3, 4, and 5.
`
`laboratory varied between —2% and —10% after doxorubicin
`treatment.
`
`dose studies carried out in one laboratory appeared to be adapt-
`able in other laboratories housing nude mice of other strains. In
`practice, only a few of the experiments had to be discarded, and
`this was due mainly to reasons of drug toxicity. However, one
`should keep in mind that body weight change may also be
`influenced by the effect of the xenograft on the host. As an
`example, median body weight change in the six human ovarian
`cancer lines studied in the same strain of mice in the same
`
`cancer patients, amsacrine is a well-known drug in the treat—
`ment of hematological malignancies, with emphasis on acute
`leukemias, while a variety of phase II trials demonstrated no
`useful activity against human solid tumors (17).
`Inactivity
`against solid tumor types was confirmed in our xenografts,
`which was further proof of the reliability of the panel.
`For brequinar sodium, our panel predicted activity against
`head and neck cancer, NSCLC, and SCLC. Preclinical analysis
`of brequinar sodium has shown the drug to be an antimetabolite
`and, as such, a potent inhibitor of the pyrimidine mitochondrial
`enzyme dihydroorotic acid dehydrogenase (18). In vitro, pro-
`Since the feasibility of this joint project appears to be well
`longed exposure was necessary for a long-lasting depletion of
`proven, the question arises as to whether this in vivo screening
`pyrimidine nucleotides and a substantial suppression of RNA
`system generates reliable data. Therefore, the reference agents
`doxorubicin and amsacrine were included. In the clinic, doxo-
`and DNA synthesis. Repletion of all nucleotides by uridine
`and/or cytidine restored cell growth. In human colon cancer-
`rubicin shows the greatest efficacy in hematological malignan—
`cies, breast cancer, ovarian cancer, sarcomas, and SCLC (16).
`bearing nude mice, the tumor tissue concentration of radiola—
`Several other tumor types are known to be occasionally respon-
`beled brequinar sodium measured approximately 50% of the
`sive, such as NSCLC and head and neck cancer. In this respect,
`1-h peak level at 24 h (19). Thus, one may expect prolonged
`exposure to the drug in viva upon daily injections given for 5
`the overall sensitivity of our human tumor xenograft panel was
`days. Indeed, in responsive tumor lines growth inhibition was
`remarkably reflective of the clinical data of doxorubicin. In
`5944
`
`HanuwlAadAnl [6AM fin.nnnnnnnnnnnnnnnnnn I AAAAAA nAAHML‘A» 0 nn4‘7 fA 4nnri Amnv:finw AAAAAZAuA». (A, f‘ nnnnn
`
`5
`
`

`

`PHASE II SCREENING IN HUMAN TUMOR XENOGRAFTS
`
`Table 5 Actirity of dwellipiium, 30—35 rug/kg i.p. on days I and 8, in human tumor lines
`No. of
`Optimal T/C
`SGD
`Tumor
`evaluable
`Maximum
`No. of
`Evaluability?
`
`line
`tumors
`BWC‘ (-%)
`toxic deaths
`96
`At day
`l—2
`l—4
`y/n
`MAXF 401
`9
`0
`0/7
`68
`21
`0.4
`0
`y
`MAXF 449
`12
`5
`0/6
`68
`21
`0.5
`0.2
`y
`MAXF 857
`12
`3
`0/6
`70
`21
`0.2
`0.3
`y
`
`CXF 158
`CXF 233
`CXF 243
`CXF 280
`
`HNX-HEp—Z
`HNX-LP
`HNx—14c
`HNX-Pl
`HNXJ4A
`
`EVXOA
`AHXOL
`LXFE 397
`LXFE 409
`LXFL 529
`LXFA 629
`NXEE 002
`CXEA 117
`
`LBXOS
`LXFS 538
`LXFS 605
`LXFS 650
`NXES 004
`WXES 322
`
`THXO
`FEMXO
`MEXF 274
`
`9
`10
`6
`10
`
`6
`1 1
`5
`8
`6
`
`7
`9
`7
`8
`9
`7
`8
`6
`
`11
`6
`8
`6
`8
`5
`
`8
`9
`7
`
`17
`26
`22
`6
`
`5
`6
`5
`4
`7
`
`NA
`5
`0
`16
`7
`7
`7
`0
`
`2
`1
`7
`11
`0
`4
`
`NA
`NA
`1
`
`0/5
`0/5
`1/4
`0/6
`
`1/5
`0/6
`0/4
`1/7
`0/4
`
`0/6
`2/7
`0/5
`0/4
`0/6
`0/5
`0/6
`0/4
`
`0/8
`1/6
`0/5
`0/6
`0/6
`1/5
`
`1/6
`1/7
`0/4
`
`53
`70
`56
`72
`
`76
`81
`52
`56
`88
`
`80
`71
`83
`19
`79
`66
`60
`76
`
`13
`43
`55
`35
`88
`86
`
`72
`70
`59
`
`28
`14
`35
`7
`
`7
`10
`21
`13
`7
`
`1 1
`23
`10
`35
`II
`7
`9
`21
`
`17
`42
`28
`11
`2
`7
`
`21
`35
`14
`
`0.5
`0
`0.8
`0.6
`
`0.4
`0.1
`0.1
`1.9
`0.1
`
`0.1
`0
`0
`0.2
`0.3
`1.3
`0.2
`0
`
`1.9
`1.0
`0
`1.6
`0.1
`0.4
`
`0.1
`0
`0.4
`
`0.6
`0.4
`0.1
`0.4
`
`NA
`0
`0
`0.2
`0.1
`
`0.3
`0.2
`0
`0
`0.3
`o
`0.3
`o
`
`1.0
`1.0
`0.1
`1.2
`0.1
`0.2
`
`0.1
`0.5
`0.4
`
`14
`Ov.Pe
`12
`0v.He
`1 2
`FMa
`7
`Ov.Gl
`8
`Ov.Ri
`10
`“(0
`' BWC. body weight change; NA, not available.
`b Test done at 30 mg/kg i.p. days 1 and 8.
`‘ 4 of 5 animals survive day 30 without significant weight loss.
`
`0.1
`0.1
`27
`81
`0/7
`8
`0.2
`1.0
`7
`68
`0/6
`4
`0.4
`0.6
`29
`63
`0/6
`2
`0
`0
`14
`77
`2/6
`9
`0
`0.6
`13
`68
`l/6
`7
`
`8
`0/5
`65
`28
`0.2
`0.3
`
`y
`n
`y
`y
`
`y”
`y”
`y”
`y”
`y"
`
`y
`y
`y
`y
`y
`y
`y
`y
`
`y
`y
`y
`y
`y
`y‘
`
`y
`y
`y
`
`y‘
`y”
`y‘
`y”
`y‘
`y”
`
`Table 6 Therapeutic eflicacy ofdoxarubicin, ammcn'ne, brequiruzr sodium, and dale/limium expressed as (It: number of human tumor lines with a T/C < 5096
`and/or a SGD > 1.0 ofthe total number ofevaluable lines (n)
`Datelliptium
`Amsacrine
`Brequinar sodium
`Doxorubicin
`SGD T/C < 50%
`T/C
`TIC
`SGD T/C < 50%
`T/C
`SGD T/C < 50%
`SGD T/C < 5096
`T/C
`
`>1.0
`SGD > LG
`<5096
`n
`<50% >1.0
`SGD > 1.0
`n
`<50% >l.0
`SGD > 1.0
`r1
`<50% >l.0
`SGD >l.0
`It
`Tumor type
`0
`0
`0
`3
`0
`0
`2
`0
`2
`0
`0
`3
`0
`0
`0
`2
`Breast
`0
`0
`0
`3
`0
`0
`0
`3
`0
`2
`0
`3
`l
`l
`l
`3
`Colorectal
`l
`0
`0
`5
`l
`- 0
`0
`5
`4
`4
`4
`3
`3
`2
`2
`5
`Head and neck
`l
`0
`l
`8
`l
`l
`0
`8
`5
`5
`5
`7
`4
`3
`3
`6
`NSCLC
`2
`2
`3
`6
`l
`O
`0
`5
`4
`4
`4
`5
`6
`4
`4
`6
`SCLC
`0
`0
`0
`3
`l
`0
`0
`4
`2
`0
`0
`3
`l
`0
`0
`3
`Melanoma
`
`Ovarian 0 6 3 3 3 6 0 0 0 6 l 0 0 6 0 0
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`most pronounced in the first 2 weeks of treatment. The mech-
`nation for the discrepancy in efficacy could possibly be found in
`anism of action also explains rapid regrowth of tumors after
`a difference between mice and humans for the degree and the
`duration of uridine depletion in tissues as a result of the inhi-
`discontinuation of drug administration. In the clinic, a number
`of phase I trials have been carried out (20—22). Mucocutaneous
`bition of dihydroorotic acid dehydrogenase (23).
`side effects and myelotoxicity were dose-limiting factors. Thus
`Datelliptium showed low activity in the panel, but in 2 of 6
`far, phase II studies in solid tumor types have failed to show
`SCLC lines its activity was significant. Datelliptium is a water-
`significant antitumor activity for brequinar sodium. An expla-
`soluble compound belonging to the ellipticine family, a group of
`5945
`
`nAlllMlAAlJnlJ (yAw.
`
`fin-‘h-‘h-‘l—VVAA nnnvi AAAAAA I nnnnnn nAnAML‘AuO fiflAV 6'“ 400’) AMA-«FAA... AAAAA:A‘;A|A IA.— (" AAAAA
`
`6
`
`

`

`PHASE ll SCREENING IN HUMAN TUMOR XENOGRAFTS
`
`
`
`LXFE 397
`
`LXFE 397
`
`
`
`a.
`
`LXFL 5 29
`_l
`
`LXFL 529
`
`100
`
`10
`
`0.1
`100
`
`10
`
`0.1
`100
`
`‘0f
`
`
`
`
`
`
`
`
`
`relativetumorvolume
`
`
`
`
`
`relativetumorvolume
`
`
`
`
`
`relativetumorvolume
`
`0.1
`100
`
`10
`
`
`
`
`
`relativetumorvolume
`
`y-{'
`4.?" a”
`\au—uJ
`LBXOS
`LBXOS
`
`_-’
`
`-
`
`n
`
`_.
`
`LXFS 605
`
`A
`
`\‘la
`
`Before general use of the panel we should await further proof
`that our human tumor xenografts will accurately predict the
`efficacy of new compounds, thus improving the efficiency of
`phase II clinical trials. Recognition of the mechanisms under-
`lying possible discrepancies in response between tumor—bearing
`mice and cancer patients is essential for a more rational use of
`the panel (30). In this respect, we are currently extending our
`experience with another four anticancer drugs, including cis-
`platin and diaziquone as reference agents.
`
`REFERENCES
`
`Shoemaker, R. H., McLemore, T. L., Abbott, B. J., Fine, D. L., Gorelik, E.,
`Mayo, J. G., Fodstad, (21., and Boyd, M. R. Human tumour xenograft models
`for use with an in vimrbased disease-oriented antitumour drug screening
`program. In: B. Winograd, M. J. Peckham, and H. M. Pinedo (eds.), Human
`Tumour Xenografts in Anticancer Drug Development, pp. 115—120. Berlin
`and Heidelberg: Springer—Verlag, 1988.
`Staquet, M. J., Byar, D. R, Green, S. 8., and Rozencweig, M. Clinical
`predictivity of transplantable tumor systems in the selection of new drugs for
`solid tumors: rationale for a three-stage strategy. Cancer Treat. Rep., 67:
`753-765, 1983.
`Venditti,
`.l. M. The National Cancer Institute antitumor drug discovery
`program, current and future perspectives: a commentary. Cancer Treat. Rep.,
`67: 767—772, 1983.
`Atassi, G., and Staquet, M. The clinical predictive value of the mouse screen-
`ing methods. [m P. Hilgard and K. Hellmann (eds.), Anticancer Drug De-
`velopment, pp. 27—34. Barcelona: .1. R. Prous Publishers, 1983.
`Corbett, T. H., Valeriote. F. A., and Baker, L. H. 15 the P388 murine tumor
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