GASTROENTEROLOGY 1991;101:303—311
`
`Establishment and Characterization of a
`
`Human Carcinoid in Nude Mice and Effect
`of Various Agents on Tumor Growth
`
`B. MARK EVERS, COURTNEY M. TOWNSEND, ]r., I. ROBERT UPP,
`ERICK ALLEN, STEPHEN C. HURLBUT, SUN WHE KIM,
`
`SRINIVASAN RAIARAMAN, POMILA SINGH,
`
`IEAN CLAUDE REUBI, and IAMES C. THOMPSON
`Departments of Surgery and Pathology, University of Texas Medical Branch, Galveston, Texas;
`and Sandoz Research Institute, Berne, Switzerland
`
`The authors have established a long-term tissue
`culture cell line [BON] derived from a metastatic
`human carcinoid tumor of the pancreas. The cells
`have been in continuous passage for 46 months.
`Tissue culture cells produce tumors in a dose-
`dependent fashion after SC inoculation of cell sus-
`pensions in athymic nude mice. BON tumors, grown
`in nude mice, are histologically identical to the
`original tumor; they possess gastrin and somato-
`statin receptors, synthesize serotonin and chromog-
`ranin A, and have a doubling time of approximately
`13 days. The antiproliferative effects of the long-
`acting somatostatin analogue, SMS 201-995 [300
`pg/kg, t.i.d.], and 2% or-difluoromethylornithine on
`BON xenografts in nude mice were examined. Tu-
`mor size was significantly decreased by day 14 of
`treatment with either agent and at all points of
`analysis thereafler until the animals were killed
`[day 33]. In addition, tumor weight, DNA, RNA, and
`protein contents were significantly decreased in
`treated mice compared with controls. Establishment
`of this human carcinoid xenografl line, BON, pro-
`vides an excellent model to study further the biologi-
`cal behavior of carcinoid tumors and the in vivo
`
`effect of chemotherapeutic agents on tumor growth.
`
`arcinoid tumors are endocrine neoplasms derived
`from neuroectodermal cells of the neural crest
`
`[GI]
`[1,2]. Primarily found in the gastrointestinal
`tract, these tumors are uncommon, but not rare, with
`
`a reported incidence in autopsy series ranging from
`0.1% to 1.4% [1]. Originally called “Karzinoide” by
`Oberndorfer [3] to describe its apparent benign na-
`
`ture, modern immunohistochemical techniques have
`allowed identification and isolation of the vasoactive
`
`substance serotonin [5-hydroxytryptamine [5-HT]] and
`a host of biogenic amines and hormones produced by
`carcinoid tumors [1,2].
`Treatment of patients with carcinoid tumors has
`been hampered by the lack of an animal model. In the
`majority of patients, treatment of metastatic carcinoid
`tumors has been directed largely toward relief of the
`debilitating sequelae of the carcinoid syndrome by
`means of blocking agents [for example, methysergide
`[4], cyproheptadine [4,5], and ketanserin [6,7]] or the
`inhibitory hormone, somatostatin [B-1 6]. Antiprolifer—
`ative agents that are commonly used to slow tumor
`growth include combinations of streptozotocin, 5-flu-
`orouracil, doxorubicin, or cyclophosphamide [5,17—
`19]. However, most carcinoid tumors respond poorly
`to cytotoxic therapy with these agents [1,4,18—19].
`In this study, we report the first in vivo establish-
`ment of a long—term xenograft cell line of a human
`pancreatic carcinoid tumor. We have characterized
`tumor growth rate in vivo, the status of gastrin and
`somatostatin receptors, the morphology, and the pro-
`duction of peptides and amines. In addition, we have
`examined possible antiproliferative effects of the long-
`acting somatostatin analogue, SMS 201-995, and oz—di—
`fluoromethylornithine [DFMO]
`[either alone or in
`combination] on growth of the carcinoid tumor.
`
`Abbreviations used in this paper: DFMO, oz-difluoromethylorni-
`thine; DMEM, Dulbecco’s modified Eagle medium; FCS, fetal calf
`serum; 5-HT. 5-hydroxytriptamine.
`© 1991 by the American Gastroenterological Association
`0016-5085/91/$3.00
`
`
`
`
`
`Roxane Labs., Inc.
`
`Roxane Labs., Inc.
`Exhibit 1030
`Page 001
`
`

`
`304 EVERS ET AL.
`
`GASTROENTEROLOGY Vol. 101, No. 2
`
`Materials and Methods
`
`Surgical Specimen Collection and
`Maintenance of Tumor Line
`
`The operative specimen of a peripancreatic lymph
`node was sterilely obtained in April 1986 from a 28-year-old
`man with metastatic carcinoid tumor of the pancreas who
`had come into the hospital with symptoms of obstructive
`jaundice and diarrhea. Before exploratory laparotomy the
`patient had not received any interventional treatment. Final
`diagnosis of a metastatic carcinoid tumor was made by
`histological examination of the lymph node and positive
`staining for neuron-specific enolase and 5-HT.
`A portion of the lymph node was washed in saline,
`minced, and tumor fragments placed in Dulbecco’s modi-
`fied Eagle medium (DMEM; Gibco, Grand Island, NY] and
`F12K [Gibco] in a 1 :1 ratio supplemented with -10% [vol/vol]
`fetal calf serum [FCS; Hyclone Laboratories, Logan, UT] and
`1% gentamicin. The cells were grown at 37°C in an atmo-
`sphere of 95% air and 5% CO2. Cells were routinely passed
`by removing the medium and overlaying the cell monolayer
`with 0.25% trypsin:0.1% ethylenediaminetetraacetic acid
`[EDTA]. The elimination of fibroblasts from the stroma of
`the tumor tissue was accomplished by brief exposures to
`0.06% trypsin:0.02% EDTA. This procedure was repeated
`until no further Fibroblast growth was observed. Tumor
`cells from passage 5 were frozen after successful in vitro
`adaptation and removal of all fibroblasts. The cell line is
`presently maintained in DMEM and F12K growth medium
`supplemented with 10% FCS; it is passed at a 1:2 ratio when
`cells reach 80% confluence. Cell cultures are routinely
`monitored for mycoplasma contamination, and no myco-
`plasma growth has been detected.
`
`Animals
`
`Male athymic nude mice [Balb/c, 20-25 g, 3-4
`weeks of age; Life Science, St. Petersburg, FL] were housed
`under specific pathogen-free conditions in a temperature-
`controlled isolation unit with 12-hour light-dark cycles in
`accordance with the National Research Council’s Guide for
`the Care and Use of the Nude Mouse in Biomedical Research
`
`[20]. The mice were fed a standard chow (Autoclavable
`Rodent Chow no. 5010; Ralston Purina, St. Louis, MO] and
`sterile water, both given ad libitum.
`
`square millimeters. Tumor areas were plotted on semiloga—
`rithmic paper, and the tumor doubling time [defined as time
`in days required for mean tumor area to double during
`logarithmic growth] was determined directly from the graph.
`On day 48, mice were killed and tumors from passage 8
`were removed for light and electron microscopy, immuno-
`histochemistry, and measurement of gastrin receptors.
`
`Light and Electron Microscopy
`
`For light microscopy, tumor tissue blocks measuring
`1 X 1 X 0.5 cm were fixed in 10% neutral buffered formalin
`for 6-8 hours, processed routinely, and embedded in par-
`affin. Sections [4 mm thick] were stained with H&E and
`examined.
`
`tissue blocks [1
`For transmission electron microscopy,
`mm3] were fixed in half-strength Karnovsky’s fixative for 4
`to 6 hours, postfixed in osmium tetroxide [1%], and embed-
`ded in Epon. Sections [70 nm thick] were stained with
`uranyl acetate and Reynolds lead citrate and examined
`with a Philips 410 electron microscope [Philips Medical
`Systems Inc., Shelton, CT].
`
`Imm unohistochemical Studies
`
`Formalin-fixed, paraffin-embedded tissue samples
`were studied by means of a three-layer immunohistochemi-
`cal method. The primary antibodies used were murine
`monoclonal antibody to chromogranin A [Boehringer Man-
`nheim, Indianapolis, IN] and monospecific polyclonal anti-
`bodies to serotonin, substance P, pancreatic polypeptide,
`vasoactive intestinal peptide, glucagon, gastrin and bombe-
`sin [all
`from Dako Corp., Santa Barbara, CA]. Tissue
`sections [4 um thick] were sequentially incubated with the
`appropriate primary antibodies, swine anti-rabbit immuno-
`globulin G [IgG] or rabbit anti-mouse IgG [1:100—1:400],
`followed by rabbit or mouse peroxidase-antiperoxidase
`complexes [12100] [Dako Corp.], with frequent washes in
`phosphate—buffered saline between incubations. The perox-
`idase reaction product was visualized by incubating with
`diaminobenzidine [0.05%] and hydrogen peroxide [0.01%].
`Controls included omission of primary antibodies and
`substitution of primary antibodies with nonimmune sera
`from the same species.
`
`Solid Tumors in Nude Mice and In Vitro
`Growth Rate
`
`BON cells [passage 8] were harvested from subcon-
`fluent cultures by a 1-minute treatment with 0.25% trypsin
`and 0.1% EDTA. Single-cell suspensions (5 X 10“, 1 X 107,
`and 2 X 107] in RPMI 1640 without serum [total volume, 0.1
`mL] were injected SC at a single site in the dermis of nude
`mice [n = 10 mice/group]. Tumor size [longest perpendicu-
`lar diameter] was measured biweekly with Vernier calipers
`[Mitutoyo Corp., Tokyo, Iapan] accurate to 0.5 mm. Surface
`areas were calculated as the product of the two greatest
`perpendicular tumor diameters and were expressed as
`
`Gastrin Bin ding Assay
`
`Tumors were quickly removed and washed with
`cold buffer A [Tris, 10 mmol/L; KCl, 2 mmol/L; MgClz, 2.5
`mmol/L; and sucrose, 0.25 mol/L; pH 7.4] containing BSB
`[1% bovine serum albumin, fraction V [Sigma Chemical Co.,
`St. Louis, MO]], 0.1% soybean trypsin inhibitor [Worthing-
`ton Biochemical Corp., Freehold, N]], and 0.1% bacitracin
`[Sigma Chemical Co.]. Tumors were then stored at — 70°C in
`an ultradeep freeze until further analysis. Specific binding
`sites for gastrin were measured on cell membranes prepared
`from tumor samples by our previously published methods
`[21,22].
`
`
`
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`Page 002
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`

`
`August 1 991
`
`HUMAN CARCINOID IN NUDE MICE 305
`
`dependent fashion [Figure 1]. After 2 X 107 BON cells
`were injected, tumors were visible by 7 days in all
`mice. When 1 X 107 cells were injected, tumors grew
`in all mice; however, they were not vis:ible until day
`10. After injection of 5 X 10“ cells, 80% of tumors
`grew and were visible by day 17. Once the tumors
`began to grow, the growth rates were similar regard-
`less of initial inoculum, and tumor-doubling time was
`approximately 13 days. All tumors grew as discrete
`encapsulated masses, without evidence of local inva-
`sion or distant metastasis.
`
`Tumor xenografts were sent to the American Type
`Culture Collection [Rockville, MD] where isoenzyme
`determination was performed by electrophoresis, con-
`firming that this was human tissue and not a sponta-
`neously occurring mouse tumor.
`
`Morphological Studies
`
`Light microscopy. The original patient’s tumor
`was composed of masses of ovoid cells arranged in a
`nonorganoid fashion in a delicate, vascular stroma.
`Cellular and nuclear pleomorphism was minimal
`[Figure 2A]. Tumors from the nude mouse exhibited a
`similar histopathologic pattern with moderate nu-
`clear pleomorphism of the cells. [Figure 2B].
`
`Somatostatin HeceptorAssay
`
`Somatostatin receptors were measured on BON tu-
`mors by autoradiography on tissue sections (10 pm thick] as
`described before in detail for various tumors,
`including
`hormone-producing gastroenteropancreatic tumors [23]. The
`iodinated Tyr“ analogue of SMS 201-995 (code-named
`204-090] was used as the radioligand.
`
`Administration of SMS 201-995 and DFMO
`
`Initially, to establish tumors, dispersed BON cells
`[passage 8, 1 X 107 cells] were inoculated SC in nude mice.
`When tumors became approximately 10 cm‘ in area, the
`mice were killed and tumors minced into 3-mmz pieces that
`were then implanted bilaterally into the flanks of 20 nude
`mice. The mice were randomly allocated to receive either
`saline (0.1 mL, IP, t.i.d.], 2% (wt/vol] DFMO (a gift from W. J.
`Hudak, Ph.D., Manager of Research Information at
`the
`Merrell Research Center, Cincinnati, OH] in drinking water,
`SMS 201-995 [300 ug/kg, IP, t.i.d.], or a combination of
`DFMO and SMS 201-995 beginning the day of tumor
`implantation and continuing until they were killed. Water
`bottles were covered to prevent light degradation. Drinking
`water was renewed every 2 days. SMS 201-995 [a gift of
`Sandoz Research Institute, Hanover, NJ] was diluted to the
`required concentration with saline.
`Mice were weighed weekly, and tumors were measured
`twice weekly by the same observer. The surface areas of the
`tumors were calculated as described above. Mice were
`
`killed on day 33, and tumors were removed, weighed, and
`frozen at —70°C until assayed for DNA, RNA, and protein
`content.
`
`Protein, DNA, and RNA Analysis
`
`Tumors were thawed, homogenized (Polytron; kine-
`matica GmbH, I(riens—Luzern, Switzerland], and extracted
`by the method of Ogur and Rosen (24). Protein content was
`determined by the method of Lowry et al. (25), with bovine
`serum albumin as standard. DNA content was measured by
`the Burton [26] modification of the diphenylamine proce-
`dure with calf thymus DNA used as the standard. RNA
`content was measured by means of the orcinol procedure
`with yeast RNA as the standard [27].
`
`20°
`
`100
`
`so
`so
`so
`
`40
`A_
`30
`‘E
`V 20
`
`BON (2 x10’)
`
`7
`BON (1 x10)
`
`6
`BON (5 X 10)
`
`A
`
`.
`
`m NU
`
`) E
`
`0§
`
`1
`
`54 3 2
`
`1o
`3e
`
`Statistics
`
`*
`
`Results are expressed as the mean : SEM. Antipro-
`liferative effects of SMS 201-995 and DFMO were analyzed
`by two-way analysis of variance. A value of P < 0.05 was
`considered significant.
`
`Results
`
`Tumor Growth in Nude Mice
`
`0
`
`1o
`
`20
`
`so
`DAYS
`
`40
`
`so
`
`so
`
`When single—cell suspensions were injected SC
`into nude mice, tumors were produced in a dose-
`
`Figure 1. Logarithmic growth curves of BON tumors injected SC
`as single-cell suspensions (5 X 10‘, 1 X 10’, 2 X 10’ cells] in the
`dermis of nude mice [n = 10 mice/group].
`
`
`
`Roxane Labs., Inc.
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`Page 003
`
`

`
`306 EVERS ET AL.
`
`GASTROENTEROLOCY Vol. ‘I01. No. 2
`
`Gostrin Binding
`
`Specific binding sites for gastrin in the range of
`10-18 fmol/mg protein with a high binding affinity
`[K] = ~ 0.134 nmol/L] were measured from a Scatch~
`and plot of the specific binding data on tumor mem-
`branes. The binding sites were specific for binding of
`gastrin and did not show any significant binding
`affinity for cholecystokinin or other unrelated pep-
`tides tested [bombesin, vasoactive intestinal peptide,
`or insulin] [data not shown].
`
`Somatostatin Binding
`
`Somatostatin receptors were localized on tu~
`tissue exclusively; however, nonhomogenous
`mor
`distribution can be observed, with tumor regions
`having higher receptor density than nontumor area.
`Binding of ”5I—[Tyr“]—SMS 201-995 was shown to be
`displaced in the nanomolar range using increasing
`concentrations of SMS 201-995 in successive tissue
`
`sections [Figure 6]. The binding was specific because
`unrelated peptides [e.g., luteinizing hormone~releas-
`ing hormone} did not compete with the ligand.
`
`Ejjfect of o—D1fluoromethylomithine and SMS
`201-995 on BON Growth
`
`There were no significant differences in final
`body weight
`in treated mice compared with the
`control group, and mice receiving SMS 201-995 and
`DFMO showed no ill effects. Food and water intake
`
`was monitored, and there were no differences in
`
`Figure 2. Light micrograph of the patienl‘s tumor [A] and the
`nude mouse tumor (B) demonstrating similar histopathologic
`characteristics (HIRE; original magnification X 500).
`
`Electron microscopy. BON tumors from nude
`mice were composed of polygonal cells arranged in
`masses in a vascular stroma. A majority of cells
`exhibited an abundance of cytoplasmic secretory
`granules that were membrane-bound, several mito-
`chondria, and profiles of endoplasmic reticulum.
`Sheaves of tonofilaments [cytokeratin] were also found
`in several cells, the features being characteristic of
`epithelial and endocrine differentiation (Figure 3].
`
`Immunohistochemicol Studies
`
`BON xenografts stain positive for serotonin
`[Figure 4) and chromogranin A granules [Figure 5).
`The staining was appreciable in approximately 50%-
`60% of the BON tumor cell population exhibiting both
`serotonin and chromogranin A irnmunoreactivity.
`Substance P, pancreatic polypeptide, vasoactive intes-
`tinal polypeptide, glucagon, gastrin, and bombesin
`were not detected in the BON tumor by irnmunohis—
`tochemical staining.
`
`
`
`Figure 3. Electron micrograph of BON tumor demonstrating
`abundant dense secretory granules interspersed with sheaves of
`cytokeratm [arrows] [original l'|'|ag|1ifica[iun x71n0)_
`
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`

`
`August 1991
`
`HUMAN CARCENOID IN NUDE MICE 307
`
`agent alone. Tumor weight of the combination—treated
`group was 23%, protein content was 18%, and DNA
`and RNA contents were 28% of the values in the
`
`control group.
`
`Discussion
`
`We have successfully established a line of
`human carcinoid tumor cells (BON) in athyrnic nude
`mice. When dispersed cells are injected SC in the
`mice, encapsulated tumors are produced that are
`histologically similar to the original patient tumor.
`BON has a stable tumor—doubling "time,
`it stains
`positive for 5-HT and chromogranin A, and it has
`retained high—affinity gastrin receptors on the cell
`membrane. In addition, we have shown somatostatin
`
`receptors by autoradiography. These characteristics
`make BON a useful model in which to study various
`treatment regimens on growth of carcinoid tumors
`and is, to our knowledge, the first long-term human
`carcinoid xenograft cell line to be established in nude
`mice. Also, BON cells grown in tissue culture exhibit
`
`Figure 4. Light micrograph of the HON tumor stained for seroto-
`nin torrowsl [counterstained with liematoxylin; original magnifi-
`cation X 1000).
`
`consumption between groups. Two mice [one control
`and one SMS 201-9Q5—treated) died during the course
`of the experiment because of traumatic injections
`[confirmed by necropsy] and were excluded from
`analysis.
`Both SMS 201-995 [300 ttg/kg, IP, t.i.d.] and 2%
`DFMO, administered either as single agents or in
`combination, significantly inhibited tumor area by
`day 14; inhibition continued to the time of killing [day
`33) [Figure 7). Tumor weight, DNA, RNA, and protein
`contents were similarly inhibited after treatment (Fig-
`ure 8). The mean tumor weight of the DFMO-treated
`group was 42% of that of the control group, protein
`content was 40%, and DNA and RNA contents were
`
`in the SMS
`53% of controls. Mean tumor weight
`201—995—treated group was 38%, protein content was
`39%, DNA content was 34%, and RNA content was
`
`44% of the values in the control group. Although
`mean values of tumor area, weight, and biochemical
`determinations were the lowest in mice treated with
`
`combination therapy, these values were not signifi-
`cantly different from those in mice treated with either
`
`Figure 5. Light micrograph of the BON tumor stained for chromo-
`granin A [arrows] [counterstained with hematoxylin; original
`miignificatifln X1000}
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`308 EVERS ET AL.
`
`GASTROEN'l”l£lx’t,')LOGY Vol. 101, N0. 2
`
`models of hu man carcinoid tumors involve transplan-
`tation of tumor chunks into the anterior chamber of
`
`the eye of rats immunosuppressed with cyclosporine
`[35,30]. Using this model,
`tumors could be n1ain-
`tained only for short periods [up to 5 weeks], thus
`preventing any definitive assessment of growth char-
`acteristics or of effects of anticancer agents on tumor
`regression. These human carcinoid xenograft tumors
`in nude mice will provide a useful model to study
`various aspects of endocrine tumor biology and will,
`we hope, allow an accurate assessment of effects of
`various chemotherapeutic regimens. However, we
`realize that we are limited to a single cell line, and
`agents that inhibit growth of BON may not suppress
`the growth of other carcinoid tumors. Ideally, addi-
`tional cell lines from other patients with carcinoid
`tumors should be established and various treatment
`
`regimens evaluated using these different tumor lines.
`Recent studies have shown that exogenous adn1inis—
`tration of pentagastrin may be useful as a provocative
`diagnostic test for patients with carcinoid tumors
`[7,37]. In this study, we found that BON cells possess
`high-affinity gastrin receptors. We have not measured
`5-HT release in vivo following pentagastrin adminis-
`tration to mice bearing BON tumors. However, prelin1—
`inary results from our ‘laboratory have shown a dose-
`dependent release of 5-HT from BON cells in vitro
`following addition of either pentagastrin or gastrin—17
`[38], suggesting a specific receptor-mediated effect of
`gastrin on 5-HT release from human carcinoid cells.
`Studies using established in vivo models would be
`useful to further examine receptor-mediated amine
`release from human carcinoid tumors.
`We found that chronic IP administration of SMS
`
`201-995 [300 pig/kg,
`
`t.i.d.) significantly inhibited
`
`
`
`*
`q
`I.
`21
`
`1
`: -
`~ I
`y .
`H
`28
`
`N
`‘ .
`§ ~
`I;
`I ,
`L..
`33
`
`In
`
`32
`.'_3
`8+
`as
`W"
`
`7
`
`14
`
`8?
`
`E E :
`
`100
`75
`
`50
`
`EJ
`<
`C‘
`2
`2 25
`O
`
`DAYS
`
`Figure 7. BON tumor area (mm‘] in relation to time from implan-
`tation comparing 2% DFMO treatment [single-hatched bars;
`n = 10 tumors], SMS 201-995 [300 ug/kg,
`IP,
`t.i.d.; double-
`hatched bars; n = 8 tumors), and the combination of SMS 201-
`995 + 2% DFMO (closed bars; n = '10 tumors] to control group
`(open bars; n = 8 tumors]. *1’ < 0.05,
`two-way analysis of
`variance.
`
`Roxane Labs., Inc.
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`
`Figure 6. Somatostatin receptors in BON: H&E-stained tumor
`section [A], autoradiogram showing total binding of ”5I-[Tyr’]-
`SMS 201-995 [B]. and autoradiogram showing nonspecific bind-
`ing [in presence of 10“’ mol/L unlabeled SMS 201-995]
`[C]
`[bar 2 1 mm].
`
`similar phenotypic changes as noted by the presence
`of secretory granules by electron microscopy. In tis-
`sue culture, BON cells secrete 5-HT [28], pancreasta-
`tin [29], and chromogranin A [30].
`Other investigators have reported establishment of
`human carcinoid tumor cells in vitro [31—33] and the
`successful transplantation, in vivo, of a gastric carci-
`noid originating from African rodents [Mastomys
`natalensis] [34]. Although valuable in studying pro-
`duction and release of various amines, these models
`
`contribute little in clarifying aspects of in vivo growth
`and behavior of human carcinoids. Previous in vivo
`
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`August 1991
`
`HUMAN CARCINOID IN NUDE MICE 309
`
`.g oO
`
`conrnou.
`
`an:SMS+nmo
`
` TUMOR
`WEIGHT(9)53
`
`
`
`
`PROTEIN(mg):2Sa
`
`RNA(mg)
`
`[g]
`Figure 8. Tumor weight
`and protein, DNA, and RNA
`content [mg] of BON tumors
`in control group (open bars;
`n = 8], DFMO group (sin-
`gle-hatched bars;
`11 = 10],
`SMS 201-995 group [double-
`hatcbed bars; n = 8], combi-
`nation of SMS 201-995 and
`DFMO [closed bars; :1 = 10]
`at sacrifice. *P < 0.05 by
`two-way analysis ofvariance.
`
`growth of BON beginning on day 14 of treatment and
`continuing until
`the mice were killed. The exact
`mechanism for this antitumor effect is not known. In
`
`this study, we have found that BON possesses soma-
`tostatin receptors, and one possibility may be a direct
`antiproliferative effect that is mediated through these
`specific receptors. Another possibility is that
`the
`effect of somatostatin may be indirect, by means of
`inhibiting release of gastrointestinal hormones, growth
`hormones, or other growth factors [i.e., epidermal
`growth factor, somatomedin C] known to stimulate
`growth of certain tumors [39]. A combination of
`effects could also be possible.
`Our study clearly shows an antiproliferative effect
`of somatostatin on experimental carcinoid tumor
`growth in nude mice and is consistent with others
`that have shown inhibition of tumor growth in exper-
`imental models of nonendocrine solid cancers [40—
`44] and endocrine tumors [-15,46]; however, the clini-
`cal effect of somatostatin in patients with carcinoid
`syndrome has been more controversial. Reports on
`the antiproliferative effect of somatostatin analogues
`have usually been based on a small number of pa-
`tients with advanced carcinoid tumors and liver
`
`metastases who were initially started on somatostatin
`treatment
`for symptomatic control. The doses of
`somatostatin and the criteria for treatment response
`vary, and the documentation of response is usually
`based on interpretation of serial computed tomogra-
`phy or liver scans. Stockmann et al. [47] reported no
`effect on the size of liver metastases in five patients
`with carcinoid tumors who were treated long-term
`with SMS 201-995 (150 Mg/Cl]. Kvols et al.
`[15]
`reported on their experience with SMS 201-995 (450
`ug/d] in the treatment of 25 patients with carcinoid
`
`syndrome. Symptoms were relieved in 22 of the
`patients; however, only 3 of 13 evaluable patients had
`apparent improvement of perfusion defects noted on
`serial liver scans. Interpretation of the antiprolifera-
`tive effect of somatostatin in some reports is further
`complicated by multiple therapeutic interventions,
`including various chemotherapeutic agents and embo-
`lization of the hepatic artery [48,49].
`A derivative of pentanoic acid, DFMO, is an irrevers-
`ible inhibitor of ornithine decarboxylase, which in
`turn catalyzes the first and rate-limiting step in
`polyamine synthesis [50]. Polyamines play an impor-
`tant role in cell growth and differentiation in rapidly
`dividing tissues and depletion of intracellular poly-
`amines by various agents can slow and eventually
`stop cell growth [51]. Studies from our laboratory
`have shown that DFMO inhibits the growth of some
`gut tumors, including a hamster pancreatic adenocar—
`cinoma [HZT] [52], mouse [MC—26] and human colon
`cancers [53,54], and human gastric cancers [55,56]. In
`our present study, DFMO, given as a single agent in
`drinking water, reduced tumor area by 42% and
`likewise inhibited tumor weight and protein, DNA,
`and RNA content after 33 days of treatment, suggest-
`ing that polyamine synthesis is important for carci-
`noid tumor growth.
`We have previously shown an additive inhibitory
`effect on human colon cancer xenografts by combin-
`ing DFMO with SMS 201-995 [54]. In our present
`study, the combination of DFMO and SMS 201-995
`produced a further decrease in mean values of BON
`growth, but these values were not significantly dif-
`ferent from those obtained with either treatment
`
`alone. However, our results suggest that DFMO or
`SMS 201-995 may be useful in treating patients with
`
`Roxane Labs., Inc.
`
`
`
`Roxane Labs., Inc.
`Exhibit 1030
`Page 007
`
`

`
`310 EVERS ET AL.
`
`GASTROENTEROLOGY Vol. 101, No. 2
`
`carcinoid tumors and represent relatively nontoxic,
`novel approaches to traditional cancer chemotherapy
`worthy of further experimentation to determine opti-
`mal dose and duration of treatment.
`
`In conclusion, we have established a transplantable
`human carcinoid cell line [BON] in nude mice. BON
`cells contain 5-HT and chromogranin A, have a
`predictable pattern of growth following tumor inocu-
`lation, and are histologically similar to the original
`tumor from the patient. SMS 201-995 and DFMO,
`given as single or combination therapy,
`inhibited
`growth of BON in nude mice. The tumor line, BON,
`will be a useful experimental model to improve our
`understanding of the biological aspects of human
`carcinoids and to study effects of various therapeutic
`strategies on growth of carcinoid tumors.
`
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