British Journal of Urology ( 1994), 74, 416-421
`
`Antitumour effect of a synthetic analogue of fumagillin on
`murine renal carcinoma
`T. MORITA, N. SHINOHARA* and A. TOKUE
`Departments of Urology and *Pathology, Jichi Medical School, Tochigi, Japan
`
`Objective To evaluate the antitumour effect of an angio(cid:173)
`inhibitory drug, a synthetic analogue of fumagillin
`(TNP-470), on murine renal carcinoma (Renea) in
`vivo and in vitro.
`Materials and methods The effect of TNP-4 70 on the
`growth of Renea cells in vitro was measured by angio(cid:173)
`genesis assay and cell counting with dye exclusion. In
`the angiogenesis assay, Renea cells were injected intra(cid:173)
`dermally and the number of blood vessels orientated
`towards the tumours was counted 3 days after tumour
`inoculation. To examine the effect of TNP-4 70 on the
`subcutaneous tumour growth and lung metastasis of
`Renea, Renea cells were injected subcutaneously or
`intravenously in BALB/c mice and they were treated
`with a subcutaneous injection every 3 days.
`Results Dose-dependent growth inhibition in vitro was
`observed with 50% inhibition occurring at 600 ng/ml.
`Angiogenesis assay revealed that administration of
`
`TNP-470 inhibited the angiogenesis induced by Renea
`in a dose-dependent manner. In the subcutaneous
`experiment, TNP-4 70 decreased the growth rate of
`established
`subcutaneous
`tumours
`rather
`than
`reduced the size of the tumour. The administration of
`TNP-4 70 in mice with lung metastasis inhibited the
`development of metastasis of Renea without weight
`loss or diarrhoea.
`Conclusion The present
`study demonstrated
`that
`TNP-4 70 had an inhibitory effect on tumour-induced
`angiogenesis and a significant anti-tumour effect on
`Renea. This suggests that TNP-4 70 could be useful in
`the treatment of renal cell carcinoma. Further studies
`are needed to clarify whether TNP-470 is more
`effective when combined with other drugs such as
`interferons.
`Keywords Renal cell carcinoma, metastasis, angiogen(cid:173)
`esis, synthetic analogue of fumagillin
`
`Introduction
`Approximately 30% of patients with renal cell carcinoma
`(RCC) have metastatic disease at the time of initial
`diagnosis [l]. Most metastatic RCC remains refractory
`to conventional hormonal treatment. chemotherapy and
`radiotherapy [2,3]. Recent clinical trials have shown
`that immunotherapy with ex-interferon (cx-IFN), interleu(cid:173)
`kin 2 (IL-2), or adoptive immunotherapy with IL-2
`and lymphokine-activated killer cells (LAK) or tumour(cid:173)
`infiltrating
`lymphocytes
`(TIL) produces occasional
`responses [4-7]. At present, however, there is no effective
`therapy for metastatic RCC, and virtually all patients not
`cured by surgical excision of their localized tumour will
`succumb to their disease. Therefore, new chemothera(cid:173)
`peutic agents whose antitumour mechanisms differ from
`those of currently available drugs are needed to improve
`the treatment of patients with RCC.
`The formation of new blood vessels in vivo, termed
`'angiogenesis', occurs during a variety of pathological
`situations, especially in the development of tumours
`
`Accepted for publication 14 March 1994
`
`416
`
`[8,9]. Good correlation between the extent of angiogen(cid:173)
`esis and the occurrence of metastasis is reported in breast
`cancer and cutaneous melanoma [10.11]. Targeting the
`tumour vasculature in the treatment of cancer therefore
`seems to be an attractive strategy. Ingber et al. demon(cid:173)
`strated that AGM-1470 (designated TNP-470 in the
`present study), one of the synthetic analogues of fumagil(cid:173)
`lin, which is a natural product of Aspergillus fumigatus
`fresenius, exhibited potent inhibitory effects on the
`growth of endothelial cells and solid tumours with
`relatively few side-effects [12]. However, there is no
`report that evaluates the effects of fumagillin analogues
`on RCC. In the present study, we have examined the
`effects of the angio-inhibitory drug, TNP-4 70, on murine
`renal carcinoma (Renea) in vitro and in vivo.
`
`Materials and methods
`
`Animals and cells
`
`Male BALB/c mice (aged 5-10 weeks) (Doken Inc ..
`Ibaragi, Japan) were used for in vivo experiments. The
`murine renal cell carcinoma line, designated Renea [ 13 j,
`
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`ANTI-TUMOUR EFFECT OF FUMAGILLIN ON MORINE RENAL CELL CARCINOMA 417
`
`was originally established and characterized by Dr G.P.
`Murphy (Roswell Park Memorial Institute, Buffalo. NY,
`USA) and supplied by Dr Fujioka (Iwate Medical School.
`Japan). Renea was maintained by serial transplantation
`in BALB/c mice. Renea cells were used which were
`maintained in vitro in DMEM (Nissui Pharmaceutical Co.
`Ltd, Tokyo, Japan) supplemented with 5% fetal bovine
`serum (PBS) and 2 mM L-glutamine in a 5%-C02
`incubator.
`
`Reagents
`
`TNP-470 (Takeda Pharmaceutical Co., Osaka, Japan)
`was stored at -80°C. For in vitro experiments, TNP-470
`was dissolved in dimethylsulphoxide (DMSO) (Seikagaku
`Co., Tokyo, Japan) diluted with culture medium to the
`desired concentrations. For in vivo experiments, TNP-470
`was dissolved in ethanol and diluted to the desired
`concentrations with 1 % gum arabic (Sigma Chemical
`Co., St Louis, USA) and normal saline.
`
`Cell proliferation studies
`
`Proliferation of cells was characterized by both cell
`counting with dye exclusion and the colourimetric
`3-( 4,5-dimethylthiazol- 2-yl) - 2,5-diphenyltetrazolium
`bromide (MIT) (Sigma Chemical Co., St Louis, USA)
`assay.
`Renea cells were seeded into 6-well tissue culture
`plates at 1 x 105 cells/well, in triplicate. After culture
`with TNP-4 70 or vehicle for 3 days, cells were detached
`with trypsin, stained with erythrosin and counted with
`a haemacytometer. A modification of the MIT assay
`originally described by Mossman [14] was used. Rapidly
`growing cells were plated at a concentration of 1000
`cells in 100 µl medium into 96-well microtitre plates
`(Costar, Cambridge, MA, USA) using a multichannel
`pipette. In preliminary experiments, seeding densities
`were determined, ensuring that cultures did not become
`confluent before conducting the assay. Cell numbers per
`microtitre well were proportional to the absorbance of
`the solubilized formazan (data not shown). After incu(cid:173)
`bation for 24 h, 100 µl of culture medium and culture
`medium containing various concentrations of TNP-4 70
`or vehicle (DMSO) were dispensed within appropriate
`wells and the plates were then incubated for 1 or 3
`days at 3 7°C in a 5%-C02
`incubator prior to the
`addition of MTT solution. The MIT working solution
`was prepared as follows: 1 mg MTT/ml phosphate
`buffered saline (PBS) was filtered with 0.45 µm filter
`units and stored at 4°C; 50 µg of MIT were then added
`to each well. After incubation for 4 h at 3 7°C, 200 µl
`supernatant were removed using a Transtar-96™
`system (Costar, Cambridge, MA, USA), leaving < 30 µl
`
`British Journal of Urology (1994), 74
`
`residual medium in each well. MTT formazan crystals
`were solubilized by adding 150 µl of 100% DMSO to
`each well. Plates were then agitated on a plate shaker
`(TAITEC, Saitama, Japan) for 5 min, following which
`immediately measured
`absorbance at 540 nm was
`with a microplate reader Model 450 (Bio-Rad Lab.,
`Richmond, CA, USA). Dose-response curves were plot(cid:173)
`ted, and the 50% inhibitory concentration (IC50 ) was
`determined graphically as the dose of drug causing a
`50% reduction in absorbance compared with control
`values. The MTT assay was performed in eight replicates
`and repeated at least three times.
`
`Angiogenesis assay
`
`The assay for tumour-induced angiogenesis was per(cid:173)
`formed as described by Kreisle and Ershler [15]. BALB/c
`mice were injected intradermally (i.d.) with 1 x 106
`Renea cells at two sites on the abdomen on day 0. Mice
`were treated with the administration of TNP-4 70 or
`vehicle consisting of ethanol and gum arable in normal
`saline on days 1 and 2. The animals were killed on
`day 3 and the skin with tumours was separated from
`the underlying tissues. Tumour-induced angiogenesis
`was quantified by counting the number of blood vessels
`orientated towards the tumour, using a dissecting
`microscope. The size of the tumour was assessed
`simultaneously by measuring the diameters of its short
`and long axes. All counts were made by the same
`observer.
`
`Experimental tumour models
`
`In the subcutaneous experiment, single cell suspensions
`of 1 x 106 Renea cells were injected into the backs of
`5-7-week-old BALB/c mice. After inoculation of Renea
`cells, tumour size and mouse weight were measured by
`the same observer, twice a week, and the estimated
`tumour volume (V) was calculated by the following
`formula: V = Width2 x Length x 0. 5. Relative
`tumour
`volumes were shown as the relative mean value (Vn/V0 ),
`VO being the mean volume at the initiation of treatment
`and Vn being at any given day. In the metastasis
`experiment, single cell suspensions of 5 x 104 viable cells
`were injected in 0.2 ml aliquots into the lateral tail vein
`of 5-7-week-old BALB/c mice. After treatment with
`TNP-470, the mice were killed for enumeration of meta(cid:173)
`static lung nodules and for weighing the lungs with
`metastases. The metastases formed discrete white nod(cid:173)
`ules on the blackened surface of the lungs insufflated
`with a 15% solution of Indian ink when fixed by Fekete's
`solution [16]. Following fixation for 24 h, the lungs were
`weighed and the number of lung nodules counted with
`the aid of a dissecting microscope.
`
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`

`418 T. MORITA, N. SHINOHARA and A. TOKUE
`
`Statistics
`
`Student's 2-tailed t-test was used to assess the statistical
`significance of differences between groups. The differ(cid:173)
`ences were considered to be statistically significant when
`a 2-tailed value of P was < 0.05.
`
`Results
`
`cells for 100 ng/ml (P<0.05 compared with controls
`treated with vehicle alone), 8 x 10 5 cells for 1 µg/ml
`(P<0.01), 5 x 105 cells for 10 µg/ml (P<0.01), and
`O.lxl04 cells for
`lOOµg/ml (P<0.01). Cytostatic
`inhibition of Renea growth was observed at concen(cid:173)
`trations < 10 µg/ml. while cytotoxicity (reduction of cell
`number below the initial plating density) was observed
`at much higher concentrations (100 µg/ml).
`
`Effect of TNP-4 70 on tire growth of Renea cells in vitro
`
`Effect of TNP-470 on tumour-induced angioge11esis in vivo
`
`Renea cells were cultured with various concentrations
`of TNP-4 70 for 1 or 3 days, and their growth was
`evaluated by both the colourimetric MTT assay and cell
`counting with dye exclusion. In the colourimetric MTT
`assay, significant growth inhibition was observed in a
`dose-dependent manner, with 50% inhibition occurring
`at 600 ng/ml after 3 days' culture with TNP-470 (Fig. 1).
`The inhibitory effect of TNP-470 was also confirmed by
`cell counting with dye exclusion. Renea cells, 1 x 1 as,
`were seeded and cultured with vehicle or various concen(cid:173)
`trations of TNP-4 70. The mean numbers of cells after 3
`days' culture were 18 x 1 as cells for vehicle, 16 x 105
`cells for 1 ng/ml. 16 x 10s cells for 10 ng/ml. 15 x 10s
`
`~
`
`Q)
`(.)
`
`C: co .c
`...
`0 50
`II) .c
`co
`Q)
`>
`·.;:;
`co
`ai
`a:
`
`*
`
`*
`
`Mice with intradermal tumours were treated with subcu(cid:173)
`taneous injection ofTNP-470 or vehicle (n= 10 for each
`group) for 2 days, and angiogenesis was assessed 3 days
`after tumour inoculation. As shown in Table 1, the
`number of blood vessels orientated towards the tumour
`was significantly decreased by treatment with TNP-4 70
`in a dose-dependent manner. Tumour volume in mice
`treated with TNP-4 70 was significantly smaller than
`in vehicle-treated mice. These findings indicate that
`TNP-470 had an inhibitory effect not only on the
`angiogenesis induced by Renea but also on intradermal
`tumour growth. Based upon the results of the angiogen(cid:173)
`esis assay, doses of 10 mg/kg and 20 mg/kg TNP-470
`were used in further studies in vivo.
`
`Effect of TNP-4 70 on the growth of subcutaneous tumours
`
`The antitumour effect ofTNP-470 on established subcut(cid:173)
`aneous tumours was examined. Treatment was initiated
`when the tumours were at least 250 mm 3 in volume.
`Tumours of similar size were matched for use in control
`and experimental groups. Mice were treated with subcut(cid:173)
`aneous injection of vehicle, 10, or 20 mg/kg TNP-470
`(n = 10 for each group) once every 3 days. TNP-
`4 70-treated mice had significantly smaller tumours than
`the vehicle-treated mice, starting with measurements
`made on day 15 (Fig. 2). TNP-470 inhibited the growth
`rate of established subcutaneous tumours in a dose(cid:173)
`dependent manner rather than reducing the size of the
`tumours. Neither weight
`loss nor diarrhoea was
`observed.
`
`0 0 10° 101 102 103 104 106 106 107
`TNP-470 concentrations (pg/ml)
`
`Fig. 1. Effect of TNP-4 70 on the growth of Renea cells in vitro
`assessed by MTT assay. Renea cells were cultured with indicated
`concentrations of TNP-4 70 for 1 day ( 0) and 3 days ( • ). The
`growth of Renea cells was assessed by MTI' assay. The results are
`expressed as a percentage of absorbance of MIT-derived formazan
`developed by the cells treated with vehicle alone (control). Each
`point. mean of three experiments performed In eight replicates.
`Bar. SD. •P<0.01 as compared with controls.
`
`Effect of TNP-4 70 on metastasis of Renea cells
`
`The effect of TNP-4 70 on the development of metastasis
`was then examined. Renea cells were injected intra(cid:173)
`venously in BALB/c mice on day 0, and the animals
`were treated with subcutaneous injection of vehicle, 10,
`or 20 mg/kg TNP-470 (n = 8 for each group) once every
`3 days from day 1 to day 28. The results shown in
`Table 2 demonstrate that TNP-470 inhibited lung met(cid:173)
`astasis of Renea cells significantly in a dose-dependent
`manner. There was no significant difference in lung
`
`British Journal of Urology ( 1994), 74
`
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`

`ANTI-TUMOUR EFFECT OF FUMAGILLIN ON MURINE RENAL CELL CARCINOMA 419
`
`Table 1. Effect or TNP-4 70 on angiogenesis
`induced by Renea
`
`Treatment
`
`Tumour volume (mm 3)
`
`No. of blood vessels
`
`Vehicle
`5 mg/kg TNP-470
`10 mg/kg TNP-470
`20 mg/kg TNP-470
`
`(n= 10)
`(n=lO)
`(n=l0)
`(n=lO)
`
`17.2±4,3*
`14.5±6.1
`10,7±5,3t
`10,6±6,1*
`
`21.3±2,3
`16.9±1.8t
`15.9±2,0t
`14.8±3.St
`
`•Mean±SD. tP<0.01 as compared with controls treated with vehicle alone. iP<0.05 as
`compared with controls treated with vehicle alone.
`
`16
`
`1/)
`Q)
`
`E 10
`::I
`0
`
`> .. ::I
`0
`E
`::I ...
`f
`'i
`"iii a:
`
`5
`
`..
`
`0 o,.____,3...__e.___9.___, ... 2--,--&--,'""e--2--,--2--4---2'""1-
`
`Davs after initiation of treatment
`
`Fig. 2. Effect ofTNP-470 on the established subcutaneous tumours
`or Renea. Renea cells ( 1 x 106 ) were injected subcutaneously into
`the backs orBALB/c mice and tumour size was measured. Tumours
`or similar size were matched for use in control and experimental
`groups (n = 10 for each group), Treatment was initiated on day 0
`when tumours were at least 250 mm 3 in volume. Mice were
`treated with subcutaneous injection or vehicle ((cid:127))
`or TNP-470
`(e. 1() mg/kg; 0, 20 mg/kg) once every 3 days. Each point,
`relative tumour volume (see Materials and Methods), Bar, SD.
`*P<0.05 as compared with controls. **P<0.01 as compared
`with controls.
`
`weights between vehicle-treated mice and TNP-4 70-
`treated mice. At no time was weight loss or diarrhoea
`observed in the TNP-4 70-treated mice.
`
`Discussion
`
`Most RCCs have typical features of malignant hypervas(cid:173)
`cularity. Several reports have demonstrated that the
`more hypovascular papillary RCC is less aggressive than
`the more vascular RCC [17]. In addition, RCCs metastas(cid:173)
`ize frequently to the lung, bone and liver, probably via
`haematogenous spread, and these metastases remain
`refractory to conventional therapy [2,3]. In view of these
`biological and clinical characteristics, angio-inhibition
`seems to be a reasonable approach in the treatment of
`metastatic RCC. In the present study, the antitumour
`effect of the angio-inhibitory drug, TNP-470, on Renea
`cells was evaluated in vitro and in vivo, and the IC 50 of
`TNP-4 70 after 3 days' culture was demonstrated to be
`600 ng/ml. Furthermore, in vivo studies revealed that
`administration of TNP-470
`inhibited angiogenesis
`induced by Renea, inhibited the growth of established
`subcutaneous tumours, and the development of lung
`metastases in a dose-dependent manner without weight
`loss or diarrhoea.
`Many experimental studies have shown that angiogen(cid:173)
`esis is essential for the growth and metastasis of solid
`tumours [8,9). If a tumour grows beyond the point
`where simple diffusion cannot nourish it (a few milli(cid:173)
`metres in diameter, about 106 cells), further expansion
`of the tumour cell population requires the induction of
`new capillary blood vessels. The induction of angiogen(cid:173)
`esis is mediated by specific angiogenic factors released
`by the tumours [9] and often by macrophages attracted
`to it [ 18]. One of the factors that stimulates angiogenesis
`is basic fibroblast growth factor (bFGF) [19). Mydlo et al.
`isolated an angiogenic heparin-binding growth factor
`homologous to bFGF from RCC [20). Chodak et al.
`
`Table 2. Inhibition or development of lung
`metastasis or Renea by TNP-470
`
`Treatment
`
`No. of lung nodules
`
`Lung weight (mg)
`
`Vehicle
`10 mg/kg TNP-470
`20 mg/kg TNP-470
`
`(n=8)
`(n=8)
`(n=8)
`
`74.2 ± 31.8*
`13.5 ± 13.0t
`12.8 ± 16.St
`
`619.2±61.8
`618.8±90.7
`548.2 ± 28. si
`
`•Mean±SD. tP<0.01 as compared with controls treated with vehicle alone. :j:P<0.05 as
`compared with controls treated with vehicle alone.
`
`British /ournal of Urology (1994), 74
`
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`

`420 T. MORITA, N. SHINOHARA and A. TOKUE
`
`showed the presence of FGF-related protein in RCC
`tissues, and demonstrated that patients with RCC had
`increased levels of bFGF-like activity in their urine [21].
`Furthermore. Fujimoto et al. detected bFGF mRNA in
`RCC tissues by Northern blot analysis, and showed that
`serum bFGF levels were elevated in patients with RCC
`[22]. The present authors have already confirmed that
`bFGF was detected immunohistochemically in Renea,
`which is a hypervascular tumour (data not shown).
`These findings suggest that bFGF as an angiogenic
`mediator plays an important role in determining the
`vascularity seen in RCC.
`Although the mechanisms underlying the antitumour
`effects of TNP-4 70 are not understood completely.
`reports suggest that TNP-4 70 exerts its antitumour
`effects primarily by acting on the tumour vasculature
`rather than the tumour cells [12]: (i) doses of TNP-470
`had to be at least 10 times higher for comparable
`inhibition of the human tumour cell lines in vitro;
`(ii) M5076 tumour cell lines. which were very sensitive
`in vivo. were found to be refractory to TNP-4 70 in vitro:
`(iii) TNP-470 had no significant effect on ascitic (less
`dependent on neovascularization) leukaemic cell growth;
`(iv J TNP-4 70 inhibited angiogenesis induced by bFGF in
`the rat sponge implantation assay [23]. or by human
`choriocarcinoma cell lines in a dose-dependent manner
`[24]. In the present study. TNP-470 inhibited not only
`the growth and metastasis of Renea but also angiogenesis
`induced by Renea where bFGF was detected immunohis(cid:173)
`tochemically. However, the IC50 of Renea cells was
`600 ng/ml, which is extremely high when compared
`with that of endothelial cells (IC50 : 10-20 pg/ml) [12].
`These findings also suggest that TNP-4 70 acts primarily
`on the tumour vasculature probably induced by angiog(cid:173)
`enic mediators. including bFGF released from Renea,
`resulting in growth inhibition of Renea.
`The in vivo antitumour effects of TNP-470 varied
`according to the different assays: the inhibitory effects
`seen in the angiogenesis assay were significant following
`treatment with low-dose TNP-470 (5 mg/kg) within a
`short period (3 days), while the effects on the established
`subcutaneous tumours were seen 15 days after initiating
`treatment with 20 mg/kg TNP-470. The difference in
`these inhibitory effects seems to be related to the differ(cid:173)
`ence in the phase of tumour formation when the treat(cid:173)
`ment was initiated: in the angiogenesis assay, treatment
`was initiated 1 day after tumour cell inoculation, while
`treatment began when the tumours were over 250 mm3
`in the assay with established subcutaneous tumours.
`Saiki et al. [25] examined the effects of an angio(cid:173)
`inhibitory drug. synthetic polymeric peptide (poly
`(RGD)), on the angiogenesis and lung metastasis of
`metastatic B16-BL6 melanoma, and showed that its
`antitumour effect was more significant when treatment
`
`was initiated earlier after tumour inoculation (when the
`tumour formation was in the early phase). In their
`report, treatment was initiated at various times following
`B16-BL6 cell inoculation. Single administration on day 1
`or day 7 after tumour inoculation caused significant
`inhibition of lung metastasis, while that on day 14 failed
`to inhibit lung metastasis. These findings suggest that
`the antitumour effect of TNP-4 70 is more significant
`when treatment begins soon after tumour inoculation.
`and may explain the differences in the in vivo inhibitory
`effects of TNP-4 70 seen in the various assays.
`In conclusion, the present study showed that TNP-470
`had a significant antitumour effect on Renea without
`weight loss or diarrhoea, suggesting that TNP-4 70 could
`be useful in the treatment of RCC. As the administration
`of TNP-4 70 alone did not result in a complete response
`under the conditions employed in this study, further
`research is required to establish whether TNP-4 70 is
`more effective when used in combination with other
`drugs such as IFNs.
`
`Acknowledgement
`
`We thank Takeda Chemical Industries Ltd for the
`generous gift of TNP-470.
`
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`ANTI-TUMOUR EFFECT OF FUMAGILLIN ON MORINE RENAL CELL CARCINOMA 421
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`
`Authors
`T. Morita, MD, Assistant Professor.
`N. Shinohara, MD, Assistant Professor.
`A. Tokue, MD, Professor.
`Correspondence: Dr T. Morita, Department of Urology, Jichi
`Medical School. Minamikawachi-machi, Kawachi-gun, Tochigi
`32904, Japan.
`
`British Journal of Urology (1994), 74
`
`NOVARTIS EXHIBIT 2072
`Breckenridge v. Novartis, IPR 2017-01592
`Page 6 of 6
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