Clinical & Experimental Metastasis 20: 407–412, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Activity of biphenyl matrix metalloproteinase inhibitor BAY 12-9566 in a human breast cancer orthotopic model Shinichi Nozaki1,2 , Sean Sissons1, Du-Shieng Chien3 & George W. Sledge, Jr.1 1 Department

of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA, 2 Department of Oral & Maxillofacial Surgery, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan; 3 Department of Pharmacokinetics and Drug Metabolism, Bayer Corporation, Pharmaceutical Division, West Haven, Connecticut, USA Received 20 September 2002; accepted in revised form 23 December 2002

Key words: angiogenesis, breast cancer, matrix metalloproteinase inhibitor, orthotopic transplant model, pulmonary metastasis Abstract Matrix metalloproteinases (MMPs) have been implicated in the invasion, metastasis, and angiogenesis associated with human cancer by mediating the degradation of extracellular matrix components. In this paper, we report data that show that BAY 12-9566, a novel inhibitor of MMPs, inhibits angiogenesis, tumor regrowth, and the growth of lung metastases. BAY 12-9566, at 15–100 µM, inhibited tubule formation by human endothelial cells in an in vitro model, but did not prevent the proliferation of endothelial and human breast cancer cells. In the MDA-MB-435 human mammary carcinoma xenograft model, in which the primary tumor is transplanted into the murine mammary fat pad, BAY 12-9566, administered daily at a dose of 100 mg/kg/day p.o. after resection of the primary tumor, inhibited local tumor regrowth by 58% without causing any toxic effect. In addition, BAY 12-9566 treatment inhibited the number and volume of lung metastases by 57 and 88%, respectively. These effects were highly correlated with the serum concentration of BAY 12-9566 at the end of treatment. The serum of the treated animals, harvested 24 h after the last treatment, and the tumor regrown at the site of tumor transplant in the treated animals, contained less protein with MMP-9 activity (as measured in a gelatin zymography assay) than the corresponding controls. However, no difference in the activity of MMP-2 was observed. Although all clinical trials in cancer involving BAY 12-9566 have been halted, this MMP inhibitor has never been used in clinical trials in breast cancer. These results suggest that the novel MMP inhibitor BAY 12-9566 maybe a useful and safe oral treatment for breast cancer, adjunctive to surgery. Abbreviations: HPLC – high-performance liquid chromatography; MC – microcarrier; MMP – matrix metalloproteinase Introduction MMPs are a family of structurally related zinc-containing enzymes involved in the degradation of extracellular matrix proteins that constitute connective tissue. The matrixdegrading activity ofMMPs has been implicated in the pathogenesis of cancer through the promotion of angiogenesis, tumor invasion and metastases [1–3]. The activity of MMPs is thought to be balanced by endogenous factors released by tumors and stromal components [2, 4]. The inhibition of these degradative processes is presently recognized as a potential strategy for cancer treatment, and has stimulated several studies to identify compounds that can inhibit MMP activity. Since treatment with antimetastatic or antiangiogenic agents may not result in a cytotoxic effect, and may require long-term administration, compounds such as Correspondence to: George W. Sledge, Jr., Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, 535 Barnhill Drive, RT 473, Indianapolis, IN 46202, USA. Tel: +1-317- 274-3550; Fax: +1-317-274-3646; E-mail: [email protected]

MMP inhibitors should preferentially be orally bioavailable. Several MMP inhibitors, including marimastat, prinomastat, solimastat, BMS 275291, metastat and neovastat are now under investigation for clinical trials in patients with cancer and rheumatoid arthritis [5, 6]. Novel compounds that exhibit this mechanism of action, however, are of great interest, particularly if they belong to a chemical class different from that of the compounds described above, in view of possible differences in pharmacological and toxicological profiles. BAY 12-9566, 4-[4-4-(chlorophenyl)phenyl]-4-oxo-2S(phenylthiomethyl) butanoic acid, is a novel, non-peptidic biphenyl MMP inhibitor that demonstrated nanomolar inhibitory activity against severalMMPs [7], and has been reported to be orally bioavailable [7, 8]. BAY 12-9566 has been found to inhibit tumor invasion in vitro and angiogenesis in vivo [7]. In addition, it has been shown to inhibit primary tumor growth and pulmonary metastases in murine and human xenograft tumor models [8, 9].

408 In the present study, we have used an MDA-MB-435 human breast cancer-athymic nude mouse system to examine the effect of BAY 12-9566 on local-regional tumor regrowth and the formation of pulmonary metastases after primary tumor resection.

Materials and methods

S. Nozaki et al. Table 1. Gradient profile of reversed-phase HPLC-UV assay. Time (min)

1% acetic acid (%)

Acetonitrile (%)

0.0 0.5 9.0 10.5 10.6 13.6

50 50 25 25 50 50

50 50 75 75 50 50

Cell culture Human MDA-MB-435 breast cancer cells (a gift from Dr Janet Price, The University of Texas M.D. Anderson Cancer Center, Houston) were cultured in minimum essential medium (Life Technologies, Grand Island, New York) that was supplemented with multi-vitamins, sodium pyrophosphate (100 mM), penicillin (105 U/100 ml), streptomycin (10 µg/100 ml) and fetal calf serum (5%) (supplements from Sigma Chemical, St. Louis, Missouri) in a 5% CO2 atmosphere at 37 ◦ C. The HUVEC umbilical vein cell kit was obtained from Clonetics (San Diego, California) and maintained according to the provided instructions.

clotting was complete, gels were equilibrated with standard medium for HUVEC cells, and following a 60 min incubation, fresh medium containing vascular endothelial growth factor (100 ng/ml, Chemicon, Temecula, California), basic fibroblast growth factor (30 ng/ml, R & D Systems, Minneapolis, Minnesota), and various doses of BAY 12-9566 were added to the fibrin matrices. The number of tubules formed was determined five days after polymerization of the gels. Human breast cancer xenograft-nude mouse model

BAY 12-9566 BAY 12-9566 was obtained from Dr Harold Kluender (Bayer Corporation, Pharmaceutical Division, West Haven, Connecticut). BAY 12-9566 is an odorless, light-yellow powder that demonstrates nanomolar inhibitory activity against severalMMPs. The inhibitor constants forMMP-2, -3 and -9 are 11, 134 and 301 nM, respectively [7]. Cell proliferation assay Cell proliferation was determined using the CellTiter Aqueous nonradioactive cell proliferation assay (Promega, Madison, Wisconsin). The cultured cells (5 × 103 cells/well) were plated into 96-well tissue culture plates (Costar, Cambridge, Massachusetts) in the presence of various doses of BAY 12-9566 dissolved in absolute ethanol. Each group had eight wells. After 48, 72, and 120 h, 20 µl/well of combined tetrazolium/phenazine methosulfate solution was added. After 60 min at 37 ◦ C in a 5% CO2 atmosphere, an absorbance reading at 450 nm was measured using Elx 800 (Bio-Tek Instruments, Winooski, Vermont). MC assay The MC assay was carried out according to the technique by Nehls et al. [10] with modifications [11]. Briefly, gelatincoated Cytodex 3 MCs (Pharmacia Biotech, Piscataway, New Jersey) were added to HUVEC cells at a final concentration of 30 cells/MC. After incubation for four days at 37 ◦ C in a 5% CO2 atmosphere, the cell coated MCs were added to fibrinogen solution (2.5 mg/ml) in 12-well tissue culture plates (Costar) at a density of 10 MCs/well. The fibrinogen solution was supplemented with aprotinin (0.15 U/ml, Sigma Chemical) and was clotted by the addition of thrombin (0.625 U/ml, Sigma Chemical). After

The xenograft model for breast cancer adjuvant setting was performed as described previously by Sledge et al. [12]. Briefly, six- to eight-week-old female BALB/c nude mice (Harlan Sprague Dawley, Indianapolis, Indiana) were used. MDA-MB-435 cells (5×105 cells/mouse) were injected into the mammary fat pads. After eight weeks, the tumors were resected under methoxyflurane anesthesia. BAY 12-9566 was suspended in 0.5% sodium carboxymethylcellulose and 0.2% Tween 80 (vehicle) and was given p.o. daily thereafter. The control mice were given vehicle only. The administrations were continued through week 15, when the mice were sacrificed. Whole blood was taken directly from the heart on Day 105 (one day after the last treatment). Circulating cells were removed by centrifugation, and sera were snap frozen at −70 ◦ C. The lungs were removed and fixed in Bouin’s solution. Macroscopically visualized tumors were enumerated and measured. The excised regrowth tumors were frozen and stored at −70 ◦ C. Tumor volume was calculated as (length) × (width)2/2. Serum concentrations of BAY 12-9566 BAY 12-9566 concentration in murine serum was determined using a reversed-phase HPLC-UV assay. This analysis was performed using a HP 1090 liquid chromatograph series II (Hewlett Packard, Palo Alto, California). Serum samples were prepared via ethyl acetate extraction followed by evaporation and reconstituted in mobile phase prior to injection onto the HPLC. For analyte quantitation, reverse phase chromatography incorporating a gradient elution was used followed by UV detection at 290 nm. The gradient profile used in this analysis consisted of varying mixtures of 1% acetic acid and acetonitrile over a run time of 13.6 min (Table 1). Retention time for BAY 12-9566 was determined to be 7.4 min. The column type used in conjunction with the

Activity of BAY 12-9566

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HPLC system was an ultrasphere octyl column (2.0 mm × 15 cm, Beckman Coulter, Inc., Fullerton, California) which processed 50–75 µl of sample per injection at a flow rate of 0.43 ml/min. Gelatin zymography of murine sera and tissue extracts Sera and tissue extracts were analyzed for collagenase activity as described by Brown et al. [13]. For the analysis of tissue extract, 20–40 mg of frozen tumors were minced with 50 µl/mg of nonreducing sample buffer. Aliquots of sera (containing 500 µg protein) or 50 µl of tissue extracts were run on discontinuous polyacrylamide gels. Statistical analysis Statistical analysis was performed using Statview software (version 4.02; Abacus Concepts, Berkeley, California). Data were evaluated by Fisher’s exact probability test, Student’s t-test, or one-factor analysis of variance (ANOVA).

Results Effect of BAY 12-9566 on in vitro growth and tubule formation BAY 12-9566 at concentrations ranging from 10 µm to 1 mM had no significant direct inhibitory effects on the proliferation of MDA-MB-435 or HUVEC cells after 48, 72, and 120 h incubation. Morphologically, there was no difference between treated and control cells (data not shown). The MC assay is a convenient in vitro angiogenesis system which allows reliable quantification of tubule formation in a three-dimensional environment [10, 11]. As shown in Figure 1A, MC generated eight tubules (control, ethanol only). No difference in tubule formation by HUVEC cells was observed when cells were incubated in the gel that was treated with BAY 12-9566 at up to 10 µM for 5 days (data not shown). BAY 12-9566 at 15–100 µM inhibited tubule formation completely (Figure 1C). In addition, under these experimental conditions, the MCs developed golf ball-like structures (Figure 1B).

Figure 1. MCs coated with HUVEC cells were cultured for five days within fibrin gel that was treated with various doses of BAY 12-9566. (A) MC generated eight tubules (control, ethanol only). (B) Golf ball-like MC without tubule formation (100 µM BAY 12-9566). (C) Quantitative analysis of effect of BAY 12-9566 on tubules. The development of tubular structures was examined in the absence or presence of 1, 10, 15, 20, and 100 µM BAY12-9566. Number of tubules per MC was calculated; 100% cell growth indicates in control. Columes, mean of three wells. Eash value was represents the averageof triplicate wells; bar, SE.

Effect of BAY 12-9566 on local-regional tumor regrowth and pulmonary metastasis An athymic mouse model of human breast cancer, previously used to evaluate the effect of anti-angiogenic agents on tumor regrowth and metastasis [12], was employed to assess the in vivo antitumor activity of BAY 12-9566. In this model, BAY 12-9566 treatment started after surgery of the primary MDA-MB-435 human mammary tumor, which was performed eight weeks after the primary tumor implant into the mammary fat pad. Daily oral administration of BAY 129566 at 100 mg/kg/day over a 7-week period did not affect the body weight of the mice (data not shown). Primary tumor regrowth occurred in 14 of 18 BAY 12-9566-treated mice

Figure 2. Effect of BAY 12-9566 treatment on MDA-MB-435 primary tumor regrowth in nude mice. Primary tumors, injected into the mammary fat pads, were resected at day 55. BAY 12-9566 at 100 mg/kg/day or vehicle was administered p.o. once daily thereafter. At day 105, the experiment was terminated. Tumor volumes are presented as mean volumes ± SE of local regional tumor regrowths. Square and triangle indicate vehicle-treated and BAY 12-9566-treated mice, respectively. P = 0.04 for difference between the treated and control tumor regrowth curves on the terminal day.

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Figure 3. Influence of serum concentration of BAY 12-9566 on its antitumor effect (A, volume of primary regrowth tumors; B, number of pulmonary metastases; C, volume of pulmonary metastases). Group 1, < 3 µg/ml; group 2, > 3 µg/ml and < 6 µg/ml; and group 3, > 6 µg/ml. Table 2. Effect of BAY 12-9566 on pulmonary metastasis.

Without pulmonary metastases Number of metastases Volume of metastases (mm3 )

Control (n = 19)

BAY 12-9566 (n = 18)

IRa (%)

P-valueb

8/19 20.8 ± 6.9c 28.7 ± 13.6

8/18 8.8 ± 2.9 3.4 ± 1.2

– 57.7 88.2

> 0.99 0.12 0.08

a Inhibition ratio = {1-(BAY 12-9566 treated group/control group)} × 100. b By Fisher’s exact probability or Student’s t-test. c Mean ± SE per mouse.

and 18 of 19 vehicle-treated controls (P = not significant). As shown in Figure 2, BAY 12-9566 inhibited the volume of the tumor that regrew by 58% (P = 0.04). In addition, daily treatment with BAY 12-9566 at 100 mg/kg/day decreased the number and the volume of pulmonary metastases by 57 and 88%, respectively (Table 2). Though these differences did not reach statistical significance, they are indicative of an inhibitory effect of BAY 12-9566 against the growth of lung metastases in this tumor model. The incidence of mice with lung metastases in control and treated mice was similar. Correlation between serum concentration and effect of BAY 12-9566 We classified the mice treated with BAY 12-9566 into three groups according to the serum concentration of BAY 129566 on the day of measurement (group 1, < 3 µg/ml; group 2, > 3 µg/ml and < 6 µg/ml; and group 3, > 6 µg/ml). As shown in Figure 3, the volume of tumor regrowth in groups 1, 2, and 3 was inhibited by 35, 66, and 78%, respectively; the number of pulmonary metastases in groups 1, 2, and 3 was inhibited by 31, 74, and 76%, respectively; and the volume of metastases in groups 1, 2, and 3 was inhibited by 83, 91, and 92%, respectively. Gelatinase activity Samples of serum and regrowth tumor from the mice treated with BAY 12-9566 or vehicle were analyzed by zymography for gelatinase activity. As shown in Figure 4, MMP-2 and MMP-9 activities were identified by molecular weight. The sera from vehicle-treated mice had higher activity levels of

Figure 4. Gelatin zymography of specimens from BAY 12-9566 or vehicle-treated mice. Numbers: 1–3, specimens from mice treated with vehicle only; 4–6, specimens from mice treated with BAY 12-9566. Identically numbered lanes indicate specimens from same mouse.

MMP-9 than sera from BAY 12-9566-treated mice. MMP9 activity was also reduced in tissue extract; this decrease in gelatinase activity was similar to that observed in sera. The levels of MMP-2 activity in vehicle-treated and BAY 12-9566-treated mice were similar.

Discussion The results reported in this paper show that BAY 12-9566 can inhibit both the primary tumor regrowth and the growth

Activity of BAY 12-9566 of spontaneous pulmonary metastases of the human mammary carcinoma MDA-MB-435, orthotopically transplanted in nude mice. BAY 12-9566, at the dose of 100 mg/kg/day, inhibited the number and size of lung metastases and the size of the tumor recurring at the surgery site, when administered orally for 15 weeks to mice in which the primary tumor had been surgically removed. The effect of BAY 12-9566 was correlated with the serum concentration of BAY 12-9566 at the end of the experiment. The antimetastatic effect of BAY 12-9566 is consistent with its ability to inhibit MMPs, particularly MMP-2 and MMP-9 [7]. The process of tumor metastasis involves the breakdown of physical barriers between a primary tumor and distant sites, which requiresMMP activity. The results presented here demonstrate that the inhibition of MMP activity can decrease the number and size of lung metastases. The incidence of mice with lung metastases in control and treated mice was similar. Previous studies have confirmed that the lung micrometastases exist eight weeks after tumor injection [14]. In addition to its antimetastatic activity, BAY 12-9566 demonstrated direct antitumor effects, as shown by its ability to inhibit the regrowth of the MDA-MB-435 human mammary carcinoma after surgery. The in vitro data presented in this paper suggest that this antitumor effect might be due in part to the ability of BAY 12-9566 to inhibit angiogenesis. BAY 12-9566 was able to inhibit the formation of tubules from HUVEC cells in vitro, while it did not have any effect on vascular endothelial cell proliferation. These results are in agreement with those previously reported by other investigators, who showed that BAY 12-9566 was able to inhibit the invasion of HUVEC cells in vitro (but not their motility or proliferation), and the angiogenesis induced by basic fibroblast growth factor in mice [7]. In the present study, MMP-9 (but not MMP-2) activity in sera and tissue extracts from BAY 12-9566-treated mice was reduced compared with control mice. In addition, MMP-9 activity was not related to primary regrowth tumor volume and BAY 12-9566 in vitro has no effect on the MMP-9 mRNA in several cell lines (data not shown). The reduction in MMP-9 activity is not due to preferential inhibition of MMP-9, but may result from an indirect effect of BAY 12-9566 on MMP-9 gene expression through an as yet undefined mechanism. The effects of BAY 12-9566 were not always dependent on the administrated dose [8, 9] but were strongly correlated with the serum concentration on the terminal day. The greatest efficacy was observed when the serum concentration of BAY 12-9566 at the end of the experiment was over 6 µg/ml (Group 3). This concentration of BAY 12-9566 is equivalent to 15 µM, which was the starting concentration for complete inhibition of tubule formation in the MC assay. In phase I clinical trials in cancer patients, BAY 129566, administered orally at the dose of 1600 mg/patient/day (800 mg twice a day), reached a plasma concentration of 150 µg/ml [15], which is well above the effective concentration observed in these studies. The mice bearing larger tumor may have had inadequate absorption or altered melabolism

411 or excretion of drug as a cause for therapeutically inadequate drug levels. The involvement of MMPs in the processes of metastasis and angiogenesis makes them appropriate targets for directed cancer therapy. Batimastat was the first MMP inhibitor to be introduced into clinical trials in cancer patients [16]. However, clinical trials of this agent were discontinued because of its low oral bioavailability [17]. Marimastat, a chemically similar agent with improved oral bioavailability, has had its use limited by the development of tendonitis and bursitis in patients. BAY 12-9566 was developed as a novel MMP inhibitor with a unique chemical structure. BAY 129566 has desirable properties for an MMP inhibitor, such as low toxicity and high oral bioavailability. Recently, all clinical trials in cancer involving BAY 12-9566 have been halted after a recommendation from an independent Data Safety Monitoring Board to close the small cell lung cancer trial with this drug due to excessive deaths in the BAY 12-9566 arm. The results from this study suggest that BAY 12-9566 may be a useful and safe ‘adjunctive to surgery’ in the treatment of breast cancer.

Acknowledgements We thank Joanne Dunn, MS, for her technical assistance, and A.M. Casazza, MD, and Lori Minasi, PhD (Bayer Corporation), for helpful discussions. This work was supported by the Catherine Peachey Fund, the Walther Medical Foundation, and the Bayer Corporation.

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