[CANCER RESEARCH 59, 1231–1235, March 15, 1999]

Correlation of Antiangiogenic and Antitumor Efficacy of N-biphenyl Sulfonyl-phenylalanine Hydroxiamic Acid (BPHA), an Orally-active, Selective MatrixMetalloproteinase Inhibitor

Ryuji Maekawa, Hideo Maki, Hiroshi Yoshida, Kanji Hojo, Hidekazu Tanaka, Tohru Wada, Naomi Uchida,Yukihiro Takeda, Hisanori Kasai, Hiroyuki Okamoto, Hiroshige Tsuzuki, Yoshikazu Kambayashi,Fumihiko Watanabe, Kenji Kawada, Ken-ichi Toda, Mitsuaki Ohtani, Kenji Sugita, and Takayuki Yoshioka1

Discovery Research Laboratories, Shionogi and Company, Ltd., 12-4 Sogisu, 5-Chome Fukushima-ku, Osaka 553-0002, Japan [R. M., H. M., H. Y., K. H., H. Ta., T. W., N. U.,Y. T., H. K., H. O., H. Ts., Y. K., F. W., K. K., M. O., K. S., T. Y.], and Department of Dermatology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan [K-i. T.]

ABSTRACT

The antiangiogenic activity and antitumor efficacy of a newly developedmatrix metalloproteinase (MMP) inhibitor were examined. N-biphenylsulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibitsMMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, anenantiomer of BPHA, was inactive against all MMPs tested. Daily oraladministration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted inpotent inhibition of tumor-induced angiogenesis, primary tumor growth,and liver metastasis. The growth inhibition activity of BPHA was 48% and45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model,respectively. BPHA also showed 42% inhibition of the liver metastasis ofC-1H human colon carcinoma cells. These results indicate that selectiveMMP inhibition is correlated with antiangiogenic and antitumor efficacyand that the selective MMP inhibitor BPHA has therapeutic potential.

INTRODUCTION

MMPs2 are a class of structurally related enzymes that function inthe degradation of extracellular matrix proteins that constitute con-nective tissue (1). MMPs are essential for tumor cells to penetrate thebasement membrane, gain access to blood vessels, exit blood vessels,and colonize at distant sites (metastasis; Refs. 2–4). Angiogenesis, aneovascularization process crucial to sustain progressive tumorgrowth by supplying oxygen and nutrients, also involves proteolyticdegradation via extracellular matrix by activated endothelial cells(5–8). The accumulated evidence has demonstrated that angiogenesisor resultant tumor vascularity could be related to metastasis andpatient survival (9–13). In many preclinical and clinical studies,increased MMP activity has been detected in a wide range of cancersincluding lung (14), prostate (15), breast (16), head and neck (17),ovarian (18), and pancreas (19) and correlated to their invasive andmetastatic potential (20–23). Therefore, MMPs may be useful targetsas a new class of inhibitory drugs.

Several novel MMP inhibitors have been identified and are pres-ently being investigated in clinical trials (24–29). A broad-type MMPinhibitor, BB-2516 (British Biotech) and AG3340 (Agouron) arepresently in Phase III trials. The selective inhibitor BAY 12–9566(Bayer) is in Phase II/III trials. Chiroscience has selective MMPinhibitors D-1927 and D-2163 in Phase I trials.

However, few studies have been focused on relationships betweenenzyme inhibitory activity of MMP inhibitors and their antitumoractivity. Recently, we have developed p.o.-active MMP inhibitors as

antitumor agents (30). We have found that BPHA strongly inhibitsMMP-2, -9, and -14, but not MMP-1, -3, or -7, and that (-)BPHA, anenantiomer of BHPA, had no inhibitory activity. Using these com-pounds, we demonstrate in this study that the MMP inhibitory activitycorrelates directly within vivo antiangiogenic and antitumor activity.

MATERIALS AND METHODS

Animals. BDF1 and C57BL/6 mice (female, 7–9 weeks of age) werepurchased from Japan SLC, Inc.(Shizuoka, Japan). Athymic BALB/c nudemice (female, 7–9 weeks of age) were purchased from CLEA Japan, Inc.(Tokyo, Japan).

Tumors. The Lewis murine lung carcinoma was obtained from the Na-tional Cancer Institute (Bethesda, MD) and are maintained by serial s.c.transplantation as tumor fragments in C57BL/6 mice. B16-BL6 murine mel-anoma, Colon 26 murine colon cancer, Ma44 human lung squamous cellcarcinoma, and C-1 human colon cancer were provided by Dr. I. J. Fidler(M. D. Anderson Cancer Center, Houston, TX), Dr. T. Tsuruo (Tokyo Uni-versity, Tokyo, Japan), Dr. T. Komiya (Habikino Hospital, Osaka, Japan), andDr. T. Kubota (Keio University, Tokyo, Japan), respectively. HT-1080 humanfibrosarcoma was purchased from American Type Culture Collection (Man-assas, VA). These cell lines were maintained byin vitro passage using Eagle’sMEM (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% FCS(Life Technologies, Inc., Rockville, MD). The C-1H human colon cancer wasestablished in our laboratory by repeat passage of C-1 liver metastasesviaintrasplenic injection. F2 murine hemangio-endothelioma was established byDr. K. Toda (31).

Chemicals. BPHA, (-)BPHA, and BB-2516 (32) were used in this study.Enzyme Assays.For MMP-1 assay, a commercially available assay kit

with natural substrate was used (Yagai, Yamagata, Japan), as described pre-viously (30). MMP-2 and -9 were purified from the culture supernatant ofHT-1080 cells (33) with enzyme activities assayed using natural substrate. AcDNA clone of MMP-14 was provided by Dr. Seiki (Tokyo University).Recombinant MMP-14 was expressed inEscherichia coli, and a 28-kDaprotein, including a catalytic core domain starting with Arg109, was purified byDEAE-Sepharose column chromatography. MMP-3 and -7 were obtained fromYagai. The enzyme activities of MMP-3, -7, and -14 were assayed in a reactionbuffer [300 mM NaCl, 10 mM CaCl2, 0.005% Brij35, 0.01% NaN3, and 50 mM

Tris-HCl (pH 7.5)] using 20mM MOAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrate. After a 90-min incubation at 25°C, fluorescence wasmeasured with excitation and emission at 320 nm and 405 nm, respectively,with a Fluoroskan Ascent (Labsystems, Helsinki, Finland). For enzyme inhi-bition assays, the enzymes were preincubated with inhibitor for 60 min.

Gelatin Zymography. Gelatin zymography was carried out as describedelsewhere (34). Briefly, the supernatant was prepared by incubating 13 106

tumor cells in a 6-cm culture dish in serum-free DMEM (Nissui Pharmaceu-tical Co.) for 24 h. The culture supernatant (10ml) was applied to nonreducedSDS-PAGE using a 7.5% gel containing 0.1% gelatin. After electrophoresis,the gel was soaked in 2.5% Triton X-100 solution at room temperature withgently shaking for 1 h. The gels were then incubated overnight in reactionbuffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM CaCl2, and 0.01%Brij-35] at 37°C and stained with Coomassie Brilliant Blue.

Tumor-induced Angiogenesisin Vivo. For tumor-induced angiogenesis,the dorsal air sac-chamber method was carried out as described previously(35). HT-1080 human fibrosarcoma cells were suspended in HBSS at a

Received 9/8/98; accepted 1/15/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Discovery Research Labora-tories, Shionogi and Company, Ltd., 12-4 Sagisu, 5-Chome, Fukushima-ku, Osaka553-0002 Japan. Phone: 81-6-6458-5861; Fax: 81-6-6458-0987; E-mail: takayuki.yoshioka@shionogi.co.jp.

2 The abbreviations used are: MMP, matrix metalloproteinase; BPHA, N-biphenylsul-fonyl-phenylalanine hydroxiamic acid.

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concentration of 53 106 cells/ml. A Millipore chamber (Millipore Co.,Bedford, MA) was filled with 0.2 ml of either a cell suspension or HBSS andimplanted s.c. into the dorsal side of mice (day 0). The compounds wereadministrated p.o. twice a day from days 0–3. At 4 days after implantation, awide rectangular incision was made in the skin on the dorsal side of anesthe-tized mice, and the skin was carefully ablated. Histological analyses andcomputer image analyses were performed, as described previously (35).

In Vivo Tumor Growth Assay. The experimental procedures have beendescribed previously (36). All experiments consisted of 5–10 mice/group. Onday 0, 23 105 B16-BL6 cells, or 23 105 F2 cells were implanted i.d. into theback of BDF1 and BALB/c nude mice, respectively. The experimental com-pounds were suspended in vehicle (saline including 0.4% Tween 80, 0.5%carboxy-methylcellulose, and 0.9% benzylalcohol) and were p.o. administereddaily from day 1. Tumor size and body weight were scored throughout eachexperiment. Growth inhibitory activity was estimated from the treated:controlratio (36).

In Vivo Liver Metastasis Model. C-1H human colon carcinoma cells(5 3 105) were injected into the spleen of BALB/c nude mice, and the spleenwas removed after tumor inoculation. The compounds were p.o. administereddaily from day 1. On day 25, the mice were sacrificed and the liver with tumornodules were excised and weighed. Allin vivostudies were performed with theapproval of the Shionogi Animal Care and Use Committee.

Statistics. The statistical significance in the present experiments was eval-uated using Dunnett’s test (37).

RESULTS

MMP Inhibitory Activity of BPHAs. The inhibitory activity ofBPHA and (-)BPHA against various human MMPs was examined (Table1). BPHA inhibited activities of MMP-2, -9, and -14 (MT1-MMP), withthe IC50 of 10–20 nM, but did not inhibit MMP-1, -3, and -7 (the IC50swere 974,.1000, and 795 nM, respectively). In contrast, (-)BPHA, anenantiomer of BHPA, had no inhibitory activity against all enzymestested. Neither BPHA nor (-)BPHA inhibited typical serine proteinases(neutrophil elastase, plasmin, trypsin, and chymotrypsin), cysteine pro-teinases (cathepsins B and L), aspartic proteinase (HIV-1 protease), ormetalloproteinase (aminopeptidase M; data not shown).

We next examined the MMP inhibitory activity of BPHAs againstmurine MMPs using gelatin zymography. As shown in Fig. 1, thegelatinase activity of MMP-2 and -9, derived from murine as well asfrom human tumor cells, was completely inhibited by BPHA at 20mM

(Fig. 1, center), but not by (-)BPHA even at 100mM (Fig. 1, right).Pharmacokinetics of BPHAs.The plasma concentration of BPHAs

after oral administration (200 mg/kg) in mice was determined by high-performance liquid chromatography. The maximum plasma concentra-tions of BPHA and (-)BPHA were 910 nM and 840 nM, respectively. Thearea under the plasma concentration curves of BPHA and (-)BPHA was0.98 mgzhr/ml and 1.07mgzhr/ml, respectively. The pharmacokineticprofiles for BPHA and (-)BPHA were, thus, similar. The plasma level ofBPHA 24 h after oral administration was;30 nM, which was higher thanthe IC50 against MMP-2, -9, and -14 (data not shown).

Correlation of Antiangiogenic Activity of BPHAs with TheirMMP Inhibitory Activity. To evaluate the antiangiogenic activity ofBPHAs, we used the dorsal air sac-chamber assay with transplantationin the dorsal side of mice of a Millipore chamber filled with human

HT-1080 fibrosarcoma, which is an angiogenesis inducer. In thecontrol experiments in which the chamber was filled with HBSS, fewnewly developed blood vessels were detected macroscopically (Fig.2A), as well as in the skin section (Fig. 3A), indicating that thenonspecific blood vessel formation induced by the dorsal air-sacprocedure was minimal. In contrast, after implantation of the chamberwith HT-1080 tumor cells, the number of blood vessels had markedlyincreased with newly formed blood vessels branching out from largeblood vessels (Figs. 2B and 3,B andE). Severe hemorrhage was alsoobserved.

In mice, p.o. administration of 200 mg/kg BPHA clearly suppressedtumor-induced angiogenesis (Figs. 2C and 3,C andF). In (-)BPHA-treated mice, however, induction of angiogenesis was not affected(Figs. 2Dand 3,D andG).

We then measured angiogenesis by analyzing vertical sections of theskin under light microscopy (Fig. 3,E-G). The total vascular area andnumber of vessels beneath themusculi cutaneus/1-mm width of skinsection were estimated using an image analyzer. The total vascular areaand number of vessels/unit width were reduced by 79% and 75% in micetreated with BPHA, respectively (Table 2;P ,0.01 compared withvehicle control). In mice treated with (-)BPHA, the MMP inhibitoryactivity-deficient enantiomer, no significant inhibition was found com-pared with the control. These results indicate that the antiangiogenicactivity of BPHAs is closely correlated with MMP inhibitory activity.

Correlation of Antitumor Efficacy of BPHAs with Their MMPInhibitory Activity. The antitumor efficacy of BPHAs was evaluatedagainst murine B16-BL6 melanoma and F2 hemangio-endotheliomaimplanted i.d. As shown in Table 3, daily p.o. administration ofBPHA, but not (-)BPHA, exerted significant (P ,0.01) growth inhi-bition in both tumor models. The growth inhibition activity of BPHAwas as potent as BB-2516 in the B16-BL6 melanoma model (Table 3).

We next examined the therapeutic efficacy of BPHA on experi-mental hepatic metastasis formed in a human colon cancer model. Theincrease in liver weight due to C-1H tumor-cell growth was inhibitedby 42% (P ,0.05) in BPHA-treated mice (Table 4). (-)BPHA, how-ever, had no inhibitory activity (Table 4). These results indicated thattumor growth inhibitory activity and the antimetastatic activity ofBPHAs were closely correlated with MMP inhibitory activity. Duringdaily treatment at the effective dose for antitumor activity (200mg/kg), BPHA did not exhibit any toxic effect on body weight loss oron hematopoietic cells (data not shown).

DISCUSSION

We have found in this study that one compound and its enanti-omer exhibit different MMP inhibitory activity. Recent elucidation

Table 1 Profile of inhibitory activity of BPHAs against human MMPs

Enzymes

IC50 (nM)

BPHA (2)BPHA

MMP-1 974 .1000MMP-2 12 .1000MMP-3 .1000 .1000MMP-7 795 .1000MMP-9 16 .1000MMP-14 17 .1000

Fig. 1. Inhibition of MMP activity by BPHAs in gelatin zymography. Gelatin zymog-raphy of culture supernatants of the Lewis murine lung carcinoma (Lane 1), B16-BL6murine melanoma (at pH 4.5;Lane 2), Ma44 human lung cancer (Lane 3), and Colon 26murine colon cancer (Lane 4). Reactions were carried out in reaction buffer only (left),reaction buffer containing BPHA at 20mM (center), or reaction buffer containing(-)BPHA at 100mM (right). The experimental protocols are described in “Materials andMethods.” Arrow, gelatinolytic band of MMP-2;arrowhead, gelatinolytic band ofMMP-9.

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of the crystallographic structures of MMP-inhibitor complexeshave demonstrated the model for enzyme-inhibitor interaction(38). In the present model, unfavorable steric repulsion between ana-substituent of hydroxamate of (-)BPHA and S1 subsite of theenzyme may explain the instability of binding between (-)BPHAand MMPs (38).

Because both tumor-derived and host stroma-derived MMPs mayplay a pivotal role in tumor growth (3), the effect of MMP inhibitoryactivity on murine MMPs is important for a therapeutic experimentalmodel. We have confirmed the cross-inhibitory effect of BPHA onmurine MMP-2 and -9 by gelatin zymography.

In this study, we demonstrated that BPHA, but not (-)BPHA, showedsignificant antiangiogenic activity, antitumor growth, and antimetastaticefficacy in various tumor models. As both compounds showed similar

pharmacokinetic profiles, the difference found in the biological activitiesof these compounds is mainly due to the inhibitory activity againstMMP-2, -9, and -14. The present study is consistent with our previousstudy showing reduced angiogenesis and tumor progression in MMP-2-deficient mice (35). These studies support the role of MMPs such asMMP-2 in angiogenesis, tumor growth, and metastasis.

BPHA selectively inhibits MMP activity, in contrast to broad-spectrum MMP inhibitors such as BB-94 and BB-2516 (26, 28).However, BPHA, which is active against MMP-2, -9, and -14, dem-onstrates similar antiangiogenic and antitumor activity as broad-spec-trum MMP inhibitors.

These findings have important implications for the therapeuticpotential of BPHA that may reduce the incidence of adverse eventssuch as pain and tenderness in joints, pain affecting shoulders and

Fig. 2. Inhibition of tumor-induced angiogenesis by BPHAs. Representative views from the inner side of the dorsal skin segment. Dorsal skin of the mice 4 days after implantationof a chamber filled with HBSS only (A) or HT-1080 cells (B-D). The implanted mice were treated p.o. daily with vehicle control (B), 200 mg/kg BPHA (C), or 200 mg/kg (-)BPHA(D). Magnitude,36.4.

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Fig. 3. Histological analysis of vertical skin sections. H&E staining of skin vertical sections 4 days after implantation of a chamber filled with HBSS only (A) or a chamber filledwith HT-1080 cells (B-G). The implanted mice were treated p.o. daily with vehicle control (B and E), 200 mg/kg BPHA (C and F), and 200 mg/kg (-)BPHA (D and G). A-D,magnification:387.5.E-G, magnification:3350.Arrowhead, blood vessels beneath themusculi cutaneus.

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hands that appeared in long-term treatment with broad-spectrumMMP inhibitors in clinical studies (39, 40).

ACKNOWLEDGMENTS

We thank Dr. H. Arita for encouragement and valuable advice, Dr. T.Kubota for providing C-1 cells, and R. Nakano for high-performance liquidchromatography analysis.

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Table 3 Correlation of in vivo antitumor efficacy with MMPI activity

Tumor CompoundaTumor volumeb

(mean6 SD) % inhibition

Vehicle control 4916 139B16-BL6 BPHA 2546 106c 48

(2)BPHA 4786 55 3BB-2516 2586 114c 47Vehicle control 5046 87

F2 BPHA 2776 52c 45(2)BPHA 4186 105 17

a 200 mg/kg, p.o.3 13 for B16, p.o.3 9 for F2.b mm3, on day 14 for B16 and on day 9 for F2.c P , 0.05, 0.01 for control by Dunnett’s test.

Table 4 Correlation of in vivo antimetastatic efficacy with MMPI activity

Tumor TreatmentaLiver weightb

(mean6 SD)Increase inliver weight % inhibition

None None 1.406 0.1 0C-1H Vehicle control 4.326 0.6 2.916 0.6C-1H BPHA 3.106 1.2 1.706 1.2c 42C-1H (2)BPHA 3.906 0.9 2.506 0.9 15a 200 mg/kg, p.o.3 24.b Gram, on day 25.c P , 0.05 for vehicle control group by Dunnett’s test.

Table 2 Antiangiogenic effect of BPHAs

Angiogenesisinducer In vivo treatment

Mean area(mm2/mm) Mean number/mm

HBSS None 368.36 354.5 2.66 3.2HT-1080 Vehicle control 6375.26 1911.6a 25.66 7.4a

HT-1080 BPHA 1350.26 1088.9 6.56 4.9HT-1080 (2)BPHA 5259.06 2185.2a 20.86 7.9a

a P , 0.01 for HBSS group by Dunnett’s test.

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1999;59:1231-1235. Cancer Res   Ryuji Maekawa, Hideo Maki, Hiroshi Yoshida, et al.   an Orally-active, Selective Matrix Metalloproteinase InhibitorN-biphenyl Sulfonyl-phenylalanine Hydroxiamic Acid (BPHA), Correlation of Antiangiogenic and Antitumor Efficacy of

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