Inhibition of Angiogenesis and Metastasis in Two Murine Models by the Matrix Metalloproteinase Inhibitor, BMS-275291

INTRODUCTION

MMPs 2 are a family of zinc-dependent endopeptidases that are responsible for the degradation of ECM proteins during normal tissue remodeling processes such as embryonic growth, wound healing, and angiogenesis (1, 2, 3) . Members of this family include collagenases, stromelysins, gelatinases, MT-MMPs and Matrilysin. MMPs are secreted by a wide variety of cell types, and their enzymatic activity is directed at the ECM, including basement membranes. During normal physiological processes, endogenous inhibitors regulate the proteolytic activities of these proteinases. If, however, this balance is disrupted and MMP levels increase, their activities directly contribute to the pathology of cancer and other disease states. Elevated levels of MMPs have been shown for many histological types of cancer, including breast, lung, prostate, colorectal, ovarian, and gastric (4 , 5) , and elevated levels of certain MMPs correlate with more rapid cancer progression in many cancer types (4) .

The growth of a solid tumor and its ability to metastasize are dependent on angiogenesis (6 , 7) . The formation of new capillaries requires the migration of endothelial cells and extensive tissue remodeling. Proteolytic enzymes, such as MMPs, must degrade the various compartments of the ECM to facilitate the remodeling of the ECM required for tumor growth, tumor angiogenesis, and metastasis. Consequently, controlling MMP activity(s) using synthetic small-molecule inhibitors (MMPIs) is attractive as a potential means to intervene in several of the rate-limiting steps in this pathway. In this study, the pharmacokinetic profile and the antimetastatic and antiangiogenic activities of BMS-275291, a novel p.o. active broad-spectrum MMPI, are described.

MATERIALS AND METHODS

Compound Preparation.

Clinical grade BMS-275291 (8) was manufactured at Bristol-Myers Squibb Co. (New Brunswick, NJ) and stored in sealed glass opaque vials at ambient temperature. For in vitro assays, BMS-275291 was suspended in DMSO containing 1.4 mm 2-mercaptoethanol and stored at −30°C. For in vivo studies, BMS-275291 was prepared daily in sterile water using sonication (15 min) at ambient temperature. BMS-275291 was administered to mice p.o. on various schedules using clean, autoclaved, stainless steel gavage needles.

Enzymatic Assays.

Human recombinant MMP-1 (collagenase 1), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (Matrilysin), and MMP-14 (MT1-MMP) were expressed in Escherichia coli as inclusion bodies and purified as described (9) . Native human MMP-9 (gelatinase B) was purchased from Boehringer Mannheim (Indianapolis, IN). MMP activities were determined in the presence of 2-mercaptoethanol (140 μm) in proximity-based substrate peptide assays (10 , 11) . MMP-2, MMP-7, MMP-9, or MT1-MMP activity was determined in the linear range using the substrate peptide, Mca-P-L-G-L-Dnp-A-R-NH₂ (Peptide International, Louisville, KY); MMP-1 activity was determined using Mca-P-L-G-L-Dpa-A-R-NH₂ (Bachem, King of Prussia, PA); and MMP-3 activity was determined using Mca-R-P-K-P-V-E-Nva-W-R-K(Dnp)-NH₂ (Peptide International). Substrate hydrolysis was measured using a fluorometer (Fluoroscan Ascent; Labsystems, Franklin, MA). The optimal MMP concentration for each proximity-based substrate peptide assay was determined empirically under reducing conditions.

Cell-based assays were used to test the effects of BMS-275291 on the shedding of TNF-α, TNF-RII, l-selectin, and IL-1-RII from the cell surface. Human peripheral blood mononuclear cells were obtained from normal blood donors. Cells (5 × 10⁵ for the TNFα and TNF-RII assays; 2 × 10⁶ for the l-selectin assays; and 1 × 10⁶ for the IL-1-RII assays) were concurrently treated with different concentrations of BMS-275291 and stimulated with mitogen [lipopolysaccharide (100 ng/ml for 22 h; E. coli serotype B8:0127; Sigma Chemical Co., St. Louis, MO) for TNFα and TNF RII assays; phorbol-13-myristate-12-acetate (250 nm for 20 min., Sigma Chemical Co.) for l-selectin assays; and IL-13 (10ng/ml for 18 h) for IL-1-RII assays]. After treatment (37°C; 5% CO₂), culture supernatants were collected and transferred to individual wells of a 96-well plate. Levels of the cell-associated molecules or receptors released in the medium were subsequently measured by ELISA (R&D Systems; Minneapolis, MN) following the manufacturer’s instructions.

The effects of BMS-275291 treatment (24 h; 37°C; 5% CO₂) on the release of the IL-6-R from the cell surface was determined as described above, except that HL-60 cells (5 × 10⁵) stimulated with phorbol-13-myristate-12-acetate (8 nm) were used. ELISA (R&D Systems) measured levels of soluble IL-6-R contained in culture supernatant.

Mouse Strains.

Female athymic (BALB/c-nu/nu) mice obtained from Harlan (Indianapolis, IN) were used in the Matrigel plug angiogenesis model. Immunocompetent C57BL/6 mice (Harlan) were used in the B16BL6 experimental lung metastasis model and pharmacokinetic studies. Mice received food and water ad libitum and were maintained in a controlled environment according to American Association of Laboratory Animal Care regulations.

Murine B16BL6 Experimental Lung Metastasis Model.

The murine B16BL6 melanoma cells used in these studies were a metastatic variant of the B16 melanoma (12) obtained from Dr. William E. Fogler (Entremed, Rockville, MD). For each assay, B16BL6 cells were harvested from low passage (≤3) cultures grown in vitro in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with heat-inactivated 10% fetal bovine serum (Life Technologies, Inc.) at 37°C under a humidified 95%:5% (v/v) mixture of air and CO₂. Cells were washed twice using sterile HBSS and suspended in HBSS to a concentration of 3 × 10⁵ viable cells/ml. Cells routinely were >95% viable at the time of inoculation. Cells were administered i.v. (6 × 10⁴ cells/mouse) into the tail vein (day 0) of syngeneic C57BL/6 mice. Compounds or vehicle were administered p.o. (9–10 mice/group) 2 h before and 2, 24, 48, and 72 h after cell injection (time 0). The health status of the mice was monitored daily. Mice were killed 10 days after tumor cell injection, and their lungs were removed and fixed in neutral-buffered formalin (10%). The number of B16BL6 colonies present on the surface of each set of lungs was determined by visual inspection using a stereoscopic dissecting microscope. Data are presented as the percentage of inhibition, defined as: . A result of ≥35% inhibition was considered active.

Murine Matrigel Plug Angiogenesis Model.

Ice-cold Matrigel (250 μl; Becton Dickinson, Bedford, MA) containing VEGF (75 ng/ml; Pepro Tech, Inc., Rocky Hill, NJ) and bFGF (300 ng/ml; Pepro Tech, Inc.) was implanted s.c. in athymic mice on day 0. The Matrigel polymerized immediately after implantation into mice, forming a gel plug. Beginning on day 0, BMS-275291 was administered p.o. once daily for 7 days at the indicated dose levels. One day after the last dose, Matrigel plugs were harvested, fixed overnight in 10% buffered formalin, embedded in paraffin, and sectioned (5-μm sections). To confirm that the migrating cells were endothelial cells, individual sections from control groups were stained with antimurine CD34 (BD PharMingen, San Diego, CA) or anti-von Willebrand factor (Dako, Carpinteria, CA) antibodies using standard avidin/biotin-based immunohistochemical techniques. All sample sections were stained with H&E, and the number of endothelial cells present in each plug section was measured using a video imaging system (Image Pro-Plus; Empire Imaging, Princeton, NJ). Fifty fields (at ×20) per plug section were randomly counted. Data are presented as the percentage of inhibition of cell migration, defined as: . A result of ≥35% inhibition was considered active.

Pharmacokinetic Studies.

BMS-275291 contains a free sulfhydryl group that in vivo has the potential to form disulfide linkages with other sulfhydryl-containing molecules. Consequently, blood samples were collected in Vacutainer tubes that contained MA:K₃EDTA. MA was used because it stabilizes the free sulfhydryl group of parent BMS-275291 to form BMS-275291-MA derivatives. These BMS-275291-MA derivatives in mouse plasma are subsequently converted (by hydrolysis) to AA derivatives, BMS-275291-AA, which can then measured directly to provide parent (active) drug concentrations.

A single dose of BMS-275291 in water was administered p.o. to 54 C57BL/6 mice of each sex at doses of 30 and 90 mg/kg. Blood samples were taken from three mice/sex/dose level at 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 h after dosing. At each sampling time point, individual blood samples were collected in Vacutainer tubes containing MA:K₃EDTA. Samples were placed on ice for ∼30 min before incubation in a water bath at 35°C for 30 min to convert BMS-275291-MA to BMS-275291-AA. Samples were subsequently extracted with 0.1 N HCl and methyl tert-butyl ether and analyzed for parent BMS-275291 using an Liquid chromatography/ tandem mass spectrometry method validated by Bristol-Myers Squibb. The standard curves were linear over the concentration range of 1–100 ng/ml and had R² values of ≥0.992. The average deviation from nominal was no more than ±6%, and the between-run precision and the within-run precision were no more than 8% relative SD. Results indicate that the assay was accurate, precise, and reproducible.

Plasma concentrations versus time profiles were analyzed using noncompartmental analysis by the MENU/PKMENU software system. The data for parent BMS-275291 was considered adequate to give approximations of the apparent peak plasma concentration (C_max) and the time to peak concentration (T_max), which were recorded directly from the experimental observations. The 24-h area under the plasma concentration-time profile, (AUC, 24 h), was calculated by a combination of the trapezoidal and log trapezoidal rules (13) .

RESULTS

In Vitro Activities of BMS-275291.

BMS-275291, a small synthetic MMPI which contains a free mercaptoacyl group as the zinc-binding group (Fig. 1) ⇓ , was rationally designed to inhibit a broad range of MMP activities while sparing those of the sheddases (14 , 15) . The IC₅₀ values (the concentration at which the activity of an enzyme is inhibited by 50%) were determined in vitro under reducing conditions using purified human MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14 and quenched fluorescent peptide substrates. These assay conditions were used to maximize the potential of the mercaptoacyl zinc-binding group to inhibit MMP activities. Consequently, the IC₅₀ values for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14 are 9, 39, 157, 23, 27, and 40 nm, respectively. Additionally, BMS-275291 does not inhibit (IC₅₀ values >30,000 nm) the in vitro activity of serine or cysteine proteases, which include chymotrypsin, elastase, plasmin, cathepsin B, trypsin, thrombin, and urokinase plasminogen activator (data not shown). In cell-based in vitro assays, BMS-275291 does not inhibit (IC₅₀ values >100,000 nm) the metalloproteases responsible for the release of TNF-α, TNF-RII, l-selectin, IL-1-RII and IL-6-R. These results therefore demonstrate that BMS 275291 is a selective, potent nanomolar inhibitor of MMP in vitro activities. Importantly, it does not inhibit closely related sheddase activities responsible for the release of cell-associated molecules.

Effects of BMS-275291 in the Murine B16BL6 Experimental Lung Metastases Model.

The in vivo efficacy of BMS-275291 was examined in a murine B16BL6 experimental lung metastases model where B16BL6 cells injected i.v. form tumor foci in the lungs. Mice were treated p.o. with BMS-275291 (10–90 mg/kg) 2 h before and 2, 24, 48, and 72 h after tumor injection. Mice were killed 10 days after tumor cell injection, and the number of clearly visible B16BL6 metastases was counted. The average number of B16BL6 metastases in the untreated mouse controls was 286 ± 45 (Study A; Table 1 ⇓ ) and 186 ± 25 (Study B; Table 1 ⇓ ). As shown in Table 1 ⇓ , a dose-dependent inhibition of tumor metastases was observed, as indicated by the reduced number of lung metastases observed in MMPI-treated mice relative to the untreated controls. In particular, treatment with BMS-275291 at 60 or 90 mg/kg resulted in ∼40% inhibition of lung metastases (Table 1) ⇓ . At these dose levels, there was no effect on body weight or any other clinical signs of toxicity. These results demonstrate that p.o.-administered BMS-275291 has antimetastatic activity that is dose dependent.

Effects of BMS-275291 in a Tumor-Independent Murine Matrigel Plug Angiogenesis Model.

BMS 275291 was examined for its ability to inhibit angiogenesis in the tumor-independent Matrigel plug angiogenesis model. Typically, in control mice, an angiogenic response is shown by the migration of a large number of endothelial cells in representative Matrigel plug sections (Table 2) ⇓ as well as by the presence of incipient blood vessels (Fig. 2a) ⇓ . As shown in Table 2 ⇓ , treatment with BMS-275291 resulted in a significant (P < 0.05) reduction in the number of endothelial cells that migrated into Matrigel plugs and a disorganized endothelial cell architecture (Fig. 2b) ⇓ relative to control animals. Treatments with BMS-275291 at doses of 30–90 mg/kg decreased endothelial cell migration by 40 and 58%, respectively (Table 2) ⇓ . These data show that once-daily BMS-275291 oral treatment for 7 days in this model results in a dose-dependent antiangiogenic effect.

Pharmacokinetics of BMS-275291.

BMS-275291 contains a free sulfhydryl group that is capable of forming disulfide linkages with itself or other sulfhydryl-containing compounds in vivo. Because parent BMS-275291 (BMS-275291 containing a free sulfhydryl group) is believed to be the physiologically active form, an analytical method was developed, using MA, to measure parent BMS-275291. The composite plasma concentration versus time profiles for parent BMS 275291 after a single oral dose of 30 or 90 mg/kg are shown in Fig. 3 ⇓ , and the pharmacokinetic parameter values are summarized in Table 3 ⇓ . On the basis of composite profiles, T_max occurred at 0.5 h after dosing. For doses increasing in the ratio of 1:3, the mean C_max values increased in ratios of 1:30 (male) and 1:27 (female), and mean AUC, 24-h values increased in ratios of 1:40 (male) and 1:31 (female). The C_max and AUC, 24-h values were similar in male and female mice, indicating no gender differences in the pharmacokinetics of BMS-275291 in mice. Overall, the results demonstrated that mice were exposed to parent BMS-275291 after a single oral BMS-275291 dose and the exposure increased in a ratio greater than the dose increment. In addition, at a dose of 90 mg/kg (an active dose in the efficacy studies), the plasma concentrations of parent BMS-275291 exceeded the in vitro IC₅₀ values for MMP-1 (4.5 ng/ml), MMP-2 (20.5 ng/ml), MMP-7 (11.5 ng/ml), MMP-9 (12.5 ng/ml), and MMP-14 (20.0 ng/ml) for at least 4 h after oral administration (Fig. 3) ⇓ . These results demonstrate that mice were exposed to unchanged, biologically active BMS-275291 after a single oral administration, and that the parent BMS-275291 plasma levels exceeded the IC₅₀ values for several MMP enzymes.

DISCUSSION

The MMPs are a family of enzymes involved in degrading and remodeling the ECM. MMP activity is tightly regulated in normal physiological states, but loss of this regulation, in diseases such as cancer, results in destruction of the ECM and subsequent tumor progression. Destruction of the ECM promotes tumor metastasis and angiogenesis, both through the direct effect of eliminating a barrier to tumor cell invasion and indirectly through the release of growth factors sequestered in the ECM that induce migration (16 , 17) . A variety of MMPIs have been shown to have activity in preclinical models of cancer (18, 19, 20, 21, 22, 23, 24, 25, 26) . Typically, in preclinical models the compounds are administered early in disease progression, and inhibition of the number and/or size of experimental metastases are observed. Several MMPIs of varying selectivity have been evaluated clinically (19 , 27, 28, 29) . To date, MMPIs have shown little clinical benefit when used as monotherapy in patients with advanced disease, and treatment with MMPIs has typically resulted in musculoskeletal side effects, including joint pain and stiffness, that limit both the dose level administered and the duration of therapy (2 , 18 , 19 , 23 , 27, 28, 29) . In the studies described here we demonstrate the preclinical antimetastatic and antiangiogenic activity of BMS-275291, a novel, broad-spectrum MMPI currently in Phase II clinical trials.

Murine B16BL6 melanoma cells, which are aggressive variants of the B16 cell line (12) , are reported to express gelatinase A (MMP-2) and MT1-MMP (MMP-14). When these cells are injected i.v. into mice, they extravasate from the bloodstream and subsequently colonize the lung (experimental metastasis). Direct injection of these cells into the circulation bypasses many of the earlier steps of the metastatic process and allows for the determination of their ability to traverse capillaries and grow in a secondary site. In this experimental metastasis model, BMS-275291 was shown to markedly inhibit the number of lung-surface metastases that developed after injection of these cells (Table 1) ⇓ . These findings are consistent with the growing body of evidence that implicates MMP activities as significant contributors to both tumor cell and endothelial cell migration. In these studies, the maximum dose level evaluated was 90 mg/kg. At this dose level there was no evidence of toxicity, suggesting that it would be possible to treat the mice with increasing dose levels. However, additional escalation of the dose was limited by the solubility of BMS-275291. Therefore, it is not clear whether increasing the dose would result in increased efficacy, or whether the 40% inhibition seen represents the maximum efficacy that can be achieved with BMS-275291 in this aggressive metastatic model.

Endothelial cells are reported to express MMPs and tissue inhibitors of metalloproteinases, which together play important roles in tissue remodeling, particularly ECM degradation. During physiological angiogenesis these inhibitors tightly regulate MMP activities. In addition, angiogenic mitogens such as VEGF and bFGF are known to modulate MMP and tissue inhibitors of metalloproteinase expression (30, 31, 32, 33, 34) . Treatment with BMS-275291 resulted in the dose-dependent inhibition of endothelial cell migration in the Matrigel plug model (Fig. 2 ⇓ , Table 2 ⇓ ). As shown in Fig. 2 ⇓ , treatment with BMS-275291 also resulted in a reduction in the number of functional blood vessels seen in the Matrigel plugs. In these studies, the once-daily BMS-275291 treatments began on the day of Matrigel implantation. In mice, after a single administration, the plasma levels of parent BMS-275291 exceeded the in vitro IC₅₀ values for MMP-1, -2, -7, -9, and -14 at least 4 h after administration at the active dose of 90 mg/kg (Table 3 ⇓ , Fig. 3 ⇓ ). The inhibition of endothelial cell migration achieved in mice at these drug exposures suggests that although the plasma levels achieved were sufficient to inhibit migration, they were not sufficient to effect complete suppression of endothelial cell migration. It is not clear whether increasing the dose level or the frequency would increase the time that the plasma concentration was above the in vitro IC₅₀ and would increase further the antiangiogenic activity observed. The dose-related inhibition of endothelial cell migration observed between 1 and 90 mg/kg suggests that greater antiangiogenic activity may be observed at higher doses. However, the limited solubility of BMS-275291 prevented additional dose escalation.

In both the B16BL6 experimental lung metastases and the Matrigel plug models, ∼50% inhibition of metastatic foci or endothelial cell migration (Tables 1 ⇓ and 2 ⇓ ) was achieved at the maximum dose level that could be administered to mice. These results may be attributable to the fact that effective drug levels in the plasma were achieved only for a few hours each day. Interestingly, the plasma exposures of BMS-275291 in patients are substantially higher than those achieved in rodent models (35) . It is also likely that other proteases, including serine, cysteine, aspartyl, and metalloproteases which are not inhibited by BMS-275291, contribute to tumor metastasis and angiogenesis (7 , 16 , 33) . These other proteolytic activities combined with those of the MMPs can directly contribute to matrix degradation and also have the potential to release sequestered growth factors present in the ECM or uncover and modify cryptic sites that modulate cellular responses (7 , 16 , 17) . Examples of these factors include the release of VEGF, bFGF, epidermal growth factor, and transforming growth factor-β from degraded matrix (5 , 7 , 16 , 17 , 36) and the exposure of cryptic sites in laminin-5 and plasminogen by proteolysis (37, 38, 39) . Additionally, MMPs can proteolytically modify nonmatrix substrate components critically involved in these processes. These include, MT1-MMP involvement in the activation of proMMP-2 (40) , posttranslational modification of fibroblast growth factor receptor 1 by MMP-2 (36) , or IL-1β (2) and heparin binding-epidermal growth factor by MMP-3 (41) . Additional clarification of the relationship of these protease activities to tumor angiogenesis and metastasis may be achieved by studies that evaluate combination therapy using different classes of proteolytic inhibitors.

Synthetic MMPIs have been reported to inhibit tumor progression and angiogenesis in a variety of in vivo models (19, 20, 21, 22, 23, 24, 25, 26) . These compounds inhibit the in vitro activities of many MMPs, and most are synthetic analogues that use hydroxamic acid as the zinc-binding group (4 , 15 , 27 , 28) . Several of these hydroxamate-based inhibitors have demonstrated a dose-limiting arthralgia/myalgia in clinical trials (27, 28, 29) , and it has been suggested that these dose-limiting side effects may result from the inhibition of MMP-1 (18) . This toxicity may also be mediated by the inhibition of a class of closely related metalloproteases referred to as the sheddases (8) , which are known to regulate the shedding of cell-surface molecules that mediate inflammatory processes such as TNF-α and TNF-RII. Clinical toxicity may also result from inhibiting the proteolytic processing of other cell-surface molecules, such as l-selectin, IL-1-RII, and IL-6R (8) . It is known that certain MMPIs affect the activities of these enzymes (42, 43, 44) . BMS-275291 was designed to inhibit a subset of MMP activities that include gelatinases, stromelysins, collagenases, and MT- MMPs and not inhibit those of the sheddases. The results presented here demonstrate that BMS-275291 is a potent broad-spectrum inhibitor of MMP activities (including MMP-1, -2, -3, -7, -9, and -14) that does not affect the activities of sheddases responsible for the in vitro release of cell-associated molecules (TNF-α and l-selectin) and receptors (TNF RII, IL-1RII, and IL-6R). The importance of sparing the activity of the sheddases was evaluated in a 3-month marmoset toxicology study. In this study, daily p.o. treatment with BMS-275291 did not result in histopathological changes in joint or tendon tissues (8) . In contrast, these adverse changes were observed in marmosets treated with MMPIs known to inhibit sheddases. Together these data support the hypothesis that inhibiting sheddase activities may contribute to the arthralgia/myalgia observed in patients treated with MMPIs that inhibit sheddases.

The results described here demonstrate that oral BMS-275291 treatment results in the inhibition of angiogenesis and tumor metastasis using two murine models, a B16BL6 experimental metastasis model and a tumor-independent Matrigel plug model. These data support the potential of this MMPI to inhibit MMP activities critical in angiogenic processes that underlie tumor cell growth and metastasis. The clinical relevance of MMP and metalloprotease (sheddase) inhibition to efficacy and toxicity should be clarified from the ongoing clinical trials of BMS-275291.