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Clinical Cancer Research Vol. 11, 3925-3929, May 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Carbamoylphosphonate Matrix Metalloproteinase Inhibitors 3: In vivo Evaluation of Cyclopentylcarbamoylphosphonic Acid in Experimental Metastasis and Angiogenesis

Reuven Reich1,3, Yiffat Katz2, Rivka Hadar1 and Eli Breuer2,3

Authors' Affiliations: Departments of 1 Pharmacology and 2 Medicinal Chemistry, and 3 David R. Bloom Center of Pharmacy, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

Requests for reprints: Reuven Reich, Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, P.O. Box 12065, 91120 Jerusalem, Israel. Phone: 972-2-6757505; Fax: 972-2-6758741; E-mail: reich{at}cc.huji.ac.il.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The spread of malignant tumor cells from a primary neoplasm to distant organs where they multiply and form new foci is the major cause of death from cancer. Despite the different modalities of cancer treatment, no effective curative therapy of metastatic lesions is available. To possess metastatic potential, a cell has to be able to invade the surrounding tissue, spread via lymphatics and/or the bloodstream, extravasate, and multiply at secondary sites. There is increasing evidence for a positive correlation between matrix metalloproteinase-2 (MMP-2) activity and tumor cell invasion. Agents blocking MMP-2 have been shown to prevent tumor cell invasion in vitro and in vivo. Inhibition of MMPs has, therefore, become the focus of considerable interest in connection with a variety of potential therapeutic applications. We have discovered a nontoxic MMP-2–selective inhibitor effective at nanomolar range on recombinant MMP. This compound, cyclopentylcarbamoylphosphonic acid, significantly inhibited cellular invasion and capillary formation in vitro. Further, i.p. or oral administration of the compound significantly reduced lung metastasis formation and s.c. tumor growth in a murine melanoma model. The effect of this novel compound on lung colonization, capillary formation, and s.c. tumor growth indicates that the compound might also be effective in treatment of primary tumor growth in reduction, or at least in prevention, of further tumor growth, thereby reducing the tumor burden of the patient by a nontoxic approach.

Key Words: MMP-2 • metastasis • MMPI • cancer • angiogenesis • melanoma • phosphonate

The malignant behavior of cancer cell is mainly based on the unrestrained invasiveness of the cells or of certain subpopulation within a given tumor, and less on extensive proliferation. The current treatments (i.e., radiotherapy, phototherapy, and chemotherapy) nonselectively attack mainly proliferating cells, whereas invasiveness remains unaffected. In malignant tumors, such antiproliferative therapies are the only existing strategies today despite their inevitable side effects on normal proliferative cells, such as the digestive tract and the immune system. The inhibition of cellular invasion is an important therapeutic target that has attracted considerable attention within the scientific community and particularly in the pharmaceutical industry (1–3).

Matrix metalloproteinases (MMP) comprise of a family of zinc-dependent enzymes that share a common functional domain structure. MMPs are characterized by their ability to degrade extracellular matrix proteins in various physiologic and pathologic conditions where rearrangement of extracellular matrix occurs (4–6). They have distinct but often overlapping substrate specificities. The basic structure of MMPs consists of the following homologous domains: a signal peptide, a prodomain that confers latency by occupying the active-site zinc and making the catalytic enzyme inaccessible to substrates, a zinc-containing catalytic site, a hemopexin domain contributing to the specificity of the enzymes, and a hinge region that links the catalytic and the hemopexin domains. Two sequence motifs are highly conserved in MMPs: (a) the consensus motif VAAHEXGHXXGXXH, found in the active site of all MMPs, which contains three histidines that coordinate with the zinc ion in the active site (4) and (b) the PRCGXPD motif located in the prodomain of MMPs. Coordination of the cysteine of this locus with the zinc ion of the active center confers latency to the proenzyme form (7). As all potentially damaging enzymes, the activity of MMPs is also tightly regulated. Extracellular activation is mediated by proteolytic activity of certain serine proteases or by some activated MMPs that selectively activate pro-MMPs as well (4).

Expression of MMPs in tumor tissues were considerably higher in both human and in experimental animal tumors compared with normal tissue. In numerous studies, high MMP levels were correlated with advanced disease stage and poor prognosis (8–14). MMP-2 and MMP-9 are expressed in most tumors; however, the pattern of MMP expression differs in the various cancer types.

It is proposed that an activated water molecule that coordinates with the zinc ion of the catalytic site attacks the carbonyl group of the scissile peptide bond of the substrate and initiates its cleavage. Several zinc-binding groups, including sulfhydryl, phosphorus acid derivatives, carboxylates, and hydroxamates have been tried as MMP inhibitors (MMPI; refs. 15, 16). These chemical groups were usually incorporated into peptidomimetic or nonpeptidomimetic type of compounds. Although all compounds developed showed substantial inhibitory effect on certain MMPs in vitro, none of these compounds passed the clinical trials thus far. First- and second-generation compounds were abandoned because of poor bioavailability or severe musculoskeletal complication. Lack of defined end-point definition for efficacy further complicated the use of these compounds clinically (17, 18). These results indicate the need for a novel approach for designing new types of MMPIs. Our group is involved in a study directed toward this goal. We found that the carbamoylphosphonic function is capable of complexing zinc ions with some selectivity (19) and we recently reported the first examples of the novel class of carbamoylphosphonates synthesized and studied as potential MMPIs (20). A group of alkylcarbamoylphosphonate and cycloalkylcarbamoylphosphonate compounds are described in our recent publication (20). We found that several of these compounds inhibit MMP-2 with a high degree of selectivity, relative to four other MMP subtypes tested. Among the compounds tested, cyclopentylcarbamoylphosphonic acid (CPCPA, cyclopentyl-NHCOPO3H2) showed especially impressive in vitro and in vivo activity in spite of its molecular structure of unprecedented simplicity. The present study describes the biological profile of this compound.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
In vitro assays
Matrix metalloproteinase-2 inhibitor. CPCPA was synthesized as previously described (20). It is a stable, white, water-soluble, crystalline solid with a melting point of 163°C to 164°C, pKa1 < 2, pKa2 = 5.35 (19).

Basement membrane invasiveness. Boyden chamber chemoinvasion assays were done as previously described (21). Matrigel (25 µg) was dried on a polycarbonate filter (PVP-free, Nucleopore, Pleasanton, CA). Fibroblast-conditioned medium (obtained from confluent NIH-3T3 cells cultured in serum-free DMEM) was used as the chemoattractant. HT-1080 human fibrosarcoma cells were harvested by brief exposure to 1 mmol/L EDTA, washed with DMEM containing 0.1% bovine serum albumin, and added to the Boyden chamber (200,000 cells). The chambers were incubated in a humidified incubator at 37°C in 5% CO2/95% air atmosphere for 6 hours. The cells that have traversed the Matrigel layer and attached to the lower surface of the filter were stained with Diff Quick (American Scientific Products, Aguada, Puerto Rico) and counted.

Chemotaxis. Chemotaxis evaluation was done in a similar way to basement membrane invasion, with the exception that the filters are coated with 5 µg collagen IV instead of Matrigel. This amount of collagen does not form a barrier to the migrating cells but rather an attachment substratum.

Analysis of matrix metalloproteinase activity. Recombinant enzymes, human MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MT1-MMP (R&D Systems, Minneapolis, MN), were incubated at four different concentrations with the relevant colorimetric or fluorescent peptide substrates (R&D Systems) for 3 hours as indicated by the manufacturer. The examined compounds were added at four to six different concentrations and the inhibitory potencies expressed in a colorimetric change were measured by an ELISA or fluorescent reader.

Endothelial capillary tube formation. Bovine primary endothelial cells (50,000) were plated on top of a thin layer of Matrigel in the presence of an angiogenic factor (basic fibroblast growth factor/vascular endothelial growth factor) and incubated for 4 to 6 hours. In this time period, the endothelial cells formed tube-like structures, which resembled the tube organization of endothelial cells in vivo. The different concentrations of the studied compound were added to the culture and the extent of tube formation was measured by an image analysis system.

In vivo assays
Murine melanoma model. Experimental metastasis was studied in the murine melanoma model. In this model, B16F10 tumor cells (50,000) were injected into the tail vein of C57Bl 6-week-old female mice. After 21 days, the metastases formed on the lungs of the mice were counted after appropriate fixation. Three groups of eight mice were used in this study. Two groups of eight mice were treated with daily (except weekends) administration of 50 mg/kg of CPCPA. One group received the compound i.p. and the other group orally in PBS. Mice were monitored for toxic symptoms.

MMP activity was determined in the excised tumors from three treated and three untreated mice. The results are incorporated in Fig. 3.



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  		Fig. 3. Dose-dependent reduction of metastasis formation by CPCPA. Mice were injected with B16F10 murine melanoma tumor cells and concomitantly treated with various i.p. doses of the compound. Columns, mean; encircled numbers, number of mice used in each group (*, P < 0.05, **, P < 0.01). Inset, MMP profile of the injected tumor cells and MMP content of tumor tissues in the lung of the mice treated with 50 mg/kg dose of CPCPA.

 
Subcutaneous tumor growth in mice. B16F10 tumor cells (200,000) were injected s.c. The mice were treated for 16 days by daily i.p. injections of 50 mg/kg of the tested compound. Tumor growth was followed daily and the tumor size was measured with a caliper and approximate tumor volume calculated. The experiment was terminated when animal stress was observed by animal care veterinarian.

MMP activity was determined in the excised tumors from three treated and three untreated mice. The results are shown in Fig. 6.



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  		Fig. 6. Effect of CPCPA on s.c. tumor formation. B16F10 murine melanoma cells were injected s.c. and the mice were treated i.p. daily with 50 mg/kg dose of CPCPA. A, untreated mice; B, CPCPA-treated mice. Tumor volumes were measured with a caliper. Inset, MMP profiles of tumor samples from treated and untreated mice.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Recently, we reported the results of screening of a series of 10 open chain N-alkylcarbamoylphosphonic acids (RNHCOPO3H2, ranging from R = C1 to C6 groups), eight N-cycloalkylcarbamoylphosphonic acids (R ranging from cyclopropyl to cyclooctyl rings), and four N, N-dialkylcarbamoylphosphonic acids as potential MMPIs (20). The results showed that among the 22 compounds examined, CPCPA is the most active inhibitor of MMP-2. This is apparent from comparing its IC50 values obtained from testing it against five different human recombinant MMPs, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MT1-MMP (Table 1). It is also evident that this compound is a highly selective inhibitor of MMP-2.


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  		Table 1. IC50 values (in µmol/L) of CPCPA against various MMPs

 
Breaching of the basement membrane by tumor cells is one of the turning points that put tumor cell dissemination beyond conventional treatment. This breaching is dependent on the presence and activity of certain MMPs, mainly MMP-2 and MMP-9. Therefore, we used the traversal of a reconstituted basement membrane by tumor cells as a measure of biological activity of the compound (21). As shown in Fig. 1, the compound inhibited the invasion of tumor cells across Matrigel-coated filters in a dose-dependent manner. The cell-associated inhibitory concentrations were higher than those presented for isolated recombinant enzymes. This discrepancy may result from the cellular environment of the invasion assay, namely ongoing synthesis of the relevant MMP and nonspecific binding or neutralization of the inhibitor. Exposure of the tumor cells to concentrations up to 200 µmol/L of CPCPA had no effect on cellular proliferation as monitored for 72 hours.



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  		Fig. 1. Effect of CPCPA on the invasion of basement membrane. HT-1080 tumor cells were challenged to traverse Matrigel-coated filters in the presence of various concentrations of the CPCPA (*, P < 0.01).

 
Further, an in vivo evaluation of the compound was initiated in parallel with a toxicity study. Previous studies using hydroxamate-based inhibitors used 50 mg/kg as the optimal dose. Therefore, we evaluated the acute toxicity of our compound at doses of 50, 250, and 500 mg/kg. C57Bl mice were injected i.p. with CPCPA in these doses for 2 weeks daily. No toxicity was observed at the 50 mg/kg group, whereas doses of 250 and 500 mg/kg showed dose-dependent toxicity after 2 or 3 days of treatment, respectively (Fig. 2). Administration of the compound at 50 mg/kg did not affect behavioral or nutritional status of the treated mice.



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  		Fig. 2. Acute toxicity of CPCPA. CPCPA was injected daily i.p. for 2 weeks. The mice were observed daily for stress signs.

 
The ability of CPCPA in reducing tumor dissemination was tested in female mice. B16F10, murine melanoma cells (50,000), were injected into the tail vein of the tested mice. Simultaneously, mice were also treated with 50 mg/kg CPCPA. The compound was administered i.p. or orally daily, except weekends, for 3 weeks. Upon sacrifice, the lungs were examined for metastatic loci. The results, presented in Table 2, show a significant reduction in metastasis formation in both i.p. and orally treated mice. The compound was also tested at lower i.p. doses and its effect on reduction of metastatic loci was found to be dose dependent (Fig. 3). Examination of the tumoral tissue in the lungs showed also a reduction of MMP content in the treated mice.


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  		Table 2. Metastases formation in CPCPA-treated mice

 
Our data indicate that the in vivo inhibition of metastasis formation elicited by CPCPA was nearly identical whether the drug was administered i.p. or orally. As the drug can be expected to be fully ionized at physiologic pH, it could hardly be predicted to be orally bioavailable to a large extent. Therefore, the possibility was raised that by administering orally, the compound might induce damage to the cellular structures of the gut and thereby facilitate absorption. To check this possibility, sections of the gut from orally or i.p. treated mice were subjected to histologic examinations. Intestine segments of the mice were examined by an expert pathologist and parameters, such as villi height, mucin content, and presence of inflammatory cells in the lamina and in the epithelium, were monitored. This examination did not uncover any tissue damage (Fig. 4).



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  		Fig. 4. Histologic sections of intestine from mice treated orally or i.p. Five-micrometer serial sections were prepared and stained with H&E. No histologic differences could be detected between the two groups at the parameters examined.

 
Growth of solid tumors depends on the formation and development of new blood vessels concomitant with the rearrangement of extracellular matrix. Information obtained by numerous laboratories directly links the expression of MMPs with these processes (22–25). The compound was evaluated in two models: an in vitro capillary formation assay and in an in vivo s.c. tumor growth model. The results show that capillary formation was inhibited dose dependently in the in vitro model of capillary formation (Fig. 5). Exposure to CPCPA had no toxic effect on endothelial cells because removal of the compound initiated tube formation (data not shown). S.c. tumor volume was also reduced significantly in the in vivo model, indicating the inhibition of an essential step in angiogenesis (Fig. 6). Examination of the tumor tissue for MMP expression also indicated reduced enzyme content in the treated mice. The in vivo model was stopped after 16 days owing to the large tumor burden observed in the control mice. Cessation of the treatment accelerated the s.c. tumor growth, indicating that the treatment was not toxic but rather tumoristatic in nature (data not shown).



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  		Fig. 5. In vitro capillary formation in the presence of CPCPA. Endothelial cells were plated on top of a Matrigel layer and treated with various doses of the compound. Capillary areas were determined by an image analysis system (*, P < 0.05, **, P < 0.01)

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The critical pathologic turning point in cancer is the initiation of local invasion leading to the dissemination of tumor cells. Tumor-induced neovascularization occurs in parallel with the transition to invasive phenotype and provides a vascular entry portal for dissemination. Invasion is an active translocation of neoplastic cells across tissue boundaries and through host cellular and extracellular matrix barriers. To achieve invasion, a cell has to couple local proteolysis with coordinated and temporally limited attachment and detachment. The biochemical mechanism of the tumor invasion is similar to that of nonmalignant cells, which are able to traverse tissue boundaries under physiologic conditions when needed. Therefore, it is assumed that the malignant cell is inappropriately utilizing physiologic invasion.

A positive correlation between tumor aggressiveness and expression of members of the metalloproteinase class of enzymes (MMPs) in tumor cell invasion and metastasis formation was established. Most invasive colonic, gastric, breast, and ovarian carcinomas have shown to overexpress MMPs, especially MMP-2 and MMP-9. The expression of these enzymes correlated with tumor aggressiveness (10, 11, 26–28).

The inhibition of MMPs is, therefore, an important therapeutic target, which has attracted considerable attention within the academic and industrial pharmaceutical laboratories. There is no MMPI approved for clinical use on the market at the present in spite of great efforts to develop one. The majority of drug candidates failed because of severe side effects that required the termination of the treatment or because of lack of efficacy, probably due to inappropriate patient choice and wrong end point selection (17, 18, 29–32).

In light of the clinical studies carried out thus far on MMP inhibitor drug candidates that did not advance beyond phase III, it is evident that MMPI treatment should be initiated very early in the progression of cancer, whereas the dissemination process is in its early phase and needs to be continued for a long period of time.

We have discovered a new, original class of highly potent, nontoxic MMPIs. As evident from our data, CPCPA selectively inhibits MMP-2, the enzyme involved and mainly responsible for the breaching of basement membranes, over a wide range of concentrations. This advantageous selectivity might help to evade undesirable side effects that would result in case other MMPs needed for other physiologic functions would also be inhibited nonspecifically (29).

CPCPA is a water-soluble molecule; therefore, it is not likely to precipitate in body cavities as suggested for the hydroxamic-derived compounds tested thus far (29, 33). Consequently, our compound is not expected to suffer from the shortcomings reported for the hydroxamic derivatives. Further, CPCPA inhibited breaching of reconstituted basement membrane in vitro, indicating that it is active in a cellular environment. In vivo studies showed a significant reduction in metastatic foci in mice. The reduction was evident regardless of the route of administration (i.p. or oral).

Toxicologic studies indicate the relative safety of the compound and the lack of structural damage to the gut upon oral administration. Kinetic data (not shown) indicate the operation of a reversible inhibition mechanism of action, which is an advantage in pharmacologic treatment. In contrast to cytotoxic treatments, MMPI-based treatment should be a lifelong treatment. The unexpected oral bioavailability of the compound is an advantage for a better compliance if turned into a drug used in the prevention and/or treatment of metastatic disease. The effects of CPCPA on capillary formation and s.c. tumor growth indicate that the compound might also be effective in treatment of primary tumor growth in reduction or at least in prevention of further tumor growth and thereby reducing the tumor burden of the patient by a nontoxic approach. These data show the superiority of this novel MMPI family and of CPCPA over the other inhibitors reported thus far.

Received 9/28/04; revised 1/11/05; accepted 1/25/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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