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Matrix Metalloproteinase Inhibitors: Do They Have a Place in Anticancer Therapy?

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In particular Section Three, below, includes this introduction.

Since MMPs contain a zinc atom in the catalytic domain and need calcium to function, a chelating compound may inhibit MMP activity. In addition, synthetic derivatives that mimic natural substrates were designed as MMP inhibitors. Several classes of structures such as carboxylic acid derivatives; heterocyclic structures; hydroxamate moieties with a peptide, peptidomimetic, or nonpeptide backbone; biphenyl moieties with nonpeptide backbone; and tetracycline analogs are the most common low-molecular-weight compounds that have in vitro inhibitory activity against MMPs (Figure 2).[97-105]

Section 1 of  4

Source

Apparent duplicate of Section 2

 

Section 2 of 4

Source

Matrix Metalloproteinase Inhibitors: Do They Have a Place in Anticancer Therapy?


 

Michelle A. Rudek, Pharm.D., Ph.D., Jürgen Venitz, M.D., Ph.D., and William D. Figg, Pharm.D.

Pharmacotherapy 22(6):705-720, 2002. © 2002 Pharmacotherapy Publications

Posted 08/16/2002

Introduction

Matrix metalloproteinases (MMPs) are a family of enzymes involved in degradation of extracellular matrix. An imbalance between MMPs and naturally occurring MMP inhibitors may cause excess extracellular matrix destruction, allowing cancer cells to invade surrounding tissues and metastasize, and permitting angiogenesis to occur. Inhibition of certain key MMPs may prevent angiogenesis, tumor growth, invasion, and metastasis. Gelatinases MMP-2 and MMP-9 are expressed during carcinogenesis and angiogenesis. Synthetic MMP inhibitors were designed to target these enzymes and potentially prevent the tumor growth and metastases associated with cancer.

 

Matrix Metalloproteinase

Cancers are cells that grow and divide without normal regulation. They can be local or metastasize. Until recently, the only treatment of metastatic tumors was cytotoxic chemotherapy. A complex process is involved in the development of metastases: invasion, intravasation of primary tumor cells, circulation, extravasation, seeding, and proliferation at distant sites.[1, 2] An essential step involves angiogenesis, recruitment of new blood vessels, for the new lesion to grow beyond 2 mm in diameter.[3] Therapy is aimed at targeting metastasis and angiogenesis. Matrix metallo-proteinases (MMPs) are a family of enzymes involved in degradation of extracellular matrix (gelatinases, collagenases) and are key for metastasis and angiogenesis (Figure 1).[1] The MMP inhibitors are a new class of compounds that have antimetastatic and antiangiogenic properties.[2]

 

Figure 1. Matrix metalloproteinases (MMPs), which are secreted by both tumor and stromal cells, are involved in degradation of extracellular matrix. This degradation is key for metastatic and angiogenic processes.

There are several ways of categorizing the MMP family; one way is according to their substrate preference (collagenase, gelatinase, stromelysin, proenzyme). Since MMPs are involved in a wide variety of processes, both beneficial and potentially harmful, tight regulation must occur in vivo. This regulation occurs during gene expression, proenzyme (pro-MMP) secretion, and endogenous inhibitor levels.[4-8] The structure of MMPs is highly conserved and contains several homologous regions.[6] All family members contain a leader domain, a propeptide domain, and a highly conserved catalytic domain.[5, 6] The catalytic domain contains a zinc atom that is thought to aid in substrate binding. A hinge domain and a hemopexin-like domain are present in all MMP family members except MMP-7, -23, and -26.[9-10] Membrane-type MMPs (MT-MMP) contain a transmembrane domain.[11, 12] Table 1 summarizes the characterized MMPs and their known substrate preferences.[13-52] As is evident, the MMP family can degrade a wide variety of substrates in extracellular matrix, in addition to being able to activate other members of the family and inactivate serine protease inhibitors.

The family also is regulated by endogenous MMP inhibitors, which include a-2 macro-globulin and tissue inhibitors of metalloproteinases (TIMPs) -1, -2, -3, and -4.[53] Both TIMPs and MMPs are secreted by stromal and tumor cells. An imbalance between MMPs and naturally occurring MMP inhibitors may cause an excess of extracellular matrix destruction, allowing cancer cells to invade surrounding tissues and metastasize, leading to angiogenesis.[54] Inhibition of certain key MMPs may prevent metastasis and subsequent angiogenesis. Although other MMPs are expressed during carcinogenesis and angiogenesis, gelatinases (MMP-2, MMP-9) have been extensively characterized.

MMP-2 (Gelatinase A)

Pro-MMP-2 has a molecular weight of 72 kDa. After cleavage, active MMP-2 has a molecular weight of 67 kDa.[18] It has a substrate preference of denatured collagen, types IV and V collagen, elastin, fibronectin, gelatin, laminin, and proteoglycan.[18, 19] Matrix metalloproteinases-14, -16, -17, and -25 and 4-aminophenylmercuric acetate (APMA) can activate pro-MMP-2, and endostatin can inhibit the activation.[12, 18, 38, 41, 49, 55] Pro-MMP-2 is not activated by serine proteases (cathepsin G, chymotrypsin, neutrophil elastase, plasmin, plasma kallikrein, thermolysin, thrombin, trypsin) or MMP-3.[18] Matrix metalloproteinase-2 can activate pro-MMP-9 and pro-MMP-13.[20, 21] Pro-MMP-2 complexes with TIMP-2, and both TIMP-1 and TIMP-2 can complex with active MMP-2 and inhibit proteolytic activity.[56] Matrix metalloproteinase-2 activity can be inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,4-dithiothreitol, 1,10-phenanthroline, sodium dodecyl sulfate, TIMP, and endostatin.[18, 55] Matrix metalloproteinase-2 is inducible by transforming growth factor-beta and not by protein kinase C activators (staurosporine, indolactam V) or a phorbol ester (12-O-tetradecanoylphorbol acetate (TPA), interferon (INF)-gamm, interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha.[34, 57-59] Matrix metalloproteinase-2 messenger RNA (mRNA), protein levels, and activity initially are upregulated, then downregulated by INF-alpha and INF-gamm.[60]

Various molecular biologic techniques have been used to determine the role of MMP-2 in both normal and cancerous cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to confirm MMP-2 expression in bladder cancer tissue and in primary culture-conditioned media from human giant cell tumor of bone.[61, 62] Zymography revealed that both proenzyme and active forms of MMP-2 are expressed in breast tumor cells and gastric cancer tissue.[63-65] Zymography and Western blots performed on cultured media from primary cultures of prostate cancer and benign prostatic hyperplasia indicated that both active and pro-MMP-2 forms were present.[66] In both these tests, the conditioned media from glioma cell cultures contained primarily inactive MMP-2, with faint traces of active MMP-2.[67] Zymography confirmed the presence of pro-MMP-2 in conditioned media from human giant cell tumor of bone and SK-N-BE, a neuroblastoma human cell line.[62, 68]

In situ hybridization for MMP-2 mRNA in human colon adenocarcinomas indicated that MMP-2 mRNA was expressed in stromal cells adjacent to the tumor but not in adenocarcinoma cells.[69] This was not the case in hepatocellular carcinoma tissue and various lung cancers, in which both stromal cells and tumor cells expressed MMP-2 mRNA.[70, 71] In malignant breast cancers, MMP-2 mRNA levels determined by in situ hybridization and immunohisto-chemistry were positive primarily in tumor cells.[64] Northern blots confirmed the presence of MMP-2 in both lung cancer and stromal cells.[71] Immunohistochemistry staining for MMP-2 in gastric cancer tissue was positive, as it was for both tumor and stromal cells associated with lung adenocarcinoma.[72, 73] In esophageal carcinomas, MMP-2 expression determined by immunohistochemistry was positively correlated with tumor invasion (p<0.05).[74] The presence of MMP-2 in medullary thyroid and nonneoplastic thyroid C cells tissue was positive as determined by immunohistochemistry.[75] Expression of mRNA of both MMP-2 and MT1-MMP were significantly higher in head and neck carcinomas cell lines than in control tissues.[76] These studies confirmed the increase of MMP-2 expression in malignant cells.

More recently, attempts were made to correlate disease prognosis and tumor tissue MMP levels. In gastric carcinoma, higher levels of MMP-2 (p<0.001) and lower levels of TIMP-2 (p<0.001) determined by immunohistochemistry were associated with a poor prognosis.[77] In primary gastric carcinoma, high total (p=0.02), active (p=0.02), and proforms (p=0.03) of MMP-2 levels in the tumor determined by zymography also were associated with poor prognosis.[78] A study of gastric carcinoma showed a higher ratio of MT1-MMP:MMP-2 mRNA in tumor:normal tissue associated with favorable prognosis (p=0.0175).[79] In head and neck squamous cell carcinoma, MT1-MMP and not MMP-2 was correlated with lymph node metastasis when comparing mRNA expression and immuno-histochemistry.[76] However, immunohisto-chemistry showed a positive correlation between mRNA levels and co-localization of MT1-MMP and MMP-2.[76] In hepatocellular carcinoma, mRNA overexpression of both MMP-2 and MMP-7 was correlated with recurrence within the first postoperative year (p=0.040).[80] In hypo-pharyngeal squamous cell carcinoma, expression of MMP-2 mRNA determined by in situ hybridization was correlated with overall survival (p<0.05).[81] In non-small cell lung carcinoma, active MMP-2 was correlated with histo-pathologic evidence of metastasis (p=0.001), but not survival.[82] In lung cancer, activation ratio of MMP-2 was again higher in patients with lymph node metastases.[83] In melanoma, MMP-2 levels determined by immunolocalization techniques in the primary tumor correlated with the occurrence of metastasis (p<0.01), although no information regarding survival was given.[84] Matrix metalloproteinase-2 mRNA in prostate cancer was overexpressed (p<0.005); and levels also correlated with a non-cure rate as defined by a rising prostate-specific antigen level during follow-up of at least 4 years (p<0.005).[85] In localized bladder carcinomas, high expression levels of MMP-2, MT1-MMP, and TIMP-2 determined by RT-PCR correlated with poor survival in a univariate analysis (p<0.0001, p<0.005, and p<0.0001, respectively); only MMP-2 and MT1-MMP were still prognostic on multivariate analysis (p=0.0305 and p=0.0073, respectively).[61]

Studies in gynecologic cancers correlated MMP-2 levels with poor prognosis. In a multiple regression analysis of mRNA levels of MMP-2 in stages I-IV breast cancer, correlation was seen between high MMP-2 levels and poor survival (p=0.009).[86] Of interest, in a smaller cohort of invasive ductal or lobular carcinomas of the breast, mRNA levels of MMP-2 were not correlated with prognosis.[87] However, mRNA levels of TIMP-2, which normally complexes with active MMP-2, were positively correlated with the development of distant metastasis (p=0.0055) and not survival (p>0.05).[87] In situ RT-PCR was used to determine MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA expression in cervical carcinoma.[88] A ratio of MMP:TIMP in both cancer (p<0.0001) and stromal cells (p=0.0009) was elevated in patients with poor prognosis.[88] In stage I squamous cervical carcinoma, increased MMP-2 expression was associated with an increase in lymph node metastases (p=0.001) and disease-free survival (p<0.001).[89] In uterine cervical squamous cell carcinomas, high levels of MMP-2 expression in tumor cells (p=0.027) and TIMP-2 in tumor (p=0.02) and stromal cells (p=0.0002) determined by both in situ hybridization and immunohistochemistry analysis correlated with poor survival in univariate analysis; only TIMP-2 was still prognostic on multivariate analysis (p=0.006).[90] High mRNA levels of MMP-2 in metastatic lesions correlated with poor survival (p=0.027) in stages III and IV ovarian epithelial cancers.[90]

MMP-9 (Gelatinase B)

The proenzyme of MMP-9 has a molecular weight of 92 kDa, and after cleavage, active MMP-9 is 64-67 kDa.[32] MMP-9 has a substrate preference of denatured collagen, types IV and V collagen, elastin, and type I gelatin.[19, 32-34] It does not degrade type I collagen, fibronectin, laminin, or proteoglycan.[33, 34] Another target of MMP-9 is IL-2 receptor-alpha (IL-2Ralpha) on activated T cells.[91] Pro-MMP-9 is activated by organomercurials (APMA), several serine proteases (alpha-chymotrypsin, cathepsin G, trypsin), and MMPs-2, -3, -7, -10, and -26.[9, 20, 27, 30, 32, 35, 92, 93] Pro-MMP-9 complexes with TIMP-1, and both TIMP-1 and TIMP-2 can complex with active MMP-9.[56] Even though the proenzyme of MMP-9 complexes with TIMP-1, it still can be activated in the presence of APMA.[34] Both TIMP-1 and TIMP-2 prevent activation of pro-MMP-9 by APMA if they are present in molar excess and complex with active MMP-9.[56] Matrix metalloproteinase-9 activity can be inhibited by 5 mM EDTA, 5 mM 1,10-phenanthroline, and endostatin.[33, 55] Matrix metalloproteinase-9 is inducible by protein kinase C activators (phorbol esters such as TPA, phorbol myristat acetate, indolactam V) and growth factors (epidermal growth factor, IL-1, INF-gamm, TNF-alpha).[33, 34, 58, 59, 94] Initially MMP-9 mRNA is upregulated, then downregulated by INF-alpha and INF-gamm.[60] Interferon-alpha stimulates the activity of MMP-9 during the time frame corresponding to the initial upregulation.[60]

As with MMP-2, MMP-9's role in cancer has been explored extensively. Zymography revealed that both active and proenzyme forms are expressed in breast tumor cells.[63] Zymography performed on the primary culture-conditioned media from human giant cell tumor of bone contained bands for inactive MMP-9, as was the case in primary cultures of lung adenocarcinomas and SK-N-BE.[62, 68, 73] Zymography performed on homogenates of breast tissue biopsies revealed a stronger presence of pro-MMP-9 and active MMP-9 in grade III malignant tissue than in nonmalignant and grades I and II malignant tissues (p<0.0001).[64] Zymography and Western blots confirmed the presence of both active and pro-MMP-9 in cultured media from primary cultures of prostate cancer.[66] The presence of MMP-9 in primary culture-conditioned media from human giant cell tumor of bone was confirmed by RT-PCR and Western blots.[62] In situ hybridization performed with human colon adenocarcinoma tissue revealed that MMP-9 mRNA was expressed in stromal cells adjacent to the tumor, but not in adenocarcinoma cells.[69] Matrix metalloproteinase-9 mRNA was expressed in tumor cells of primary colorectal carcinoma and lung cancer tissue, but not in the normal stroma.[71, 95] In malignant breast cancers, in situ hybridization and immunohistochemistry for MMP-9 mRNA were positive, primarily in tumor cells.[64] Immunohistochemical staining for MMP-9 was positive in both tumor and stromal cells associated with lung adenocarcinoma; immuno-histochemical staining for MMP-9 in medullary thyroid and nonneoplastic thyroid C cells tissue also was positive.[73, 75] These studies confirm overexpression of MMP-9 in malignant tissues.

More recent attempts were made to correlate prognosis of various malignancies and MMP-9 levels. A high tumor:normal ratio (> 5.0) of mRNA levels of MMP-9 was associated with higher recurrence (p=0.0001) and shorter survival (p=0.0002) in patients with colorectal cancer.[95] In primary gastric carcinoma, high total (p=0.04) and pro-forms of MMP-9 (p=0.006) levels determined by zymography indicated poor prognosis, whereas high levels of active MMP-9 (p=0.02) in adjacent mucosa were associated with a good prognosis.[78] In hypopharyngeal squamous cell carcinoma, expression of MMP-9 mRNA determined by in situ hybridization was not correlated with survival.[81] In non-small cell lung carcinoma, active MMP-9 had a negative correlation with prognosis (p=0.017).[82] Matrix metalloproteinase-9 mRNA levels were overexpressed in prostate cancer (p<0.05) and also correlated with a non-cure rate (p<0.005).[85]

Studies in gynecologic cancers correlated MMP-9 levels with poor prognosis. In a multiple regression analysis of mRNA levels of MMP-9 in stages I-IV breast cancer, an inverse correlation was seen between survival and MMP-9 levels (p=0.006).[86] In a smaller cohort of invasive ductal or lobular carcinomas of the breast, mRNA levels of MMP-9 were not correlated with prognosis; however, mRNA levels of TIMP-1 (which normally complexes with active MMP-9) were correlated with the presence of lymph node metastasis (p=0.0067), development of distant metastasis (p=0.014), and decreased survival (p=0.020).[87] In situ RT-PCR was used to determine MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA expression in cervical carcinoma.[88] An inverse relationship exists with the ratio of MMP:TIMP in both cancer (p<0.0001) and stromal cells (p=0.0009) and prognostic status.[88] In squamous cell carcinoma of the cervix, MMP-9 mRNA did not correlate with survival (p>0.05).[96] In stages III and IV ovarian epithelial cancers, high mRNA levels of MMP-9 in tumor cells correlated with poor survival on univariate analysis (p=0.012); MMP-9 was still prognostic on multivariate analysis (p=0.011).[96]

Overall, the number of patients in studies that attempted to correlate MMP-2 and MMP-9 levels and survival was relatively small. Larger, prospective studies would have to be performed to confirm conclusively that MMP levels and MMP activity affect cancer prognosis. Nonetheless, there appears to be sufficient evidence that MMP-2 and MMP-9 may play a role in survival and are therapeutic targets for cancer, and that their inhibition may be therapeutically useful.

 

Matrix Metalloproteinase Inhibitors

Since MMPs contain a zinc atom in the catalytic domain and need calcium to function, a chelating compound may inhibit MMP activity. In addition, synthetic derivatives that mimic natural substrates were designed as MMP inhibitors. Several classes of structures such as carboxylic acid derivatives; heterocyclic structures; hydroxamate moieties with a peptide, peptidomimetic, or nonpeptide backbone; biphenyl moieties with nonpeptide backbone; and tetracycline analogs are the most common low-molecular-weight compounds that have in vitro inhibitory activity against MMPs (Figure 2).[97-105]

 

Figure 2. Structural formulas of matrix metalloproteinase inhibitors: (A) hydroxamate, (B) peptide-hydroxamate, (C) tetracycline. The Rx groups are various chemical substituents, and AA represents a peptide substituent.

A peptide backbone with a hydroxamate moiety mimics naturally occurring substrates for MMPs. Several variations of hydroxamic acid derivatives with a dipeptide backbone were studied to elucidate the structure-activity relationship for MMP-1, -2, -3, and -9 inhibition.[98] Hydrogen bonding (a large, planar, fused-ring, hydrophilic aryl substituent) must occur in two locations between the MMP and substrate. The hydroxamate moiety acts as a 1,4 bidentate ligand for the zinc atom in the active site of MMPs, and is more potent than carboxylic acid analogs that can achieve only a 1,3-bidentate ligand or a monodentate interaction. A nonpeptide biphenyl structure that does not contain the hydroxamate moiety loses activity against MMP-1 but retains activity against MMPs-2, -3, and -9.[106] The presumed mechanism of action of tetracyclines is the chelating calcium or zinc at the active site.[107-109]

Matrix metalloproteinase inhibitors that have entered clinical trials for an oncologic indication include prinomastat (AG3340; Agouron/Pfizer), BAY 12-9566 (Bayer Corp.), batimistat (BB-94; British Biotech, Ltd,), BMS-275291 (formerly D2163; Celltech/Bristol-Myers Squibb), marimastat (BB 2516; British Biotech, Ltd./Schering-Plough), MMI270(B) (formerly CGS-27023A; Novartis), and Metastat (COL-3; CollaGenex). Table 2 compares pharmacokinetic characteristics of these agents.[110-123]

Batimastat

This was the first MMP inhibitor to go into clinical trials. Batimastat, a synthetic compound with a peptide-like backbone and hydroxamate moiety, is a broad-spectrum MMP inhibitor with in vitro concentrations required to produce 50% of maximum inhibition (IC50s) of MMPs-1, -2, -3, -7, and -9 from 0.5-10 ng/ml.[110] The agent is poorly soluble and was administered intra-peritoneally in several clinical trials.

In the original phase I trial, batimastat 600, 1200, or 1800 mg/m2 was administered every 4 weeks in patients with advanced cancer.[110] The mean maximum plasma concentration (Cmax) was 805, 1225, and 1570 ng/ml for the three doses, respectively, which are all above IC50s for this compound. Time of maximum concentration (Tmax) occurred between 24 and 48 hours after administration, with residual concentrations in systemic circulation after 4 weeks. Terminal half-life, apparent steady-state volume of distribution (Vdss/F), and apparent total clearance (ClT/F) ranged from 349-743 hours (14.5-30.9 days), 0.7-5.4 L, and 0.000013-0.000117 ml/minute, respectively. Plasma protein binding appeared to be approximately 96% in these nine patients. Adverse effects included abdominal pain, bradycardia, constipation, diarrhea, fatigue, fever, hypotension, nausea, and vomiting. Four patients had stable disease of 3-8 months duration. Due to its poor solubility and undesirable pharmacokinetic profile, and with the advent of marimastat, an orally bioavailable MMP inhibitor, batimastat was assessed only in malignant ascites after this phase I trial.

In a phase I-II trial in nine patients with malignant ascites, a one-time dose of batimastat 600 or 1050 mg/m2 was administered, replacing 500 ml of ascites fluid.[124] The mean Cmax over both doses was approximately 1500 ng/ml and occurred within 4 hours of administration. Residual systemic batimastat was present for up to 6 weeks. Adverse effects included abdominal pain, nausea, postural hypotension, pyrexia, scrotal edema, and vomiting. One patient died within 4 days of the dose secondary to a pulmonary embolus. Clinical response in five patients consisted of decreases in abdominal girth, drainage frequency, and weight.

In the last study of batimistat, 23 patients with malignant ascites were administered 150, 300, 600, 1050, or 1350 mg/m2 as a one-time dose after ascites fluid was drained.[111] The Cmax was 300-1500 ng/ml at all doses and occurred within 1, 4, and 24 hours of administration at 150 mg/m2, 300 mg/m2, and all other doses, respectively. Residual systemic batimastat was present for up to 4 weeks. The terminal half-life appeared to be 458 hours (19.1 days). Adverse effects included abdominal pain, bowel obstruction, diarrhea, fatigue, fever, nausea, and vomiting. Seventy percent of these patients did not require drainage within 28 days, and this figure dropped to 22% by 112 days.

No other clinical trials of this agent have been published since 1998.

Marimastat

Marimastat, which also contains a peptide-like backbone and a hydroxamate moiety, is an orally available, broad-spectrum MMP inhibitor with in vitro IC50s of MMPs-1, -2, -3, -7, -9, and -12 between 1 and 76 ng/ml.[123] The pharmacokinetics were linear for both Cmax and area under the curve (AUC) after single-dose administration of up to 200 mg in healthy male volunteers.[123] Plasma accumulation after twice-daily dosing was not significant, consistent with the terminal half-life of 8-10 hours. In 1998 a meta-analysis was performed to assess the biologic activity of marimastat in 415 patients in 6 clinical trials (2 each in patients with advanced colorectal and ovarian cancer, 1 each in patients with pancreatic and prostate cancer).[125] Biologic effect was defined as less than 0% (full) or less than 25% (partial) rise in the respective tumor marker over 28 days and was not based on traditional oncologic end points such as tumor progression documented radiographically. The analysis revealed that marimastat exerts a full or partial biologic effect at dosages greater than 20 mg/day (p=0.01, Cochran-Mantel-Haenszel test).[125] The dose-limiting toxicity was a time-dependent and dose-dependent musculoskeletal triad (arthralgia, myalgia, tendinitis). Other toxicities in these trials were ascites, disseminated carcinoma, chills, cholangitis, dizziness, dyspnea, edema, fatigue, fever, gastrointestinal (anorexia, nausea, vomiting, diarrhea, constipation), gastrointestinal hemorrhage, headache, heartburn, hepatic toxicity, hypercalcemia, hyperglycemia, rash, and shortness of breath.[117, 125, 126]

In a phase I trial of 12 patients with advanced lung cancer, the maximum tolerated dosage was 50 mg twice/day, with dose-limiting toxicity being inflammatory polyarthritis.[117] The terminal half-life, Vdss/F, and ClT/F in these patients were 4.9 ± 2.3 hours, 224.8 ± 98.6 L, and 5278 ± 3323 ml/minute, respectively. Eight patients were withdrawn for progressive disease (by standard criteria); the remaining four had stable disease and eventually were withdrawn due to toxicity. No changes in plasma MMP-2 and MMP-9 were observed.

From the results of another clinical trial in 64 patients with advanced pancreatic cancer, a dosage of 5, 10, or 25 mg twice/day was suggested since the rate of rise of tumor marker (CA 19/9) levels was less than with other dosages of marimastat.[126] This study investigated the early biologic activity of marimastat and compared changes in CA 19/9 levels before the study with those after the first 28 days of marimastat therapy. Of 30 patients who received marimastat for longer than 28 days, 33% developed musculoskeletal events (pain, stiffness, tenderness), and 50% of them required dosage reduction. A dose-dependent increase in Cmax was observed at all dosages except 10 mg once/day. This study was conducted before the approval of gemcitabine; published results state that comparison trials with marimastat are continuing in both the United States and Europe.[126]

Preliminary data of marimastat were presented for several cancers: in 555 patients with small cell lung cancer, 162 with glioblastoma multiforme or gliosarcoma, and 11 with metastatic breast cancer in combination with doxorubicin and docetaxel.[127-129] A placebo-controlled trial was conducted in patients with small cell lung cancer who responded to first-line chemotherapy.[127] Overall, median survival was 9.5 months, although there appeared to be no advantage to marimastat in overall response (using standard criteria). A complete response or partial response was observed in 32.5% (90/277) or 62.8% (174/277) of patients treated with marimastat 10 mg twice/day versus 32.4% (90/278) or 66.2% (184/278) of patients treated with placebo. The main toxicity was musculo-skeletal and required dosage modification and withdrawal from treatment in 40.8% (113/278) and 46.2% (128/278) of patients, respectively. In the placebo-controlled trial in patients with glioblastoma multiforme or gliosarcoma who had response to first-line therapy (either radiotherapy or surgery), marimastat 10 mg twice/day did not improve survival compared with placebo.[128] A small subset of patients chose to continue and received procarbazine-lomustine-vinblastine (PCV) chemotherapy in addition to the original randomized treatment. A survival benefit was seen in 27 patients who received the combination of marimastat and PCV. Musculoskeletal toxicities led to dosage reductions. A 75% response rate was reported in 11 patients treated with a combination of doxorubicin and docetaxel plus marimastat 20 mg twice/day.[129] Toxicities reported were known toxicities of either chemotherapeutic agents or marimastat as a single agent. A less myelotoxic regimen of doxorubicin and docetaxel is being explored.[129]

A study with marimastat in non-small cell lung cancer was terminated when it failed to show clinical benefit, although a trial in patients with resected pancreatic cancer continues.[130] Long-term follow-up of a study in patients with gastric carcinoma suggests a survival benefit for marimastat versus placebo.[130]

Results with marimastat in clinical trials are mixed. It appears that benefits are more noticeable in long-term trials with adjunct chemotherapy than in single-agent trials. A potential criticism of most of these trials is the soft end point of biologic effect versus the harder end point of disease progression by standard criteria (radiography). Another concern is dose-dependent musculoskeletal toxicity in most patients who continued marimastat long term, which is how MMP inhibitors would be administered. The agent did not change total or active plasma MMP-2 or -9 consistently in any study.

Prinomastat

A nonpeptidic hydroxamate, AG-3340 is a selective MMP inhibitor with in vitro activity (dissociation constant of the enzyme-inhibitor complex [Ki]) against MMPs-1, -2, -3, -9, -13, and -14 at concentrations less than 0.1 ng/ml.[121, 131] Preclinically, it had greatest efficacy in a colon cancer tumor model when a minimum plasma concentration was maintained, versus targeting an AUC0-24 or Cmax.[132] This oral MMP inhibitor has linear pharmacokinetics at doses up to 200 mg (100 mg twice/day) and low plasma protein binding (69%) in patients with advanced cancer.[132] When combined with mitoxantrone given every 3 weeks and continuous prednisone, the Cmax of AG-3340 25 mg twice/day was not affected and was either 513 ± 264 ng/ml after prednisone or 554 ± 295 ng/ml after prednisone and mitoxantrone.[120] The terminal half-life was 2-3 hours, with Tmax within 1 hour after oral dosing.[121]

Patients with breast cancer randomized to receive AG-3340 5 or 25 mg twice/day had no response, although those with low plasma vascular endothelial growth factor (VEGF) and urine pyridinoline levels tended to have stable disease after 2 months of treatment.[133] The addition of AG-3340 5 or 10 mg twice/day to a combination of mitoxantrone and prednisone did not enhance efficacy in patients with chemo-therapy-naïve hormone-refractory prostate cancer (HRPC).[134] In patients with chemotherapy-naïve non-small cell lung cancer, the addition of AG-3340 5, 10, or 15 mg twice/day to a combination of paclitaxel and carboplatin did not enhance efficacy. Adverse effects of AG-3340 are reversible musculoskeletal symptoms (arthralgia, joint swelling, joint stiffness, myalgia, tendinous contracture) and neuropathy.[121, 131, 133-135]

Clinical trials of AG-3340 combined with standard chemotherapy in HRPC and non-small cell lung cancer were halted since primary efficacy end points were not achieved.[136] Although AG-3340 was meant to be a specific MMP inhibitor with an improved pharmaco-kinetic profile and picomolar Ki values, the compound has limitations, notably musculo-skeletal symptoms. Its future appears to be in early intervention and potentially in combination with chemotherapy or radiation in other carcinomas.

BAY 12-9566

Another MMP inhibitor, BAY 12-9566, is an oral nonpeptide biphenyl and has activity against MMPs-2, -3, and -9, with Ki values of 4.5-123.7 ng/ml.[112, 113] In a phase I clinical trial, 21 patients with solid tumors were enrolled to 47, 28-day courses of BAY 12-9566.[137] The maximum tolerated dosage was not determined since steady-state concentrations increased less than dose-proportionally with total daily dose and were within expected biologically relevant levels. Toxicities were abdominal pain, alopecia, anemia, anorexia, bruising, diarrhea, dizziness, fatigue, headache, hepatic function abnormalities, hyperglycemia, nausea, numbness, dose-dependent thrombocytopenia, upper respiratory infections, ureteric obstruction, and vomiting.[112, 113, 137] Plasma MMP-2 (pro-MMP-2, and pro-MMP-2 complexed with TIMP-2), MMP-9 (pro-MMP-9, and pro-MMP-9 complexed with TIMP-1), TIMP-2 levels (TIMP-2, and TIMP-2 complexed with active MMPs), and active MMP-9 levels were assessed with respect to time and steady-state BAY 12-9566 plasma concentrations.[137] After 29 days of treatment, relative changes in MMPs-2 and -9 from baseline were unchanged (p=0.66 and p=0.59, respectively); however, the percentage change in TIMP-2 did reach statistical significance (p=0.046), increasing with increasing daily doses. Active MMP-9 was below the limits of quantification for each sample.

Similar results were reported in later phase I clinical trials.[112, 113] Stable disease was the best response in 62% of patients receiving treatment.[113] Due to saturable absorption, 800 mg twice/day was determined to be the acceptable phase II starting dosage. The terminal half-life was estimated to be 60.7 hours based on the ratio of C24hr:Css.[112] Plasma protein binding of BAY 12-9566 was 99.99%.[113] The agent had no effect on potential angiogenic surrogate markers plasma VEGF, plasma basic fibroblast growth factor (bFGF), urinary deoxypyridinoline, and urinary pyridinoline.[112] It increased disease progression and mortality in patients with small cell lung cancer compared with placebo. As a result, all trials with this compound were halted.[138, 139]

MMI270(B)

This oral, nonpeptidic, hydroxamate MMP inhibitor has in vitro activity against MMPs-1, -2, -3, -9, and -13, with IC50s of 2.4-19.7 ng/ml.[118, 119] Seventeen patients were enrolled in a phase I study to determine the effect of food on the pharmacokinetics of MMI270(B) 150, 400, and 600 mg.[119] The AUC0-8 hr did not change, although Cmax was decreased and Tmax was increased when the agent was administered after a light breakfast. To date, there is no recommendation with regard to administering the compound with food.

In a phase I study conducted in 92 patients with various cancers, the maximum tolerated dosage was 300 mg twice/day due to a dose-dependent rash experienced by 19.6% of patients.[118] Other toxicities were a musculo-skeletal syndrome (arthralgia, mylagia, tendinitis) in 42.4% of patients and nausea in 16.3%. A total of 19 patients (20.7%) had stable disease at 3 months and continued MMI270(B) for up to 406 days. Absorption was rapid, with a median Tmax of 0.58 hour, and half-life was short (range 0.6-7.6 hrs, median 1.6 hrs). Both Cmax and AUC were linear with dosages from 50 mg/day up to 600 mg 3 times/day. Several end points were assessed with direct measurements (MMP and TIMP levels) and indirect measure-ments (bFGF, collagen breakdown products, lysozymal proteases, serine peptidases, TNF-alpha convertase, vascular cell adhesion molecule-1, VEGF). Of these markers, only increases in bFGF, MMP-2, MMP-9, TIMP-1, and TIMP-2 were correlated with Cmax (p=0.02 for MMP-2, p=0.048 for MMP-9) and AUC (p=0.03 for bFGF, p=0.0004 for MMP-2, p=0.002 for TIMP-1, p=0.02 for TIMP-2). Urinary excreted collagen breakdown products were decreased at 2 weeks (pyridinoline with Cmax p=0.01 and AUC p=0.04; deoxypyridinoline with Cmax p=0.02) but rebounded by 4 weeks.

Preliminary reports from a phase I trial of MMI270(B) (various dosages) in combination with 5-fluorouracil (5-FU) and folinic acid in 20 patients with colorectal cancer indicated that this regimen was tolerable.[140] Adverse effects were similar to those when the agent was administered alone. Ten percent of patients had partial responses, and 50% had stable disease as their best response. Although the agent showed positive outcomes in the phase I trial, it is a broad-spectrum MMP inhibitor that may have long-term musculoskeletal complications (hypothesized to be due to inhibition of MMP-1), similar with those that appear to plague marimastat. Although the phase I open-label study with 5-FU and folinic acid appears promising, such a study would have to be conducted in a double-blind, randomized fashion looking for clinical end points, rather than biologic end points.

BMS-275291

An oral nonhydroxamate MMP inhibitor, BMS-275291, showed in vitro activity against a wide range of MMPs, with IC50s less than 20 ng/ml for MMPs-2 and -9.[114, 141] The compound was designed to spare sheddases, which are closely related to the metalloprotease family of enzymes and are thought to be responsible for musculoskeletal adverse effects of broad-spectrum MMP inhibitors.[115, 141] In 40 healthy subjects, no changes in collagen breakdown product excretion and no dose-limiting toxicity were discerned.[115] In 44 patients with cancer, BMS-275291 was well tolerated; toxicities in the phase I trial included arthralgia, subcapsular cataracts, dyspnea, headache, hepatic toxicity (elevated aspartate aminotransferase), myalgia, rash, and tenosynovitis.[141] The phase II dosage was determined to be 1200 mg/day since trough plasma levels of BMS-275291 exceeded in vitro IC90s of MMPs.[114, 141]

Two studies in patients with cancer and in healthy subjects revealed different pharmaco-kinetics for BMS-275291.[114, 115] The agent has free sulfhydryl groups that form disulfides in vivo. The pharmacokinetics of the parent com-pound is dose proportional for dosages up to 1200 mg/day in healthy subjects, and 2400 mg/day in patients with cancer with respect to Cmax; in healthy subjects, exposure is nonlinear, with a less than proportional increase in patients with cancer. An almost opposite phenomenon occurs with regard to the total drug (BMS-275291 plus reducible disulfides). The pharmacokinetics reveal nonlinearity with total drug AUC in healthy subjects; this relationship is linear in patients with cancer. Plasma protein binding of BMS-275291 is between 46% and 77%.[114]

Metastat

By eliminating the dimethylamino, methyl, and hydroxy functionalities on the basic tetracycline structure to produce metastat, the antimicrobial properties of the molecule were eliminated while MMP inhibitory properties were retained.[142] The proposed mechanism of action for metastat, 6-demethyl-6-deoxy-4-dedimethylaminotetracycline, is pleotropic and includes inhibition of MMPs due to divalent cation chelation of zinc at the active site of the enzyme, downregulation of production of pro-MMPs, inhibition of oxidative activation of pro-MMPs, increase in degradation of pro-MMPs, induction of apoptosis, inhibition of production of secretory nonpancreatic phospholipase A2, inducible nitric oxide (NO) synthase and NO, inhibition of production of TNF-alpha and IL-8, and reduction of the expression of a serine proteinase (trypsinogen-2).[104, 142-154] Metastat induces potent MMP inhibition of the 2, 9, and 14 isozymes.[40, 99, 155] It may have anti-angiogenic and antimetastatic activity, as evidenced by its cytotoxic, antiinvasive, and MMP inhibitory properties.

In a phase I clinical trial conducted at the National Cancer Institute, the maximum tolerated dosage was 70 mg/m2/day administered orally; dose-limiting toxicity was phototoxicity.[116] Disease stabilization for longer than 40 months, 8 months, and 6 months were seen in hemangioen-dothelioma, Sertoli-Leydig cell tumor, and fibrosarcoma, respectively. Non-dose-related toxicities were anemia, anorexia, constipation, dizziness, elevated liver function tests, fatigue, fever, headache, heartburn, nausea, vomiting, neurotoxicities, and three cases of drug-induced lupus erythematosus. Metastat has nonlinear pharmacokinetics, thought to be due to saturable absorption. The median single-dose half-life, ClT/F, and Vd/F were 56.7 hours, 0.0077 L/hr/kg, and 0.63 L/kg, respectively. Plasma protein binding is 94.5%, with most binding due to albumin (MA Rudek, unpublished data, 2001). In assessing potential pharmacodynamic markers, there was a statistically significant relationship between changes in plasma MMP-2 levels and cumulative doses of metastat when patients with progressive disease were compared with those with stable disease or toxicity (p=0.042).

Metastat was administered in another phase I clinical trial.[156] This trial has accrued 26 patients to date, with enrollment continuing.[157] Toxicities include anemia, asthenia, fatigue, photosensitivity, and skin hyperpigmentation.[156, 158, 159] In November 2000, it was noted that one patient with leiomyosarcoma had stable disease for 9 months.[158]

A third phase I clinical trial was conducted in 18 patients with Kaposi's sarcoma related to acquired immunodeficiency syndrome (AIDS) through the AIDS Malignancy Consortium. Patients were administered metastat 25, 50, or 70 mg/m2/day.[160] Ninety-four percent of these patients were receiving antiretroviral therapy and had failed earlier Kaposi's sarcoma treatment. The median duration of metastat therapy was 9.5 weeks, with one patient remaining in the study as of this writing. Nine patients terminated therapy for toxicity, and eight terminated for progressive disease. The dose-limiting toxicity was photosensitivity; other toxicities were arthralgia, fatigue, fever, headache, myalgia, nausea, pain, and pruritus. The overall response rate was 44%, with one complete response and seven partial responses. Due to the response rate in this trial, a future phase II clinical trial is planned in patients with Kaposi's sarcoma, with a dosage of 25 mg/m2/day. In addition, a phase I-II clinical trial in patients with high-grade anaplastic astrocytoma, anaplastic oligodendroglioma, or glioblastoma multiforme is being conducted through New Approaches to Brain Tumor Therapy.[157]

 

Conclusion

The MMP inhibitors offer a new approach to the treatment of cancer. A potential limitation of narrow-spectrum MMP inhibitors' clinical usefulness may be their complex interaction with a wide variety of substrates that have unknown effects in cancer. For example, the agents may affect the release of TNF-alpha by MMPs-14 and -17. If this occurs, TNF-alpha will not be available to exert its biologic effect.[11] Limitations of broad-spectrum MMP inhibitors include musculoskeletal adverse effects that can hamper long-term therapy.

Synthetic MMP inhibitors under development have been plagued with problems similar to those of other noncytotoxic therapies. They do not have antiproliferative properties and therefore are not expected to result in responses assessed by traditional cytotoxic clinical trials.[161] Instead, stable disease is accepted as the best response. The traditional paradigm of randomized phase III clinical trials that pits the test agent against standard therapy and looks for equality or superiority may not occur with these compounds unless the end points of the trials change.

Marimastat, prinomastat, and BAY 12-9566 were assessed in phase III clinical trials using the current paradigm, and most failed. Marimastat showed hints of efficacy when given early in disease progression and in combination with cytotoxic chemotherapy. Metastat, MMI270(B), and BMS-275291 are still in early stages of development and may benefit from knowledge gained from MMP inhibitors that failed in late stages of development. Preliminary results with MMI270(B) and BMS-275291 look promising, although the potential for long-term toxicities plaguing marimastat are of concern since both are broad-spectrum inhibitors. The agent BMS-275291 is being developed rationally in an attempt to determine dosages based on preclinical data rather than maximum tolerated dosage. Metastat's clinical development may be hastened by the high degree of photosensitivity, drug-induced lupus, and sideroblastic anemia in the small population in phase I clinical trials. These effects ultimately may be problematic when assessed in larger populations. If any MMP inhibitor proves efficacious in a phase III clinical trial and is approved by the Food and Drug Administration, its role will more than likely be in combination with more aggressive therapy to keep cancer either in check or in a palliative setting.

 

Tables

Table 1. Matrix Metalloproteinase Family


 

MMP Pseudonym

Substrates

1

Collagenase-1, interstitial C

alpha1-Antichymotrypsin, types I, II, III, VII, and X collagen, gelatin, alpha1-proteinase inhibitor[13-17]

2

Gelatinase A

Denatured collagen, types IV and V collagen, elastin, fibronectin, gelatin, laminin, proteoglycan, pro-MMP-9, pro-MMP-13[18-21]

3

Stromelysin-1

alpha1-Antichymotrypsin, carboxymethylated transferrin, type IV collagen, type I procollagen, fibronectin, gelatin, laminin, alpha1-proteinase inhibitor, proteoglycan, pro-MMP-1, pro-MMP-7, pro-MMP-9[17, 22-25, 27-30]

7

Matrilysin

Casein, type IV collagen, elastin, fibronectin, type I gelatin, laminin, nidogen, proteoglycan, pro-MMP-1, pro-MMP-9[11, 26, 27]

8

Collagenase-2, neutrophil C

Aggrecan, type I collagen, fibrinogen, gelatin[28, 29, 31]

9

Gelatinase B

Denatured collagen, types IV and V collagen, elastin, type I gelatin[19, 32-34]

10

Stromelysin-2

Carboxymethylated transferrin, casein, gelatin, pro-MMP-1, pro-MMP-7, pro-MMP-8, pro-MMP-9[30, 35]

11

Stromelysin-3

Not known

12

Macrophage metalloelastase

Apolipoprotein(a), factor XII, fibrinogen[31, 36]

13

Collagenase-3

alpha1-Antichymotrypsin, types I, II, and III collagen, factor XII, fibrinogen, gelatin, plasminogen activator inhibitor-2[28, 29, 31, 37]

14

MT1-MMP

Aggrecan, types I, III, and III collagen, cassein, factor XII, fibronectin, fibrinogen, gelatin, laminin, alpha1-macroglobulin, nidogen, perlecan, alpha1-proteinase inhibitor, pro-MMP-2, pro-MMP-13, proteoglycan, large tenascin-C, pro-TNF-alpha, vitronectin[11, 21, 27, 31, 38-40]

15

MT2-MMP

Aggrecan, fibronectin, laminin, nidogen, perlecan, large tenascin-C, pro-TNF-alpha[11]

16

MT3-MMP

Pro-MMP-212

17

MT4-MMP

Fibrin, fibrinogen, gelatin, pro-MMP-2, pro-TNF-alpha[41, 42]

18

 

Not known[43]

19

 

Casein, type IV collagen, fibrin, fibronectin, fibrinogen, gelatin, laminin, nidogen, large tenascin-C[44]

20

Enamelysin

Amelogenin[45]

21

 

Not known[46]

23

 

Not known[10]

24

MT5-MMP

Gelatin, pro-MMP-2[47, 48]

25

MT6-MMP, leukolysin

Type IV collagen, fibrin, fibronectin, gelatin, pro-MMP-2[49, 50]

26

Endometase, matrilysin-2

Type IV collagen, fibronectin, fibrinogen, type I gelatin, a1-proteinase inhibitor, pro-MMP-9[9, 51]

28

Epilysin

Casein[46, 52]

MMP = matrix metalloproteinase; MT = membrane-type; TNF = tumor necrosis factor.


 

Table 2. Clinical Pharmacokinetics of Matrix Metalloproteinase Inhibitors in Patients with Cancer


 

Agent
(route)

Dosage

Cmax
(ng/ml)

Half-life
(hrs)

PPB
(%)

Vd/F
(L)

Cl/F
(ml/min)

Remarks

Batimastat
   (i.p.)[110, 111]

150 mg/m2
1800 mg/m2

300
1570

349-743

96

0.7-5.4

0.00001-0.00012

Poorly solubility, not orally bioavailable

BAY 12-9566
   (oral)[112, 113]

100 mg q.d.
400 mg q.d.
800 mg b.i.d.

50,000
50,000
160,000

60.7

99.99

--

--

Nonlinear pharmacokinetics (possibly due to saturable absorption)

BMS-275291
   (oral)[114, 115]

600 mg/day
2400 mg/day

-

~24-50a

46-77

--

--

Linear pharmacokinetics up to 1200 and 2400 mg/day in healthy subjects and patients with cancer, respectively

Marimastat
   (oral)[117, 123]

25 mg b.i.d.
50 mg b.i.d.
100 mg b.i.d.

184
196
540

3.7-11.3

--

66-444

1600-12633

Linear pharmacokinetics up to 200 mg (single dose) in healthy subjects

Metastat
   (oral)[116]

36 mg/m2/day
70 mg/m2/day

1036
2465

23.7-144.4

94.5

490.9-2222.7b

81.8-668.1b

Linear pharmacokinetics up to 70 mg/m2/day; potential saturable absorption

MMI270(B)
   (oral)[118, 119]

150 mg b.i.d.
600 mg b.i.d.

941.9
9155.9

1.6

--

383.8c

2771c

Linear pharmacokinetics up to 600 mg t.i.d.

Prinomastat
   (oral)[120]

25 mg b.i.d.

513

2.0-3.0

69

91d

422d

Linear 100 mg b.i.d.

i.p. = intraperitoneal;Cmax = maximum plasma concentration; PPB = plasma protein binding; Vd/F = apparent volume of distribution; Cl/F = apparent clearance.
aBased on statement that steady state was achieved by day 15 in patients with cancer or day 7 in healthy subjects.[114, 115] Calculated as time to reach steady state equals 7 times half-life.[122]
bBased on 75-kg person. Both ClT/F (apparent total clearance) and Vdpss/F (apparent pseudo-steady-state volume of distribution) are corrected for plasma protein binding.
cBased on fasting The AUC0-8 hr = 2422 ± 1246 ng·hr/ml at a dose of 400 mg.[119] Estimated using ClT/F = dose/AUC0-infinity and Vdpss/F = (ClT/F)/ke.[122]
dBased on historical data of AG-3340 AUC = 987 ± 366 ng·hr/ml at a dose of 25 mg.[120] Calculated using ClT/F = dose/AUC0- and Vdpss/F = (ClT/F)/ke.[122]


 

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Funding Information
 

Funded in part by the Office of Minority Health, National Institutes of Health, Bethesda, Maryland.

Reprint Address
 

William D. Figg, Pharm.D., Molecular Pharmacology Section, Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 5A01, MSC1910, 9000 Rockville Pike, Bethesda, MD 20892; e-mail: wdfigg@helix.nih.gov.



 

From the Clinical Pharmacology Research Core, Medical Oncology Clinical Research Unit Center for Cancer Research (Drs. Rudek and Figg), and the Molecular Pharmacology Section, Cancer Therapeutics Branch (Dr. Figg), National Cancer Institute, National Institutes of Health, Bethesda, Maryland; and the Department of Pharmaceutics, School of Pharmacy, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia (all authors).
 

 

Section 3 of 4

Source

Matrix Metalloproteinase Inhibitors
from Pharmacotherapy

 

Matrix Metalloproteinase Inhibitors

 

Since MMPs contain a zinc atom in the catalytic domain and need calcium to function, a chelating compound may inhibit MMP activity. In addition, synthetic derivatives that mimic natural substrates were designed as MMP inhibitors. Several classes of structures such as carboxylic acid derivatives; heterocyclic structures; hydroxamate moieties with a peptide, peptidomimetic, or nonpeptide backbone; biphenyl moieties with nonpeptide backbone; and tetracycline analogs are the most common low-molecular-weight compounds that have in vitro inhibitory activity against MMPs (Figure 2).[97-105]

 

Click to zoom

Figure 2. (click image to zoom) Structural formulas of matrix metalloproteinase inhibitors: (A) hydroxamate, (B) peptide-hydroxamate, (C) tetracycline. The Rx groups are various chemical substituents, and AA represents a peptide substituent.

 

A peptide backbone with a hydroxamate moiety mimics naturally occurring substrates for MMPs. Several variations of hydroxamic acid derivatives with a dipeptide backbone were studied to elucidate the structure-activity relationship for MMP-1, -2, -3, and -9 inhibition.[98] Hydrogen bonding (a large, planar, fused-ring, hydrophilic aryl substituent) must occur in two locations between the MMP and substrate. The hydroxamate moiety acts as a 1,4 bidentate ligand for the zinc atom in the active site of MMPs, and is more potent than carboxylic acid analogs that can achieve only a 1,3-bidentate ligand or a monodentate interaction. A nonpeptide biphenyl structure that does not contain the hydroxamate moiety loses activity against MMP-1 but retains activity against MMPs-2, -3, and -9.[106] The presumed mechanism of action of tetracyclines is the chelating calcium or zinc at the active site.[107-109]

Matrix metalloproteinase inhibitors that have entered clinical trials for an oncologic indication include prinomastat (AG3340; Agouron/Pfizer), BAY 12-9566 (Bayer Corp.), batimistat (BB-94; British Biotech, Ltd,), BMS-275291 (formerly D2163; Celltech/Bristol-Myers Squibb), marimastat (BB 2516; British Biotech, Ltd./Schering-Plough), MMI270(B) (formerly CGS-27023A; Novartis), and Metastat (COL-3; CollaGenex). Table 2 compares pharmacokinetic characteristics of these agents.[110-123]

Batimastat

This was the first MMP inhibitor to go into clinical trials. Batimastat, a synthetic compound with a peptide-like backbone and hydroxamate moiety, is a broad-spectrum MMP inhibitor with in vitro concentrations required to produce 50% of maximum inhibition (IC50s) of MMPs-1, -2, -3, -7, and -9 from 0.5-10 ng/ml.[110] The agent is poorly soluble and was administered intra-peritoneally in several clinical trials.

In the original phase I trial, batimastat 600, 1200, or 1800 mg/m2 was administered every 4 weeks in patients with advanced cancer.[110] The mean maximum plasma concentration (Cmax) was 805, 1225, and 1570 ng/ml for the three doses, respectively, which are all above IC50s for this compound. Time of maximum concentration (Tmax) occurred between 24 and 48 hours after administration, with residual concentrations in systemic circulation after 4 weeks. Terminal half-life, apparent steady-state volume of distribution (Vdss/F), and apparent total clearance (ClT/F) ranged from 349-743 hours (14.5-30.9 days), 0.7-5.4 L, and 0.000013-0.000117 ml/minute, respectively. Plasma protein binding appeared to be approximately 96% in these nine patients. Adverse effects included abdominal pain, bradycardia, constipation, diarrhea, fatigue, fever, hypotension, nausea, and vomiting. Four patients had stable disease of 3-8 months duration. Due to its poor solubility and undesirable pharmacokinetic profile, and with the advent of marimastat, an orally bioavailable MMP inhibitor, batimastat was assessed only in malignant ascites after this phase I trial.

In a phase I-II trial in nine patients with malignant ascites, a one-time dose of batimastat 600 or 1050 mg/m2 was administered, replacing 500 ml of ascites fluid.[124] The mean Cmax over both doses was approximately 1500 ng/ml and occurred within 4 hours of administration. Residual systemic batimastat was present for up to 6 weeks. Adverse effects included abdominal pain, nausea, postural hypotension, pyrexia, scrotal edema, and vomiting. One patient died within 4 days of the dose secondary to a pulmonary embolus. Clinical response in five patients consisted of decreases in abdominal girth, drainage frequency, and weight.

In the last study of batimistat, 23 patients with malignant ascites were administered 150, 300, 600, 1050, or 1350 mg/m2 as a one-time dose after ascites fluid was drained.[111] The Cmax was 300-1500 ng/ml at all doses and occurred within 1, 4, and 24 hours of administration at 150 mg/m2, 300 mg/m2, and all other doses, respectively. Residual systemic batimastat was present for up to 4 weeks. The terminal half-life appeared to be 458 hours (19.1 days). Adverse effects included abdominal pain, bowel obstruction, diarrhea, fatigue, fever, nausea, and vomiting. Seventy percent of these patients did not require drainage within 28 days, and this figure dropped to 22% by 112 days.

No other clinical trials of this agent have been published since 1998.

Marimastat

Marimastat, which also contains a peptide-like backbone and a hydroxamate moiety, is an orally available, broad-spectrum MMP inhibitor with in vitro IC50s of MMPs-1, -2, -3, -7, -9, and -12 between 1 and 76 ng/ml.[123] The pharmacokinetics were linear for both Cmax and area under the curve (AUC) after single-dose administration of up to 200 mg in healthy male volunteers.[123] Plasma accumulation after twice-daily dosing was not significant, consistent with the terminal half-life of 8-10 hours. In 1998 a meta-analysis was performed to assess the biologic activity of marimastat in 415 patients in 6 clinical trials (2 each in patients with advanced colorectal and ovarian cancer, 1 each in patients with pancreatic and prostate cancer).[125] Biologic effect was defined as less than 0% (full) or less than 25% (partial) rise in the respective tumor marker over 28 days and was not based on traditional oncologic end points such as tumor progression documented radiographically. The analysis revealed that marimastat exerts a full or partial biologic effect at dosages greater than 20 mg/day (p=0.01, Cochran-Mantel-Haenszel test).[125] The dose-limiting toxicity was a time-dependent and dose-dependent musculoskeletal triad (arthralgia, myalgia, tendinitis). Other toxicities in these trials were ascites, disseminated carcinoma, chills, cholangitis, dizziness, dyspnea, edema, fatigue, fever, gastrointestinal (anorexia, nausea, vomiting, diarrhea, constipation), gastrointestinal hemorrhage, headache, heartburn, hepatic toxicity, hypercalcemia, hyperglycemia, rash, and shortness of breath.[117, 125, 126]

In a phase I trial of 12 patients with advanced lung cancer, the maximum tolerated dosage was 50 mg twice/day, with dose-limiting toxicity being inflammatory polyarthritis.[117] The terminal half-life, Vdss/F, and ClT/F in these patients were 4.9 ± 2.3 hours, 224.8 ± 98.6 L, and 5278 ± 3323 ml/minute, respectively. Eight patients were withdrawn for progressive disease (by standard criteria); the remaining four had stable disease and eventually were withdrawn due to toxicity. No changes in plasma MMP-2 and MMP-9 were observed.

From the results of another clinical trial in 64 patients with advanced pancreatic cancer, a dosage of 5, 10, or 25 mg twice/day was suggested since the rate of rise of tumor marker (CA 19/9) levels was less than with other dosages of marimastat.[126] This study investigated the early biologic activity of marimastat and compared changes in CA 19/9 levels before the study with those after the first 28 days of marimastat therapy. Of 30 patients who received marimastat for longer than 28 days, 33% developed musculoskeletal events (pain, stiffness, tenderness), and 50% of them required dosage reduction. A dose-dependent increase in Cmax was observed at all dosages except 10 mg once/day. This study was conducted before the approval of gemcitabine; published results state that comparison trials with marimastat are continuing in both the United States and Europe.[126]

Preliminary data of marimastat were presented for several cancers: in 555 patients with small cell lung cancer, 162 with glioblastoma multiforme or gliosarcoma, and 11 with metastatic breast cancer in combination with doxorubicin and docetaxel.[127-129] A placebo-controlled trial was conducted in patients with small cell lung cancer who responded to first-line chemotherapy.[127] Overall, median survival was 9.5 months, although there appeared to be no advantage to marimastat in overall response (using standard criteria). A complete response or partial response was observed in 32.5% (90/277) or 62.8% (174/277) of patients treated with marimastat 10 mg twice/day versus 32.4% (90/278) or 66.2% (184/278) of patients treated with placebo. The main toxicity was musculo-skeletal and required dosage modification and withdrawal from treatment in 40.8% (113/278) and 46.2% (128/278) of patients, respectively. In the placebo-controlled trial in patients with glioblastoma multiforme or gliosarcoma who had response to first-line therapy (either radiotherapy or surgery), marimastat 10 mg twice/day did not improve survival compared with placebo.[128] A small subset of patients chose to continue and received procarbazine-lomustine-vinblastine (PCV) chemotherapy in addition to the original randomized treatment. A survival benefit was seen in 27 patients who received the combination of marimastat and PCV. Musculoskeletal toxicities led to dosage reductions. A 75% response rate was reported in 11 patients treated with a combination of doxorubicin and docetaxel plus marimastat 20 mg twice/day.[129] Toxicities reported were known toxicities of either chemotherapeutic agents or marimastat as a single agent. A less myelotoxic regimen of doxorubicin and docetaxel is being explored.[129]

A study with marimastat in non-small cell lung cancer was terminated when it failed to show clinical benefit, although a trial in patients with resected pancreatic cancer continues.[130] Long-term follow-up of a study in patients with gastric carcinoma suggests a survival benefit for marimastat versus placebo.[130]

Results with marimastat in clinical trials are mixed. It appears that benefits are more noticeable in long-term trials with adjunct chemotherapy than in single-agent trials. A potential criticism of most of these trials is the soft end point of biologic effect versus the harder end point of disease progression by standard criteria (radiography). Another concern is dose-dependent musculoskeletal toxicity in most patients who continued marimastat long term, which is how MMP inhibitors would be administered. The agent did not change total or active plasma MMP-2 or -9 consistently in any study.

Prinomastat

A nonpeptidic hydroxamate, AG-3340 is a selective MMP inhibitor with in vitro activity (dissociation constant of the enzyme-inhibitor complex [Ki]) against MMPs-1, -2, -3, -9, -13, and -14 at concentrations less than 0.1 ng/ml.[121, 131] Preclinically, it had greatest efficacy in a colon cancer tumor model when a minimum plasma concentration was maintained, versus targeting an AUC0-24 or Cmax.[132] This oral MMP inhibitor has linear pharmacokinetics at doses up to 200 mg (100 mg twice/day) and low plasma protein binding (69%) in patients with advanced cancer.[132] When combined with mitoxantrone given every 3 weeks and continuous prednisone, the Cmax of AG-3340 25 mg twice/day was not affected and was either 513 ± 264 ng/ml after prednisone or 554 ± 295 ng/ml after prednisone and mitoxantrone.[120] The terminal half-life was 2-3 hours, with Tmax within 1 hour after oral dosing.[121]

Patients with breast cancer randomized to receive AG-3340 5 or 25 mg twice/day had no response, although those with low plasma vascular endothelial growth factor (VEGF) and urine pyridinoline levels tended to have stable disease after 2 months of treatment.[133] The addition of AG-3340 5 or 10 mg twice/day to a combination of mitoxantrone and prednisone did not enhance efficacy in patients with chemo-therapy-naïve hormone-refractory prostate cancer (HRPC).[134] In patients with chemotherapy-naïve non-small cell lung cancer, the addition of AG-3340 5, 10, or 15 mg twice/day to a combination of paclitaxel and carboplatin did not enhance efficacy. Adverse effects of AG-3340 are reversible musculoskeletal symptoms (arthralgia, joint swelling, joint stiffness, myalgia, tendinous contracture) and neuropathy.[121, 131, 133-135]

Clinical trials of AG-3340 combined with standard chemotherapy in HRPC and non-small cell lung cancer were halted since primary efficacy end points were not achieved.[136] Although AG-3340 was meant to be a specific MMP inhibitor with an improved pharmaco-kinetic profile and picomolar Ki values, the compound has limitations, notably musculo-skeletal symptoms. Its future appears to be in early intervention and potentially in combination with chemotherapy or radiation in other carcinomas.

BAY 12-9566

Another MMP inhibitor, BAY 12-9566, is an oral nonpeptide biphenyl and has activity against MMPs-2, -3, and -9, with Ki values of 4.5-123.7 ng/ml.[112, 113] In a phase I clinical trial, 21 patients with solid tumors were enrolled to 47, 28-day courses of BAY 12-9566.[137] The maximum tolerated dosage was not determined since steady-state concentrations increased less than dose-proportionally with total daily dose and were within expected biologically relevant levels. Toxicities were abdominal pain, alopecia, anemia, anorexia, bruising, diarrhea, dizziness, fatigue, headache, hepatic function abnormalities, hyperglycemia, nausea, numbness, dose-dependent thrombocytopenia, upper respiratory infections, ureteric obstruction, and vomiting.[112, 113, 137] Plasma MMP-2 (pro-MMP-2, and pro-MMP-2 complexed with TIMP-2), MMP-9 (pro-MMP-9, and pro-MMP-9 complexed with TIMP-1), TIMP-2 levels (TIMP-2, and TIMP-2 complexed with active MMPs), and active MMP-9 levels were assessed with respect to time and steady-state BAY 12-9566 plasma concentrations.[137] After 29 days of treatment, relative changes in MMPs-2 and -9 from baseline were unchanged (p=0.66 and p=0.59, respectively); however, the percentage change in TIMP-2 did reach statistical significance (p=0.046), increasing with increasing daily doses. Active MMP-9 was below the limits of quantification for each sample.

Similar results were reported in later phase I clinical trials.[112, 113] Stable disease was the best response in 62% of patients receiving treatment.[113] Due to saturable absorption, 800 mg twice/day was determined to be the acceptable phase II starting dosage. The terminal half-life was estimated to be 60.7 hours based on the ratio of C24hr:Css.[112] Plasma protein binding of BAY 12-9566 was 99.99%.[113] The agent had no effect on potential angiogenic surrogate markers plasma VEGF, plasma basic fibroblast growth factor (bFGF), urinary deoxypyridinoline, and urinary pyridinoline.[112] It increased disease progression and mortality in patients with small cell lung cancer compared with placebo. As a result, all trials with this compound were halted.[138, 139]

MMI270(B)

This oral, nonpeptidic, hydroxamate MMP inhibitor has in vitro activity against MMPs-1, -2, -3, -9, and -13, with IC50s of 2.4-19.7 ng/ml.[118, 119] Seventeen patients were enrolled in a phase I study to determine the effect of food on the pharmacokinetics of MMI270(B) 150, 400, and 600 mg.[119] The AUC0-8 hr did not change, although Cmax was decreased and Tmax was increased when the agent was administered after a light breakfast. To date, there is no recommendation with regard to administering the compound with food.

In a phase I study conducted in 92 patients with various cancers, the maximum tolerated dosage was 300 mg twice/day due to a dose-dependent rash experienced by 19.6% of patients.[118] Other toxicities were a musculo-skeletal syndrome (arthralgia, mylagia, tendinitis) in 42.4% of patients and nausea in 16.3%. A total of 19 patients (20.7%) had stable disease at 3 months and continued MMI270(B) for up to 406 days. Absorption was rapid, with a median Tmax of 0.58 hour, and half-life was short (range 0.6-7.6 hrs, median 1.6 hrs). Both Cmax and AUC were linear with dosages from 50 mg/day up to 600 mg 3 times/day. Several end points were assessed with direct measurements (MMP and TIMP levels) and indirect measure-ments (bFGF, collagen breakdown products, lysozymal proteases, serine peptidases, TNF-alpha convertase, vascular cell adhesion molecule-1, VEGF). Of these markers, only increases in bFGF, MMP-2, MMP-9, TIMP-1, and TIMP-2 were correlated with Cmax (p=0.02 for MMP-2, p=0.048 for MMP-9) and AUC (p=0.03 for bFGF, p=0.0004 for MMP-2, p=0.002 for TIMP-1, p=0.02 for TIMP-2). Urinary excreted collagen breakdown products were decreased at 2 weeks (pyridinoline with Cmax p=0.01 and AUC p=0.04; deoxypyridinoline with Cmax p=0.02) but rebounded by 4 weeks.

Preliminary reports from a phase I trial of MMI270(B) (various dosages) in combination with 5-fluorouracil (5-FU) and folinic acid in 20 patients with colorectal cancer indicated that this regimen was tolerable.[140] Adverse effects were similar to those when the agent was administered alone. Ten percent of patients had partial responses, and 50% had stable disease as their best response. Although the agent showed positive outcomes in the phase I trial, it is a broad-spectrum MMP inhibitor that may have long-term musculoskeletal complications (hypothesized to be due to inhibition of MMP-1), similar with those that appear to plague marimastat. Although the phase I open-label study with 5-FU and folinic acid appears promising, such a study would have to be conducted in a double-blind, randomized fashion looking for clinical end points, rather than biologic end points.

BMS-275291

An oral nonhydroxamate MMP inhibitor, BMS-275291, showed in vitro activity against a wide range of MMPs, with IC50s less than 20 ng/ml for MMPs-2 and -9.[114, 141] The compound was designed to spare sheddases, which are closely related to the metalloprotease family of enzymes and are thought to be responsible for musculoskeletal adverse effects of broad-spectrum MMP inhibitors.[115, 141] In 40 healthy subjects, no changes in collagen breakdown product excretion and no dose-limiting toxicity were discerned.[115] In 44 patients with cancer, BMS-275291 was well tolerated; toxicities in the phase I trial included arthralgia, subcapsular cataracts, dyspnea, headache, hepatic toxicity (elevated aspartate aminotransferase), myalgia, rash, and tenosynovitis.[141] The phase II dosage was determined to be 1200 mg/day since trough plasma levels of BMS-275291 exceeded in vitro IC90s of MMPs.[114, 141]

Two studies in patients with cancer and in healthy subjects revealed different pharmaco-kinetics for BMS-275291.[114, 115] The agent has free sulfhydryl groups that form disulfides in vivo. The pharmacokinetics of the parent com-pound is dose proportional for dosages up to 1200 mg/day in healthy subjects, and 2400 mg/day in patients with cancer with respect to Cmax; in healthy subjects, exposure is nonlinear, with a less than proportional increase in patients with cancer. An almost opposite phenomenon occurs with regard to the total drug (BMS-275291 plus reducible disulfides). The pharmacokinetics reveal nonlinearity with total drug AUC in healthy subjects; this relationship is linear in patients with cancer. Plasma protein binding of BMS-275291 is between 46% and 77%.[114]

Metastat

By eliminating the dimethylamino, methyl, and hydroxy functionalities on the basic tetracycline structure to produce metastat, the antimicrobial properties of the molecule were eliminated while MMP inhibitory properties were retained.[142] The proposed mechanism of action for metastat, 6-demethyl-6-deoxy-4-dedimethylaminotetracycline, is pleotropic and includes inhibition of MMPs due to divalent cation chelation of zinc at the active site of the enzyme, downregulation of production of pro-MMPs, inhibition of oxidative activation of pro-MMPs, increase in degradation of pro-MMPs, induction of apoptosis, inhibition of production of secretory nonpancreatic phospholipase A2, inducible nitric oxide (NO) synthase and NO, inhibition of production of TNF-alpha and IL-8, and reduction of the expression of a serine proteinase (trypsinogen-2).[104, 142-154] Metastat induces potent MMP inhibition of the 2, 9, and 14 isozymes.[40, 99, 155] It may have anti-angiogenic and antimetastatic activity, as evidenced by its cytotoxic, antiinvasive, and MMP inhibitory properties.

In a phase I clinical trial conducted at the National Cancer Institute, the maximum tolerated dosage was 70 mg/m2/day administered orally; dose-limiting toxicity was phototoxicity.[116] Disease stabilization for longer than 40 months, 8 months, and 6 months were seen in hemangioen-dothelioma, Sertoli-Leydig cell tumor, and fibrosarcoma, respectively. Non-dose-related toxicities were anemia, anorexia, constipation, dizziness, elevated liver function tests, fatigue, fever, headache, heartburn, nausea, vomiting, neurotoxicities, and three cases of drug-induced lupus erythematosus. Metastat has nonlinear pharmacokinetics, thought to be due to saturable absorption. The median single-dose half-life, ClT/F, and Vd/F were 56.7 hours, 0.0077 L/hr/kg, and 0.63 L/kg, respectively. Plasma protein binding is 94.5%, with most binding due to albumin (MA Rudek, unpublished data, 2001). In assessing potential pharmacodynamic markers, there was a statistically significant relationship between changes in plasma MMP-2 levels and cumulative doses of metastat when patients with progressive disease were compared with those with stable disease or toxicity (p=0.042).

Metastat was administered in another phase I clinical trial.[156] This trial has accrued 26 patients to date, with enrollment continuing.[157] Toxicities include anemia, asthenia, fatigue, photosensitivity, and skin hyperpigmentation.[156, 158, 159] In November 2000, it was noted that one patient with leiomyosarcoma had stable disease for 9 months.[158]

A third phase I clinical trial was conducted in 18 patients with Kaposi's sarcoma related to acquired immunodeficiency syndrome (AIDS) through the AIDS Malignancy Consortium. Patients were administered metastat 25, 50, or 70 mg/m2/day.[160] Ninety-four percent of these patients were receiving antiretroviral therapy and had failed earlier Kaposi's sarcoma treatment. The median duration of metastat therapy was 9.5 weeks, with one patient remaining in the study as of this writing. Nine patients terminated therapy for toxicity, and eight terminated for progressive disease. The dose-limiting toxicity was photosensitivity; other toxicities were arthralgia, fatigue, fever, headache, myalgia, nausea, pain, and pruritus. The overall response rate was 44%, with one complete response and seven partial responses. Due to the response rate in this trial, a future phase II clinical trial is planned in patients with Kaposi's sarcoma, with a dosage of 25 mg/m2/day. In addition, a phase I-II clinical trial in patients with high-grade anaplastic astrocytoma, anaplastic oligodendroglioma, or glioblastoma multiforme is being conducted through New Approaches to Brain Tumor Therapy.[157]

Section 4 of  4

Source

Matrix Metalloproteinase Inhibitors
from Pharmacotherapy

Conclusion

The MMP inhibitors offer a new approach to the treatment of cancer. A potential limitation of narrow-spectrum MMP inhibitors' clinical usefulness may be their complex interaction with a wide variety of substrates that have unknown effects in cancer. For example, the agents may affect the release of TNF-alpha by MMPs-14 and -17. If this occurs, TNF-alpha will not be available to exert its biologic effect.[11] Limitations of broad-spectrum MMP inhibitors include musculoskeletal adverse effects that can hamper long-term therapy.

Synthetic MMP inhibitors under development have been plagued with problems similar to those of other noncytotoxic therapies. They do not have antiproliferative properties and therefore are not expected to result in responses assessed by traditional cytotoxic clinical trials.[161] Instead, stable disease is accepted as the best response. The traditional paradigm of randomized phase III clinical trials that pits the test agent against standard therapy and looks for equality or superiority may not occur with these compounds unless the end points of the trials change.

Marimastat, prinomastat, and BAY 12-9566 were assessed in phase III clinical trials using the current paradigm, and most failed. Marimastat showed hints of efficacy when given early in disease progression and in combination with cytotoxic chemotherapy. Metastat, MMI270(B), and BMS-275291 are still in early stages of development and may benefit from knowledge gained from MMP inhibitors that failed in late stages of development. Preliminary results with MMI270(B) and BMS-275291 look promising, although the potential for long-term toxicities plaguing marimastat are of concern since both are broad-spectrum inhibitors. The agent BMS-275291 is being developed rationally in an attempt to determine dosages based on preclinical data rather than maximum tolerated dosage. Metastat's clinical development may be hastened by the high degree of photosensitivity, drug-induced lupus, and sideroblastic anemia in the small population in phase I clinical trials. These effects ultimately may be problematic when assessed in larger populations. If any MMP inhibitor proves efficacious in a phase III clinical trial and is approved by the Food and Drug Administration, its role will more than likely be in combination with more aggressive therapy to keep cancer either in check or in a palliative setting.

 

Funding Information
 

Funded in part by the Office of Minority Health, National Institutes of Health, Bethesda, Maryland.

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