Matrix metalloproteinase inhibitors*/an emphasis on gastrointestinalmalignancies

Ian Chau, Anne Rigg, David Cunningham *

Gastrointestinal Unit, Department of Medicine, Royal Marsden Hospital, Downs Road, Sutton, London, Surrey SM2 5PT, UK

Accepted 9 February 2002

* Corresponding author. Tel.: �/44-208-661-3156; fax: �/44-208-643-9414.

E-mail address: dcunn@icr.ac.uk (D. Cunningham).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

2. The importance of proteases for tumour growth and metastasis . . . . . . . . . . . . . . . . 152

2.1. The matrix metalloproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

2.1.1. Structure of matrix metalloproteinases . . . . . . . . . . . . . . . . . . . . . . . . 154

2.1.2. Genomic and post transcriptional control of MMPs . . . . . . . . . . . . . . . . 155

2.2. Tissue inhibitors of metalloproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

2.2.1. Structure of TIMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

2.2.2. Interaction between MMPs and TIMPs . . . . . . . . . . . . . . . . . . . . . . . 159

2.2.3. Other functions of TIMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

3. Therapeutic approaches to alter the MMP/TIMP balance in cancer . . . . . . . . . . . . . . . 161

3.1. Synthetic MMP inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

3.2. Gene therapy using TIMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

4. Problems encountered in designing MMPI clinical trials . . . . . . . . . . . . . . . . . . . . . 164

4.1. Tools to assess efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

4.1.1. Biomarker assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

4.1.2. Histological assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

4.1.3. Non invasive imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

4.2. Clinical trial design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

5. Matrix metalloprotenase inhibitors in clinical development . . . . . . . . . . . . . . . . . . . . 167

5.1. Batimastat (BB-94) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

5.2. Marimastat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

5.2.1. Phase II studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

5.2.2. Phase III studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

5.2.3. Combination with other cytotoxic and angiogenesis therapy . . . . . . . . . . . . 169

5.2.4. Adjuvant therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

5.2.5. Safety profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

5.3. BAYER 12-9566 (Tanomastat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

5.4. MMI270 (CGS27023A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

5.5. AG3340 (Prinomastat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

5.6. COL-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

5.7. BMS-275291 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Critical Reviews in Oncology/Hematology 45 (2003) 151�/176

www.elsevier.com/locate/critrevonc

1040-8428/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 1 0 4 0 - 8 4 2 8 ( 0 2 ) 0 0 0 1 5 - X

Abstract

Gastrointestinal malignancies are the commonest sites of human cancer collectively. Improved understanding of tumour biology

in the last few decades has allowed the identification of cellular pathways responsible for the autonomous growth and replication in

cancer cells. There is considerable preclinical evidence implicating matrix metalloproteinases (MMPs) in cancer dissemination and

tumour angiogenesis. Effective MMP inhibitors (MMPIs) may, therefore, hold an important key in the treatment of gastrointestinal

cancers. MMPIs are cytostatic agents and traditional values of tumour regression may not be the best measures of treatment

efficacy. Biological correlation studies are increasingly being incorporated into the early development of these agents, but many of

these studies lack preclinical validation and are often chosen on availability rather than biological plausibility. Disappointing results

with many MMPIs that have entered phase III testing so far would prompt for identification of reliable surrogate biomarkers and

incorporation of functional imaging in the clinical development of matrix metalloproteinase inhibitors in gastrointestinal

malignancies. In this review, the integral part in which MMPs are involved in cancer growth and metastases will be presented.

This is then followed by a discussion of the challenges that clinicians are facing in assessing the efficacy of MMPIs and finally a

review of the clinical studies of the synthetic MMPIs in development.

# 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Matrix metalloproteinases; Matrix metalloproteinase inhibitors; Metastasis; Angiogenesis; Cytostatic agents

1. Introduction

Gastrointestinal malignancies are the commonest sites

of human cancer collectively. Colorectal, oesophageal,

gastric and pancreatic cancers are among the top ten

cancer killers in the world accounting for over 2.5

million cases in 2000 [1]. Five year survivals are poor

in these diseases ranging from 40 to 60% in colorectal

cancers to less than 5% in pancreatic cancers [2]. Newer

cytotoxic drugs such as irinotecan, oxaliplatin, gemci-

tabine, taxanes and oral fluopyrimidines have at best

made a very modest (if any) impact on the survival of

these patients. There is, therefore, an urgent need to

identify novel agents to complement these tumoricidal

drugs. Improved understanding of tumour biology in

the last few decades has allowed the identification of

cellular pathways responsible for the autonomous

growth and replication in cancer cells. Several strategies

have emerged to target these abnormal processes

therapeutically. These targets include tumour angiogen-

esis, genetic mutation encoding signal transduction

pathway, overexpression of membrane receptors (e.g.

epidermal growth factor receptor family) and molecules

involved in tumour growth and metastasis. There is

considerable preclinical evidence implicating matrix

metalloproteinases (MMPs) in cancer dissemination

and tumour angiogenesis. Effective MMP inhibitors

(MMPIs) may, therefore, hold an important key in the

treatment of gastrointestinal cancers. In this review, the

integral part in which MMPs are involved in cancer

growth and metastases will be presented. This is then

followed by a discussion of the challenges that clinicians

are facing in assessing the efficacy of MMPIs and finally

a review of the clinical studies of the synthetic MMPIs in

development.

2. The importance of proteases for tumour growth and

metastasis

An integral feature of cancer is local invasion

accompanied by spread to distant sites: the process of

metastasis. Much work has been done to investigate the

mechanisms by which the metastatic process occurs.

Steps in this process are the escape of malignant cells

from the primary tumour, entry of the cells into the

vascular or lymphatic circulation (intravasation), survi-

val and transport in the circulation, escape of the cells

from the circulation (extravasation) and the growth of

cells at the new site to form a secondary tumour.

Mechanisms of avoiding immune destruction are re-

quired at all stages.

Proteolysis in the tumour environment degrades the

components of the extracellular matrix (ECM) and

basement membranes, allowing tumour cells to invade.

The key proteases of the ECM are the serine proteases

(plasmin) and the MMPs as these enzymes function at

neutral pH. There is considerable evidence that in-

creased levels of plasmin and MMPs occur in tumours

and that the level directly correlates with the tumour

stage [3�/8]. Rat embryo fibroblasts over-expressing

MMP-9 caused more metastases in nude mice than the

unmodified cells [9]. Likewise, rat bladder carcinoma

cells over-expressing MMP-2 produced an increased

area of lung metastases when injected into nude mice

[10].

As it became obvious that proteases were crucial for

tumour metastasis it was of interest to identify the

specific cellular components that were producing these

enzymes. Initially, it was expected that the tumour cells

alone would produce the proteases. However, a different

picture emerged when human tumour specimens were

studied. For human colon cancer, urokinase plasmino-

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176152

gen activator (uPA) was expressed by stromal fibro-

blasts around the tumour whereas uPA receptor (uPAR)

was expressed by cancer cells adjacent to the stroma and

tumour-infiltrating macrophages [11,12]. Plasminogen

activator inhibitor 1 (PAI-1) was shown to be present in

endothelial cells in tumour stroma, but not endothelial

cells of normal tissue [13]. It has been proposed that this

PAI-1 distribution may protect the cancer tissue from

uPA-mediated matrix degradation. This fits with the

observation from clinical studies that high serum PAI-1

levels correlate with a poor prognosis [5,14].

A very similar pattern is seen with MMP expression in

colorectal tumours. MMP-2 is restricted to fibroblast-

like stromal cells, as are MMP-3 and membrane-type 1-

matrix metalloproteinase (MT1-MMP). MMP-9 is pre-

sent in macrophages at the invasive edges of the tumour

and MMP-7 is found in the cancer cells themselves [15�/

17]. There is also evidence that tissue inhibitors of

matrix metalloproteinases (TIMPs) are overexpressed

by some tumours and that, like PAI-1, they may be

protecting the cancer from excessive protease activity.

Pancreatic carcinomas have been shown to over-express

several MMPs. MMP-2 and MMP-9 were expressed in

75% of pancreatic tumour samples [18] and the level of

MMP-2 has been directly correlated with the degree of

disruption of the basement membrane [19]. MMP-2 was

found to be present in both tumour epithelial cells and

the cellular elements of the stroma. MT1-MMP, MMP-

7, TIMP-1 and TIMP-2 expression was confined to the

tumour epithelium [20]. Ultimately, it is the net balance

between the proteases and their inhibitors that is critical

to their effect on the environment.

It would appear that cancer cells recruit stromal cellsvia growth factors and control the specific proteases that

they produce. Therefore, the ability to invade is a result

of the properties of the stromal cells as well as the

cancerous cells themselves.

There is additional work with knock-out mouse

models. Lewis lung carcinoma and B16F10 melanoma

cells injected into MMP-2-deficient mice showed re-

duced lung metastasis, although, the tumour cellsthemselves did produce some MMP-2 [21].

The overall conclusion from these studies is that the

majority of cancers over-express a variety of proteases

and inhibitors. It is the stromal cells within a tumour

that are integral to the production of proteases under

the control of the tumour cells. The involvement of the

stromal cells may explain the pattern of organ metas-

tasis for a particular tumour and why there is often alatent period between the appearance of a primary

tumour and the development of metastases. Fig. 1

shows a schematic representation of the metastatic

process and the points at which MMPs are involved.

2.1. The matrix metalloproteinases

The MMPs are a family of zinc-dependent endopep-

tidases that degrade various components of the ECM.

Fig. 1. A schematic representation of the metastatic process and the points at which MMPs are involved. Adapted from Chambers and Matrisian

[147].

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 153

Twenty MMPs have been identified to date. Theseenzymes function at physiological pH and are either

secreted or membrane-bound. There are three main

groups of MMPs: collagenases that degrade fibrillar

collagens at a specific locus of the helix, stromelysins

that degrade proteoglycans and glycoproteins, and

gelatinases that degrade non-fibrillar and denatured

collagens (gelatins). The family members can be cate-

gorised depending on substrate specificity, cellularsources and inducibility (Table 1).

2.1.1. Structure of matrix metalloproteinases

All the MMPs share a basic structure. This consists of

a signal peptide, a propeptide domain that has to becleaved to ensure enzyme activity, a catalytic domain

with a zinc-binding site and a C-terminal domain. The

MMP family members differ in the presence or absence

of additional domains that are involved in activities such

as membrane binding, inhibitor binding and substrate

specificity (Fig. 2).

The signal peptide sequence directs the translated

protein to the endoplasmic reticulum. The N-terminalpropeptide domain is 77�/87 amino acids in length and

contains a conserved amino acid sequence PRCGXPDV

(proline/arginine/cysteine/glycine/ (valine or aspara-

gine)/proline/aspartic acid/valine). The cysteine residue

is unpaired and able to interact with the zinc atom at the

catalytic site of the enzyme. This cysteine�/zinc interac-

tion maintains the proenzyme in an inactive state.

MMPs share a common catalytic domain containing

the HEYGH motif (histadine/glutamic acid/phenylala-

nine, leucine, or isoleucine/glycine/histadine) responsible

for binding zinc that is essential for functional enzymatic

activity.

Specific MMPs have additions or deletions to this

basic structure as illustrated in Fig. 2. For example both

gelatinases MMP-2 and MMP-9 have a fibronectin-like

region inserted into the catalyic domain and MMP-9 has

an additional collagen-like region situated at the C-

terminal end of the catalytic domain. These additional

domains are thought to be involved in binding of

gelatins as they resemble the gelatin-binding domains

of fibronectin.

The C-terminal domain appears to mediate binding of

the enzyme to its substrate although it does not actively

cleave in the absence of the catalytic domain. Also the

C-terminal domain appears to be involved in binding of

the TIMPs as loss of this domain prevents proMMP-2

binding to TIMP-2 and MMP-2 binding to TIMP-1 or

TIMP-2 [22,23]. The functions of TIMPs will be

Table 1

MMPs and their cellular sources and major substrates

MMP Other names Cell sources Major substrates

1 Collagenase-1 Connective tissue cells Collagen I, II, III, VII, VIII, X, gelatins, aggrecan

2 Gelatinase A Most cell types and tumour cells Gelatin I, II, III collagen IV, V, VII, X, fibronectin, elastin, plasminogen

3 Stromelysin-1 Connective tissue cells, macrophages Proteoglycan, fibronectin, laminin, collagen III, IV, V, IX, gelatin I, III,

IV, V, activates procollagenase

7 Matrilysin, PUMP1 Immature monocytes, measngial Gelatin I, III, IV, V, proteoglycan, fibronectin

8 Collagenase-2 Neutrophils, chondrocytes, synovial

fibroblasts, endothelial cells

Collagen I, II, III, aggrecan core protein

9 Gelatinase B Monocytes, connective tissue cells Gelatin I, V, collagen IV, V

10 Stromelysin-2 Fibroblasts, macrophages Gelatin I, III, IV, V, collagen III, IV, V, activates procollagenase

11 Stromelysin-3 Stromal cells of tumours Serine protease inhibitors such as a1-proteinase inhibitor and a1-

antitrypsin

12 Metalloelastase Macrophages Elastin, fibronectin

13 Collagenase-3 Connective tissue cells, tumour cells Collagen I, II, III, IV, X, XI, gelatin, laminin, tenascin, aggrecan,

fibronectin

14 MT1-MMP Malignant and stromal cells within a

tumour

Pro-MMP2, proteoglycan, fibrillar collagen, gelatin, fibronectin,

laminin, vitronectin, aggrecan

15 MT2-MMP Collagen I, II, III, gelatin, fibronectin, laminin, vitronectin, aggrecan,

ProMMP2

16 MT3-MMP Pro-MMP2, fibronectin. Collagen III

17 MT4-MMP ND

18 Collagenase 4 ND

19 Placenta, lung, panreas, ovary, spleen,

intestine

ND

20 Enamelysin Odontoblastic cells Dental amelogenin

21 XMMP (xenopus) ND

22 CMMP (chicken) ND

23 MT5-MMP Brain, kidney, pancreas, lung ProMMP2

Note the MMPs initially described as 4, 5 and 6 were found to be identical to other family members, hence 4, 5 and 6 are redundant nomenclature.

ND, not determined.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176154

discussed in Section 2.2. Four membrane-bound MMPs

have been described. They differ from the secreted

MMPs in that they have a transmembrane domain atthe carboxy terminus and a recognition site for the

furin-like enzymes at the end of the propeptide domain.

Therefore, membrane-bound MMPs can be activated in

the intracellular compartment by Golgi-associated

furin-like proteases [24]. Of the four, MT1-MMP has

been most extensively studied [25]. MT1-MMP is over-

expressed in a variety of human tumours and its level

correlates with the activation rate of MMP-2 in thetissues.

2.1.2. Genomic and post transcriptional control of MMPs

The genes encoding MMPs are not usually continu-

ously expressed, but become transcriptionally activewhen tissue remodelling is required for physiological

or pathological reasons. MMP gene transcription can be

induced by a variety of factors including cytokines,

growth factors, and exposure to components of the

ECM (Table 2). Intracellular signal transduction trig-

gered by the cell surface interactions with growth

factors/cytokines, other cells or the ECM leads to the

phosphorylation of transcription factors via the mito-

gen-activated protein kinase (MAPK) and serine/threo-

nine kinase pathways.

One of the most stringent levels of control over MMP

enzymatic function is the activation of the proenzyme

that takes place in the extracellular space. In all cases

this involves the ‘cysteine-switch’ hypothesis of cleavage

of a proportion of the propeptide domain so that the

cysteine�/zinc interaction is disrupted and a H2O mole-

cule can interact with the zinc atom. This then allows

auto-catalytic cleavage of the remainder of the propep-

tide domain. The majority of proMMPs are activated by

plasmin. Pro-urokinase plasminogen activator (pro-

uPA) and pro-tissue plasminogen activator (pro-tPA)

are secreted by cells in a latent form and bind to cell

Fig. 2. Domain structure of the members of the matrix metalloproteinase family. The conserved motif of the propeptide domain, PRCGXPD, is

involved with maintaining latency of the pro-enzyme. The zinc-binding motif of the catalytic domain is HEYGH. MMPs 2 and 9 have an additional

fibronectin-like domain in the catalytic domain, and MMP9 has another extra domain called the collagen-like region.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 155

Fig. 3. The ‘cysteine-switch’ hypothesis of matrix metalloproteinase latency and activation. The cysteine in the propeptide domain links to zinc

causing latency of the enzyme. In the presence of physical agents such as SDS (sodium dodecyl sulphate), hypochlorous acid (HOCl) or

aminophenylmercuric acetate (APMA) disruption of cysteine-zinc bond occurs so that the zinc atom becomes exposed. Alternatively, proteases such

as plasmin cleave the propeptide domain allowing hydrolysis of the zinc. The enzyme undergoes auto-catalytic cleavage to remove the propeptide

domain permanently.

Table 2

Factors inducing and repressing MMP expression

Type Inducing agents Repressive agents

Cytokines and growth factors Epidermal growth factor

Fibroblast growth factor b

Interferon aInterleukin 1a Platelet-derived growth factor

Tumour necrosis factor-a

Transforming growth factor-bInterferon b, g

Chemical agents Colchicine

Mitomycin C

Prostaglandin E

Lipopolysacchariden

Cyclic AMP Organomercurials e.g. concanavalin A

Retinoic acid

Glucocorticoids

Oestrogen progesterone

Physical agents Heat shock

UV light

Factors at cell surface Cell fusion

Integrin receptor antibody

Iron

Urate, calcium pyrophosphate & hydroxyapatite crystals

Miscellaneous Viral transformation Oncogenes Adenovirus-5 E1a gene

Adapted from Woessner [145] and Goetzl et al. [146].

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176156

surface receptors. Once bound to the receptors they are

activated and able to convert extracellular plasminogen

into plasmin [26,27]. Plasmin is then able to cleave a

portion of the propeptide domain of proMMPs as

demonstrated in Fig. 3.

Unlike the other MMPs, MMP-2 appears to be

refractory to ‘classical’ activators such as plasmin and

is activated by a unique cell-mediated mechanism. A

series of experiments using recombinant MT1-MMP

immobilised on agarose beads to mimic the cell surface

showed that MT1-MMP binds TIMP-2 and in turn the

MT1-MMP/TIMP-2 complex acts as a receptor for

proMMP-2 [28]. Then an adjacent free MT1-MMP

molecule cleaves the propeptide domain from the

proMMP2/TIMP2/MT1-MMP complex. Thus when

levels of TIMP-2 are low some MT1-MMP molecules

form the complex with TIMP-2 and proMMP-2 whileother MT1-MMP molecules are free and able to activate

proMMP2. However, when TIMP-2 levels are high all

the MT1-MMP molecules bind TIMP-2 (and

proMMP2) so there are no adjacent free molecules to

activate the proMMP2. Therefore, activation of

proMMP-2 requires at least two MT1-MMP molecules,

one that functions as the receptor and another as an

activator (Fig. 4).There is evidence that MMP-13 is activated by MT1-

MMP and MMP-2. Kinetic studies show that MMP-13

does not bind to the C-terminal domain of TIMP-2

suggesting that the mechanism is different to that for

proMMP2 [29]. MT-MMPs are known to be activated

in the intracellular space by furin-like proteases.

2.2. Tissue inhibitors of metalloproteinases

To date, four TIMPs have been identified (Table 3).

TIMP-1 was first described in 1985 and its sequence

found to be identical to the growth factor erythroid-

potentiating activity [30]. Four years later TIMP-2 was

identified [31]. TIMP-3 was initially cloned from Rous

sarcoma virus transformed chick embryo fibroblasts [32]

and subsequently the human homologue has been

identified [33]. TIMP-4 has now been cloned fromhuman and murine genomes [34,35]. TIMP-1 and

TIMP-2 are secreted proteins located in the vicinity of

the cell surface and the ECM. TIMP-3 is unusual in that

it appears to be bound to components of the ECM.

These molecules are specifically able to inhibit the

activity of the MMPs. All four TIMPs can inhibit all

forms of active MMPs. In addition, TIMP-1 can bind to

Fig. 4. Activation of pro-MMP2. Pro-MMP2 forms a triple complex

with TIMP2 and MTI-MMP at the cell surface to aallow an adjacent

free MTI-MMP molecule to cleave toe propeptide domain of pro-

MMP2. If excessive TIMP2 is present pro-MMP2 cannot be activated

as all the MT1-MMP molecules have TIMP2 bound. Zn, MMP

catalytic domain; Hx, MMP hemopexin-like domain; N, TIMP2 N-

terminal domain; C, TIMP2 C-terminal domain.

Table 3

General characteristics of the tissue inhibitors of metalloproteinases

TIMP1 TIMP2 TIMP3 TIMP4

Messenger RNA

(kb)

0.9 1.1, 3.5 2.2, 2.5, 4.4 0.9, 1.4, 2.1,

4.1

Amino acids 184 194 188 195

Molecular

weight (kDa)

28 glycosylated 21 24 glycosylated 22

Complex forma-

tion

ProMMP9 ProMMP2 ProMMP2 �/ ECM ProMMP2

MMPs inhibited All All All All

Localisation Soluble Soluble ECM Soluble

Gene Xp11.23-11.4 17q2.3-2.5 22q12.1-13.2 3p25

Regulation Induced by TPA TGFb IL-1 & Il-6, serum

retinoic acid progesterone oncostatin M bFGF

EGF PDGF

Constitutive upregulated by

cAMP and retinoic acid

Induced by TPA dexamethasone

TGFb serum oncostatin M

Maximum mur-

ine

Ovary Placenta Kidney Heart

Tissue expres-

sion

Bone Brain

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 157

and stabilise the pro-enzyme form of MMP-9

(proMMP-9) and likewise TIMP-2 for proMMP-2.

2.2.1. Structure of TIMPs

Although these proteins have been described as

inhibitors for MMPs their role extends beyond this

with effects on cell morphology, enhanced growth of

certain cell types while being pro-apoptotic and anti-angiogenic for others. TIMP-2 is also implicated in the

activation of proMMP2.

TIMP proteins consist of 184�/195 amino acids.

TIMPs contain 12 conserved cysteine residues that

divide the protein into two distinct domains, each with

three internal disulphide-bonded loops. This six loop

structure confers stability to pH and temperature [36].

TIMPs consist of two domains: the N-terminal domain

that can bind to the active site of a MMP molecule and

the C-terminal domain that has the ability to bind to aregion of the C-terminal domain of certain MMPs [37].

2.2.2. Interaction between MMPs and TIMPs

The binding of a TIMP molecule to an activatedMMP is straightforward with the TIMP N-terminal

domain inhibiting the MMP active site. The inhibition is

a 1:1 stoichiometric relationship. It is estimated that

TIMP-1 has a high binding affinity Kd of 10�9�/10�11

M for most MMPs [38]. Binding constants and associa-

tion rate constants suggest that there is no accumulation

Fig. 5. The interactions between active MMP2 and TIMP1 or TIMP2. The upper panel illustrates the binding of TIMP1 to activate MMP2 in a 1:1

stoichiometric manner with the MMP catalytic domain interacting with the TIMP1 N-terminal domain thus blocking enzymatic activity. The lower

panel illustrates the in teraction between active MMP2 and TIMP2 showing that as well as the blockade of the MMP catalytic domain by TIMP2 N-

terminal domain, there an electrostatic interaction between the TIMP2 and MMP2 C-termini.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176158

of intermediate products and that the interaction is a

simple bimolecular collision. All the MMPs were

inhibited by the N-terminal domain of either TIMP-1

or TIMP-2 [37]. Mutants of TIMP-1 showed that therewas no one site within the TIMP-1 N-terminal domain

that is critical to the interaction, and suggested that

there are numerous sites of contact between MMP and

TIMP-1 [39]. Of interest is the observation that TIMP-2

inhibits MMP-2 more rapidly than TIMP-1. However, if

the C-terminus of the TIMP-2 molecule is deleted then

its ability to inhibit MMP-2 is reduced 4-fold. Also if

full-length TIMP-2 and MMP-2 interact in different saltconcentration, increasing ionic strength leads to decreas-

ing TIMP-2 inhibition, whereas salt concentrations have

no effect on TIMP-1, nor TIMP-2 lacking the C-

terminus [40]. This suggests that there is a charged

region at the C-terminal end of the TIMP-2 molecule

that enables docking of the TIMP-2 to MMP-2 in the

correct alignment (Fig. 5). There may also be electro-

static interactions between TIMP-2 and MMP-2 N-terminal domains but this phenomenon has not been

fully described. In addition to the charged C-terminus of

the TIMP-2 C-terminal domain, there have been ob-

servations that if the whole C-terminal domain is

removed from either TIMP-1 or TIMP-2 then the

inhibitory ability of these molecules for MMP-2 is

reduced. It is thought that the interaction between the

TIMP C-terminal domain and the MMP-2 C-terminaldomain is distinct from the TIMP-2-specific binding site

in the C-terminal domain of proMMP2 [41].

Recently, the crystal structure of the complex formed

between human MMP-3 and TIMP-1 has been discov-

ered [42]. The catalytic domain of an MMP is known to

have an active site cleft that is relatively flat on the left-

hand side, contains the catalytic zinc at the centre and

has a deep pocket on the right-hand side. When asubstrate approaches the catalytic domain of an MMP it

becomes fixed to the MMP by inter-main chain hydro-

gen bonds. Its scissile peptide bond carbonyl group is

then directed towards the catalytic zinc to be polarised.

In this protease-substrate encounter the water molecule

localised to the catalytic zinc is squeezed between the

two and attacks the scissile peptide of the substrate. As a

result the substrate decomposes into two fragments.The relationship between TIMP-2 and proMMP2/

MMP-2 has been the most extensively investigated as it

is known that TIMP-2 can bind to either the catalytic

domain active site or the C-terminal domain. Mutant

proMMP2 lacking a C-terminal domain cannot bind

TIMP-2 [41]. Therefore, when TIMP-2 binds to

proMMP2 it must be via the C-terminal domain as the

catalytic site is blocked by the propeptide domain.Recent mutagenesis studies demonstrate that TIMP-2

binds to the upper surface of the proMMP2 C-terminal

domain at the junction of hemopexin modules III and

IV [43]. ProMMP2 complexed to TIMP-2 via C-

terminal domain interactions can be activated by

organomercurials such as concavalin A. The active

MMP-2 still complexed to TIMP-2 only has 5�/10% of

the gelatinolytic activity of free activated MMP2 [41].The active MMP-2/TIMP-2 complex is in equilibrium

between the inhibited and non-inhibited forms of the

complex with a tendency toward inhibition [40]. There is

evidence that when the active site becomes available

after activation that the TIMP-2 is now in a position to

bind there resulting in a completely and irreversibly

inactivated complex. It is not yet clear whether both

TIMP-2 domains may interact with the two binding siteson MMP-2 simultaneously (Fig. 6). It is thought that the

interaction between TIMP-1 and proMMP-9 is of a

similar nature [44].

2.2.3. Other functions of TIMPs

There appears to be increasing evidence that TIMPs

have additional activities to MMP inhibition. TIMP-1

was originally identified as the growth factor witherythroid-potentiating activity that stimulates the

growth of erythroid precursor cells [30,45]. This function

of TIMP-1 is thought to be distinct from its activity as

an MMP inhibitor. TIMP-1 and TIMP-2 have been

shown to potentiate growth for a number of human cell

types in vitro, including aortic smooth muscle, skin

epithelia, gingival fibroblasts, Raji Burkitt’s lymphoma

and MCF-7 breast adenocarcinoma [46]. Overall thereported growth effects have been modest with only

1.5�/2.5-fold increase in proliferation as assessed by 3H

thymidine incorporation or DNA content. Overall the

reported growth effects have been modest with only

1.5�/2.5 fold increase in proliferation as assessed by 3H

thymidine incorporation of DNA content. A recent

study has demonstrated that a chimeric protein of

TIMP-1 and green fluorescent protein (GFP) binds tothe cell surface of MCF-7 breast adenocarcinoma cells,

but not HBL-100 breast epithelial cells. After 72 h of co-

culture the TIMP-GFP had localised to the nuclei of the

MCF-7 cells [47]. There has also been a suggestion that

TIMP-1 acts as an autocrine growth factor in the

fibrosis of systemic sclerosis [48].

TIMPs and synthetic protease inhibitors are able to

inhibit angiogenesis. In vitro there is a reduction ofendothelial cell proliferation in the presence of TIMP-1

and TIMP-2. In vivo, angiogensis is abrogated by

TIMP-1, TIMP-2 and TIMP-3. Studies include work

with normal endothelial cells in the chick yolk sac

membrane and endothelial cells in the context of a

tumour [49�/54]. TIMP-2 was shown to inhibit the

proliferation of cultured endothelial cells while the

protease inhibitor BB94 did not, suggesting thatTIMP-2 may be having effects beyond the role of

protease inhibition [52]. Kaposi’s sarcoma is a highly

vascular tumour and in vivo experiments demonstrated

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 159

that TIMP-2 is able to significantly reduce Kaposi’s

sarcoma angiogenesis [49].

An alternative explanation as to why TIMPs can limit

tumour growth as well as tumour invasion is that the

MMPs are known to proteolytically activate latent

growth factors in the ECM [55]. TGFa and IGF-II are

complexed with components of the ECM that renders

them inactive. Theoretically, TIMPs by inhibiting

MMPs would be reducing the level of active growth

factors in the extracellular space. It is thought that the

relationship between cells and an intact ECM exerts a

restriction on cell growth [56]. Therefore, inhibiting

degradation of the ECM may be preserving the growth

restriction signals. This gives another potential sphere in

which TIMPs could limit cell growth and proliferation.

It is possible that in addition to a limiting effect on cell

proliferation, the TIMPs are also having an effect on the

fate of cells by promoting or limiting apoptosis. A panel

of Burkitt’s lymphoma cell lines was examined for

TIMP-1 expression. It was noted that cell lines that

Fig. 6. The interactions between proMMP2 and TIMP2. TIMP2 and proMMP2 C-termini are able to bind. If the proMMP2 is now activated by

removal of the propeptide domain an equilibrium exists between full inhibition of the active MMP2 by TIMP2 and the free active MMP2 molecule.

The inhibited form of MMP2 is favoured.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176160

were TIMP1 positive were resistant to cold shock-

induced apoptosis. The resistance to apoptosis was

reversed with anti-TIMP-1 antibodies. TIMP-1 up-regulation induced expression of Bcl-XL but not Bcl-2

[57]. There have been several experiments with transfec-

tion of tumour cells with TIMP-1, TIMP-2 or TIMP-3

using a variety of delivery vectors. TIMP-3 has been

shown to be pro-apoptotic in melanoma, cervical

carcinoma and vascular smooth muscle cells [58,59].

TIMP-3-mediated cell death is independent of MMP

inhibitory activity as the synthetic inhibitor BB-94 doesnot have the same effect.

3. Therapeutic approaches to alter the MMP/TIMP

balance in cancer

The functions of the MMP enzymes and their

inhibitors TIMPs have been described. Given that

disturbances of the levels of the MMPs and TIMPsare implicated in tumour growth and metastasis it is

logical to attempt to correct the balance by reducing the

level of MMP and/or increasing the level of TIMP.

There are several potential points of intervention as

illustrated in Fig. 7. The majority of attention has been

focused on the inhibition of the MMP enzyme by

synthetic inhibitors or TIMP gene therapy and these

will be discussed in detail. There is some experimentalevidence of intervention in the earlier steps of the MMP

production pathway. For example, squamous cancer

cells transfected with an antisense expression construct

for the transcription factor E1AF produce less MMP-1,

MMP-3 and MMP-9 and this correlates with reduced

invasion by the tumour cells in vitro and in vivo [60].Another point of intervention is the use of synthetic

furin inhibitors to prevent the intracellular activation of

the MT-MMPs by furin-like enzymes [61].

3.1. Synthetic MMP inhibitors

One approach to limit the activity of the MMPs in

cancer has been the development of synthetic inhibitors.

The first generation of MMPIs was designed to mimicthe part of the collagen peptide sequence that is initially

cleaved by MMP-1. Therefore, the inhibitor fits tightly

into the active site of the MMP in a stereo-specific

manner. The inhibitor includes a zinc-binding group

such as hydroxamate or carboxylate. This then binds to

the zinc at the MMP active site. The compound

marimastat (British Biotech) is an example of a pep-

tide-mimetic MMP inhibitor with a hydroxamate zinc-binding group. Marimastat is a broad-spectrum inhibi-

tor for the MMP family with low nanomolar IC50s

against all the MMPs except MMP-3.

Most of the peptide-mimetic MMPIs have poor oral

bioavailability so research focused on the development

of a non-peptidic inhibitor based on the newly available

X-ray crystallographic information about the MMP

active site. CGS 27023A (Novartis), AG3340 (Agouron)and BAY 12-9566 (Bayer) are examples for these non-

peptidic inhibitors. As yet it is not possible to design an

MMP inhibitor specific for just one MMP, but com-

Fig. 7. Potential levels of intervention to alter the MMP/TIMP balance with therapeutic intent.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 161

pounds can be made that favour the inhibition of MMPs

with a ‘deep pocket’ at the active site (MMP-2, MMP-9,

MMP-3) at the expense of MMPs with ‘shallow pockets’

(MMP-1 and MMP-7). AG3340 is a compound thatappears to preferentially inhibit the gelatinases, while

Ro 32-3555 (Roche) inhibits MMP-1 fifty times more

potently than MMP-2.

Many of the synthetic MMPIs have been widely

tested for a variety of cancer in vivo models. There is

much evidence that they can inhibit the growth of both

primary tumours and metastatic deposits. Animals were

established with a primary tumour adjacent to themammary fat pad. The animals were then commenced

on batimastat (British Biotech) for short (7 days) or long

(58 days) courses just before the primary tumour was

resected. Animals treated with the short course of

batimastat had a reduction of pulmonary metastases

compared with untreated controls, but still developed

lymphatic metatases. However, the animals that re-

ceived the long course of batimastat did not developlymphatic metastases as any tumour cells remained as

silent micrometastases [62].

The effect of synthetic MMPIs on human cancers

established as xenografts in nude mice has also been

investigated. In vivo models of intra-peritoneal human

colorectal and ovarian carcinomas in mice have shown

significant reductions in tumour burden and ascites, and

increased survival when batimastat was administered asan intra-peritoneal agent [63�/65]. The ‘selective’ gelati-

nase inhibitor AG3340 has shown tumour inhibition for

human gliomas, human colon carcinomas and Lewis

lung carcinomas in vivo [66,67]. Both broad-spectrum

MMPIs like batimastat and ‘selective’ MMPIs like BAY

12-9566 have shown anti-angiogenic activity when tested

in a matrigel implant model [68�/70]. Another interesting

approach has been to combine a synthetic MMPinhibitor with a cytotoxic drug producing additive

benefits without a significant increase in toxicity. This

has been observed for batimastat in combination with

cisplatin in a human ovarian cancer model, AG3340 in

combination with carboplatin for a pulmonary metas-

tasis model, and for CT1476 (Celltech) with cisplatin or

cyclophosphamide in Lewis lung carcinoma models

[3,71,72].The first MMP inhibitor to be tested in a cancer

clinical trial was batimastat for patients with ascites

secondary to ovarian cancer. Batimastat had to be

administered parenterally as it has poor oral bioavail-

ability. Newer MMPIs such as marimastat, AG3340

(prinomastat), BAY 12-9566 (tanomastat), Ro 32-3555,

Col 3 and CGS27023A demonstrate high plasma con-

centrations after oral administration. There has beeninterest in developing peptide inhibitors to the MMPs.

Synthetic peptides mimicking the conserved

PRCGXPDV motif of the propeptide inhibited proteo-

lytic activity and inhibited invasion of tumour cells. A

recent report has demonstrated that by screening a

library of peptides a candidate that specifically inhibits

gelatinases MMP-2 and MMP-9 could be identified. The

candidate peptide is cyclic and contains the motifHWGF. In the presence of this peptide tumour growth

and invasion are reduced in vitro and in vivo [73].

Synthetic MMPIs have shown promising pre-clinical

evidence of anti-tumour effects in a variety of solid

tumour models. The availability of oral formulation

allows chronic administration in human subjects. These

agents have been brought forward for large scale clinical

testing (Section 4).

3.2. Gene therapy using TIMPs

As an alternative to synthetic MMPIs several groups

have opted to utilise the TIMPs themselves as anti-

cancer agents. Initially recombinant TIMP proteins

were used, but the approach has evolved into the gene

transfer of cDNA encoding the required TIMP gene into

the target cells. A variety of gene transfer vectors havebeen used to achieve this. The advantage of TIMP gene

therapy is definitive MMP inhibition by the naturally

occurring substance that can be produced in excess by

cells at the desired location. Genetic manipulation of

TIMP levels locally may also avoid the toxicity asso-

ciated with synthetic MMPIs. With the advent of tissue/

tumour specific promoters and targeted vectors gene

delivery is becoming increasingly more accurate.The first reports of the use of TIMPs as anti-cancer

agents centred on recombinant TIMP-1. It was demon-

strated that the recombinant protein interfered with the

invasion of B16-F10 melanoma cells through an amnio-

tic membrane. Interestingly, the tumour cells were able

to adhere to the membrane but appeared to be less able

to invade through it in the presence of TIMP-1. The

same melanoma cells were then injected into the tail veinor subcutaneous tissue of mice and intra-peritoneal

recombinant TIMP-1 was administered. TIMP-1 sig-

nificantly reduced the number of lung metastases

following tail vein injection of melanoma cells, but

those tumours that did develop were of the same size as

tumours in the control mice. This suggested that TIMP-

1 was inhibiting the invasion step rather than tumour

growth and this was corroborated by the fact that thesubcutaneous tumours were not significantly affected by

recombinant TIMP-1 [74]. A highly metastatic rat

embryo cell line transformed with H-ras (4R cells) had

reduced ability to colonise the lungs after tail vein

injection into nude mice if recombinant TIMP-1 was

administered into the peritoneal cavity. Again, if lung

metastases did develop they were the same size as those

in control mice [75]. Detailed in vitro studies of theeffects of recombinant TIMP-2 on tumour cell lines

indicated that there was an inhibition of gelatinolytic

activity that was independent of cell growth rates.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176162

TIMP-2 did not appear to interfere with cell attachment

nor mobility but did prevent the invasion of tumour

cells through a porous membrane [76,77]. Recombinant

TIMP-1 and TIMP-2 were shown to reduce angiogen-esis in chick yolk sac membranes that had been induced

with spermine [54].

DeClerck et al. transfected 4R cells with a plasmid

expressing TIMP-2 from a constitutive promoter. When

given as an intravenous injection to nude mice only one

4R TIMP2-expressing clone demonstrated a reduction

in the number of lung metastases formed, although this

clone was the highest TIMP-2 expresser. Another cohortof nude mice received 4R-TIMP2 cells in one flank and

parental 4R cells in the other flank as subcutaneous

injections. The TIMP2-expressing cells formed signifi-

cantly smaller tumours than the control cells. Control

tumours had invaded through the abdominal muscle

into the peritoneal cavity. However, the TIMP-2 tu-

mours were confined to the subcutaneous tissue and

surrounded by a capsule of dense connective tissue.When parental cells were implanted and recombinant

TIMP-2 was injected around the tumour site a similar

peri-tumoral connective tissue capsule was seen con-

firming that local TIMP-2 production is causing this

response [78]. This reduction in tumour growth as well

as reduction of invasion contrasts with the findings for

recombinant TIMP proteins. Similarly in mice, TIMP1-

expressing tumour cells injected intravenously producedfewer and smaller lung tumours [79]. Another group

used videomicroscopy to observe the movement of B16-

F10 melanoma cells across the chick embryo chorioal-

lantoic membrane. They found B16-F10 cells over-

expressing TIMP-1 extravasated as quickly as parental

cells across the membrane, but led to smaller and fewer

established metastases at 7 days [80]. Again there was

the suggestion that TIMPs were exerting their effect bylimiting tumour growth post-extravasation.

A highly metastatic human melanoma cell line

M24net was transfected by electroporation with a

eukaryotic expression vector containing the TIMP-2

cDNA under the control of the CMV promoter. High

TIMP2-expressing clones were identified by reverse

zymography. In vitro the high-expressing clones had a

reduced growth rate when embedded in a three-dimen-sional collagen matrix compared with the wild-type

cells. However, on collagen-coated plates there was no

reduction in growth rate. If the collagen concentration

on the plate was increased the growth-inhibitory effect

was seen again. This suggested that type I collagen

might suppress the early growth of M24net cells and

that elevated TIMP-2 levels perpetuate the growth

inhibition by preventing localised collagen degradation.It was also noted that the TIMP2-expressing clones

maintained morphology with multiple dendrites when in

the collagen matrix. In a murine model, TIMP2-

transfected M24net tumours grew significantly more

slowly than wild-type M24net tumours. There was no

evidence of tumour-associated capsules around the

TIMP2-expressing tumours in contrast to the findings

of other experiments. All tumours metastasised to thelungs and lymph nodes at equal rates although by

reverse zymography it could still be shown that TIMP-2

was being over-expressed [81].

The same group went on to develop retroviral vector

producing cells (VPCs) that release retroviruses encod-

ing the TIMP-2 cDNA under the control of the retro-

viral LTR. H-ras transformed rat embryo fibroblasts

were mixed with irradiated VPCs at ratios of 1:5 and1:10 and injected into the flanks of nude mice. There was

a significant reduction in the growth of tumours injected

with VPCs releasing TIMP-2 compared with VPCs

secreting galactosidase. Transduction efficiency of tu-

mour cells was 13%. In addition, the TIMP2-exposed

tumour cells were confined to the subcutaneous layer

and had apparently been unable to invade through

muscle while the control tumours had invaded throughthe muscle wall to the peritoneal cavity [82]. Recent data

shows that TIMP-1 or TIMP-2 can be successfully

delivered to the peritoneum of nude mice by an

adenoviral vector. Nude mice harbouring human pan-

creatic carcinomas had significantly improved survival if

treated with the adenoviral-TIMP-1 or TIMP-2 vector

than untreated controls [83].

There have been recent reports of TIMP-3 being usedfor gene therapy and also inhibiting invasion in vitro

[58,59] when delivered by an adenoviral vector. As yet

there are no in vivo data for TIMP-3. Another area of

interest has been that TIMPs may not be inducing an

anti-tumour effect by pure MMP inhibition. The effects

of TIMPs on apoptosis and angiogenesis have already

been discussed and are relevant to the gene transfer of

TIMP cDNA to target cells.Gene therapy targeting TIMPs may avoid toxicity

associated with synthetic MMPIs. However, there are

limitations to gene therapy, which include low efficiency

of gene transfer, poor specificity of response and

methods to accurately evaluate responses, and lack of

truly tumour-specific targets at which to aim. As with all

new therapies, we are climbing a steep learning curve in

terms of encountering treatment-related toxicities, aswell as profound ethical and regulatory issues. Preclini-

cal data are promising, but they will require confirma-

tion by large scale clinical testing.

4. Problems encountered in designing MMPI clinical

trials

One of the major obstacles for clinical trials ofsynthetic MMPIs in cancer patients is that the agents

are tumoristatic and so traditional values of tumour

regression may not be the best measures of treatment

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 163

efficacy. The trials conducted to date have focused on

tumour marker levels, MMP concentrations, growth

factors such as VEGF and urinary levels of collagen

breakdown products-all of which has led to debateregarding the value of these surrogate markers [84]. The

most objective endpoint would be survival, but as in

accordance with clinical trial ethics, synthetic MMPIs

have mainly been tested in patients with advanced

cancer for whom any treatment is unlikely to have a

great impact.

4.1. Tools to assess efficacy

Although survival would remain as the most objective

endpoint, two other approaches have been adopted to

assess the efficacy of MMPIs. Use of serum tumour

markers and serial histological assessment have been

used as surrogate markers of the biological activity of

the underlying tumour. Serum tumour markers have

been used for measurement of tumour activity anddetection of recurrence for a number of years. Carbohy-

drate antigen (CA) 19-9 and carcinoembryonic antigen

(CEA) are the two most commonly used markers in

gastrointestinal cancer. A change of CA19-9 has been

shown to predict response and survival in patients with

inoperable carcinoma of pancreas. [85�/87] Most of

these studies, however, involved only small number of

patients. In addition, interpretation of CA19-9 levelchanges is particularly problematic in patients with

obstructive jaundice [88] and a proportion of patients

with advanced pancreatic cancer may develop a degree

of biliary obstruction over the course of their treatment.

Serial measurement of CEA levels can add to the clinical

decision-making process in patients with metastatic

colorectal cancer. However, the presence of tumour

clone that do not express CEA marker would result in alack of rising CEA levels in some patients with

progressive disease. Therefore although tumour marker

measurement can reflect biological effects (BE) of the

antitumour agents, these markers are still suboptimal in

their sensitivity and specificity.

The second approach to assess biological activity of

MMPIs would involve serial macroscopic and micro-

scopic assessment of tumour, but this requires sites ofdisease which are readily accessible for repeated biop-

sies. Upper gastrointestinal endoscopy has relatively few

associated morbidity and mortality and the primary

tumour is amenable to biopsy during the procedure.

This strategy has therefore been adopted in MMPI trials

in gastric cancer. This approach would be impractical in

pancreatic cancer patients as the primary tumours are

often difficult to biopsy. In colorectal cancers, serialcolonscopies or tumour biopsies from colon or liver

would not be acceptable in large scale clinical trials in

view of their associated morbidity.

4.1.1. Biomarker assessment

In general, the implementation of correlative biologi-

cal studies has been a rather disorganised process in

which assays, that are often chosen on the basis ofavailability rather than relevance, are utilised in clinical

studies without a strong rationale and with minimal, if

any, preclinical validation [89]. In the clinical studies of

MMPIs, plasma concentration of angiogenic growth

factors such as vascular endothelial growth factor

(VEGF) and basic fibroblast growth factor (bFGF),

urinary secretion of collagen degradation products such

as pyridinoline and deoxypyridinoline as well as plasmaMMP activity have been measured as surrogate markers

for the BEs of MMPIs. However, results from these

biological correlative studies have often been inconclu-

sive (Table 4). This may be due to the lack of preclinical

evidence supporting that the MMPI being studied

impacts on these parameters or that changes in plasma

concentration of a given marker correlates with changes

in tumour tissue. Most investigators determined MMPexpression and secretion at the protein level using

ELISA. Duivenvoorden et al. tried to overcome the

limitations of these assays through the measurement of

the true activity of MMPs found in human plasma

sample using gelatin enzymography and fluorimetric

degradation assays [90]. Fluorimetric degradation assay

is truly quantitative in contrast to the method of gelatin

enzymography, but this assay determines total gelati-nase activity and is thus unable to distinguish between

the activities of a single MMP. By using gelatin

enzymography in combination with densitometry, the

authors could make a distinction between individual

MMP activity and at least semi-quantitative results

could be obtained using this approach.

4.1.2. Histological assessment

The development of MMPI would be accelerated bythe availability of reliable and reproducible surrogate

end-points to determine efficacy of treatment. As

discussed before, there is now growing evidence that

MMPs and TIMPs are involved in angiogenesis [91].

Indeed MMP activity is an early event in the angiogenic

response and much effort has been put in to identify

specific MMPs that mediate the angiogenic response in

order to target MMPIs to disrupt tumour neovascular-isation and subsequent dissemination.

The most widely used method to assess neovascular-

isation in human neoplasm is the quantitative analysis

of intratumoral microvessel density (IMD) using im-

munohistochemical methods and specific markers for

endothelial cells [92].

Assessment of IMD can be performed using three

different methods: (a) IMD in vascular hot spotsaccording to Weidner’s method [93]; (b) Chalkly count-

ing [94]; (c) multiparametric computerised image analy-

sis system [95�/98]. These methods all identify the

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176164

Table 4

Biological correlation studies performed in MMPI clinical trials

Authors MMPIs /cyto-

toxic drugs

Number of

patients

Tumour sites Biomarkers Comments

Wojtowicz-Praga

et al. [116]

Batimastat 9 All sites Plasma MMP-2 and MMP-9 Zymography used. Not useful as surrogate markers

Nemunaitis et al.

[118]

Marimastat 415 Pancreas, colorec-

tal, ovary and

prostate

Serum CA19-9, CEA, CA125 and PSA BE and PBE increased with dose escalation

Rosemurgy et al.

[120]

Marimastat 64 Pancreas Serum CA 19-9 Reductions in CA19-9 rate of rise at doses of 5, 10 and 25mg twice

daily

Primrose et al.

[119]

Marimastat 70 Colorectal Serum CEA BE or PBE more pronounced in twice daily dosing

Tierney et al. [122] Marimastat 35 Stomach and oe-

sophago-gastric

cancers

Histology: Degree of tumour differentiation; presence

of chronic �/ acute inflammation; amount of stroma

and evidence of necrosis

No change in degree of differentiation; no trend for acute or chronic

inflammatory infiltrate; 2 patients had increase in fibrous/tumour

ratio

Carmicheal et al.

[125]

Marimastat/

gemcitabine

31 Pancreas Serum CA19-9 9 patients had decrease in CA19-9

Rowinsky et al.

[110]

BAY12-9566 21 All sites Plasma MMP-2 and MMP-9 and TIMP-2 ELISA used. No consistent changes in MMP-2 and MMP-9 levels.

TIMP-2 seemed to increase in a dose-dependent manner

Erlichman et al.

[108]

BAY12-9566 13 All sites Plasma VEGF and bFGF; urinary pyridinoline and

deoxypyridinoline

ELISA used. No significant changes in VEGF, bFGF, urinary

pyridinoline and deoxypyridinoline crosslinks with BAY 12-9566

Duivenvoorden et

al. [90]

BAY12-9566 29 All sites Plasma MMP-2 and MMP-9 Gelatin enzymography and fluorimetric degradation assay (FDA)

used. FDA showed decreased plasma gelatinolytic activity with

BAY12-9566, but could not distinguish individual MMP activity

Rudek et al. [138] COL-3 35 All sites Plasma MMP-2, MMP-9; serum VEGF and bFGF Potential significant relationship between MMP-2 level and

cumulative doses of COL-3 when patients with progressive disease

were compared with patients with stable disease or patients with

toxicity. No relationships seen between MMP-9, VEGF or bFGF

with COL-3

Munoz-Mateu et

al. [141]

COL-3 26 All sites MMP-2 and MMP-9 in plasma, peripheral blood

mononuclear cell (PBMC) and skin

ELISA used for plasma; western blot for PBMC and immunohis-

tochemistry for skin. Plasma MMP-9 ¡/ in 11/18 patients; MMP-2 ¡/

in 6/16 patients. PBMC MMP-9 ¡/ in 5/7 patinets. Skin MMPs

showed no correlation

Levitt et al. [132] MMI270 92 All sites Plasma MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2,

bFGF, VEGF, VCAM-1,suPAR, CATB, CATH and

TNFa; urinary pyridinoline and deoxypyridinoline

Dose response increase of MMP-2 and TIMP-1. No correlations

with other biomarkers

BE, Biological effect (see text for definition); PBE, Partial biological effect (see text for definition); ELISA, Enzyme-linked immunoadsorbent assay; VEGF, Vascular endothelial growth factor;

bFGF, Basic fibroblast growth factor; VCAM-1, Vascular cell adhesion molecule-1; CATB, Cathepsin B; CATH, Cathepsin H; SuPAR, Soluble urokinase plasminogen activator receptor; TNFa,

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vascular hot spot defined as the area of highest

neovascularisation within a tumour section. Such highly

neovascular areas may occur anywhere within the

tumour but are generally located near its edges.The Weidner method and Chalkley point counting are

reproducible and require little time for assessment of

IMD with low associated costs. However, both methods

require manual counting which may give rise to inter-

observer variability particularly for the selection of the

vascular hot spot. In an attempt to automate the

counting procedure, improve the reproducibility and

reduce the variability associated with manual counting,computerised image analysis system has been developed.

However, the systems still require a high degree of

operator interaction and are more expensive than

manual counting.

Although histological microvessel density technique is

the current gold standard to characterise tumour

angiogenesis, it may not be ideal for clinical purposes,

because, it requires histological material and does notassess dynamic angiogenic activity. In vivo functional

imaging would, therefore, be a much more attractive

tool in clinical studies.

4.1.3. Non invasive imaging

Non invasive imaging methods, capable of providing

information on the physiological changes occurring in atumour during therapy, are being developed. Since

many cytostatic agents such as MMPIs are being tested

in clinical trials, there is an increasing need to utilise

functional measures of response rather than changes in

lesion dimension [99]. These functional imaging techni-

ques can be used to measure tumour blood flow, tumour

blood volume, changes in tumour metabolism and

changes in tumour vascular density. Among thesetechniques, positron emission tomography (PET) and

magnetic resonance imaging (MRI) appear to hold early

promises.

PET has been used in a variety of clinical settings to

measure both blood flow and blood volume [100�/102],

as well as rates of glucose metabolism [103,104]. Several

studies have demonstrated malignant lesions undergo

elevated glycolysis when compared with normal tissues.

Increased uptake of [18F] fluorodeoxyglucose (18FDG)

in cancer tissues has been demonstrated in several

studies, therefore, allowing PET evaluation. Why tu-

mours accumulate glucose is only partly understood.

The energy requirement for rapidly dividing cells

obviously plays a role. In addition, if the blood flow

to a tumour is decreased, the supply of oxygen and

nutrients should be decreased as well. The decrease in

oxygen should result in a shift from aerobic to anaerobic

metabolism, which may also have an impact on glucose

metabolism of the tumour cells. For these reasons, it

may be important to measure both tumour blood flow

and metabolism. The ability of PET scanning to per-

form such functional imaging (i.e. assessing both

tumour physiology and biochemical function) may

hold future promise as an important adjunct to conven-

tional imaging methods such as CT, which portray

primarily anatomy, in the assessment of new MMPIs.

Dynamic enhanced MRI involves the rapid adminis-

tration of a gadolinium-based contrast agent followed

by rapid analysis of signal intensity. Patterns of MRI

contrast uptake within tumour correlate with micro-

vessel density. Although this correlation may have some

variability, it allows investigators to monitor relative

changes in microvessel density, although absolute den-

sities cannot be determined. Using image-processing

algorithms established for the detection of malignancy

by MRI, it might be possible to assess changes in

perfusion and microvessel density over time in lesions

being treated with angiosuppressive agents.

Functional MRI is based on imaging differences in

oxygenated and deoxygenated haemoglobin. Carbogen

is a gas mixture of 95% O2 and 5% CO2 and allows

measurement of tissue oxygenation and perfusion with

MRI after administration to patients. Carbogen may

have diagnostic value, because, tissue oxygenation may

Table 5

Challenges in developing MMPI

Phase of clinical

study

Potential problems in trial design

Phase I Dose finding: Biological dose vs. maximum toler-

ated dose based on toxicity

Phase II Efficacy: Tumour regression vs. progression-free.

Clinical benefit vs. valid biological end points

Phase III Disease setting: Metastatic disease vs. minimal

volume disease (i.e. adjuvant or maximal response

after chemotherapy). Treatment strategies: MMPI

vs. placebo vs. chemotherapy alone vs. combination

of chemotherapy and MMPI

Table 6

Matrix metalloproteinase inhibitors (MMPI) tested in gastrointestinal

malignancies

MMPI MMP Inhibition Recommended doses for

phase II and III testing

Batimastat MMP-1,-2, -3, -7 and -9 No further testing

Marimastat MMP-1,-2, -3, -7 and -9 5, 10 and 25 mg b.i.d.

Prinomastat

(AG3340)

MMP-2, -3, -9 and -13 5, 10 and 15 mg b.i.d.

BAY 12-9566 MMP-2, -3 and -9 800 mg b.i.d.

CGS27023A Broad spectrum 300 mg b.i.d.

COL-3 MMP-2 and -9 36 mg/m2 per day

BMS275291 Broad spectrum including

MMP-2 and -9; shed-

dases sparing

1200 mg/day

b.i.d., twice daily.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176166

reflect on the angiogenesis stimulus and response to

therapy [105,106].

To assess efficacy of MMPIs, it would require parallel

effort to develop valid assessment tools that arepractical in clinical trials. At present, the search for

sensitive and specific biomarkers for assessing the

efficacy of MMPI continues. Histological assessment is

still the gold standard for angiogenesis assessment, but

non invasive functional imaging techniques holds early

promise and may represent a significant advance.

4.2. Clinical trial design

Novel clinical trial designs are needed to properly

assess the potential beneficial effects of MMPIs (Tables

5 and 6). Unlike the cytotoxic agents, many MMPIs in

development do not cause unacceptable toxicity at doses

that achieve concentrations with desirable BEs. In

designing phase I studies for MMPI, the traditional

goal for achieving maximum tolerated dose based on

toxicity is no longer appropriate, rather the optimumbiological dose required to obtain the hypothesised

cellular effects needs to be determined. The use of these

biological end points is based on preclinical data, but as

discussed before, the measurement of these end points in

clinical trials is often based on availability rather than

plausibility. It may be difficult to define and validate an

appropriate end point to measure and agents may have

additional antitumour mechanisms than those initiallyhypothesised [107]. Furthermore, the measurement of

plasma concentrations of MMPs in clinical studies is

often carried out, but this does not necessarily reflect the

expression of MMPs at the tumour site. Obtaining

adequate tumour tissues for testing MMP expression is

difficult to achieve in large-scale clinical trials. As a

result, the actual BEs of MMPI at the tumour site may

not be able to be determined.Although one assumes that higher doses of agents, if

tolerable, will provide at least the same effect, if not

more, there is no rationale to escalate the dose any

further if pharmacokinetic analysis implies saturable

absorption beyond a certain dose. The phase II�/III

doses of BAY 12-9566 and prinomastat were both

determined from this rationale using pharmacokinetic

data [108,109]. As there was no valid surrogate markersof BE or any knowledge about the effect of prolonged

inhibition of MMPs on MMP regulation and cross

reactivity, the investigators related plasma steady state

trough levels of BAY12-9566 to the inhibiting concen-

trations for the target MMPs i.e. MMP-2, MMP-3 and

MMP-9 [108�/110]. These trough levels were of multiple

orders of magnitude higher than the inhibiting concen-

trations for the target MMPs. As BAY12-9566 was99.9% protein bound, these plasma concentrations

included both free and bound drug. It was unclear

what levels of free drug were required to achieve optimal

concentrations of drug in tumour tissue and inhibition

of MMPs in the tumour milieu.

Phase II trial designs are also problematic for MMPI

although many agents appear to move directly fromphase I to phase III. Rather than assessing tumour

shrinkage, a different clinical end point (e.g. progression

free survival) may be more meaningful. One could still

use a standard two stage design that targets progression

free survival or time to treatment failure. However, one

potential problem in a two-stage design with an out-

come that takes 6 months or 1 year to evaluate is that

the accrual of the first stage can be completed for sometime before it is known whether there are sufficient

clinical efficacy to continue on to the second stage of the

trial. One of the solutions would be allowance for a

slight over-accrual to the first stage [111] or incorporat-

ing tumour response as an additional criterion to

proceed to second stage such as the Zee’s design [112].

Many MMPIs moved directly to large definitive phase

III testing where survival is the primary endpoint. Mosttrials reported so far failed to make any impact on

survival either compared with chemotherapy or in

combination with chemotherapy. These trials are re-

source intensive in terms of patients, funding and

planning time. As such the pharmaceutical companies

and the clinicians have learnt a very dear lesson. Before

moving onto large scale phase III trials, smaller

randomised screening phase II�/III trials could beplanned to determine optimum situations in which these

MMPIs can be tested.

5. Matrix metalloprotenase inhibitors in clinical

development

5.1. Batimastat (BB-94)

The development of metalloproteinase inhibitors has

been hindered originally by the lack of non-invasive

route of administration. Batimastat (BB-94), one of the

first MMPI developed, was administered directly into

peritoneum or pleural space of patients with malignant

effusion [113�/116]. As batimastat can only be adminis-

tered invasively, widespread use is impractical and its

development has been halted.

5.2. Marimastat

It is a broad spectrum MMPI, which inhibits all the

major MMPs (collagenase, stromelysin, gelatinase A,

gelatinase B and matrilysin). It is the most extensively

tested MMPI among all the current available MMPIs. It

has a good oral bioavailability in contrast to batimastat.In healthy volunteers marimastat was well tolerated at

doses of up to 200 mg twice daily for a week, with a

small rise in transaminases being the only abnormality

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 167

noted in a minority of the patients [117]. Cancer patients

registered higher plasma concentrations of marimastat

than healthy volunteers for the same dosage possibly

due to increased plasma protein binding or alteredhepatic/renal function. Some cancer patients who re-

ceived marimastat for 1�/2 months were noted to

experience arthralgia, tendonitis or myalgia. These side

effects were reversible [118]. About 60% of the drug was

metabolised by the liver and 40% excreted unchanged

via the kidney.

5.2.1. Phase II studies

Six studies have been conducted to assess effects of

marimastat in advanced cancers using serum tumour

markers. A combined analysis of all six studies have

been reported involving 415 patients [118]. Patients with

advanced, serologically progressive pancreatic, color-

ectal, ovarian and prostatic cancers were recruited into

six nonrandomised, dose ranging, multicenter clinical

trials in North America and Europe. Two studies wereconducted in advanced colorectal cancer, one in pan-

creatic cancer, two in ovarian and one in prostatic

cancers. CEA and CA19.9 were used as tumour markers

for colorectal and pancreatic cancer studies. Histologi-

cally proven cancers were required for patients entering

the colorectal study whereas that was not a prerequisite

for pancreatic cancer study. Patients were selected for

these studies on the basis of serum tumour markersabove prespecified levels (�/5 ng/ml CEA and �/37 Iu/

ml CA19-9) and values rising by 25% or more over 4-

week period before entry into the study. An exception

was the North American colorectal cancer study, in

which the required rate of rise of CEA was 25% over 12

weeks. All patients had failed conventional first-line

treatment where offered.

Patients were recruited in sequential dose groups ofeight to ten patients starting at 25 mg twice a day with a

view to dose escalate to 100 mg twice daily. It became

apparent that doses beyond 50 mg twice daily were

poorly tolerated. Thereafter, doses were titrated down-

ward and patients were recruited at 10 and 5 mg twice

daily and 5�/25 mg once daily. Patients received

marimastat for 4 weeks, except in the North American

colorectal cancer study, in which treatment was for 12weeks. Patients who benefited from marimastat treat-

ment were allowed into a long term continuation arm in

which they could continue for as long as clinical benefits

continued.

A BE was defined as a rate of rise of tumour marker

at the end of treatment period of B/0% (i.e. tumour

marker level no greater than that on day 0). A partial

biological effect (PBE) was defined as a rate of rise oftumour marker of between 0 and 25%. Any patients

with rise of tumour markers �/25% were classified as

non-responders.

However, practical difficulties during these studies

meant that blood samples were not always taken at

scheduled times or according to the protocol. A pre-

analysis algorithm was devised that selected those

tumour marker measurements that adhered most closely

to the protocol for analysis. Both per-algorithm and

intention-to-treat data were presented in the paper.

About 54% were eligible for tumour marker response

analysis per algorithm. Only 75% of patients were

included in the intention-to-treat analysis with a total

of 103 patients excluded from any analysis. These

studies represented an unconventional way of presenting

clinical data and interpretation was, therefore, proble-

matic.

A total of 131 patients and 64 patients were enrolled

into colorectal and pancreatic cancer studies, respec-

tively. Although one of the colorectal studies and the

pancreatic cancer study was published separated

[119,120], results in this paper were presented as

combined analysis for all solid tumour groups. Propor-

tion of patients showing a BE and PBE increased as

dose increased. No correlation was found between

trough levels of marimastat and BE or PBE. Survival

was significantly different in favour of those patients

showing a BE or PBE, but patients suffering early

deaths are excluded from the nonresponders to avoid an

exaggeration of the poorer survival curve. Based on the

combined analysis of biological activity described,

pharmacokinetic data and the observation of dose

related musculoskeletal pain, dose range of 5�/25 mg

twice daily was identified for future studies.

The analysis in pancreatic cancer has been published

separately in full [120]. The most pronounced reduction

in CA19-9 rate of rise was observed at a dose of 10 mg

twice daily. As expected, no objective tumour response

was observed on CT scan. Overall median survival was

160 days and 1 year survival was 21%. The group of

patients was heterogeneous with regard to exposure to

cytotoxic agents and therefore, it would be difficult to

make direct comparison with published studies of

cytotoxic chemotherapy. No correlation between anti-

gen response and survival was found.

Another multicentre phase II study of marimastat in

advanced pancreatic cancer evaluated 113 patients in

three different doses. The majority of patients received

25 mg once daily. Survival was better in patients with

stable or falling CA19-9 level than those with rising

CA19-9 (245 vs. 128 days, respectively). Half of the

patients who completed the study had stabilisation or

reduction in pain, mobility and analgesia scores [121].

The European colorectal study has also been pub-

lished in full [119]. A total of 70 patients were recruited.

Highest percentages of patients achieving BE and PBE

were found in 10 and 50 mg twice daily dose groups.

Changes in CEA were more substantial in the higher

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176168

dose groups and more evident in twice daily dosing of

marimastat.

Tierney et al. reported a study of marimastat in

gastric cancer [122]. Patients with inoperable, histologi-cally proven primary or recurrent gastric or gastro-

oesophageal carcinoma were enrolled. Evidence of

biological activity for marimastat was assessed by

changes in the endoscopic and histological appearance

of the tumour. Study medication dose was originally

intended to be 50 mg twice daily for 28 days, but was

subsequently reduced to 25 mg once daily after realisa-

tion of higher trough level in these groups of patientscompared with healthy volunteers and the development

of musculoskeletal events (MSE) at higher doses. All

patients underwent endoscopic examination at days 0

and 28. Thirty-five patients were enrolled in this study.

No macroscopic changes in tumour size were noted. Ten

patients had a definite increased fibrous cover. Micro-

scopically, no changes in degree of differentiation of

tumour, necrosis, inflammatory infiltrate of fibroustissue were noted. Although authors remarked that the

apparent increase in fibrotic tissue in the gastric tumours

of some patients was one of the most striking evidence

of marimastat’s biological activity, no direct indication

of tumour apoptosis or arrest of angiogenic process was

shown. This again emphasises the importance of better

histological and molecular markers in assessing this

novel class of cytostatic drugs.

5.2.2. Phase III studies

Bramhall et al. reported a randomised study compar-

ing marimastat to gemcitabine as first line therapy in

patients with non resectable pancreatic cancer [123]. As

discussed before, phase II studies have shown the

feasibility of a dose range of 5�/25 mg twice daily for

marimastat, but no single dose has emerged as the most

efficacious. This study, therefore, randomised patientsinto three different doses of marimastat (5, 10 and 25 mg

twice daily) or gemcitabine. Comparison between mar-

imastat and gemcitabine was open-labelled, whereas

comparison between the marimastat dose levels were

double-blind. A total of 414 patients were enrolled.

There was no difference in overall survival between the

marimastat 25 mg group and the gemcitabine group.

Overall survivals of marimastat 5 and 10 mg groupswere poorer than gemcitabine. The multivariate analysis

using Cox proportional hazard model confirmed this. In

addition, a statistically worse progression free survival

was found with each of the three marimastat treatment

groups compared with gemcitabine. Further exploratory

analyses revealed that patients who received marimastat

enjoyed better survival if they had non-metastatic

disease (stages I, II and III) compared with metastaticdisease (stage IV). Tolerability was similar except more

MSE were reported with marimastat and more haema-

tological toxicity with gemcitabine.

Limited data are available on a further study of

gemcitabine with or without marimastat (10 mg twice

daily) in advanced pancreatic cancer. About 239

patients have been recruited and the combination ofmarimastat and gemcitabine failed to show a significant

improvement in survival. Moreover analyses of second-

ary end-points did not reveal significant benefit attribu-

table to marimastat. No quality of life advantages were

seen, but there was a trend indicating that patients with

less extensive disease responded better to marimastat.

Marimastat has also been tested in a randomised trial

in gastric cancer [124]. Three hundred and sixty-sevenpatients were recruited in 37 centres. Patients with

inoperable gastric carcinoma were randomised to mar-

imastat 10 mg twice daily or placebo. The primary

endpoint was overall survival and the secondary end-

points were progression-free survival, overall survival

for patients who received chemotherapy, quality of life

and safety. There was a trend towards survival advan-

tage in the marimastat group at censored date and withfurther follow up a significant survival advantage

emerged in the marimastat group. In subset analysis,

patients who received prior chemotherapy had better

survival in the marimastat group, especially in those

who had no evidence of distant metastasis. Progression

free survival showed similar trend. About 1 year survival

was 20% for marimastat and 14% for placebo. This is

the first randomised trial that has shown a favourablesurvival outcome for patients treated with MMPI in

gastrointestinal cancers.

5.2.3. Combination with other cytotoxic and angiogenesis

therapy

No combination therapy trials have been published in

full including the aforementioned phase III study with

gemcitabine in advanced pancreatic cancer. Phase I dose

escalation study of marimastat with gemcitabine inunresectable pancreatic cancer was reported by Carmi-

chael et al. [125]. Sequential dose levels of marimastat of

5, 10, 15 and 20 mg twice daily with gemcitabine at 1000

mg/m2 weekly for 3 out of 4 weeks were studied. Thirty-

one patients were enrolled and grade 3 and 4 toxicities

included deranged liver function, myelosuppression and

back pain. About two responses out of 11 were noted

and six had stable disease.Another study combining 5-FU with marimastat was

reported [126]. Thirteen patients with solid tumour

malignancies were enrolled. 5-FU was administered

either by continuous infusion (300 mg/m2 per day) or

by bolus Mayo schedule (425 mg/m2 per day with

leucovorin 20 mg/m2 per day for 5 days every 4 weeks).

Marimastat was started 1 week prior to 5-FU. The

starting dose was 5 mg twice daily escalating to 10 mgtwice daily. Only one patient with grade 3 neck pain was

noted who had a past history of cervical spondylosis and

toxicity resolved with discontinuation of marimastat

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 169

and subsequent retreatment with 50% dose reduction

was well tolerated.

Marimastat has also been combined with other

putative antiangiogenesis therapy. Jones et al. reportedon the use of marimastat 10 mg twice daily, dalteparin

and captopril in 17 patients with colorectal and other

solid tumours [127]. Plasma VEGF, thrombin�/antith-

rombin complexes (TAT) and tissue factor (TF) were

measured at baseline and after 1 month of treatment as

surrogate markers of angiogenesis. This combination

was well tolerated with the most common toxicity being

musculoskeletal pain. VEGF fell in five patients. TATand TF increased in five patients. These data are

preliminary, but support the increasing recognition of

the need to employ alternative biochemical and mole-

cular outcome measures in assessing these novel drugs.

5.2.4. Adjuvant therapy

Preclinical and clinical studies suggested that marima-

stat works best in patients with less extensive disease and

smaller tumour volume. MMPI would, therefore, beideal in adjuvant setting. Its favourable tolerability as

oral medication would also be an distinct advantage for

long term use. No published data have been identified in

gastrointestinal tumours. However, Miller et al. re-

ported a randomised phase II trial of adjuvant marima-

stat in patients with early breast cancer [128]. Patients

with high risk node negative or node positive breast

cancer were randomised to receive either 5 or 10 mgmarimastat twice daily for 12 months. Marimastat was

given either as single agent following completion of

adjuvant chemotherapy with doxorubicin and cyclopho-

sphamide or concurrently with tamoxifen. Sixty-three

patients were recruited so far. Main toxicity encountered

was musculoskeletal with 17% of patient required dose

interruptions or reductions. Recurrence and overall

survival have not been reported, but the data would betoo premature at this stage.

5.2.5. Safety profiles

The most commonly reported adverse events of

marimastat were MSE, particularly arthralgia, myalgia

and back pain. These events appeared to be dose-

related. The symptoms very often started in the small

joints in the hand, as well as the shoulder girdle, oftenon the dominant side. If dosing continued unchanged,

the symptoms would spread to involve other joints as

well. These symptoms responded poorly to non-steroi-

dal anti-inflammatory drugs. These side effects also

became more frequent beyond 28 days of treatment,

particularly at doses of 25 mg twice daily and higher.

MMP activation is known to be important in wound

healing and bone resorption. There is increasing evi-dence that MMPs are important in the pathogenesis of

rheumatoid arthritis. Not surprisingly marimastat,

being a broad spectrum MMPI, would interfere with

the normal bone remodelling process. However, these

MSE often subside following a brief period of absti-

nence and lower restarting dose of marimastat.

Safety review of marimastat has been presented withover 1109 patients enrolled in four ongoing placebo

controlled studies [129]. About 10 mg twice daily dose

has been utilised in these ongoing studies. At least 70

patients received more than 6 months of therapy. MSE

emerged as the only observable toxicity. About 5% of

patients required discontinuation of therapy due to

adverse events. 66.3% of patients receiving marimastat

developed any MSE with 3.1% classified as seriousadverse events. There was a high level of background

symptoms in these patients with advanced cancers as

evidenced by the high percentage of MSE in the placebo

group. Other toxicities noted in phase II studies such as

liver dysfunction have not been confirmed.

5.3. BAYER 12-9566 (Tanomastat)

This is a novel nonpeptidic biphenyl compound whichinhibits MMP-2 and MMP-9. Four phase I studies have

now published [108�/110,130]. Doses up to 1600 mg per

day were well tolerated with biologically relevant plasma

steady state concentrations. The predominant toxicities

were asymtomatic thrombocytopenia, anaemia and

hepatic transaminase elevation. There is a lack of

MSE with BAY 12-9566. A dose of 800 mg twice a

day was recommended in further studies.Biological correlative studies were undertaken during

these phase I studies. There was no consistent effects of

BAY 12-9566 on the plasma concentrations of MMP-2

and MMP-9 over the continuous dosing period at any

dose schedule level using ELISA assay. However,

plasma levels of TIMP-2 seemed to increase in a dose-

dependent manner [109]. In a separate study, changes in

plasma VEGF, bFGF and urinary pyridinoline anddeoxypyridinoline crosslinks were measured and no

significant association was found with BAY12-9566

administration [108]. Duivenvoorden et al. showed

that BAY12-9566 was effective in lowering the plasma

MMP-2 and MMP-9 activity using fluorimetric gelati-

nase assay [90].

In a phase III study in chemonaıve patients with

metastatic pancreatic carcinoma, a dose of 800 mg twicea day was used [131]. Patients were randomised between

gemcitabine (G) and BAY 12-9566 (BAY). Patients who

progressed on BAY 12-9566 were permitted to cross

over to gemcitabine but not vice versa. Study was

terminated prematurely after the second interim analysis

when 277 patients had been recruited. Two hundred and

thirty-nine deaths had occurred at final analysis. Base-

line patient characteristics were balanced. Haematolo-gical toxicity was significantly more frequent in

gemcitabine arm. There were no treatment related

deaths. However, median progression free survival and

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176170

overall survival were both superior in gemcitabine

compared with BAY 12-9566 [Median progression free

survival: 3.4 (G) vs. 1.68 months (BAY) P�/0.0001

Median overall survival: 6.67 (G) vs. 3.74 months(BAY) P�/0.0001]. One third of the patients who

progressed on BAY 12-9566 received gemcitabine sal-

vage. Multivariate analysis identified treatment with

BAY 12-9566, pain, metastatic disease, elevated aspar-

tate transaminase and alkaline phosphatase to be

adverse prognostic markers in this study. In view of

these highly significant adverse results in this study and

excessive deaths occurring in the BAY 12-9566 arm inanother small cell lung cancer trial, all clinical studies

with BAY 12-9566 had been suspended temporarily.

5.4. MMI270 (CGS27023A)

This is another orally administered, broad spectrum

MMPI with antitumour activity in preclinical models. It

inhibits MMP-1, 2, 3, 9 and 13. In a phase I study, 92

patients were enrolled (26 with colorectal and five withgastric cancers) [132]. No myelosuppression was seen.

Two main non-haematological toxicities were maculo-

papular rash and MSE. The rash was symmetrical

affecting trunk and arms generally. The maximum

tolerated dose was 300 mg twice daily. At all dose levels

higher than this, there was a marked increase in both the

incidence and severity of rash. Biological correlative

studies were undertaken. Although there was a dose-response increase of MMP-2 and TIMP-1 with MMI-

270, no correlation was seen with other biomarkers

being investigated (see Table 4). Clear interpretation of

the data was problematic reinforcing the need for better

surrogate marker for MMPIs.

Another phase I study in combination with 5-FU and

folinic acid (according to de Gramont’s regimen) has

been reported in which 18 patients with colorectalcancer were enrolled. Eleven had received previous

fluropyrimidine chemotherapy. Grade 3 toxicity oc-

curred in MSE. Two partial response and ten stable

diseases were seen. It was feasible to administer

MMI270 at 300 mg twice daily in combination with 5-

FU and leucovorin [133].

5.5. AG3340 (Prinomastat)

It is a potent selective inhibitor for MMP-2 (gelati-

nase A), MMP-3, MMP-9 and MMP-13. As MMP-1 is

believed to be associated with joint related toxicities,

prinomastat has been designed selectivity to inhibit

MMPs that are most commonly associated with growing

and invasive tumours while sparing the musculoskeletal

system. Phase I studies showed that doses below 25 mgtwice daily were well tolerated. Prinomastat has also

been tested in combination with a number of chemother-

apy drugs in solid tumour and toxicities attributable to

prinomastat was minimal [134,135]. Although no phase

III trials have been reported in GI tumours, two phase

III studies were presented recently in advanced non-

small cell lung cancer (NSCLC) [136] and metastatichormone refractory prostate cancer (HRPC) [137]. Both

studies involved concomitant administration of prino-

mastat with chemotherapy compared with chemother-

apy alone. When prinomastat at different doses was

combined with paclitaxel and carboplatin in chemonaıve

patients with advanced NSCLC, no differences were

observed among treatment groups in overall or 1-year

survival, progression-free survival, symptomatic pro-gression free survival or response rate. Adding prinoma-

stat to cytotoxic drug combination did not, therefore,

enhance efficacy. Similar results were found when

different doses of prinomastat were added to mitoxan-

trone and prednisolone in chemonaıve patients with

metastatic HRPC.

5.6. COL-3

Tetracycline and its derivatives inhibit collagenase

activity in a range of cell types. Probable mechanisms

for inhibiting MMP include divalent cation chelation of

zinc at the active site of the MMPs, down regulation of

the production of the proenzyme, inhibition of the

oxidative activation of the proenzyme and an increase

in the degradation of the proenzyme. COL-3 wasproduced by modifying the basic tetracycline structure.

The anitmicrobial properties of the molecule were

eliminated whereas the MMP inhibitory properties

were retained. COL-3 is a competitive inhibitor of

MMP-2 and MMP-9 [138].

In a phase I study, 35 patients with advanced

refractory metastatic cancer were recruited [138]. Cuta-

neous phototoxicity was dose-limiting at 98mg/m2 perday. As grade 2 phototoxicity was observed at the first

dose level, mandatory routine use of sunblock with a

sun protection factor of 28 was implemented with dose

level 2. The sunblock used protected against ultraviolet

(UV)A and UVB. In addition, sun avoidance and

covering sun-exposed areas with clothing were advised.

The phototoxicity resolved with time at lower doses

despite continuation of treatment. Two other unusualside effects were noted with COL-3. Three patients

developed drug induced lupus [139]. These patients

developed sunburnlike eruptions accompanied by fever

and a positive antinuclear antibody titer. Two out of

three had positive antihistone antibody levels and

arthralgia. One patient had marked systemic manifesta-

tions including pulmonary infiltrates. Another unusual

side effect was the development of sideroblastic anaemiain three patients confirmed on bone marrow examina-

tion [140]. Efficacy appeared to be more marked in

patients with tumours of nonepithelial origin.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176 171

Plasma MMP-2 and MMP-9 and serum VEGF and

bFGF levels were determined using ELISA kit in this

study. There was a possible significant relationship

between changes in plasma MMP-2 levels and cumula-tive doses of drug when progressive disease patients

were compared with those with stable disease.

In another phase 1 study with 26 patients, principal

toxicities were photosensitivity skin reactions and asth-

enia [141]. Correlative biological studies were performed

assessing MMP-2, MMP-9 expression in peripheral

blood mononuclear cells and skin biopsies using ELISA,

Western Blot and immunohistochemical techniques.Plasma concentrations of MMP-2 and MMP-9 were

decreased in a proportion of patients as well as the

expression of MMP-9 in peripheral blood mononuclear

cells. No changes were observed in MMPs expression in

serial skin biopsies.

5.7. BMS-275291

This is a novel, non-hydroxamate MMPI designed toinhibit a broad spectrum of MMPs (including MMP2

and MMP9) and spare sheddases. Sheddase inhibition is

hypothesised to induce the dose limiting arthritis

observed with earlier hydroxamate based MMPIs. In

healthy subjects, BMS-275291 was safe and very well

tolerated across all dose levels [142]. In cancer patients,

maximum tolerated dose was not reached in two phase I

studies [143,144]. One thousand and two hundred mgper day was chosen for phase II studies as the trough

concentration of BMS-275291 exceed IC90s (concentra-

tion that causes 90% of enzyme inhibition) of multiple

MMPs.

6. Conclusion

MMPs are novel targets for gastrointestinal malig-

nancies. Matrix metalloproteinase inhibitors remain an

important and potentially useful class of drugs for

cancer therapy, but they have not clearly demonstrated

clinically significant activity when used as single agents

in patients with advanced stage gastrointestinal cancers.

They have not been adequately tested in early stage

(adjuvant) settings or in combination with chemother-apy or radiation, where their impact may be the greatest.

The recent lack of success from large phase III MMPI

studies in solid tumours would obviously dampen the

enthusiasm of both the clinicians and the pharmaceu-

tical industries in investing further MMPI studies.

However, the superior results of marimastat in advanced

gastric cancer patients who have received chemotherapy

are encouraging. The challenges in future studies wouldlie at targeting the correct matrix metalloproteinase at

the correct time point in tumour growth and metastasis,

and the correct sequence of using MMPIs with other

cytotoxic agents. Identification of reliable surrogate

biomarkers and incorporation of functional imaging

will be of high priority in the clinical development of

matrix metalloproteinase inhibitors in gastrointestinalmalignancies.

Reviewers

Nicholas R. Lemoine, Professor of Molecular Pathol-

ogy, Head of Molecular Oncology, Imperial Collage

School of Medicine, Department of Cancer Medicine,Hammersmith Campus, Du Cane Road, London W12

0NN, UK.

Dr Arnaud Roth, Department de chirurgie, Onco-

chirurgie, Hopitaux Universitaires de Geneve 24, rue

Micheli-du-Crest, CH-1211 Geneve 4, Switzerland.

Professor K. Kesteloot, K. U. Leuven. Centre for

Health Sciences & Nursing Research, Kapucynenvoer

35, B-3000 Leuven, Belgium.

Acknowledgements

The Organizing Committee of the ICACT Conference

on ‘New Drugs in GI Malignancies’ held from 30

January to 1 February 2000 in Paris.

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Biography

Dr D. Cunningham is a Counsultant in Medical

Oncology at the Royal Mardsen Hospital in London

and Surrey, and he is Head of the GI and Lymphoma

Units. He qualified from Glasgow University and was a

Cancer Research Campaign Fellow there before moving

to London where he completed his training. Before

talking up his current post, he was Senior Lecturer in the

Institute of Cancer Research. His main research inter-

ests are clinical trials in GI cancer and lymphoma, along

with molecular therapies.

I. Chau et al. / Critical Reviews in Oncology/Hematology 45 (2003) 151�/176176