Heart Failure Reviews, 9, 63–79, 2004
C 2004 Kluwer Academic Publishers. Manufactured in The Netherlands

Matrix Metalloproteinase Inhibitor Development and the Remodeling of Drug Discovery J. Thomas Peterson Cardiovascular Pharmacology, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA

Abstract. Collagen turnover is a slow process on a biologic timescale with a t 12 of 20–27 days that is mediated primarily by the matrix metalloproteinases (MMPs). Low collagen metabolism is not due to an intrinsically low Km of MMPs, but rather due to a highly regulated system of activity. Despite the stability of collagen and MMPs, the articles in this special addition illustrate the importance of this enzyme family in the disease process leading to congestive heart failure. Like MMPs, drug development is a tightly regulated process, and the successful turnover of MMP inhibitors into a marketed drug has also been a slow process on a pharmaceutical timescale. Since the discovery of the archetypal MMP (type 1 collagenase) over four decades ago by Gross and Lapierre, most major pharmaceutical companies have had MMP inhibitor programs for a variety of indications. Despite decades of research, tens of thousands of compounds synthesized and screened, and billions of dollars spent in clinical studies—Periostat® (doxycycline hyclate, CollaGenex Pharmaceuticals Inc.) is the only collagenase inhibitor to be successfully launched. In addition, Periostat’s approval is currently limited to periodontal disease. This article focuses on some of the lessons to be learned from the failure of so many MMP inhibitors across so many indications, and what potential exists for MMP inhibitors as a drug class, especially for heart failure. Key Words. matrix metalloproteinase inhibitor, MMP, collagenase, drug discovery, drug development, attrition, heart failure

Introduction ICI (now AstraZeneca) is attributed as initiating the search for an orally bioavailable small molecule matrix metalloproteinase (MMP) inhibitor in the late 1970’s for the treatment of arthritis [1]. The project was driven by the hypothesis that MMP inhibition would slow or reverse the remodeling events (collagen degradation) causing arthritis rather than acting primarily as a palliative [2]. Since the seminal work at ICI, there has been an expansion in the number of known MMPs to over 26 identified enzymes [3], as well as potential therapeutic indications (rheumatoid arthritis, osteoarthritis, osteoporosis, periodontal disease, metastatic tumorogenesis and neoplastic growth, aneurysm, atherosclerosis, and heart failure). Drug discovery also changed substantially during this period, moving from a reliance

on bioassay screens (e.g., reduction in cartilage degradation by MMP inhibitors [4,5]) to the use of recombinant enzymes [6,7]; utilization of chombichem arrays [8], NMR [9], and computational chemistry [10,11] to accelerate rationale drug design; the advent of mass screening to identify hits [12]; the use molecular biology both to identify new targets [13] and provide confidence in rationale; the development of in silico pharmacokinetic profiling [14] and cell based systems to assess pharmacokinetics and in vitro toxicology screens [15]; the recognition that biomarkers can greatly enhance the chance of successful drug development [16–18]; and the quantum leap in development costs [19] and perceived risk [20]. Continued investment in MMP inhibitor development has been sustained despite increased costs and a growing litany of failed clinical trials (Table 1) by the potential market for developing a new drug class useful in the life-long treatment of rheumatoid or osteoarthritis, metastatic tumor and neoplastic growth, osteoporosis, periodontal disease, aneurysm, atherosclerosis, and/or heart failure. Compelling MMP inhibitor efficacy in animal models has also maintained interest among medical and pharmaceutical researchers. What then has stymied the clinical success of MMP inhibitors (e.g., inappropriate animal models, poor PK, unavoidable toxicology, poor clinical trial design, lack of human efficacy), and does Periostat, the only approved collagenase inhibitor, represent an opportunity to generate a new generation of MMP inhibitors that avoid the problems of the past?

Gene Transfer and the Case for MMP Inhibitors The case for developing small molecule inhibitors of MMPs starting in the 1990’s was greatly enhanced by an array of gene transfer and

Address for correspondence: J.T. Peterson, Cardiovascular Pharmacology, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. Tel.: 734-6227189; Fax: 734-622-1480; E-mail: [email protected] 63

Discontinued Phase III in Canada Europe US and Phase II in Japan

Phase III

Marimastat BB-2516 Marimastat, Softgel TA-2516 Strommex

BAY-12-9566

Discontinued Phase III

Structure

Batimastat, iv

Product name/ Synonyms

Bayer

British Biotech Tanabe Seiyaku RP Scherer Schering Plough

British Biotech

Originator/ Licensee

Table 1. Matrix metalloproteinase inhibitors discontinued while in clinical development

Gel-A, Gel-B, Stromelysin

Broad spectrum MMP inhibitor

MMP inhibitor

Mechanism

Arthritis, Cancer

Cancer, Other

Cancer

Indication(s)

British Biotech discontinued development of marimastat for the treatment of cancer, due to the lack of efficacy in trials (Press release, British Biotech, 13 Feb. 2001). Produced tendinitis like side-effect (MSS). All clinical trials stopped after results showed BAY12-9566 was performing worse than placebo in small cell lung cancer. Bayer says it has no plans for the further development of Bay 12-9566 for any indication. Produced MSS.

Poor oral bioavailability. (Superseded by marimastat.)

Comments

64 Peterson

Phase III

Phase III

Phase II

Psovascar Arthrovas Neovastat AE-941 (shark cartilage derivative)

Ilomastat Galardin

D-5410

Celltech–Chiroscience

Glycomed– Ligand

Aeterna

MMPs

Collagenase Inhibitor

Collagenase inhibitor Gelatinase inhibitor

Arthritis, IBD

Cancer, Macular Degeneration

Psoriasis, Arthritis, Cancer

(Continued on next page.)

A phase III trial for moderate-to-severe psoriasis began in Nov. 99. In Phase I/II monotherapy trials, Psovascar has shown preliminary signs of efficacy with no serious side-effects during the first 3 months of the trial. Of 30 evaluable patients, 3 of the 8 patients treated at the highest dose have shown significant improvement in their condition. Aeterna has discontinued indications other than cancer (Neovastat formulation) for AE-941, as of May 2000) Now in development for treatment of inflammastory respiratory diseases such as smoking-related emphysema and COPD (Press release, Arriva, 12 Feb. 2000). It was previously under development (Phase 2) by Glycomed (Ligand) for ophthalmological indications as an angiogenesis inhibitor, but development was discontinu Discontinued from phase II trials for IBD due to poor bioavailability. It was previously discontinued from trials for arthritis for the same reason. Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery 65

Phase I/II

Phase I

Phase I

CGS-27023A MM-270 MMI-270

BB-2983 GI-245402 GI-5402

Solimastat BB-3644

Discontinued Phase II

Structure

RS-13-0830

Product name/ Synonyms

Table 1. (Continued ).

British Biotech Schering Plough

British Biotech Glaxo Wellcome

Novartis

Roche

Originator/ Licensee

MMP inhibitor TNF antagonist

Gel-A, TNF

Collagenase, Gelatinase, Stromelysin

Collagenase-3, Gelatinase, Stromelysin

Mechanism

Cancer, Other

Arthritis, IBD

Cancer

Arthritis

Indication(s)

Demonstrated efficacy in multispecies arthritis models. May have been superseded by Agouron compound. Produced MSS. Phase I study in 60 colorectal cancer and melanoma patients: 17 maculopapular rash, 3 discontinuing use (Almost all patients had some rash on therapy.) Arthralgia/myalgia (i.e., MSS) developed in 24 patients7 had nausea. Anti-tumor effects not seen Side-effects in long-term preclinical toxicity studies. Tendonitis (i.e., MSS), and other nonspecified toxicities that weren’t limited to any one tissue. GW declined an option for follow-up compounds. British Biotech is dropping other MMPIs in its portfolio including its second generation BB-3644, which was in Phase I. (Bioventure-View, March 08, 2001)

Comments

66 Peterson

Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery

recombinant protein studies which showed that increasing MMP activity is pathological, and decreasing MMP activity is therapeutic in models of cancer, arthritis, and left ventricular remodeling. MMP activity was manipulated in these studies either by overexpression, deletion, or administration of recombinant MMPs or the endogenous tissue inhibitors of matrix metalloproteases (TIMPs, 21–30 kDa). The TIMPs are potent (picomolar IC50/Ki) regulators of in vivo MMP activity [21–23], and four homologs (TIMPs-1 to -4) have been identified to date [24]. TIMP-1 anti-sense depletion of mouse 3T3 cells has a tumorigenic effect [25]. Transgenic mice that constitutively express interstitial collagenase within the epidermis of the skin have an increased susceptibility to tumorigenesis and produced tumors at lower doses of TPA as compared with wildtype mice [26]. Transgenic mice that express an autoactivating form of MMP-3 (i.e., stromelysin) exhibit spontaneous development of premalignant and malignant lesions in the mammary glands unlike nontransgenic littermates [27]. Expression of constitutively active human MMP-13 using a cartilage-specific promoter to target joints of transgenic mice result in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA [28]. TIMP-1 gene deletion results in LV dilation and hypertrophy compared to wildtype mice [29], and accelerates post-MI remodeling [30]. Furthermore, expression of MMP-1 in mice, a species which lacks type 1 collagenase in its genotype, leads to left ventricular remodeling and ultimately results in cardiac dysfunction [31]. TIMP-1 overexpression increases resistance to experimental metastasis [32,33], decreases chemically induced carcinogenesis in skin [34], and reduces tumor growth and angiogenesis [35]. Overexpression of TIMP-1 reduces joint destruction in a model of rheumatoid arthritis [36]. Finally, administration of recombinant TIMP-1 reduces both experimental metastasis [37], and the incidence of ventricular aneurysm following myocardial infarction [38]. The cell types critical in maintaining the proper homeostatic balance of MMP activity has not clearly elucidated. For example, MMP-9 gene deletion reduces keratinocyte hyperproliferation at all neoplastic stages in transgenic mice [39]. However, this response can be can be reconstituted by bone marrow transplantation from wild-type mice [39]. This study demonstrates that cells of hematopoietic origin, and not just the epidermis are critical to maintaining MMP activity mediating squamous carcinogenesis. Although the studies cited above provide strong confidence in rationale, the magnitude and direction of MMP activity change was not directly measured. In addition, TIMPs have been ascribed

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a few other functional roles beyond MMP inhibtion, and that increased TIMP expression can also increase MMP expression [40]. Despite these caveats, the consistency, variety, and number of studies strongly supports the role of elevated MMP activity in several human diseases. Therefore, it would seem myopic given the weight of the molecular data above, or prescient based on the toxicology intrinsic to broad-spectrum MMP inhibitors that has subsequently been revealed that Merck discontinued its MMP inhibitor program when it was discovered that the gene deletion of MMP-3 did not alter arthritis in a mouse model [41].

The Musculoskeletal Syndrome Ironically, a class of drugs developed to treat arthritis, among other conditions, induces a tendonitis-like fibromylagia or musculoskeletal syndrome (MSS) in humans. In a clinical study using marimastat, MSS events requiring dose modification were not observed during the first 28 days of dosing [42]. However, MSS occurred among a substantial number of patients who continued in the long-term continuation protocol. MSS events were dose related and consisted of joint pain, stiffness, edema, skin discoloration, and reduced mobility. The symptoms usually started in the small joints of the hand, as well as the shoulder girdle, typically on the dominant side. If dosing continued unchanged, the symptoms would spread to involve other joints as well. Treatment with nonsteroidal anti-inflammatory agents did not alleviate symptoms. A total of 10/30 patients in the long-term continuation protocol developed MSS judged to be drug-related. The symptoms were severe enough in 5/10 of the patients exhibiting MSS that dose was reduced. The time to onset of musculoskeletal toxicity for the five patients with severe events varied from 56 days (75 mg twice daily) to 199 days (25 mg daily). In another marimastat study, patients with gastric cancer developed arthralgia and joint stiffness. In addition, subcutaneous skin thickening of the palmar surface of the hands, associated with contracture of the digits was observed [43]. These changes were described as resembling Dupuytren’s contacture, a thickening of the deep tissue which passes from the palm into the fingers, and eventually results in the fingers being pulled into the palm. The mean time to sideeffect onset was 45 days, but were to a large extent reversible following the discontinuation of marimastat [43]. Other clinical studies have verified that MSS is dose and time-related; involves joints in the hands, arms and shoulders; is reversible following discontinuation of dosing; and unresponsive

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to analgesics and NSAIDs [44,45]. One group reported that MSS pain begins in the hands [42], however, another group reported that pain started in the shoulders and extends down to the hands which become edematous [46]. The exact sequence of events may depend on the drug used and the dosing regimen. The plasma drug concentrations necessary to produce efficacy for batimastat, marimastat, CGS-27023A and Prinomastat also produced MSS [47,48]. Clinical studies in which MMP inhibitor treatment was not efficacious may have resulted because the therapeutic index was not clearly defined, and too low a dose was employed so as to avoid MSS. One study supporting this hypothesis reported that drug treated cancer patients with MSS showed a significant increase in survival time compared to those which did not [49]. However, increased survival in drug treated patients exhibiting more severe MSS may have also resulted from an increased sensitivity to an indirect (non-MMP) effect. Several hypotheses based on a lack of selectivity have been advanced to explain MSS. An early hypothesis was that inhibition of MMP-1 (type 1 collagenase) activity induced MSS. Table 2 shows the structures and the diversity in the in vitro inhibition profiles of four major types of MMP inhibitors that have been clinically evaluated: hydroxymates, carboxylates, thiols, and the tetracycline analogs (doxycyclines). Marimastat is a potent broad-spectrum MMP inhibitor excluding its activity against MMP-3 (stromelysin type 1), Prinomastat has picomolar potency against MMP-2 (gelatinase A) and MMP-13 (collagenase type 3) but is relatively impotent against MMP-7 (matrilysin), Trocade is a potent collagenase inhibitor, CGS 27023A is a balanced inhibitor, and RS-130830 has picomolar activity against MMP-2 and MMP-13 inhibitor with low activity against MMP-1 and MMP-7. All of these inhibitors were reported to produce MSS including the “deep-pocket” MMP inhibitors prinomastat (Agouron) and tanomastat (Bayer) which disproves the MMP-1 hypothesis of MSS [50]. The carboxylate BAY 12-9566 a relatively selective MMP-2 inhibitor produces MSS while it is unclear whether Rebimastat, which is under phase II for the treatment of cancer, also generates MSS. The doxycyclines are the only clinically effective MMP inhibitors not limited by MSS. A more recent hypothesis is that inhibition of “sheddase” activity attributed to non-MMP “shallow pocket” metalloproteinases such as the adamalysins has been implicated in MSS [51]. Figure 1 illustrates the difference in the human S1’ binding domain size between a small pocket (MMP-1) and large pocket MMP (MMP-13) MMP as well as the sheddases Adamalysin II and tumor necrosis factor alpha converting enzyme (TACE).

The MMP inhibitor, BMS-275291, which has reduced activity against sheddases does not appear to produce MSS [52]. A contrary study reports that broad-spectrum MMP inhibitors with additional ability to block sheddases such as tumor necrosis factor-α convertase (TACE) do not induce MSS [47]. However, in an recently described rodent model of MSS [53], the hydroxymate MMP inhibitor PD 0200126 which is inactive against sheddases produces MSS like effect in rats (unpublished data). MSS is quantified in this model by scoring the presence and magnitude of various clinical signs and histological changes. The presence of a compromised ability to rest on their hind feet, high-stepping gait, reluctance or inability to move and hind paw swelling is noted. Histological changes such as soft tissue and bony changes, increased epiphyseal growth plate, synovial hyperplasia and increased cellularity in the joint capsule and extra capsular ligaments are measured [53]. Figure 2 shows a hematoxylin and eosine stained cross-section of the knee joint from a control (A, 10× magnification) and a marimastattreated (9.375 mg/kg/day) rat (B, 10× magnification) at the end of 2-week experimental period. Note the thickened growth plate, and synovial deterioration in the marimastat-treated rat. A moderate inflammatory cell infiltrate is also present in this model [53]. These changes are dose and time-dependent, and are reversible following the termination of dosing. The MSS produced by marimastat was greater in magnitude than that produced by PD 0200126, and it took a longer period of exposure to PD 0200126 to produce a mild MSS (unpublished data). This model provides both a screen to assess MSS potential and therapeutic index. Identifying the mechanism of MSS has been complicated by the different functional role for a specific gene across species. For example, mutation of MMP-2 causes an arthritis-like syndrome in humans which involves carpal and tarsal osteolysis, osteoporosis, palmar and plantar nodules. This pathology is distinct from that of MMP inhibitor induced MSS [54]. The deletion of the MMP-2 gene in mice is not reported to result in similar joint defects. Deletion of MMP-2 has a different consequence in humans vs. mice. Studies of MMP-9 and MMP-14 deficient mice suggest a role for these MMPs in one or more events associated with MSS (growth plate remodeling and endochondral bone formation, release of angiogenic factors, neo-vascularization, apoptosis, and ossification. Deletion of the MMP-9 gene in mice produces growth plate enlargement, due to a pronounced increase in the zone of chondrocyte maturation and hypertrophy [55]. MMP-14 (membrane type-1 MMP) gene deletion in mice produces joint defects including endochondral ossification

Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery

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Table 2. In vitro inhibition profiles of four major types of MMP inhibitors that have been clinically evaluated: hydroxymates, carboxylates, thiols, and the tetracycline analogs (doxycyclines) Compound

Status & indication

MMP inhibition (IC50 , or Ki∗ )

Matrix Metalloproteinase Inhibitors Evaluated in Clinical Trials Hydroxamic Acid ZBG Phase III, Cancer

MMP-1 MMP-2 MMP-3 MMP-7 MMP-9

5 nM 6 nM 200 nM 20 nM 3 nM

Phase III, Cancer

MMP-1 MMP-2 MMP-3 MMP-7 MMP-13

8.2 nM 0.083 nM 0.27 nM 54 nM 0.038 nM

Phase III (withdrawn), Arthritis

MMP-1 MMP-2 MMP-3 MMP-9 MMP-13

3nM 154 nM 527 nM 59 nM 3 nM

Phase I (withdrawn), Cancer

MMP-1 MMP-2 MMP-3 MMP-9

Phase II (withdrawn), Arthritis

MMP-1 MMP-2 MMP-3 MMP-7 MMP-13

70 nM∗ 0.054 nM∗ 5.2 nM∗ 240 nM∗ 0.17 nM∗

Phase III (withdrawn), Cancer

MMP-1 MMP-2 MMP-3 MMP-9 MMP-13

>5000 nM 11 nM 143 nM 301 nM 1470 nM

BB-2516 (Marimastat)

AG 3340 (Prinomastat)

Ro 32-3555 (Trocade) 33 nM 20 nM 43 nM 8 nM

CGS 27023A

RS-130,830 Carboxylic Acid ZBG

BAY 12-9566 (Continued on next page.)

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Table 2. (Continued ). Compound

Status & indication

MMP Inhibition (IC50 , or Ki∗ )

Phase II, Cancer

MMP-1 MMP-2 MMP-3 MMP-9 MMP-13

Thiol ZBG 25 nM 41 nM 157 nM 25 nM 4 nM

BMS 275291 (Rebimastat) Tetracycline Anologs Phase II, Cancer

Metastat Marketed for Gingivitis/Periodontal Disease

Periostat

Fig. 1. Illustration of the difference in the human S1’ binding domain size between a small pocket (MMP-1) and large pocket MMP (MMP-13) MMP as well as the sheddases Adamalysin II and tumor necrosis factor alpha converting enzyme (TACE).

defects, osteopenia, fibrosis of soft tissues and arthritis [56]. These changes are similar to those observed following chronic marimistat treatment in rats [53]. However, the soft tissue changes in both MMP-9 and MMP-14 knockout mice are characterized by fibrosis rather than the fibroblast hy-

perplasia observed following MMP inhibitor treatment. It is not clear what relevance the growth plate changes in mice have to MSS in humans given that epiphysis occurs at puberty while the average age of patients in MMP inhibitor trials is above 40 years.

Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery

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Fig. 2. Hematoxylin and eosine stained cross-section of the knee joint from a control (A, 10× magnification) and a marimastat-treated (9.375 mg/kg/day) rat (B, 10× magnification) at the end of 2-week experimental period in this rodent model of matrix metalloproteases inhibitor induced musculoskeletal syndrome (MSS). Note the thickened growth plate, and synovial deterioration in the marimastat-treated rat.

Perhaps the most convincing evidence that MSS is not due to MMP inhibition, per se, comes from experiments TIMP gene expression experiments. NMR studies indicate that the binding mode between TIMP homologs and MMPs are similar. The N-terminal side-chain (Thr2 ) of TIMP-1 [57] and TIMP-2 [58] have both been shown to extend into the S1’ pocket containing the catalytic Zn2+ of MMP-3. The direct catalytic inhibitors of

MMPs also target the catalytic Zn2+ within the S1’ pocket, and presumably should share similar biological properties with the TIMPs. Overexpression of TIMP-1 and TIMP-3 is protective in mouse models of rheumatoid arthritis [36,59]. In one study, systemic treatment with significantly reduced paw swelling and increased grip strength compared to control groups. Radiographic assessment also demonstrated a significant reduction

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of joint destruction in the AdTIMP-1 group which was confirmed by histologic analyses showing reduced formation of pannus and erosions [59]. Therefore, MSS appears to be the result of non-selectivity (i.e., the inhibition of some other metalloproteases), or the combined inhibition of a combination of several critical MMPs.

Learnings from Cancer Phase III Trials and Biomarkers The complete failure of MMP inhibitors in phase III cancer trials is puzzling given the number of reports indicating that MMP activity is directly related to tumorigenesis in a variety of mouse cancer models across several independent laboratories. These disappointing clinical results have led many investigators to conclude that MMP inhibitors have no therapeutic benefit in human cancer. In part, the lack of clinical efficacy, despite abundant proof-of-concept may reflect the inadequacy of the animal models available (pancreatic adenocarcinoma and small-cell lung cancer in particular). Classical animal models of subcutaneous or intravenous injection of human tumor cells into immunodeficient mice are insufficient to evaluate the activity of cytostatic agents such as MMP inhibitors because they do not adequately recapitulate host-tumor interactions. Finally, MMP inhibitor clinical studies have typically targeted late stage cancer patients while most preclinical studies manipulated MMP activity prior to the induction of cancer. When batimastat is given at advanced tumor stages in mice no efficacy is observed [60]. This result suggests that the therapeutic benefit of MMP inhibition declines with cancer progression thus providing one explanation for the lack of efficacy in phase III MMP inhibitor cancer trials. Dose-limitations imposed by MSS mentioned in the previous section may also have contributed to the lack of success for MMP inhibitors in cancer clinical trials. A critical liability in MMP inhibitor cancer clinical trials has been the lack of an adequate target inhibition biomarker, or knowledge of the relationship between target inhibition and efficacy. Ideally, a concentration-effect relation based on a biomarker of target inhibition can be generated in phase I studies which can serve as a guide for dose-selection and administration schedule for phase II trials. Validation and selection of the best disease related biomarker(s) in phase II studies would provide a valuable tool to identify patients and monitor efficacy in large-scale phase III trials. This lack of an adequate biomarker calls into question whether proper exposure was achieved. Because MMP inhibitors are cytostatic (cells are growth-arrested but viable) rather than cyto-

toxic (cells are killed), conventional measures of efficacy such as reduction in tumor size could not be used to monitor drug activity. The rate of increase of serum tumor markers was used as a disease or level II related biomarker strategy to guide dose selection for [44]. This approach was criticized because the rate of change in serum tumor marker levels do not necessarily reflect tumor regression [61]. As a consequence of these and other issues, phase I trials were often followed immediately by phase II/III combination trials without the benefit of efficacy information from smaller studies. In addition to tumor markers, the use of MMP expression has met with mixed success based on phase II results. Analysis of circulating gelatinase levels by zymography is not predictive of prognosis in cancer [62]. MMP-9 downregulation by col-3, a tetracycline derivative, does correlate with efficacy [63]. Biomarkers may not only be invaluable in understanding how to derive optimal biological dose and dosing schedule, but may also be used preclinically to define target identification. For example, MMP-11 and MMP-14 expression levels are negative prognostic indicators in small-cell lung cancer, and this tumor type has undetectable expression of MMP2 [64]. Tanomastat, which targets MMP-2 and lacks MMP-11 may have not been the best choice for this type of cancer. In fact, tanomastat treatment accelerated mortality in patients with pancreatic and small-cell lung cancer which lead to the withdrawal of this drug for both arthritis and cancer [65,66]! Marimastat did not reduce survival in a similar patient group suggesting that the mortality observed with tanomastat was not due to MMP inhibition, per se. However, a few animal studies have shown that under certain conditions MMP inhibition may be detrimental rather than therapeutic. MMP inhibitor treatment in mice increases the number of liver micrometastases from breast and lymphoma cells [67]. MMP-9 knockout mice exposed to human papilloma virus 16 tumors; although fewer fewer squamous cell carcinomas are evident in knockout mice their tumors are more aggressive than that of wildtype mice [39]. MMP upregulation has been shown to result in the increased conversion of plasminogen to angiostatin which in turn decreases the vascularization of transplanted tumors [67,68]. Therefore, MMP inhibitors under certain conditions may increase tumor vascularization thus enhancing tumorigenesis. The ultimate biological effect of MMP inhibition on cancer appears to be dependent on disease stage as well as the balance of tumor suppressing (e.g., induction of apoptosis) and tumor-promoting (e.g., decreased angiostatin) activities. The lack of an adequate biomarker(s) for cytostatic agents such as MMP inhibitors has made phase 1/II doserange finding problematic. Dosing limitations

Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery

imposed by side-effects (MSS in particular) may have lead to the selection of doses with marginal efficacy. In the final analysis, the lack of MMP inhibitor clinical efficacy in cancer seems to be a result of “too little - too late”.

Periostat (Doxycyline Hyclate)—The Only Clinically Approved MMP Inhibitor Periostat® (CollaGenex Pharmaceuticals Inc.), also know as doxycycline, is a tetracycline analog which lacks anti-bacterial activity, but inhibits collagenase activity. Periostat was approved by the FDA in September 1998, and still remains the only registered collagenase inhibitor. Periostat (20 mg po, bid) when used as an adjunct to scaling and root planing (SRP), a deep cleaning of the teeth and gums, has been demonstrated to slow the progression of adult periodontitis more effectively than SRP alone. Periodontitis, commonly referred to as gum disease, is an infectious disease estimated to occur in approximately a third of American adults over the age of 35, and is the leading cause of adult tooth loss. The first-line therapy for periodontitis, SRP, primarily address the bacterial component of the disease. When indicated, SRP may be supplemented by antimicrobial drugs. Periostat is a synthetic tetracycline derived from chlortetracycline which was isolated from the soil bacterium streptomyces aureofacians. The tetracyclines were initially modified to increase their antibacterial effectiveness while Periostat was selected because of its weaker antibacterial activity compared to other tetracyclines. Two observations served as a conceptual bridge leading to Periostat. First, Golub et al. in 1990 reported that gingival tissues excised during surgery contained significantly less collagenase when patients received low doses of doxycycline two weeks prior to surgery [69]. Golub hypothesized that suppressing MMPs should result in less periodontal disease by reducing the loss of connective tissues. In addition, out of the 13 MMPs known in 1990, tetracycline appeared to have greater selectivity for the MMPs responsible for fibrillar collagen turnover (i.e., interstitial collagenase or MMP-1, and neutrophil collagenase or MMP-8) [69]. The second observation leading to Peroistat was that the tetracycline molecule was determined to have two distinct regions—one zone where antimicrobial activity is derived, and the other zone with the property to lower MMP activity. Periostat efficacy in adult periodontitis is mediated by decreasing MMP activity which breaks down the supporting structures (gums, ligaments, bone) that hold the teeth in place rather than an antimicrobial action. The novelty of this approach is that it seeks

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to interrupt the progress of periodontal disease by increasing host resistance rather than eliminating the microbial challenge that induces the inflammatory response. Evidence that increased MMP activity originates from host vs. bacterial cells is supported by immunohistochemical analysis which shows that MMP-8 immunopositivity is associated with PMN and epithelial cells while MMP-13 immunopositive cells are located in the basement membrane zone and macrophage-like cells in samples taken from periodontal patients [70]. In the first set of clinical trials using low dose doxycycline, an analysis of sites with the largest lesions (pockets ≥3 mm) revealed that low dose doxycycline had significant benefit [71,72]. In the second clinical study, a randomized, multi-center, double-blind, nine month phase III study involving 190 subjects with periodontal disease, a subantimicrobial dose doxycycline (20mg bid, PO) plus SRP significantly improved clinical attachment level by up to 52 percent and reduced pocket depth by as much as 67 percent compared to SRP plus placebo. These benefits were observed as early as three months into the study and were maintained over the nine-month period of the trial [73]. Progressive improvements in both clinical endpoints were demonstrated in patient groups over 9 months of active treatment, and this benefit was maintained during a 3-month post-treatment period. The maintenance of drug benefit following discontinuation of dosing indicates a normalization of MMP activity following the termination of drug treatment. The attachment level changes in the doxycycline group indicate that the greatest drug gains occurred during the first three months of treatment. Clinical studies indicate that the clinical utility of Periostat in periodontitis is limited to the 15% of the population in which conventional periodontal therapies are not totally successful, a much smaller market than hoped for. The clinical studies also raise a number of issues regarding the use of Periostat. First, the attachment level gains are modest in these studies (∼0.7 mm), with the greatest gain in the deepest sites. However, the gains are only half of the gains experienced in deeper pockets treated with SRP and systemic tetracyclines at an antimicrobial level. In addition, both clinical trials indicate a significant effect in only the severest of defects, antibiotics would be most indicated in aggressive disease. It may be that a combination of low dose doxycycline following systemic antibiotics and SRP would have an additive effect, but no data is available to support this approach. Second, compliance may be an issue for chronic doxycycline treatment. The label for Periostat lists side-effects as including (but not limited to): diarrhea, heartburn, joint pain,

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Fig. 3. Doxycycline (PD 0108627-0002) is a weak direct inhibitor of MMPs (IC50 > 100 µM for MMP-1, MMP-2, MMP-3∗ , MMP-7, and MMP-9). Data courtesy of Dr. Adam Johnson, Pfizer Research and Development, Ann Arbor.

and nausea. Chronic oral doxycycline treatment (100mg bid) in a phase II resulted in 5/33 patients with abdominal aortic aneurysm exhibiting significant treatment-related side effects [74]. In another study, although 90% patients receiving doxycycline reported taking their medication as directed 16% actually managed this level of compliance [75]. The limited utility patient compliance may explain why Periostat sales did not exceed $31 million in 2001 [76].

Doxycycline—Direct Catalytic Inhibitor of MMPs? It is not clear whether the predominant mechanism by which Periostat decreases MMP activity is due to a direct effect, or due to an indirect decrease in collagenase expression. Using a panel of recombinant MMPs in a thiopeptilide assay described elsewhere [6,7], we have shown doxycycline to be a weak inhibitor of MMPs as shown in Figure 3. However, one property of doxycycline which may potentiate its efficacy in periodontal disease is its reported ability to bind to the calcified surfaces of tooth roots [77]. Tissue drug concentration can be as much as 5× greater than that found in blood. In addition, the gradual release of doxycycline from teeth in active form also may contribute to increased exposure, the maintained effectiveness during the post-treatment period. Therefore, it is possible that gingival concentrations of Periostat are sufficient to directly inhibit MMPs.

Periostat and the Treatment of Cardiovascular Disease The clinical utility of Periostat is being assessed in clinical studies for a variety of medical disorders such as dermatologic disorder, rosacea, and

postmenopausal osteoporosis, and acute coronary syndrome. One rationale for evaluating the effect of Periostat in the setting of coronary heart disease originates from a report that periodontal inflammation is associated with an increased risk of heart disease and stroke [78]. The link between periodontal coronary disease was confirmed in a subsequent study of 1,147 men, and this link was interpreted as indicating that periodontal diseases, which are chronic Gram-negative infections, represent a previously unrecognized risk factor for atherosclerosis and thromboembolic events [79]. This association has been hypothesized to result from an underlying inflammatory response trait predisposing individuals to develop both periodontal disease and atherosclerosis. In this scenario periodontal disease produces endotoxins and cytokines which initiate and exacerbate atherogenesis and thromboembolic events [79]. The relative risk for coronary heart disease, fatal coronary heart disease, and stroke are up to 2.8 times greater for those with periodontal disease vs. those without [79]. One potential mechanism linking periodontal and coronary disease involves periodontal bacteria gaining entry into the systemic circulation, and bacteremia causing changes in blood vessel walls that lead to atherosclerosis. Recently, in a study of 50 human specimens removed from carotid arteries, periodontal pathogens were present in all specimens, with 26% being Porphyromonas gingivalis [80]. It is suspected that these microorganisms play a role in the development and progression of atherosclerosis, leading to coronary vascular disease. In a study using mice, oral exposure P. gingivalis resulted in the spread of bacteria into the bloodstream and ultimately the aorta, aortic inflammation ensued and accelerated atherosclerosis was evident by 17 weeks [81]. These results provided supporting evidence that oral infection can accelerate atherosclerotic lesion progression in the aorta. Cytokines are involved in the destruction of both periodontal tissue and alveolar bone. They can stimulate increased production of C-reactive protein (CRP), an important marker of systemic inflammation. Patients with both CVD and periodontal disease had significantly higher mean CRP levels (8×) compared to healthy control patients with neither disease [82]. When the disease group was provided periodontal treatment (SRP) CRP levels dropped 65% by 3 months post-treatment. This effect persisted at 6 months post-treatment. Another recent study 50 patients who were randomized to either a six month subantimicrobial oral dose of Periostat (20 mg, bid) or placebo control [52]. At enrollment, the two treatment arms had similar demographic and clinical characteristics, including age, sex, and frequency of hypertension, diabetes, smoking, prior cardiac

Matrix Metalloproteinase Inhibitor Development Remodeling of Drug Discovery

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Table 3. MMP-inhibitors evaluated for cardiovascular application(s) Product name/ Synonyms

Structure

Originator/ Licensee

PD166793

Pfizer (Parke-Davis)

CP-471474

Pfizer

RS-11-3456

Roche

RS-132908

Roche

PGE7113313

Proctor & Gamble

MT1-MMP inhibitors, 3-D

3-D Pharmaceuticals

KB-R-7785

Kanebo

Nephrostat CMT

CollaGenex

MMP inhibitors, Washington University Doxycycline, Washington matrilysin inhibitor

Washington University

history, extent of coronary disease, presentation with acute myocardial infarction or unstable angina, and need for a percutaneous coronary intervention. Doxycyline significantly reduced CRP levels by 45.8% compared to baseline values at the six-month follow-up period. Drug treatment was also associated with a 33.5% reduction in interleukin-6 and a 50% reduction in MMP-9 ac-

Comments Administration was found to attenuate LV dilation in SHHF rats and paced pigs. Discontinued after preclinical toxicology because of glomelular lesions following chronic dosing in rats. Administration was found to attenuate early LV dilation following experimental MI in mice. Not developed because of low oral bioavailability. Broad spectrum MMPI; has been evaluated in animal models of aneurysm and arthritis. It showed no evidence of fibroplasia after 10 day oral dosing in rats up to 200 mpk Under development for CHF. Data relating to the effects of RS-132908 on constrictive remodeling post-angioplasty in hyperlipidemic FHHL rabbits were presented at the 11th International Meeting of Vascular Biology (Geneva, September 2000). Rabbits were dosed orally once daily with 50 mg/kg for four weeks starting at the time of angioplasty. At the end of the treatment period, the minimal lumen area was 40% larger in the inhibitor group compared to placebo rabbits. (Iddb, 9/00). Under development for CHF. Effective in paced pig HF model [89]. 3-D Pharmaceuticals developing inhibitors of membrane-type 1 matrix metalloproteinase (MT1-MMP) inhibitors for the treatment of restenosis and atherosclerosis. Attenuates cardiac hypertrophy in a pressure-overloaded model in C57BL mice. KB-R7785 treatment also lowered mortality after from 27.9% in vehicle to 10%. Reduces aortic aneursysm in rats. Significantly prolongs survival diabetic rats.

Researchers from Washington U. treated 8 patients with doxycycline, 100 mg bid for 1 week, prior to surgery for abdominal aortic aneurysms. Following surgery, a >5X reduction in MMP-9 RNA in diseased tissue was observed in the treated patients

tivity ( p < 0.05). Low-dose doxycycline was safe with no discontinuations due to treatment-related side effects. However, there was no difference between the low-dose doxycycline and placebo groups in the composite endpoint of cardiovascular death, myocardial infarction, or troponinpositive unstable angina. Brown et al. suggested that their study may have been too short to permit

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Table 4. MMP inhibitors in clinical development Drug name

Therapeutic area

Stage

Company

Mechanism

1 Periostat 2 Neovastat

Periodontitis Cancer/Psoriasis

Launched Phase 3

Collagenex Aeterna Laboratories

3 Dermostat 4 CPA-926

Acne Arthritis

Phase 2 Phase 2

5 DPC-333 6 S-3304 7 Rebimastat

Arthritis Cancer Cancer

Phase 2 Phase 2 Phase 2

Cancer Cancer CHF Arthritis

Phase 2 Phase 1 Phase 1

Collagenex Kureha Chemical Sankyo Bristol Myers Squibb Shionogi Celltech Bristol Myers Squibb Collagenex NIH Pfizer Ono

MMP-1 inhibitor Collagenase & Gelatinase inhibitor, VEGF Receptor-2 antagonist Collagenase inhibitor MMP inhibitor

8 CMT-3 9 CP-471358 10 ONO-4817

adequate endpoint assessment. Finally, Brown et al. hypothesized that positive feedback loop may exist with systemic infection and inflammation accelerating underlying atherothrombosis, inducing myocardial injury, resulting in IL 6 elevation which stimulates hepatic CRP synthesis which exacerbates atherothrombosis (in part through MMP upregulation) and thus ultimately predisposing to plaque destabilization and additional myocardial injury. It is not clear what mechanism(s) account for CRP and IL 6 reductions, nor whether continued Periostat treatment would have a significant and substantial effect on cardiovascular morbidity and mortality.

Will There Be a Next Generation of MMP Inhibitors? Poor bioavailability limited the first generation of MMP inhibitors (e.g., batimastat, D-5410, and Galardin) [75], and side-effects (primarily MSS) limited the second generation of MMP inhibitors [53]. The issue of confidentiality makes it difficult to assess preclinical drug development, however, Table 3 shows MMP inhibitors evaluated in preclinical cardiovascular disease models. Three types of compounds are represented: MMP catalytic site inhibitors (PD0166793, CP-471474, and RS-11-3456), chemically modified tetracyclines (doxycylcine and Nephrostat); and gene therapy (adenoviral TIMP-1). With the exception of doxycycline, no other MMP has advanced into Phase II efficacy evaluation. As mentioned above, chemically modified tetracyclines apparently lower MMP activity by decreasing MMP expression, and possibly by a weak direct inhibition of MMPs in vivo [83–85]. In addition, the clinical efficacy of chemically modified tetracyclines against any form of cardiovascular disease (e.g., atheroscelerosis, aneurysm, or heart failure) has yet to be demonstrated. Although adenoviral ther-

MMP & TNF Convertase Inhibitor Gelatinase & MMP-14 inhibitor Gelatinase inhibitor Collagenase inhibitor MMP-2 inhibitor MMP-8 & MMP-13 inhibitor

apy may eventually prove useful for angiogenesis or restenosis, it is unsuited for heart failure treatment because it is too transient. Peak protein expression following transfection within the myocardium typically occurs within 1-week and declines quickly thereafter [86,87]. The success of even one of the 10 drugs currently under clinical evaluation (Table 4) will generate tremendous interest in developing a new generation of MMP inhibitors. Demonstration of safety and efficacy for a specific chemotype(s) will provide the rationale for re-investment in this area, especially given the potential market for MMP inhibitors across a variety of diseases. The availability of a small animal model to evaluate MSS potential provides a technique to assess the therapeutic index preclinically instead of in a Phase III trial. Finally, the therapeutic effect of many drugs depends on pleotropism (i.e., drug action on multiple targets) [88]. Although the matrix degrading abilities of the MMPs are likely to be important, these activities alone do not account for the diversity of biologic responses modulated by the enzyme family. MMP activity on non-matrix substrates (e.g., chemokines, growth factors, growth factor receptors, adhesion molecules) are also potentially critical for disease progression. If one MMP inhibitor achieves clinical success this could serve as the basis for a parachute strategy to develop a new generation of MMP inhibitors with superior pharmacological properties and efficacy.

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