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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To characterize the clinical and histopathologic changes in a rat model of broad-spectrum matrix metalloproteinase (MMP)–induced musculoskeletal syndrome (MSS), and to facilitate research into the causes and treatments of MSS in humans.

Methods

Male Lewis rats weighing 150–180 gm were administered 10–30 mg of the broad-spectrum MMP inhibitor marimastat over a 2-week period via surgically implanted subcutaneous osmotic pumps. The animals were monitored and scored for the onset and severity of MSS, using clinical and histologic parameters.

Results

Marimastat-treated rats exhibited various clinical signs, including compromised ability to rest on their hind feet, high-stepping gait, reluctance or inability to move, and hind paw swelling. Histologically, marimastat-treated rat joints were characterized by soft tissue and bone changes, such as increased epiphyseal growth plate, synovial hyperplasia, and increased cellularity in the joint capsule and extracapsular ligaments. The severity of MSS, as judged by clinical criteria (2 blinded observers using 3 clinical parameters), paw volume, and histologic score, was nearly identical. The observed changes were indistinguishable from those reported for primate models and mimic MSS in humans.

Conclusion

This simple and sensitive model of MSS is an attractive alternative for studying the pathology of MSS.

The matrix metalloproteinases (MMPs) constitute a gene family comprising more than 20 members whose function is to degrade extracellular matrix (ECM) components. Because of the potential of MMPs to degrade ECM, overexpression of MMPs in pathologic states such as arthritis, cancer, and atherosclerosis is believed to be responsible for the matrix destruction observed in these disorders (1–4).

The ECM degradation function of MMPs is also important for mediating various physiologic processes, including growth and development, wound healing, and postpartum uterine involution (4–5). Membrane type 1 (MT1)–MMP–deficient mice are reported to develop dwarfism, osteopenia, and arthritis (6). Similarly, mutation of the MMP-2 gene is known to cause dwarfism, multicentric osteolysis with crippling arthritic changes, and marked osteoporosis. However, MMP-2–deficient mice develop normally, except for a subtle delay in their growth (7, 8).

The function of MMPs is not restricted to ECM degradation. They also mediate other biologic processes, such as cell migration, cleavage, and activation of growth factors and other proteinases (7, 9–11). Data on MMP-9 and MT1-MMP–deficient mice suggest a role for these MMPs in 1 or more events associated with growth plate remodeling and endochondral bone formation, including release of angiogenic factors, neovascularization, apoptosis, and ossification (6, 12).

MMPs have been considered excellent targets for drug development for both arthritis and cancer, because inhibition of their activity has resulted in disease-modifying capabilities in animal models. In particular, broad-spectrum MMP inhibitors such as marimastat and batimastat have been shown to be efficacious in a wide array of animal models of inflammation and cancer (13–15). The clinical utility of the broad-spectrum MMP inhibitors, including batimastat, marimastat, CGS-27023A, and prinomastat, has been restricted, however, by a musculoskeletal side effect observed in humans (16–17). The side effect is characterized by a variety of clinical signs, including joint stiffness, inflammation, and symptoms manifested as pain in the hands, arms, and shoulders. The musculoskeletal syndrome (MSS) or tendinitis that occurs at or near the time when effective pharmacologic concentrations of these drugs are reached is dose- and time-dependent but is reversible shortly after stopping the drug treatment (4, 18).

Several hypotheses have been proposed to explain the mechanism of MSS. One early hypothesis was that inhibition of MMP-1 (collagenase 1) activity was responsible for MSS. Conversely, other reports suggest that collagenase-selective and gelatinase-selective inhibitors have less of an association with MSS (16). It has also been reported that broad-spectrum MMP inhibitors with additional ability to block sheddases, such as tumor necrosis factor α convertase (TACE), do not induce MSS (16). At present, the mechanism of this toxicity is not clearly understood.

To facilitate research into the causes of the side effect and to possibly screen for compounds that do not cause this side effect, efforts have been made to develop animal models that mimic human MSS. Marmosets are perhaps the species most commonly used for evaluating the propensity of MMP inhibitors to induce MSS. However, because marmosets are primates and require prolonged dosing before exhibiting clinical signs of MSS, they may not be the ideal species for compound screening. It was recently reported that infusion of marimastat into rats for ∼14 days results in joint changes that are histologically and clinically similar to those seen in marmosets (16). Thus, the rat appears to be a more practical species in terms of both the compound requirement and the time needed for completion of a study.

Currently, very little published information is available on either the marmoset model or the rat model of MSS. Thus, the purpose of this study was to characterize the histologic and clinical changes in rat knee joints following administration of the broad-spectrum MMP inhibitor marimastat.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Dose-response and time-course studies.

Male Lewis rats (Charles River, Wilmington, MA) weighing 150–180 gm were administered a total of either 10, 20, or 30 mg/animal (3.125, 6.250, and 9.375 mg/kg/day, respectively) of marimastat (synthesized at Ann Arbor Laboratories, Pfizer Inc., Ann Arbor, MI) dissolved in equal parts DMSO and water over a 2-week period via surgically implanted subcutaneous Alzet osmotic pumps (5 μl/hour) (Alza, Palo Alto, CA). The control group received vehicle alone (50% DMSO/50% water). Animals in the time-course experiments were administered 30 mg of marimastat for various time periods (1–14 days). Both the dose-response and time-course studies included 6 animals per group.

In vivo procedures and observations.

Animal treatment groups were coded for blinded behavioral scoring. Each day, the animals were monitored and scored by 2 blinded observers for the onset and severity of MSS, based on the clinical scoring system developed previously (19). Briefly, the animals received a score between 0 (normal) and 2 (resting on no feet) for resting posture, between 0 (normal) and 2 (avoids use of both hind feet) for gait, and between 0 (normal movement) and 3 (very reluctant to move) for willingness to move when stimulated. For each animal, the total score obtained over the 2-week experimental period was used as a measure of the severity of MSS; scores for each treatment group were averaged. In the time-course experiments, however, the scores were averaged each day and were presented as daily scores. Paw volumes were measured at the end of the 2-week experimental period using a Buxco (Troy, NY) plethysmometer. Both hind paw volumes were measured and averaged.

Histology.

At necropsy, the hind limbs from all rats were collected for histopathologic evaluation of the knee and ankle joints. Tissues were fixed in 10% neutral buffered formalin. Fixed tissues were decalcified and trimmed sagittally. One-half of each joint was embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Knee sections were also subjected to proliferating cell nuclear antigen (PCNA) staining for assessment of fibroblast proliferation. Coded sections were evaluated according to a semiquantitative grading scheme consisting of 8 microscopic parameters for knee joints and 3 for ankle joints. The study pathologist was blinded to the identity of the treatment groups. Scores ranged from 0 (no pathologic abnormality) to 4 (very pronounced changes). Scores for each rat and treatment were averaged.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Clinical assessment.

Rats dosed with marimastat differed markedly from control animals in terms of their resting posture, gait, and willingness and/or ability to move. The animals' hind feet appeared to be most affected by the drug treatment, as evidenced by their swollen paws, reluctance or inability to rest on their hind feet, and characteristic high-stepping gait. Furthermore, these animals were lethargic, had a hunched appearance, and refused to move when prodded gently. The treated animals also exhibited a reduction in body weight gain. The animals that received vehicle exhibited normal behavior throughout the experimental period.

As assessed by 2 blinded observers, marimastat-treated animals in any of the treatment groups exhibited no clinical signs until day 7 or 8. From then on, a time-dependent increase in the clinical scores of marimastat-treated animals was observed (Figure 1). Clinical scores for each of the marimastat-treated groups were significantly higher than those for controls (P < 0.05) (Figure 2). In concordance with the clinical scores, the marimastat-treated animals also exhibited dose-dependently higher hind paw volumes than did controls (results not shown).

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Figure 1. Development of the musculoskeletal syndrome in rats administered marimastat (total dose 30 mg/animal [9.375 mg/kg/day]), as judged by the clinical criteria described in Materials and Methods. Bars show the mean and SEM.

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Figure 2. Dose-related increases in clinical scores for the musculoskeletal syndrome (MSS) in marimastat-treated rats. Values are the mean and SEM of the total scores obtained over the 2-week experimental period. ∗ = P <0.05 versus control, by Dunnett's multiple comparison test.

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Gross and histopathologic changes.

Upon gross examination, the joints of control rats were easily distinguished from those of marimastat-treated rats. The growth plates of the marimastat-treated animals were thickened and more friable than those of control animals. Histologically, treated rats had a group of abnormal findings that could be separated into effects on bone and effects on soft tissues. The most striking change was observed in the growth plate: the growth plates of marimastat-treated animals were enlarged compared with those of control animals (Figures 3A and 3B). The growth plate enlargement was attributable to a pronounced increase in the zone of chondrocyte maturation and hypertrophy (Figure 3B). The proliferative zone was of normal thickness (Figure 3B). Newly formed metaphyseal trabecular bone, subjacent to the growth plate, was also thickened and disorganized in treated animals (results not shown).

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Figure 3. Histologic sections of the growth plate (area between arrows) in the knee joints of control (A) and marimastat-treated (B) rats at the end of the 2-week experimental period. Note the thickened growth plate of the marimastat-treated rat. (Hematoxylin–eosin stained; original magnification × 40 in A; × 20 in B.) JS = joint space; AC = articular cartilage.

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Synovial cells lining the joint space increased in both size and number, forming a vascular pannus over the cartilage in treated animals (Figure 4B). The articular cartilage in these animals was not altered, however. The synovium of control animals was normal (Figure 4A). The rat knee has an adipose tissue pad adjacent to the synovium at the lateral articular junction (Figure 5A). This tissue was focally fibrotic in treated animals (Figure 5B). In marimastat-treated animals, the joint space itself had slightly increased amounts of proteinaceous fluid and fibrin compared with the amounts in controls.

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Figure 4. Histologic sections showing the articular cartilage and the synovium in the knee joints of control (A) and marimastat-treated (B) rats. Note the thin, flat, 1–2-layer synovium lining the joint space in the control rat (arrow in A) and the multilayered synovium, with polygonal cells (arrow in B), and vascular pannus over articular cartilage (double arrow in B) in the marimastat-treated rat. (Hematoxylin–eosin stained; original magnification × 200.)

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Figure 5. Histologic sections showing adipose tissue beneath synovium in the knee joints of control (A) and marimastat-treated (B) rats. Although the adipose tissue is unremarkable in the control rat (circle in A), it is fibrotic, with dense collagen matrix extending well beneath the synovial surface in the treated rat (circle in B). (Hematoxylin–eosin stained; original magnification × 100.)

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Increased cellularity was observed in the soft tissue of the joint capsule and in the extracapsular ligaments of marimastat-treated animals, as compared with controls (Figure 6A and 6B). The cell nuclei were large, with dispersed chromatin, and mitotic figures could be identified. The impression of reactive, proliferating fibroblasts was supported by PCNA immunohistochemical staining, which revealed many more cycling cells in the treated animals than in controls (Figure 6C). The fibroblastic proliferation was associated with scattered lymphocytes as well as focal collections in the soft tissues. Ankle joints exhibited primarily soft tissue and synovial changes (results not shown).

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Figure 6. Histologic sections showing the joint capsule in the knee joints of control (A) and marimastat-treated (B) rats. Note the scattered, condensed fibroblast nuclei in dense collagen matrix in the control animal and the marked increase in fibroblast cellularity and nuclear size, with perivascular aggregates of lymphocytes (arrows in B) in the treated rat. (Hematoxylin–eosin stained; original magnification × 200 in A, × 400 in B.) A histologic section of the joint capsule of a marimastat-treated rat (C) shows proliferating cell nuclear antigen immunohistochemical staining of the joint capsule. Note the positively stained (brown) large fibroblast nuclei, indicating cycling cells (original magnification × 400).

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Histopathology scores in treated animals increased in a dose-dependent manner (Table 1). The parameters with the most dynamic range of scores were growth plate thickening, subphyseal trabecular thickening, synovial hyperplasia/hypertrophy, and capsule fibroplasia. None of the control animals had any positive scores for any parameter. In contrast to evaluation of the knee, ankle joint evaluation did not separate treatment groups well (results not shown), although controls were distinctly separated from treated animals (results not shown).

Table 1. Histologic scores in the knee joints of rats administered marimastat*
ParameterControlMarimastat dose, mg/kg/day
9.3756.2503.125
Growth plate thickening03.02.81.7
Subphyseal trabecular thickening02.71.71.0
Articular surface pannus01.50.70
Synovial hyperplasia/hypertrophy02.52.21.0
Synovial fibrosis of adipose tissue01.71.50.5
Capsule fibroplasia02.52.51.0
Soft tissue lymphocytic infiltrate01.71.50.8
Fibrin in joint space01.31.30.2
Total knee score016.814.25.8

Interestingly, the severity of MSS, as judged by clinical criteria (2 blinded observers using 3 clinical parameters), by paw volume, and by histologic score, was nearly identical (Figure 2 and Table 1).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Marimastat is a prototype of the broad-spectrum MMP inhibitors that were used in clinical trials based on their efficacy in preclinical studies but were found to induce MSS (20–24). MSS is characterized by varying degrees of stiffness and pain in the hands, arms, and shoulders (25). Unfortunately, other than clinical parameters, no other information is available to evaluate MSS in humans and to understand the underlying pathology. Thus, development of an animal model predictive of MSS in humans has been critical to the development of broad-spectrum MMP inhibitors and to the understanding of the pathophysiology of MSS.

Given the lack of information on the pathology of MSS as it occurs in humans, preclinical studies have focused on histologic and clinical changes in animals—frequently in primates (marmosets and rhesus monkeys) and more recently in rats—treated with compounds that are known to cause MSS in humans. Unfortunately, very little of this information has been published. In this study, we examined the clinical and histologic characteristics of a rat model of marimastat-induced MSS.

The marimastat-treated rats in this study exhibited various clinical signs, including compromised ability to rest on their hind feet, high-stepping gait, reluctance or inability to move, and hind paw swelling. Histologically, the joints of rats treated with marimastat were characterized by growth plate and subphyseal trabecular thickening, synovial hyperplasia and hypertrophy, formation of articular surface pannus, fibrosis of perisynovial adipose tissue, soft tissue fibroplasias, lymphocyte infiltrates, and fibrin within the joint space (Figures 3–6). Deposition of matrix was not increased, other than what would be expected from the proliferation of fibroblast-like cells. Furthermore, the severity of MSS, as judged by clinical criteria (2 blinded observers using 3 rating parameters), by hind paw volume, and by blinded pathologic examination, was almost identical (Figure 2 and Table 1).

It has been reported that marimastat induces MSS in marmoset and rat models of MSS, and that the histologic and clinical changes observed in the 2 models are similar (16). Monkeys treated with broad-spectrum MMP inhibitors are known to exhibit dose-dependent contracture of appendicular joints, swelling of the knee joints, and disuse of the legs (the animals pull themselves around with their arms [personal communication]). Except for growth plate changes, almost all other joint-related histologic changes observed in rats have also been observed in monkeys (personal communication).

It is likely that the growth plate changes observed in this study are related to the age of the animals used (young animals undergoing active remodeling of their growth plates), because these changes were less significant or absent when the study was performed in older rats (results not shown). It is unclear whether the growth plate changes share the same mechanism as the soft tissue changes in the joint, and whether they have any bearing on the MSS observed in humans, given that previous trials of MMP inhibitors in humans have involved adults, whose growth plates are closed. Thus, it appears that the rat model of MSS is indistinguishable from the primate model. Compared with primate models, however, the rat model has several advantages: low cost, ease of setup, and shorter time to complete a study (2 weeks versus several months). Thus, the rat model can potentially be used for screening compounds with the potential to induce MSS and for studying the mechanism by which broad-spectrum MMP inhibitors induce MSS.

Several hypotheses have been proposed regarding the mechanism of MMP inhibitor–induced MSS. An early hypothesis, probably one of chemical convenience, was that inhibition of MMP-1 (collagenase 1) activity was responsible for MSS (16). Fortunately, MMP-1 has a unique binding pocket that allowed for ready exclusion of this enzyme from the inhibitory profile of MMP inhibitors. However, lack of MMP-1 inhibition has failed to correlate in any meaningful way with protection from MSS. Indeed, as seen in the present study and in other investigations, rodents dosed with broad-spectrum MMP inhibitors develop the same histologic changes as those observed in marmosets, but the rodent genome lacks MMP-1 altogether (16). Furthermore, it has been reported that collagenase-selective and gelatinase-selective inhibitors are not associated with MSS (16). For instance, patients treated with trocade, a specific inhibitor of collagenases 1, 2, and 3, exhibited no clinical signs of MSS, although the doses chosen were sufficiently low to avoid this side effect, and to date, trocade has demonstrated no efficacy against RA (26).

Another hypothesis that has been put forth is that broad-spectrum MMP inhibitors with additional ability to block sheddases such as TACE do not induce MSS (24). In the present study, however, marimastat, a broad-spectrum MMP inhibitor with the ability to block TACE activity, induced MSS (27). More experimental data regarding the role of sheddases in MSS are needed.

Clearly, a better understanding of the relative contributions of various MMPs to MSS toxicity is needed. It may be difficult to obtain this type of information from studies on MMP inhibitors, because essentially all of these studies have used broad-spectrum MMP inhibitors. Rather, the degree of MSS induced by these broad-spectrum MMP inhibitors has varied significantly with simple changes in chemical structure (data not shown). One must recognize that many MMP inhibitors block the action not only of MMPs, but also of other metal-dependent enzymes (dependent upon potency of the enzyme and blood concentrations of the inhibitor), albeit to a lesser degree. MMPs represent <10% of metal-dependent proteinases and <1% of metal-dependent proteins.

Studies on MMP expression suggest that the tissues involved in MSS can potentially express the whole spectrum of MMPs. Synovium and the cartilage–pannus junction in rats have been shown to express high levels of stromelysin and collagenase when subjected to collagen-induced arthritis (28). Using immunolocalization techniques, MMP-9 was detected in synovial macrophages, fibroblasts, and neutrophils in rats with adjuvant-induced arthritis (AIA) (29). Expression of MMP-3 and MMP-13 messenger RNA in the synovial tissue of rats with AIA has been reported (30). Enzyme assays and Western blot techniques have revealed the presence of MMP-2 as well as MMP-9 in paw tissue extracts prepared from rats with AIA. Similarly, expression of several MMPs (including MMP-9, MMP-8, MMP-13, and MMP-14) has been observed in, or immediately adjacent to, the epiphyseal growth plates of rats (31–35). In addition, MMP-3 is reported to be involved in the activation of transforming growth factor β1 in rat growth plate chondrocytes (36). Thus, based on the information on MMP expression in rat joints, it is possible to speculate that multiple MMPs could be involved in the marimastat-induced joint pathology observed in the present study. Thus, it appears that the studies on MMP expression or broad-spectrum MMP inhibitors do not discriminate the relative roles of individual MMPs in MSS.

Data from recent gene deletion studies on individual MMPs have offered additional information on the molecular mechanisms of MSS (37). Data from MT1-MMP−/− mice suggest that disruption of the MT1-MMP gene results in endochondral ossification defects, osteopenia, fibrosis of soft tissues, and arthritis (37). The soft tissue changes observed in our study as well as in the MT1-MMP−/− mice were characterized by increased cellularity of synovial tissue, synovial capsules, and ligaments. Although severe ankylosis resulting from progressive fibrosis of articular soft tissues was observed in the knee joints of MT1-MMP−/− mice, soft tissues in the present study were characterized mainly by fibroplasia. Although vascular pannus over the cartilage was observed, and the adipose tissue pad adjacent to the synovium was focally fibrotic, the cartilage was not altered in the rats, unlike in MT1-MMP−/− mice (37). Thus, the degree of severity of the soft tissue findings was much greater in the MT1-MMP−/− mice than in the marimastat-treated rats.

In terms of changes in the growth plate, a markedly lengthened zone of hypertrophic chondrocytes was observed in the growth plates of animals treated with marimastat. In contrast, the hypertrophic zones in the growth plates of MT1-MMP mice were essentially normal in postnatal life (6, 28). The development of epiphyseal (secondary) centers of ossification was disrupted in the mutant mice (6, 28), but no such findings were observed with marimastat. It has been suggested that the endochondral ossification process in the growth plate may be different from that in the epiphysis, perhaps requiring gelatinase B in the case of the growth plate but not in the case of the epiphyseal cartilage (12). Thus, based on different histologic findings, it does not appear that MT1-MMP is responsible for marimastat-induced musculoskeletal side effects. Moreover, it is not known whether MT1-MMP−/− mice display the clinical signs of MSS. It should be noted that our study evaluated rodents that underwent normal musculoskeletal development until drug administration, whereas MTI-MMP−/− mice have been subject to the absence of MT1-MMP since conception. Therefore, it remains possible that some of the histologic changes observed in the 2 studies represent different stages of an MT1-MMP–mediated pathology. However, we consider this to be a remote possibility.

Comparison of data from the present study with those regarding MMP-9/gelatinase B−/− mice indicates that the growth plate changes observed in the 2 studies are similar (12). In both studies, progressive lengthening of the growth plates was observed. The enlargement of growth plates was attributable to a pronounced increase in the zone of chondrocyte maturation and hypertrophy, while the proliferative zone was of normal thickness (12). Thus, it appears that the growth plate changes observed in the present study could be the result of inhibition of gelatinase B by marimastat. We are aware of no report that MMP-9−/− mice exhibit soft tissue changes such as those observed in the present study. Therefore, although it is likely that the MMP-9 inhibition by marimastat may be responsible for the growth plate changes, the soft tissue changes and perhaps the clinical signs are unrelated to inhibition of MMP-9.

In humans, mutation of the MMP-2 gene is known to cause a multicentric osteolysis and arthritis syndrome. Reported clinical findings, including carpal and tarsal osteolysis, osteoporosis, and palmar and plantar nodules, however, appear to be distinct from those reported for patients with MSS (8). Moreover, MMP-2–null mice are not reported to have joint defects. These data do not support the idea of involvement of MMP-2 in MSS. Even when all available data are combined, the relative role of individual MMPs in MSS remains unclear.

In summary, marimastat treatment resulted in several musculoskeletal side effects in the joints of rats, including soft tissue and bone changes. The histologic changes in soft tissue and the clinical signs observed in this study are indistinguishable from those observed in primate models, and perhaps mimic MSS in humans. The observed growth plate changes indicate that broad-spectrum MMP inhibitors can also disrupt endochondral ossification of the growth plates, thereby interfering with long-bone growth. It is possible that the mechanisms of broad-spectrum MMP inhibitor–mediated MSS and growth plate changes are different.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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