Elsevier

Bone

Volume 22, Issue 1, January 1998, Pages 33-38
Bone

Original Articles
In Vitro Sensitivity of the Three Mammalian Collagenases to Tetracycline Inhibition: Relationship to Bone and Cartilage Degradation

https://doi.org/10.1016/S8756-3282(97)00221-4Get rights and content

Abstract

There are at least nine tetracycline (TC) analogs (both antimicrobial and nonantimicrobial) with documented capacity to inhibit, both in vitro and in vivo, the connective tissue degrading activity of matrix metalloproteinases (MMPs). Of the three MMPs that can degrade native helical collagens, MMP-13 (initially identified as rat osteoblast and human breast cancer collagenase, and now known to also be expressed by human cartilage and bone cells) is the most sensitive to TC inhibition (IC50 values in vitro generally less than 1 μg/mL); the TCs inhibit both the collagenolytic as well as the gelatinolytic activity of this enzyme. The IC50 for MMP-8 (neutrophil collagenase) in vitro ranges from 15 to 86 μg/mL depending on assay conditions and choice of TC, whereas inhibition of the fibroblast enzyme (MMP-1) generally requires levels in excess of 200 μg/mL (except for CMT-3). The TC compounds that are highly effective against MMP-13 in vitro are also highly inhibitory of glycosaminoglycan release from interleukin-1-stimulated cartilage explants in culture. The current data correlate well with: (i) literature values for TC inhibition of bone resorption by isolated osteoclasts; (ii) inhibition by TCs of avian tibial resorption in organ culture; and (iii) the dramatic ability of TCs to inhibit bone destruction in many rat models (rats have only MMP-8 and MMP-13, and no MMP-1). By carefully selecting a TC-based MMP inhibitor and controlling dosages, it should be possible to inhibit pathologically excessive MMP-8 and/or MMP-13 activity, especially that causing bone erosion, without affecting the constitutive levels of MMP-1 needed for tissue remodeling and normal host function; in this regard, three newly developed CMTs (especially CMT-8 and, to a lesser extent, CMT-3 and -7) appear to be most effective.

Introduction

The superfamily of matrix metalloproteinases (MMPs, or matrixins) includes three neutral proteinases (collagenases) capable of degrading native, helical collagens of several genetically distinct subtypes. The first such enzyme was discovered in 1962 and reported in the classic work of Gross and Lapierre. The enzyme was identified as that which caused resorption of the tadpole tail during metamorphosis and is now referred to as MMP-1 or fibroblast-type collagenase (collagenase-1). MMP-1 is believed to be the constitutive enzyme responsible for tissue remodeling, but has also been found to be produced in elevated amounts in certain disease processes. A second collagenase, MMP-8, or neutrophil-type collagenase, is found in the specific granules of polymorphonuclear leukocytes, but has also been found to be expressed by non-PMN lineage mesenchymal cells such as chondrocytes and fibroblasts.4, 37Recently, a third form of mammalian collagenase, now called MMP-13 (or collagenase-3), was cloned from human breast cancer and found to have 86% sequence homology with rat osteoblast collagenase.10, 22This collagenase, which degrades type II collagen more efficiently than types I or III as well as degrading proteoglycan, and which also degrades gelatin much more efficiently than MMP-1 or MMP-8,9, 23, 29is also expressed in human bone and cartilage cells and has been implicated in bone and cartilage destruction as occurs in rheumatoid and osteoarthritis.6, 19, 20, 26, 30, 41

Pathologically excessive levels of collagenase have been identified in a wide variety of disease states characterized by connective tissue, including bone, breakdown. Although the potential therapeutic utility of a collagenase inhibitor was recognized within a few years after the discovery of MMP-1, it is somewhat surprising that there is no pharmacologic product currently available for therapeutic use specifically designed as an MMP inhibitor. One explanation cited for this therapeutic deficiency is the need to find inhibitors specific for the species of collagenases/MMPs associated with inflammatory and degenerative disease processes that would not interfere with physiologic tissue remodeling.

An extensive literature now exists on the inhibitory capabilities of various tetracycline (TC) analogs toward MMPs; the findings have been documented for enzymes from more than 60 tissue sources in at least 26 independent laboratories (see references 14, 17, 18and [32]for reviews). Rat models have been extensively used for much of this work; periodontal disease, diabetes, adjuvant arthritis, and estrogen-deficient osteoporosis are but four examples of rat models of disease affecting bones in which TCs have been shown to inhibit collagenase and/or connective tissue degradation. In view of the recent studies demonstrating that MMP-8 and MMP-13 are the major inducible forms of excess collagenase in inflammatory processes,11, 36and the finding that rats do not have MMP-1 but do express MMP-13, the current study was undertaken to compare the relative inhibitory capacities of both the antimicrobial and nonantimicrobial TCs toward the three mammalian collagenases.

Section snippets

Tetracyclines

The MMP-inhibitory effect of TCs was first demonstrated using the semisynthetic antibiotic TCs known as minocycline (Min) and doxycycline (Dox), which are readily available commercially for antimicrobial use; for the current studies, these two TCs were obtained either from Sigma (St. Louis, MO) or Pfizer (Groton, CT). Subsequent studies led to the development of a series of TC analogs modified to eliminate their antimicrobial capability but preserve their capacity to inhibit MMPs; these have

Results

Table 2 displays eight sets of data relating to TC inhibition of bone or cartilage degradation; six parameters are reported here for the first time and are compared with two sets of data from the literature. Three compounds, especially CMT-8 (and to a lesser extent, CMT-3 and CMT-7), stand out as having the highest across-the-board potency. CMT-5, the pyrazole derivative devoid of Zn++-binding capacity, is essentially inactive as an MMP inhibitor. CMT-1, the first nonantibacterial TC to have

Discussion

Collagenases and other MMPs have long been implicated in disorders that result in destruction of bone and/or cartilage, and the search for therapeutically useful inhibitors of collagenases has spanned three decades. Although many low-molecular-weight MMP inhibitors have been developed, none has been brought to market. The discovery that certain TCs, including the commercially available antibiotics minocycline and doxycycline, can inhibit at least five different MMPs (MMP-1, -2, -8, -9, and -13,

Acknowledgements

Note added in proof: In contrast to the findings in the current study, Lindy et al. (Arth Rheumatol 40:1391–1399; 1997) just reported that “autoactive” MMP-13 was relatively insensitive to TC inhibition in vitro like MMP-1. These observations, apparently contradictory to the potent ability of TC analogs (e.g., CMT-8) to inhibit MMP-13 in vitro, may be reconciled as follows: in the current study, the CMTs were added to the MMP-13 together with APMA, the latter used to activate the pro-form of

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