Barry R. Rifkin

Blocking Periodontal Disease Progression by Inhibiting Tissue-Destructive Enzymes: A Potential Therapeutic Role for Tetracyclines and Their Chemically-Modified Analogs

Tetracyclines (TCs) have wide therapeutic usage as antimicrobial agents; these drugs (e.g., minocycline, doxycycline) remain useful as adjuncts in periodontal therapy. However, TCs also have non-antimicrobial properties which appear to modulate host response. In that regard, TCs and their chemically-modified analogs (CMTs) have been shown to inhibit the activity of the matrix metalloproteinase (MMP), collagenase. The activity of this enzyme appears crucial in the destruction of the major structural protein of connective tissues, collagen. Such pathologic collagenolysis may be a common denominator in tissue destructive diseases such as rheumatoid and Osteoarthritis, diabetes mellitus, bullous dermatologic diseases, corneal ulcers, and periodontitis. The mechanisms by which TCs affect and, possibly, diminish bone resorption (a key event in the pathogenesis of periodontal and other diseases) are not yet understood. However, a number of possibilities remain open for investigation including the following: TCs may 1) directly inhibit the activity of extracellular collagenase and other MMPs such as gelatinase; 2) prevent the activation of its proenzyme by scavenging reactive oxygen species generated by other cell types (e.g. PMNs, osteoclasts); 3) inhibit the secretion of other collagenolytic enzymes (i.e. lysosomal cathepsins); and 4) directly affect other aspects of osteoclast structure and function. Several recent studies have also addressed the therapeutic potential of TCs and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. Furthermore, the latter drug (CMT) was not associated with the emergence of TC-resistant microorganisms. In human clinical trials, low-dose doxycycline therapy substantially reduced collagenase activity in the gingiva and GCF, and prevented the loss of attachment in adult periodontitis. Clearly, the non-antimicrobial properties of TCs have enormous medical and dental therapeutic potential since these drugs can inhibit the activity of MMPs and their degradation of non-osseous and osseous connective tissues. J Periodontol 1993; 64:819-827.

Cathepsin B and L activities in isolated osteoclasts.

Cathepsin B and L activities were examined with chicken osteoclasts isolated by sequential filtration and inhibitors were added to disaggregated rat osteoclasts on cortical bovine bone. Z-Phe-Phe-CHN2, a selective inhibitor of cathepsin L, at 1, 5, and 10 microM, inhibited bone resorption by rat osteoclasts 50, 85, and 100 per cent and, in chicken osteoclasts, cathepsin L activity was comparably inhibited. Cathepsin L in avian osteoclasts was also 25-fold higher than cathepsin B. Chicken osteoclasts treated with Z-Phe-Ala-CHN2, a generalized cysteine proteinase inhibitor, had both cathepsins inhibited to the same extent. Cathepsin L may play a key role in resorption.

Effects of Tetracyclines on Bone Metabolism

The anti-resorptive properties of tetracyclines (TCs) and their non-antimicrobial, chemically modified analogues (CMTs) have enormous therapeutic potential in medicine and dentistry. Osseous destructive diseases associated with excessive mammalian collagenase (matrix metalloproteinase) activity and collagen breakdown include malignancy, arthritis, and periodontitis. However, apart from the significant antimatrix metalloproteinase effects of TCs, TCs/CMTs are also potent inhibitors of osteoclast function (i.e., anti-resorptive). Thus, TCs can affect several parameters of osteoclast function and consequently inhibit bone resorption by (1) altering intracellular calcium concentration and interacting with the putative calcium receptor; (2) decreasing ruffled border area; (3) diminishing acid production; (4) diminishing the secretion of lysosomal cysteine proteinases (cathepsins); (5) inducing cell retraction by affecting podosomes; (6) inhibiting osteoclast gelatinase activity; (7) selectively inhibiting osteoclast ontogeny or development; and (8) inducing apoptosis or programmed cell death of osteoclasts. TCs/CMTs, as anti-resorptive drugs, may act similarly to bisphosphonates and primarily affect osteoclast function.

CMT-8/clodronate combination therapy synergistically inhibits alveolar bone loss in LPS-induced periodontitis.

Earlier studies have reported that a chemically modified doxycycline (CMT-8) administered as a monotherapy inhibited alveolar bone loss in a lipopolysaccharides(LPS) injected rat periodontitis model. 1,2 Teronen et al. reported that clodronate, a bisphosphonate, also inhibited the activity of MMP-8, the predominant collagenase in inflamed gingival tissue and in gingival crevicular fluid in human adult periodontitis. 2 In the present study the effects of the combination of subtherapeutic levels of chemically modified doxycycline (CMT-8) and a bisphosphonate (clodronate) were investigated in a LPS-induced periodontal disease model. This model, described here, was published by Ramamurthy et al. 1 and involves the injection of E. coli endotoxin into the gingival tissues. Injection of endotoxin into the gingiva produces marked inflammation in the periodontium, pathologically elevated levels of tissuedestructive matrix metalloproteinases (MMPs), leading to severe alveolar bone resorption and bone loss around the affected teeth.

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ “receptor”. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca2+] response to an application of 5 mM-[Ni2+]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the osteoclast Ca2+ “receptor”.

The effect of tetracyclines on quantitative measures of osteoclast morphology

We report the effects of the tetracycline analogues 4-dedimethylaminotetracycline (CMT-1) and minocycline on osteoclast spreading and motility. Both agents influenced the morphometric descriptor of cell spread area, ϱ, producting cellular retraction or an R effect (half-times: 30 and 44 minutes for CMT-1 and minocycline, respectively). At the concentrations employed, the tetracycline-induced R effects were significantly slower than, but were qualitatively similar to, those resulting from Ca2+ “receptor” activation through the application of 15 mM-[Ca2+] (slopes: −1.25, −0.18, and −4.40/minute for 10 mg/l-[CMT-1], 10 mg/l-[minocycline] and 15 mM-[Ca2+], respectively). In contrast, the same tetracycline concentrations did not influence osteoclast margin ruffling activity as described by μ, a motility descriptor known to be influenced by elevations of cellular cyclic AMP. Thus, the tetracyclines exert morphometric effects comparable to changes selectively activated by occupancy of the osteoclast Ca2+ “receptor” which may act through an increase in cytosolic [Ca2+].

The effect of phenytoin on parathyroid hormone stimulated cAMP activity in cultured murine osteoblasts

Cells were isolated by sequential collagenase digestion from the parietal segments of one day old mice (Swiss albino BNL strain) and characterized for osteoblast parameters by alkaline phosphatase histochemistry and bovine parathyroid hormone (bPTH-(1-34] induced cAMP activity (protein binding assay). Phenytoin (DPH) reduced PTH stimulated cAMP activity nearly 3-fold in the presence and nearly 1.5-fold in the absence of added calcium. In the absence of PTH, DPH exerted no significant effect. Bay-K-8644, a calcium channel activator, appeared to approximate the PTH stimulation of cAMP activity, even in the presence of DPH. This study demonstrates that DPH has a direct effect on PTH stimulated cAMP activity in cultured murine osteoblasts.

The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells.

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase.

Gelatinases/type IV collagenases in jaw cyst expansion.

Jaw cysts are destructive lesions of the facial skeleton that occur frequently. The expansile growth of cysts has been extensively studied, but the molecular mechanism(s) responsible for cyst expansion is still unclear. Interstitial collagenase can initiate the destruction of extracellular collagenous matrix (ECM), and collagenolytic activity has been demonstrated in radicular cyst and keratocyst homogenates.14 Tissue degradation also involves other members of the matrix metalloproteinase (MMP) family, namely, the gelatinases/type IV collagenases. In this study we characterize the molecular forms of gelatinases/type IV collagenases present in radicular cysts utilizing zymographic analysis and Western blotting. In addition, we studied the effects of different compounds with regard to regulation-activation and inhibition-an jaw cyst gelatinases as well as 92-kDa polymorphonuclear leukocyte

Collagenase and Other Osteoblast Enzymes

Publisher Summary This chapter focuses on osteoblast collagenase and related matrix metalloproteinase (MMPs) and their putative roles in skeletal remodeling as degradative enzymes; along with a discussion of osteoclast MMPs. Bone remodeling depends on the precise regulation of bone resorption and formation. Thus, an imbalance between these processes may lead consequently to pathologic bone loss. Osteoblasts synthesize a collagen-containing matrix which mineralizes to form mature bone. Collagen is the major component of the bone matrix; it represents at least 90% of the organic matrix of bone and is mostly type I collagen. Interstitial collagenase (matrix metalloproteinase- 1, MMP- 1) activity, as well as the activity of other MMPs (for example, gelatinases [MMP-2 and MMP-9] and stromelysin [MMP-3]), may be important in both the normal and pathologic remodeling and resorption of the bone collagen extracellular matrix. The activity of the bone cell MMPs is regulated in vivo by the tissue inhibitors of MMPs (TIMPs). Two independent studies showed the role of matrix metalloproteinases as not only significant but also, perhaps, novel and distinct in resorption. PTH-induced resorption in fetal rat limb bones was associated with the production of the MMPs, collagenase and gelatinase B, and inhibited with recombinant TIMP-1. A discussion of the plasmin and plasminogen activator system and its role in bone remodeling is presented in the chapter.