L.M. Golub

Tetracyclines Inhibit Connective Tissue Breakdown by Multiple Non-Antimicrobial Mechanisms

A seminal experiment involving a germ-free rat model of connective tissue breakdown (followed soon thereafter by a series of in vitro studies) identified an unexpected non-antimicrobial property of tetracyclines (TCs). This ability of TCs to inhibit matrix metalloproteinases (MMPs) such as collagenase was found to reflect multiple direct and indirect mechanisms of action, and to be therapeutically useful in a variety of dental (e.g., adult periodontitis) and medical (e.g., arthritis, osteoporosis, cancer) diseases. The site on the TC molecule responsible for its MMP-inhibitory activity was identified which led to the development of a series of chemically modified non-antimicrobial analogs, called CMTs, which also have therapeutic potential but do not appear to induce antibiotic side-effects. Longitudinal double-blind studies on humans with adult periodontitis have demonstrated that a sub-antimicrobial dose of doxycycline (previously reported to suppress collagenase activity in the periodontal pocket) is safe and effective and has recently been approved by the FDA as an adjunct to scaling and root planing.

A Non-antibacterial Chemically-modified Tetracycline Inhibits Mammalian Collagenase Activity

Tetracyclines (including the semi-synthetic analogues, minocycline and doxycycline) are considered useful adjuncts in periodontal therapy because they suppress Gram-negative periodontopathogens. Recently, these antibiotics were found to inhibit mammalian collagenase activity, a property which may also be of therapeutic value. It has been suggested that the anti-collagenase properties of the tetracyclines are independent of their antibiotic efficacy. To advance this hypothesis further, we chemically converted tetracycline hydrochloride to its non-antimicrobial analogue, de-dimethylaminotetracycline. This chemically-modified tetracycline (CMT), although no longer an effective antibiotic, was found to inhibit the in vitro activity of collagenase from partially purified extracts of human rheumatoid synovial tissue and rachitic rat epiphysis. In a preliminary in vivo study, pathologically-excessive collagenase in skin and gingiva was induced by rendering adult male rats diabetic, and the oral administration of CMT to these rats significantly reduced the excessive collagenase activity in both tissues. Moreover, CMT administration did not affect the severe hyperglycemia in these rats but did prevent, at least in part, the diabetes-induced loss of body weight, skin weight, and skin collagen mass; these effects suggest a lack of toxicity in this animal model. A proposed clinical advantage of CMT over conventional tetracyclines, in the treatment of diseases characterized by excessive collagenolytic activity, is the lack of development of antibiotic-resistant micro-organisms during prolonged use. However, the consideration of clinical trials to support this hypothesis must await further laboratory and extensive toxicity tests.

Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells

Doxycycline is a commonly used broad-spectrum antibiotic. Recently, it has been shown that it also inhibits the activity of mammalian collagenases and gelatinases, an activity unrelated to its antimicrobial efficacy. In this study, we show that doxycycline not only inhibits MMP-8 and MMP-9 (gelatinase B) activity, but also the synthesis of MMPs in human endothelial cells. Doxycycline (50 μM) completely inhibited the phorbol-12-myristate-13-acetate (PMA)-mediated induction of MMP-8 and MMP-9, as measured by Western blotting and gelatin zymography, respectively. The inhibition was also observed at the mRNA level. No effect was observed on the expression of MMP-2 and of the MMP inhibitors TIMP-1 and TIMP-2. Chemically modified tetracyclines (CMTs) showed an inhibition similar to that of doxycycline, albeit less efficient. These observations demonstrate that endothelial cells display a specific regulation of MMPs, which may have implications for the pharmaceutical interaction in angiogenesis and angiogenesis-related diseases.

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

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 microgram/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 micrograms/mL depending on assay conditions and choice of TC, whereas inhibition of the fibroblast enzyme (MMP-1) generally requires levels in excess of 200 micrograms/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.

Periodontopathic potential of two strains of Porphyromonas gingivalis in gnotobiotic rats

Germ-free rats were monoinfected with Porphyromonas gingivalis strains 381 or A7A1-28 for 42 or 84 days. Both strains induced substantial destruction of alveolar bone and soft tissue when compared to non-infected controls, but the patterns were different. Strain A7A1-28 was associated with increased activity of host collagenase and gelatinase at 42 days, whereas the activity was elevated to a lesser extent at 84 days. Strain 381 showed a moderate increase in host proteinase activity at 42 days, and this remained unchanged until day 84. Strain A7A1-28 was associated with more bone loss than strain 381 by a morphometric analysis that detects horizontal bone loss in the maxilla. Strain 381 was associated with more bone loss than strain A7A1-28 by a radiographic method that detects vertical intrabony defects in the mandible. Infection with one strain gave rise to serum and salivary antibodies strongly reactive to the infecting strain and moderately reactive to antigens from the other strain. This indicates that some antigenic similarity exists between the strains and that there are also strain or perhaps serotype differences in antibody responses induced by infection. Thus two strains of P. gingivalis differing in antigenicity and pathogenicity in the mouse model of the subcutaneous abscess cause substantial periodontal destruction in the germ-free rat. The disease pattern is, however, different, with strain A7A1-28 inducing mostly horizontal bone loss and strain 381 mostly vertical.

Tetracycline Prevents Cancellous Bone Loss and Maintains Near-Normal Rates of Bone Formation in Streptozotocin Diabetic Rats

The skeletal consequences of streptozotocin-induced (STZ) diabetes in the rat are characterized by decreased bone formation and, consequently, reductions in bone mass. Given the ability of tetracyclines to inhibit the breakdown of connective tissue collagen in experimental diabetes (and in other diseases), we examined the potential of this drug to prevent the osteopenia associated with STZ diabetes. To evaluate drug efficacy, the cortical and trabecular bone histomorphometry were analyzed and compared between vehicle-treated control and diabetic rats and control and diabetic rats treated orally with 20 mg/day of minocycline, a semisynthetic tetracycline. In addition, blood and urine glucose, body weight change, tibia lengths, cortical bone densities, and bone ash content were compared. At the end of the 26 day experimental period, diabetic (D) and minocycline-treated diabetic (MTD) rats were polyuric with reduced body weights and significantly elevated blood and urinary glucose levels (p < 0.01). Compared to control (C) and minocycline-treated control (MTC) animals, the periosteal and cancellous bone formation in the D rats had virtually ceased (p < 0.001), and the cancellous bone mass in the tibial metaphysis was reduced 47% (p < 0.01). In contrast, bone formation rates in the MTD animals were increased compared to the D rats (p < 0.001), while cancellous bone areas in the MTD animals were essentially equivalent to those observed in the C and MTC groups. Moreover, growth plate thickness, reduced 43% in the D rats, was preserved in the diabetic animals treated with minocycline. These results demonstrate that minocycline treatment of the streptozotocin diabetic rat maintains normal bone formation, normalizes growth plate thickness, and prevents cancellous bone loss.

Tetracyclines inhibit protein glycation in experimental diabetes.

Glycation of proteins, which is accelerated in the diabetic state, has been implicated in many of the long-term complications of diabetes. This process can be inhibited by members of the tetracycline family of compounds. This novel finding is supported by studies conducted on drug (streptozotocin)induced Type I and genetic (ZDF/Gmi-fa/fa) Type II diabetic rats. These animals were orally gavaged daily with 5 mg of doxycycline and a variety of non-antimicrobial chemically modified tetracycline derivatives for time periods of 3 weeks to 11 months, while control untreated diabetic and nondiabetic animals were gavaged with vehicle alone (2% CMC). Blood and tissue samples were collected and analyzed for glucose and glycated proteins. None of the treatments had any effect on the severity of hyperglycemia or the intracellular glycation of hemoglobin of either Type I or II diabetic animals. However, the tetracycline analogues did affect the extracellular glycation of several proteins such as those found in the serum as well as skin collagen. In the Type II (ZDF) animals, initial mortality (3-5 months) was seen only in the doxycycline-treated animals, associated with infection by tetracycline-resistant micro-organisms, which was eventually surpassed by mortality rates in the untreated diabetics (6-9 months). CMT treatment not only decreased mortality but also increased longevity in the Type II diabetic animals, most likely by preventing the development of a number of long-term complications of uncontrolled diabetes, including glycation of proteins, that eventually lead to the demise of untreated diabetic animals.

Tetracycline/flurbiprofen combination therapy modulates bone remodeling in ovariectomized rats: Preliminary observations

The loss of trabecular bone in the ovariectomized (OVX) rat provides a useful experimental model of postmenopausal osteoporosis. In this study, two bone-modulating compounds, an NSAID (flurbiprofen: FBP) and a chemically modified nonantimicrobial tetracycline (CMT), were tested either individually or in combination in this model. Ninety days after OVX, 6-month-old female rats were distributed into the following groups: sham-operated controls, untreated OVX, CMT-treated OVX (5 mg P.O./day), FBP-treated OVX (0.3 mg P.O./day), and combination (CMT plus FBP)-treated OVX (COMBO) groups. Untreated 3-month-old rats were used as pretreatment group. After 21 days of therapy, the dissected distal femurs were processed for light and fluorescence microscopic and backscattered electron microscopic examinations. Net trabecular bone values showed that all the treatment groups lost trabecular bone over the 111 day protocol compared to pretreatment group. In the untreated OVX rats, trabecular bone volume/unit area was reduced by 56% compared to that in the sham-operated controls, this bone loss associated with increased numbers of osteoclasts (p < 0.05). Cortical bone volume was, however, not significantly reduced in OVX rats. Both FBP-alone and COMBO therapy showed marginal, but significant, (p < 0.05, p < 0.01, respectively) inhibition of trabecular bone loss, and osteoclast numbers were also decreased (p < 0.05). Both CMT alone and COMBO therapy appeared to increase bone deposition (p < 0.01) at the endosteal surfaces of cortical bone. These results suggest that, in this animal model, (a) cortical bone volume increases by CMT; (b) FBP inhibits osteoclastic bone resorption in the trabecular area, and (c) a combination of these drugs may synergistically prevent bone loss.

The development of an altered gingival crevicular microflora in the alloxan-diabetic rat

The microflora of the rat gingival crevice were examined at various time intervals after inducing experimental diabetes. A variety of Gram-positive and Gram-negative cocci and short rods were isolated from the normal rat crevice. Within a week after alloxan administration, Leptotrichia buccalis was detected for the first time. Other features in the diabetic animals included an increased frequency of isolation of Proteus spp., Lactobacillus spp. and decrease of Escherichia coli. Occasionally, a decrease was also observed in Bacteroides spp. and Streptococcus spp. No difference was observed between diabetic and non-diabetic animals in the isolation of Selonomonas, Campylobacter, Bifidobacterium and Actinomyces. Plaque accumulation was markedly increased in the diabetic rats. It was concluded that the change in gingival microflora resulted from diabetes-induced alterations in the sulcular environment (such as increased substrate levels, e.g. glucose and urea, and decreased oxygen) and that the microbiological changes preceded the depending of the periodontal pocket.

HPLC determination of a chemically modified nonantimicrobial tetracycline: Biological implications

Chemically modified tetracycline (4-de-dimethylamino tetracycline), like commercially available tetracyclines, is known to inhibit experimentally induced pathologic collagen breakdown. A method for measurement of chemically modified tetracycline in small volumes (50 microliters) of rat serum was developed using reversed-phase HPLC; this was necessary because this tetracycline analog lacks antimicrobial activity and, therefore, cannot be measured with standard bioassays. This method uses the same solution for extraction and elution thus providing a simple and rapid assay for both drugs. Using this technique, the concentration of chemically modified tetracycline and tetracycline were determined in rat serum at different times after oral administration. The serum concentration of chemically modified tetracycline was much higher than that for tetracycline, and its serum half-life was greater. The IC50 of chemically modified tetracycline and tetracycline, as inhibitors of collagenase from rat polymorphonuclear leukocytes, was determined and found to be 4.1 x 10(-8) M (0.02 micrograms/ml) and 2.4 x 10(-4) M (120 micrograms/ml), respectively. Based on the serum levels of these drugs after oral administration, and their IC50 values, chemically modified tetracycline is potentially a far more potent inhibitor of excess collagenase activity than tetracycline, during pathologic conditions, and may have the added advantage of not producing some of the typical complications of long-term antibiotic therapy.