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Background: Periodontal disease (PD) and rheumatoid arthritis (RA) share many common pathophysiologic features, but a clinical relationship between the two conditions remains controversial, in part because of the confounding effects of anti-inflammatory drug therapy universally used in the latter disease. To further explore this issue, inflammatory arthritis was induced in rats to determine the effect on gingival biomarkers of inflammation and tissue destruction and to investigate the effect of a therapeutic intervention devoid of conventional anti-inflammatory properties., Methods: Adjuvant arthritis (AA) was induced in Lewis male rats by injecting mycobacterium cell wall in complete Freund's adjuvant using standard techniques. One group of animals was treated by induction of systemic tissue inhibitor of matrix metalloproteinases (TIMP-4). At 3 weeks, arthritis severity was recorded and both paw and gingival tissues were collected for matrix metalloproteinase activity (MMP) and cytokine analysis. In addition, the maxillary jaws were removed for assessment of periodontal bone loss., Results: The development of arthritis was associated with elevated joint tissue MMPs, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta levels compared to control rats. In the gingival tissue of the untreated arthritic rats, gelatinase, collagenase, TNF-alpha, and IL-1beta were also elevated compared to control rats. Periodontal bone loss and tooth mobility were also increased significantly (P <0.05) in untreated arthritic rats. All parameters improved after TIMP-4 gene therapy., Conclusions: To our knowledge, this is the first study to report an association between experimental systemic arthritis in rats and elevated gingival tissue MMPs, cytokine levels, and periodontal disease. Reversal of these changes with TIMP-4 gene therapy strengthens the pathophysiologic correlation between systemic and local disease.

Dextran sulfate sodium-induced inflammatory bowel disease in mice resembles human ulcerative colitis. In inflammatory bowel diseases matrix metalloproteinases contribute to tissue degradation. Laminin-5 is an anchoring filament protein in the basement membrane area that can be cleaved by matrix metalloproteinases. We investigated the expression of matrix metalloproteinases-2 and -8 and laminin-5 gamma2-chain in dextran sulfate sodium-induced mice by immunohistochemistry and in situ hybridization. Matrix metalloproteinase-8 expression was evidenced in the colon surface epithelial cells and the protein was more abundant in dextran sulfate sodium-induced mice colon. Matrix metallproteinase-2 and laminin-5 gamma2-chain colocalized in the colon surface epithelial cells and in the basement membrane zone as demonstrated by double immunostaining. In dextran sulfate sodium-induced colon, matrix metalloproteinase-2 immunoreactivity was detected in epithelial cells in the lower parts of the crypt and surrounding the degraded crypts. Matrix metalloproteinase-2 and -8 could participate in the local epithelial inflammatory processes and tissue destruction. The presence of laminin-5 gamma2-chain indicates alternative anchoring mechanisms in the colon, a compartment devoid of hemidesmosomes.

Background: Chemically modified tetracyclines (CMTs), devoid of antimicrobial activity, inhibit pathologically elevated collagenase activity both in vivo and in vitro. In the current study, doxycycline and 5 different CMTs were tested to prevent matrix metalloproteinase (MMP)-dependent periodontal tissue breakdown in an animal model of periodontitis., Methods: Adult male rats received intragingival injections with either 10 microl of physiologic saline or Escherichia coli endotoxin (1 mg/ml) every other day for 6 days and were distributed into 8 treatment groups (12 rats/group): saline (S), endotoxin alone (E), E + CMT-1, E + CMT-3, E + CMT-4, E + CMT-7, E + CMT-8, and doxycycline. All animals were treated daily with 1 ml of 2% carboxymethyl cellulose (CMC) alone or containing one of the above-mentioned CMTs (2 mg/day) orally. The gingival tissues were removed, extracted, and assayed for gelatinase (GLSE). Some rat maxillary jaws from each treatment group were fixed in buffered formalin and processed for histology and immunohistochemistry for the cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6, and MMP-2 and MMP-9., Results: Endotoxin injection induced elevated GLSE activity (functional assay and osteoclast-mediated bone resorption), the former identified as predominantly MMP-9 (92 kDa GLSE) by gelatin zymography. All 6 tetracyclines (2 mg/day) inhibited periodontal breakdown in the following order of efficacy: CMT-8 > CMT- 1 > CMT-3 > doxycycline > CMT-4 > CMT-7. Immunohistochemistry was positive for TNF, IL-1, and IL-6 in the inflammatory cells from untreated endotoxin rat tissues, whereas treatment with CMTs decreased the number of immuno-positive stained cells for cytokines and MMPs. The in vivo efficacy of these drugs varied with CMT structure and was significantly correlated with bone resorption: r2 = -0.77, P<0.01; gelatinase inhibitory activity: r2 = -0.84, P <0.01; and serum drug concentrations., Conclusion: Since both conventional (antimicrobial) and non-antimicrobial tetracyclines inhibited periodontal bone resorption induced by endotoxin injection, MMP-mediated bone loss in this model can be prevented by inhibition of MMPs.

Periodontal disease is characterized by excessive host collagenase resulting in loss of gingival and periodontal ligament collagen and adjacent alveolar bone. Intragingival endotoxin injection induces a model of periodontal disease characterized by rapid bone loss with biochemical features similar to that of naturally occurring adult periodontitis. CH1766, a peptide with a zinc binding moeity which fits into the active site of the enzyme, and CH6631, a hydroxamic acid derivative with aryl-substituted sulphonamide residues, are inhibitors of matrix metalloproteinases (MMPIs) with differing inhibitory profiles as characterized by in vitro assays. In this study, endotoxin was injected into the gingivae of rats which were then treated orally with either 3 mg/kg or 30 mg/kg of one of the two inhibitory compounds. The gingival tissues were assessed for collagenase and gelatinase activity, plus three different pro-inflammatory cytokines. In addition, alveolar bone height in defleshed jaws was studied by computerized morphometric analysis and scanning electron microscopy. Both drugs reduced active and/or total MMP activity, in many cases to normal, and also partially normalized cytokine levels as well. A dose-response effect was seen with regard to amelioration of lipopolysaccharide-induced alveolar bone loss with both drugs. Other than studies with tetracyclines, this is the first report of beneficial effects of MMPIs in a model of periodontal disease, strongly suggesting that this class of agents could bring therapeutic benefit to patients with this disorder, and that periodontal disease can be used as a model to demonstrate in vivo efficacy of this class of drugs.

Estrogen deficiency is associated with impaired cutaneous wound healing. Remodeling of the extracellular matrix in wound healing involves the action of matrix metalloproteinases on basement membrane zone components, especially laminin-5. We studied the effects of estrogen and a potent matrix metalloproteinase inhibitor, chemically modified non-antimicrobial tetracycline, CMT-8, on wound healing in ovariectomized rats. At the tissue level, laminin-5 gamma2-chain expression was decreased and the migration-inductive 80 kDa form of laminin-5 gamma2-chain was absent in ovariectomized rats when compared with sham and CMT-8- or estrogen-treated ovariectomized animals as detected by Western blotting. The highest levels of gelatinolytic activity (matrix metalloproteinase-2 and -9) were found in sham animals. Levels were reduced in ovariectomized rats and were lowest after treating ovariectomized rats with CMT-8 or estrogen as analyzed by functional activity assay and zymography. The total amount of membrane type 1-matrix metalloproteinase was unchanged in all groups. We conclude that CMT-8 and estrogen can promote wound healing in ovariectomized rats, not only by normalizing wound bed total collagen content and structure, but also by recovering the expression and processing of key molecules in wound healing, i.e., laminin-5 gamma2-chain. This study shows, for the first time, the role of estrogen and CMT-8 in laminin-5 gamma2-chain modulation in vivo.

Background: Tetracyclines, particularly doxycycline (Doxy), and their non-antimicrobial chemically-modified derivatives (CMTs) inhibit the activities of human matrix metalloproteinases (MMPs), and reduce the severity and progression of periodontal disease in animal models and humans. In this study, the effects of Doxy and CMT-1, -3, and -5 on proteolytic, serpinolytic, and progelatinase-B activation activities of potent periodontopathogens were studied., Methods: The effect of Doxy and CMTs (0.5 to 50 microM) on proteolytic activities were investigated by incubating bacteria with chromogenic substrates or human serum albumin. A collagenolytic fraction of Porphyromonas gingivalis was used to evaluate the effect of these substances on collagenolytic (type I collagen) and serpinolytic (alpha1-proteinase inhibitor) activities. Lastly, the effect of Doxy on progelatinase-B (pro-MMP-9) activation by purified proteinases from P. gingivalis and Treponema denticola was investigated by SDS-PAGE/Western immunoblotting., Results: Doxy and CMTs, except CMT-5 which lacks the structural elements required for cation chelation, inhibited Arg- and Lys-gingipain activities as well as collagenolytic activity of P. gingivalis. Doxy and CMTs did not markedly affect the chymotrypsin-like activity of T. denticola but inhibited its trypsin-like activity. In addition, degradation of human serum albumin by cells of P. gingivalis and T. denticola was strongly inhibited by Doxy and CMT-1. Doxy and CMT-1 also inhibited the inactivation of alpha1-proteinase inhibitor (serpinolytic activity) by a collagenolytic fraction of P. gingivalis. Lastly, Doxy prevented the latent to active conversion of human neutrophil progelatinase-B (pro-MMP-9) by Arg-gingipains A/B of P. gingivalis but not by the chymotrypsin-like proteinase of T. denticola., Conclusions: Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response.

Background: Chemically modified non-antimicrobial tetracyclines (CMTs) have been shown to inhibit pathologically elevated collagenase (and other matrix metalloproteinase, MMP) activity and bone resorption in vivo and in vitro., Methods: In the current study, suboptimal doses of CMT-8 (a non-antimicrobial chemically modified doxycycline) and a bisphosphonate (clodronate, an anti-bone resorption compound) were administered daily, either as a single agent or as a combination therapy, to rats with experimental periodontitis induced by repeated injection of bacterial endotoxin (LPS) into the gingiva. At the end of the 1-week protocol, the gingival tissues were dissected, extracted, and the extracts analyzed for MMPs (collagenases and gelatinases) and for elastase, and the defleshed jaws were morphometrically analyzed for alveolar bone loss., Results: LPS injection significantly (P<0.001) increased alveolar bone loss and increased collagenase (MMP-8), gelatinase (MMP-9), and elastase activities. Treatment of the LPS-injected rats with suboptimal CMT-8 alone or suboptimal clodronate alone produced slight reductions in the tissue-destructive proteinases and no significant reductions in alveolar bone loss. However, a combination of suboptimal CMT-8 and clodronate "normalized" the pathologically elevated levels of MMPs, elastase, and alveolar bone loss, indicating synergistic inhibition of tissue breakdown in this animal model of periodontitis., Conclusions: Combination of a CMT and a bisphosphonate may be a useful treatment to optimally suppress periodontal destruction and tooth loss and in other tissue-destructive inflammatory diseases such as arthritis.

Diabetes mellitus in rats is characterized by excessive activity of several matrix metalloproteinases (MMPs), notably collagenase(s) and gelatinase(s), in skin, gingiva, and other tissues. A number of tetracyclines (TCs), both antimicrobial compounds as well as chemically modified non-antimicrobial TC analogues (CMTs) are known to possess potent inhibitory activity against these enzymes. Three conventional antimicrobial TCs and six CMTs were used in this study. In vitro, doxycycline was shown to possess higher inhibitory capacity (i.e. lower IC(max)) against diabetic rat skin collagenase than either minocycline or tetracycline HCl. Addition of excess zinc partially reversed the proteinase inhibition by TCs. In vivo, using rats made diabetic with streptozotocin (STZ), oral administration of various TCs led to decreased weight loss and substantial reductions in the activity of both skin collagenase and skin gelatinase (primarily MMP-9, 92 kDa) without affecting blood glucose. Using an in vitro spectrophotometric technique, the Zn(++) reactivity of several CMTs was assessed and found to be positively related to the potency of these compounds as MMP inhibitors. One particular CMT (CMT-5, pyrazole analogue), which is neither antimicrobial nor capable of binding metal cations, did not inhibit the MMPs. TCs have potential utility in management of diabetic complications mediated by excessive activity of MMPs.

CMT-3 is a NON-ANTIMICROBIAL tetracycline (TC), chemically modified to enhance its collagenase-inhibitory property. This property is therapeutically useful in treating diseases such as periodontitis, cancer and arthritis. CMT-3 was labeled with tritium [(3)H] at Carbon 7. Four adult male Sprague-Dawley rats (350--400 g body weight) were gavaged once with a mixture of cold CMT-3 and [(3)H] CMT-3 (750 microCi). An additional four rats were gavaged for 2 days with cold CMT-3(15 mg/Kg/day) and on the third day the rats were gavaged with a mixture of cold and [(2)H] CMT-3 (750 microCi); and all 8 rats were placed in the metabolic cages. Blood samples were collected from the tail at multiple intervals from 1--14 hr after [(3)H] CMT-3 administration. At 14 hr, the rats were anesthetized, euthanized and various tissues including visceral organs were removed and weighed. The contents of GI tracts were emptied and added to the fecal pellets and weighed. The urine samples were collected and volume measured. Each tissue or organ was minced finely with scissors and 100 mg of tissue was digested in 1 ml of Tissue-solv (Packard Lab), for 4 hrs at 37 degrees C and each sample was diluted up to 10 ml of distilled water. A 100 microl aliquot was taken and diluted with an equal volume of glacial acetic acid, 10 ml of Atom-lite was added and counted for radioactivity in a liquid scintillation spectrometer. This biodistribution study revealed that over 14 hrs, 54% and 3% of [(3)H] CMT-3 were excreted in the feces and urine, respectively. The serum [(3)H] CMT-3 count reached its maximum value at about 12 hours. The tissues retained the CMTs as follow: muscle (23%); skin (2.41%); bone (1.72%); and the brain retained 0.21% of the label. The radioactive CMT-3 in the visceral organs is as follows: GI tract - its contents (8.9%); heart (0.41%), testis (0.41%); lungs >(0.16%); spleen (0.08%); liver (0.03%); kidneys > (0.02%).

Background: Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils. Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS). We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB., Methods and Results: Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration. CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05). These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity., Conclusions: All pathological changes typical of ALI after CPB were prevented by CMT-3. Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity. This study suggests that strategies to combat ARDS should target terminal neutrophil effectors.

Estrogen deficiency in the postmenopausal (PM) female is the major cause of osteoporosis and may contribute to increased periodontal disease, including alveolar bone loss, seen in these women. In the current study, an animal model of PM osteoporosis, the OVX adult female rat, was studied to determine: (i) the relationship between periodontal breakdown and skeletal bone loss, and (ii) the effect of CMT-8 on gingival collagenase and bone loss. OVX rats were daily gavaged with CMT-8 (1, 2, or 5 mg/rat) for 28 or 90 days; non-OVX rats and those gavaged with vehicle alone served as controls. Elevated collagenase activity, assessed using [3H-methyl] collagen as substrate in the presence or absence of APMA, was seen in the gingiva of the OVX rats, and CMT-8 therapy suppressed this effect. Western blot revealed a similar pattern for MMP-8 and MMP-13 concentrations. The changes in the gingival collagenase activity paralleled changes in periodontal bone loss, which, in turn, reflected trabecular bone density changes. Preliminary studies on PM humans administered sub-antimicrobial tetracycline as a matrix metalloproteinase inhibitor are under way.

We examined the effects of a nonantimicrobial tetracycline analogue, CMT-8, on bone loss and osteoclasts in ovariectomized (OVX) rats. Three-month-old female rats were OVX, and, one week later, distributed into three groups: sham-operated non-OVX controls, untreated OVX controls, and CMT-8-treated OVX rats. After 145 days of daily drug administration (p.o.), the femurs were dissected and examined histologically. Ovariectomy markedly decreased trabecular and cortical bone volume in the metaphyses compared to sham-operated controls. Treating the OVX rats with CMT-8 produced a significant inhibition of trabecular and cortical bone loss and induced new bone formation, in which connectivity of the trabecular struts was increased by bridging the adjacent longitudinal bone trabeculae. Ultrastructurally, CMT-8 reduced ruffled border formation in osteoclasts, while it caused no structural impairment in osteoblasts. To further evaluate the effects of CMT-8 on the resorbing activity of osteoclasts, osteoclasts were cultured on dentine slices pretreated with CMT-8 at concentrations of 2, 10, or 50 micrograms/ml, and resorption lacuna formation on the dentine surface was found to be reduced, dose-dependently, by the bound CMT-8. Our results suggest that CMT-8 therapy effectively inhibits post-ovariectomy bone loss not only by inducing new bone formation, but also by inhibiting osteoclastic bone resorption, and that CMT-8 binding to bone may provide a prolonged release delivery of this anti-resorptive therapy.

Both Type I and Type II diabetes mellitus (DM) have been associated with unusually aggressive periodontitis. Accordingly, rat models of both types of DM were used to study (i) mechanisms mediating this systemic/local interaction and (ii) new pharmacologic approaches involving a series of chemically modified tetracyclines (CMTs) that have lost their antimicrobial but retained their host-modulating (e.g., MMP-inhibitory) properties. In vitro experiments on tissues from Type I DM rats demonstrated that several of these CMTs were better matrix metalloproteinase (MMP) inhibitors than was antibacterial doxycycline (doxy), except for CMT-5, which, unlike the other MMP inhibitors, was found not to react with zinc. Data from in vivo studies on the same rat model generally supported the relative efficacy of these compounds: the CMTs and doxy were found to inhibit MMP activity, enzyme expression, and alveolar bone loss. To examine other long-term complications such as nephropathy and retinopathy, a Type II (ZDF) model of DM was studied. Treatment of these DM rats with CMT-8 produced a 37% (p < 0.05), 93% (p < 0.001), and 50% (p < 0.01) reduction in the incidence of cataract development, proteinuria, and tooth loss, respectively; whereas the doxy-treated ZDF rats showed little or no effect on these parameters. CMT treatment decreased mortality of the Type II ZDF diabetic animals, clearly indicating that CMTs, but not commercially available antibiotic tetracyclines (TCs), may have therapeutic applications for the long-term management of diabetes.

The effect of a new non-antimicrobial analog of tetracycline (CMT-8) on bone loss in ovariectomized (OVX) rats was examined. Three-month-old female rats were ovariectomized, and one week later, were distributed into 3 groups: sham-operated non-OVX controls, vehicle-treated OVX controls, and CMT-8-treated OVX rats. After 145 days of daily CMT-8 administration, the intact femurs were dissected and examined by several histological and histomorphometric techniques. OVX significantly (p < 0.01) decreased trabecular bone volume by 53.4% in the metaphyses compared with sham-operated controls. CMT-8 therapy produced a significant (p < 0.05) inhibition of trabecular bone loss and also induced bone formation in the OVX rats. Of interest, the newly synthesized bone in the CMT-treated OVX rats was found to increase the "connectivity" of the trabecular "struts" by bridging the adjacent longitudinal bone trabeculae, forming dense, plate-like bone trabeculae. These results strongly suggest that long-term CMT-8 therapy effectively inhibits bone loss after OVX, not only by inhibiting bone resorption but also by inducing new bone formation in the trabecular areas of long bones.

Delayed wound healing is one of the complications of diabetes mellitus, exhibited by increased wound collagenase and decreased granulation tissues. The current study compared wound healing in normal and diabetic rats, and the effects of topically applied 1% or 3% concentrations of chemically modified tetracycline-2 (CMT-2) on 6-mm circular full-thickness skin wounds healed by secondary intention. On day 7 after wounding, tissues were removed for biochemical analysis and histology. The wound granulation tissue hydroxyproline was less in the untreated diabetic rat with increased collagenase and gelatinase. Treating the diabetic rat wounds with 3% CMT-2 increased the wound hydroxyproline and decreased activities of gelatinase and collagenase. There was a delay in wound filling by granulation tissue in diabetic rats. In CMT-2-treated diabetic rats, the volume of granulation tissue was greater than that in untreated diabetic rats. CMT-2 appears to normalize wound healing in diabetic rats and may be a valuable adjunct in the treatment of chronic wounds.

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.

The incidence of root caries has been found to increase as the population ages and as edentulism becomes less prevalent due to improved dental awareness and care, and as exposure of roots due to gingival recession has also increased in the elderly. The mechanism of root caries is thought to be mediated by both bacterial and mammalian proteases produced by plaque and the periodontal tissues, respectively. In the current study, a rat model of periodontal disease was used in which gnotobiotic rats were infected intra-orally with a periodontal pathogen (P. gingivalis). Infecting the rats with P. gingivalis increased the collagenase activity in the gingival tissue in association with severe alveolar bone loss. Treating P. gingivalis-infected rats with doxycycline or CMT-1 prevented the destruction of the periodontium by MMPs, thus preventing exposure of roots to subgingival bacterial plaque and host tissue collagenases and the subsequent development of root caries. In addition, a low-dose doxycycline (LDD, 20 mg bid, non-antimicrobial dose) for 3 months was used in humans predisposed to increased root caries as the result of heavy use of smokeless (chewing) tobacco, causing gingival recession, subgingival plaque accumulation with Gram-negative bacteria, increased gingival crevicular fluid flow (GCF), and elevated GCF collagenase. Daily administration of LDD in smokeless tobacco patients reduced the GCF collagenase and prevented the further development of root caries.

Collagenase and gelatinase are matrix metalloproteinases (MMPs) which play an important role in tissue destruction in arthritic joints. Studies have demonstrated that tetracyclines can inhibit MMPs and prevent tissue destruction independent of their antimicrobial activity. The purpose of this pilot study is to assess the potential therapeutic role of Doxycycline in patients with advanced osteoarthritis of the temporomandibular joint (TMJ). This ongoing investigation includes patients with a diagnosis of osteoarthritis of the TMJ based on clinical and diagnostic imaging findings, symptoms (localized TMJ pain, limited mobility, dysfunction) for a minimum of 36 months, and failure of previous non-surgical and surgical modalities to alleviate the symptoms. A synovial fluid sample is collected by a saline injection and aspiration technique, followed by diagnostic arthroscopy. Patients are placed on Doxycycline 50 mg BID for three months and then undergo repeat diagnostic arthroscopy and synovial fluid collection. The samples are stored at -80 degrees C. Collagenase activity is determined by a combination of SDS-polyacrylamide gel electrophoresis and fluorography and calculated based on the percentage of collagen alpha chains that are degraded into alphaA breakdown products. Three patients have completed the three-month course of Doxycycline thus far, and 5 joints with osteoarthritis have been analyzed. All patients were female (mean age = 35, mean duration of symptoms = 132 months) and had undergone previous bilateral arthroscopies. One patient had undergone unilateral arthroplasty. The mean collagenase activity showed 55% collagen lysis prior to Doxycycline treatment and 19% after three months of therapy. The mean gelatinase activity was 28% prior to Doxycycline treatment and 7% after three months of therapy. The mean interincisal opening was 33 mm initially and 41 mm after three months of Doxycycline. Subjectively, two of the three patients reported significant improvement in their overall symptoms, which they had not experienced over the previous three years. One patient did not experience any change in symptoms, in spite of a marked reduction in collagenase activity from 86.4% to 9.6%. Because of the very small numbers of patients enrolled in this pilot study so far, no statistically significant differences could be appreciated. However, the dramatic reduction in collagenase activity in these patients, with a long history of TMJ symptoms from osteoarthritis, suggests the potential promising role of Doxycycline in the management of osteoarthritis, and further investigation is warranted.

Objective: We studied the relative ability of 6 different chemically modified non-antimicrobial analogs of tetracycline (CMT) to inhibit human and chicken matrix metalloproteinases (MMP) in vitro. The ability of tetracycline and its analogs to inhibit MMP appears to depend on the Ca++/Zn++ binding site at C11 (carbonyl oxygen) and C12 (OH group) of the molecule, which is lacking in CMT-5, the pyrazole derivative of tetracycline. This significant property of CMT-5 was used to differentiate between the effects of CMT on already active MMP versus the oxidative activation of latent MMP (pro-MMP)., Methods: Cultured chicken osteoclast conditioned medium and purified human neutrophil progelatinase (MMP-9) and pro-collagenase (MMP-8) were assayed for proteinase activities using gelatin and collagen, respectively. The pro-MMP were activated either by preincubation with 1 mM aminophenylmercuric acetate (APMA) or 100 microM sodium hypochlorite (NaOCI). CMT were added either to the preincubation mixtures together with NaOCl or after activation of pro-MMP with NaOCl., Results: All CMT tested, except CMT-5, inhibited APMA or NaOCl activated pro-MMP. However, CMT-5 (like the other CMT), inhibited the oxidative activation of pro-MMP by NaOCl when added together by scavenging the reactive oxygen species. The degradation of type-I collagen by chicken osteoclast conditioned medium was probably due to MMP-2 and/or MMP-13., Conclusion: Oxidative activation of pro-MMP may be crucial during soft tissue/bone destruction in the inflammatory diseases, including the arthritides. Our results indicate that the Ca++/Zn++ binding site of CMT is not essential for inhibition of the oxidative activation of pro-MMP.

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.

Tissue remodeling is a dynamic state in which a balance is achieved between the proteolytic breakdown and synthesis of the extracellular matrix. Type I collagen is a major component of the gingival connective tissue (GCT) and the periodontal ligament (PDL) throughout development, while type XII collagen has been found in the mature forms of these tissues. The purpose of this study was to investigate the effects of periodontitis on the expression of type I and XII collagen and subsequently to investigate the effects of doxycycline (DOXY) and chemically modified non-antimicrobial tetracycline (CMT-1) on the expression of these molecules in this model. Adult barrier-raised male Sprague-Dawley rats were inoculated with Porphyromonas gingivalis obtained from humans to create the experimental periodontitis. The animals with the P. gingivalis-induced periodontitis were then split into the following groups: Group A served as infected untreated controls (PGI group); group B was treated with doxycycline (DOXY group); and group C was treated with chemically modified tetracycline-1 (CMT-1 group). Group D contained uninfected animals that served as uninfected controls (NIC group). The expression of type I and XII collagen mRNAs was examined by in situ hybridization in each group, with the co-expression of these molecules representing mature and functional gingival connective tissue. In the NIC group, cells hybridized with digoxygenine-labeled cDNA probes encoding rat alpha2(I) or alpha1(XII) collagens were found distributed uniformly throughout the periodontal connective tissue. The PGI group showed little hybridization in the areas of infection, while both the DOXY and CMT-1 groups showed co-expression of the alpha2(I) and alpha1(XII) probes in the GCT and coronal part of the PDL. This study demonstrates that doxycycline and CMT-1 moderate or reduce the inhibitory effects of periodontal infection on the expression of type I and type XII collagen mRNAs. These results suggest that doxycycline and a form of non-antimicrobial tetracycline, chemically modified tetracycline-1, can reduce periodontal destruction by reversing the inhibitory effect of periodontal infection on collagen synthesis.

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.

Sulfasalazine is widely used in rheumatoid arthritis and inflammatory bowel diseases. The mechanisms of its activity have not been elucidated. In leukocytes, sulfasalazine and its analogue, CL 42A, inhibited the formation of leukotrienes and possibly of the second messenger compounds at the level of phospholipase C. Partial inhibition of interleukin-lbeta (IL-1beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) was also found. Since the synthesis of eicosanoids is induced by phospholipase A2 and since secretory phospholipase A2 (sPLA2) is proinflammatory, we investigated the impact of sulfasalazine and related compounds on mRNA, protein synthesis, and release of sPLA2 from osteoblasts. Sulfasalazine and CL 42A markedly inhibited extracellular release of sPLA2. The impact of sulfasalazine was evident at 50 microM (P < 0.001) and maximal at 400 microM, and that of CL 42A at 10 microM (P < 0.001) and 200 microM, respectively. Split products of sulfasalazine, 5-aminosalicylic acid (400 microM) and sulfapyridine (400 microM), had no impact. The effect of sulfasalazine and CL 42A was evident regardless of whether the cells were stimulated with IL-1beta/TNF-alpha, lipopolysaccharide/forskolin, or dibutyryl-cAMP. Sulfasalazine and CL 42A did not alter the level of sPLA2 mRNA. Exposure of stimulated fetal rat calvaria osteoblasts (FRCO) to sulfasalazine did not show accumulation of the intracellular sPLA2 protein as tested by western blot; however, enzymatic activity of PLA2 in disrupted cells was definitely increased. Thus, the impact is on the post-transcriptional release of sPLA2 rather than on the synthesis. There was also an increase in the extracellular release of prostaglandin E2 from FRCO exposed to sulfasalazine or to CL 42A. In contrast, sulfasalazine had no effect on the extracellular release of gelatinase from the cells or on mRNA of cytosolic PLA2 or cyclooxygenase 2. We conclude that the anti-inflammatory activity of sulfasalazine may be related, in part, to the selective inhibition of the extracellular release of proinflammatory sPLA2.

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.

Specially-formulated low-dose doxycycline (LDD) regimens have been found to reduce collagenase activity in the gingival tissues and crevicular fluid (GCF) of adult periodontitis subjects in short-term studies. In the current, double-blind, placebo-controlled study, adult periodontitis patients were administered for 6 months a "cyclical" regimen of either LDD or placebo capsules; and various clinical parameters of periodontal disease severity, and both collagenase activity and degradation of the serum protein, alpha 1-PI, in the GCF were measured at different time periods. No significant differences between the LDD- and placebo-treated groups were observed for plaque index and gingival index. However, attachment levels, probing depth, and GCF collagenase activity and alpha 1-PI degradation were all beneficially and significantly (P < 0.05) affected by the drug regimen. We propose: 1) that LDD inhibits tissue destruction in the absence of either antimicrobial or significant anti-inflammatory efficacy; and 2) that long-term LDD could be a useful adjunct to instrumentation therapy in the management of the adult periodontitis patient.

We previously reported that both local and systemic factors relevant to the pathogenesis of periodontal disease can increase gingival collagenase activity in rats. Since the degradation of extracellular matrix is an essential feature of periodontal disease and this tissue breakdown requires multiple enzyme interactions, the current study was carried out to determine the effects of bacterial endotoxin (LPS) (a local factor) and diabetes (a systemic factor) on a panel of matrix-degrading enzymes (collagenase, gelatinase, elastase, and beta-glucuronidase) in the gingiva of rats. In addition, the effects of therapy with a semisynthetic tetracycline (minocycline) were investigated. Ten male, Sprague-Dawley rats were made diabetic by IV injection of streptozotocin. Four of the ten rats then received minocycline (10 mg/day) by oral gavage on a daily basis for 3 weeks. Nineteen nondiabetic rats served as controls and 9 of them received 10 microliters of E. coli LPS (10 mg/ml) by injection into the labial gingiva every other day during the last week of the study. The other 10 nondiabetic rats were sham injected with saline into the gingiva. At the end of the 3 week experimental period, gingival tissue and skin were dissected from each rat and extracted for enzyme analysis. Our results showed that diabetes markedly increased the four matrix-degrading enzyme activities in both gingiva and skin. In contrast, local LPS injection increased these enzyme activities in the gingiva alone. Systemic therapy with minocycline completely ameliorated these elevated enzyme levels in diabetic rats in both gingiva and skin. Minocycline added in vitro to the enzyme assay systems containing skin extract from diabetic rats also inhibited collagenase and gelatinase activities, but no inhibition was observed for elastase and beta-glucuronidase activities, indicating that the MMPs and other enzymes were inhibited by minocycline, during diabetes, by indirect and indirect mechanisms, respectively.

Objective: To identify a mechanism by which a matrix metalloproteinase (MMP) inhibitor might act synergistically with other agents to decrease MMP activity and thereby lessen the radiologic severity of adjuvant arthritis., Methods: Rats with adjuvant arthritis were treated with either flurbiprofen (FBP) or tenidap (TDP), along with 4-dedimethylaminotetracycline (CMT-1), a potent MMP inhibitor. Indices of inflammatory severity and of radiologic destruction were assessed and compared to serum and bone levels of the MMP inhibitor., Results: Combination therapy with the MMP inhibitor plus either of the other drugs led to synergistic improvement in radiologic severity. For example, CMT-1 combined with TDP reduced radiologic severity 45% while decreasing collagenase and gelatinase activities by 61 and 72%, respectively, more than doubling bone CMT-1 levels (7.6 micrograms/g to 16.4 micrograms/g). FBP had similar effects., Conclusion: MMP inhibitors need access to the arthritic joint to interact with their target enzymes. Concomitant antiinflammatory therapy is required to assure drug entry into the inflamed tissues.

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and alpha-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blotting revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (< 60 kD) were detected by alpha-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl).(ABSTRACT TRUNCATED AT 250 WORDS)

We previously reported that low-dose doxycycline (DOXY) therapy reduces host-derived collagenase activity in gingival tissue of adult periodontitis (AP) patients. However, it was not clear whether this in vivo effect was direct or indirect. In the present study, inflamed human gingival tissue, obtained from AP patients during periodontal surgery, was extracted and the extracts partially purified by (NH4)2SO4 precipitation. The extracts were then analyzed for collagenase activity using SDS-PAGE/fluorography/laser densitometry, and for gelatinase activity using type I gelatin zymography as well as a new quantitative assay using biotinylated type I gelatin as substrate. DOXY was added to the incubation mixture at a final concentration of 0-1000 microM. The concentration of DOXY required to inhibit 50% of the gingival tissue collagenase (IC50) was found to be 16-18 microM in the presence or absence of 1.2 mM APMA (an optimal organomercurial activator of latent procollagenases); this IC50 for DOXY was similar to that exhibited for collagenase or matrix metalloproteinase (MMP)-8 from polymorphonuclear leukocytes (PMNs) and from gingival crevicular fluid (GCF) of AP patients. Of interest, Porphyromonas gingivalis collagenase was also inhibited by similar DOXY levels (IC50 = 15 microM), however the collagenase activity observed in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific alpha A (3/4) and alpha B (1/4) collagen degradation fragments. In contrast, the inhibition of collagenase purified from culture media of human gingival fibroblasts (MMP-1) required much greater DOXY levels (IC50 = 280 microM). The predominant molecular forms of gelatinolytic activity presented in the AP patients gingival tissue extracts were found to closely correspond to the 92 kD PMN-type gelatinase (MMP-9) although small quantities of 72 kD fibroblast-type gelatinase (MMP-2), and some other low molecular weight gelatinases, were also detected. The IC50 of DOXY versus gingival tissue gelatinolytic activity was estimated at 30-50 microM measure using either type I gelatin zymography or the biotinylated type I gelatin assay. We conclude that MMPS in inflamed gingival tissue of AP patients, like those in GCF, originate primarily from infiltrating PMNs rather than resident gingival cells (fibroblasts and epithelial cells) or monocyte/macrophages, and that their pathologically-elevated tissue-degrading activities can be directly inhibited by pharmacologic levels of doxycycline.

Tetracyclines have been widely used as adjuncts in periodontal therapy due to the antimicrobial efficacy of these drugs. Recently, their ability to inhibit host-derived matrix metalloproteinases (collagenase and gelatinase) and bone resorption in organ culture has also been invoked as a therapeutic rationale. The current study was undertaken to determine whether tetracyclines can inhibit alveolar bone loss in vivo due to a non-antimicrobial action of these drugs. Experimental periodontitis was induced by inoculating adult, male Sprague-Dawley rats with P. gingivalis (strain 381) following kanamycin/ampicillin pretreatment. Doxycycline, non-antimicrobial chemically-modified tetracycline (CMT-1) and vehicle alone were administered daily to 3 infected groups of rats (n = 6 rats per group; each group housed in a sterilized inflatable isolator) beginning 10 days after P. gingivalis inoculation. The control group (n = 6; non-infected rats) received only vehicle. After 5 weeks of daily drug administration by gastric intubation, the experiment was terminated and blood samples were taken from each animal to determine antibody levels against P. gingivalis. Plaque samples were collected from each group of animals before and after P. gingivalis inoculation and at the end of the experiment for microbiological examination. The jaws were removed from each rat, defleshed and then analyzed morphometrically and radiographically to assess bone loss. Serum antibody levels against P. gingivalis were significantly elevated in the 3 infected groups compared to the non-infected controls. This, together with the microbiologic findings, indicated that these groups of rats were infected with P. gingivalis.(ABSTRACT TRUNCATED AT 250 WORDS)

3H-labeled chemically modified, non-antimicrobial tetracycline (4-de-dimethyl-aminotetracycline; CMT) was injected intraperitoneally into neonatal rats and the animals killed at 20 min, 2 or 4 h after isotope injection. The binding of 3H-CMT in the calvariae was localized by light and electron microscopic autoradiography. At 20 min after injection, a high level of bound 3H-CMT was localized in the cells of the periosteum including the osteoblasts and preosteoblasts. The newly formed bone matrix was also heavily labeled by silver grains. At the ultrastructural level, many silver grains appeared over the cytoplasm of osteoblasts and preosteoblasts, particularly along the plasma membranes, in the periosteum. At 2 and/or 4 h after isotope injection, the number of silver grains over the cells was markedly reduced by that over the bone matrix appeared unchanged. These results indicate that, in addition to the mineral phase of bone, CMT directly binds to (or is taken up by) osteogenic cells and is then rapidly metabolized by these cells.

Tetracyclines have nonantimicrobial properties that appear to modulate host response. In that regard, tetracyclines and their nonantimicrobial chemically modified analogues (chemically modified tetracycline molecules [CMTs]) inhibit the extracellular activity of mammalian neutrophil and osteoblast collagenases. The activity of this matrix metalloproteinase appears crucial in the destruction of collagen. Apart from its anticollagenase effect, tetracyclines are also potent inhibitors of osteoclast function. Several recent studies have also addressed the therapeutic potential of tetracyclines 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. CMT was not associated with the emergence of resistant microorganisms. In human double-blind clinical trials, low-dose doxycycline therapy substantially reduced collagenase activity in the gingival and crevicular fluid, and prevented the loss of attachment in adult periodontitis without the emergence of doxycycline-resistant microorganisms. Tetracyclines and CMTs have enormous therapeutic potential because these drugs can inhibit the activity of matrix metalloproteinases as well as osteoclast function, and thus prevent the degradation of osseous connective tissues in periodontal as well as arthritic diseases.

An earlier study indicated that a chemically-modified non-antimicrobial tetracycline (4-de-dimethylaminotetracycline; CMT-1) can inhibit excess collagenase activity in the connective tissues of diabetic rats, however, the optimum oral dose and resulting serum concentration were not determined. In the current study, adult male Sprague-Dawley rats (body weight approx. 350 g) were made diabetic by streptozotocin injection and administered by oral gavage either 0, 1, 2, 5, or 10 mg CMT-1 per day. After 3 weeks of drug therapy, the rats were killed and gingiva, skin, and serum collected. The tissues were 1) extracted, partially purified and analyzed for collagenase activity using [3H-methyl] collagen as substrate and SDS-PAGE/fluorography; 2) extracted in neutral salt and dilute acid solutions (4 degrees C) to assess collagen solubility; and 3) analyzed for hydroxyproline to determine tissue (skin) collagen mass. Serum was analyzed for glucose and CMT-1 concentration, the latter by HPLC. Inducing diabetes dramatically increased both gingival and skin collagenase activity and reduced skin collagen mass by 69.8%. Increasing the oral dose of CMT-1 progressively increased the serum concentration of the drug from 0.6-6.5 micrograms/ml and progressively decreased the excessive collagenase activity in gingiva and skin (p < 0.01 vs untreated diabetics). Although skin collagen mass tended to be increased at all oral doses of CMT-1, only the 5 mg dose effect was statistically significant (p < 0.01). The diabetes-induced reduction in collagen solubility, a classic abnormality (reflecting excessive collagen crosslinking) of this disease, was also normalized by CMT-1 therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCl), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCl also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCl. HOCl (5 microM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106-01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50-400 microM) and chemically modified tetracycline, CMT-1 (100-400 microM), significantly inhibited collagenase activity 50-90% and 40-80%, respectively, in the presence of 5 microM HOCl. Concentrations of 6-25 microM doxycycline and 10-50 microM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCl2 or 50 microM ZnCl2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCl and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.

Streptozotocin-induced, insulin-deficient diabetic adult rats were daily administrated either minocycline or a chemically-modified non-antimicrobial tetracycline (CMT) by oral gavage for a 3-week time period; untreated diabetic and non-diabetic rats served as controls. On day 21, all rats received an intravenous injection of 3H-proline followed by perfusion fixation with an aldehyde mixture at 20 minutes and 4 hours after isotope injection. The upper and lower mandibles of these rats were dissected and processed for quantitative electron microscopic autoradiography to study 3H-proline utilization by fibroblasts in the periodontal ligament (PDL) of molars. In the non-diabetic controls, at 20 min after 3H-proline injection, radioprecursor was incorporated by the Golgi-RER system of PDL fibroblasts. At the 4-h time period, most of the label was present over the collagen fibers around these cells. In contrast, PDL fibroblasts in the untreated diabetic rats showed marked abnormalities ultrastructurally and minimal uptake (20 min) and secretion (4 h) of labeled proline. At both time periods, in both minocycline- and CMT-treated diabetic rats, fibroblasts were structurally more normal and the radioprecursor was localized in the fibroblasts and the PDL matrix in a pattern similar to that seen in the control rats. These results suggest that the diabetes-induced structural abnormalities and suppression of synthesis and secretion of protein (presumably collagen and its precursor) by PDL fibroblasts can be restored to near-normal by administration of a tetracycline and that this effect is mediated by a non-antimicrobial property of this family of antibiotics.

Tetracycline antibiotics (TETs) have a recently discovered novel action: inhibition of extracellular metalloproteinase activity, especially that of collagenase and gelatinase. This property, now confirmed in 8 different laboratories using > 40 tissue sources, includes natural and semi-synthetic TETs as well as a chemically modified TET (CMT) devoid of antimicrobial activity. We have used 14C-Tyr biosynthetically labelled intracellular proteins in L-6 myoblast culture as a test system to assess intracellular proteolysis. Starvation accelerates proteolysis, which can be suppressed by agents such as insulin or serum. Minocycline, doxycycline, and CMT all retarded the rate of intracellular protein degradation in a dose dependent manner. These agents also demonstrated marked synergism with insulin. A CMT derivative (pyrazole) stripped of one of its metal chelation sites and lacking anti-collagenase activity, also lost its antiproteolytic effect. CMT at physiologic concentrations (< or = 5 micrograms/ml) had no effect on protein synthesis, but at 15 micrograms/ml (pharmacologic), a suppressive effect was noted. These findings demonstrate that TETs can inhibit protein degradation as well as synthesis in a mammalian muscle-derived cell line.

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.

Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease.

Tetracyclines are potent inhibitors of 2 major matrix metalloproteinases which have been implicated in connective tissue degradation: collagenase and Type IV collagenase/gelatinase. We directly identified these enzyme activities in extracts of inflamed paw tissue from rats with adjuvant arthritis. Oral tetracycline therapy suppressed metalloproteinase activity in arthritic tissue, but even very high doses failed to exhibit substantial antiinflammatory efficacy (reduced joint swelling and paw diameter). Flurbiprofen, a conventional nonsteroidal antiinflammatory drug, reduced inflammatory indices as expected. The combination of the 2 agents administered orally completely inhibited collagenase activity, significantly inhibited gelatinase activity and produced substantial normalization of radiographic joint damage, far greater than either drug alone. Tetracycline inhibition curves in vitro suggest that the collagenase in this tissue is not of fibroblast origin. Tetracycline derivatives might be useful adjuncts to prevention of tissue damage in chronic inflammatory arthritides.

Streptozotocin-induced, insulin-deficient diabetic rats were administrated either minocycline (MC) or a chemically modified non-antimicrobial tetracycline (CMT) by oral gavage for a 3-week period; untreated diabetic and nondiabetic rats served as controls. On day 21, all rats received an intravenous injection of 3H-proline followed by perfusion fixation with an aldehyde mixture at 20 minutes and 4 hours after isotope injection. The parietal bones of these rats were dissected and processed for quantitative electron microscopic autoradiography to study 3H-proline utilization by osteoblasts. At 20 minutes after 3H-proline injection, radioprecursor was incorporated by the Golgi-RER system of the osteoblasts in the periosteal surface of the control rats. At the 4-hour time period, most of the label was present over the collagen fibers of the osteoid. In contrast, the flattened bone-lining cells in the untreated diabetic rats showed minimal uptake (20 minutes) and secretion (4 hours) of labeled proline. In both MC and CMT-treated diabetic rats, the radioprecursor was localized in the osteoblasts and osteoid matrix in a pattern similar to that seen in the control rats at both 20 minutes and 4 hours after isotope injection. Labeling of the osteoid by the radioprecursor was greater as a result of CMT treatment than during minocycline treatment. These results suggest that the diabetes-induced suppression of synthesis and secretion of protein (presumably collagen and its precursor) by osteoblasts can be restored to near-normal levels by administration of tetracycline(s) and that this effect is mediated by a non-antimicrobial property of these antibiotics.

The present study was designed to further understand the role of PTH on the secretion of the neutral metalloproteinases, collagenase and gelatinase, from the rat osteosarcoma clonal cell line, ROS 17/2.8. Semiconfluent cells were treated with bovine parathyroid hormone, b-PTH-(1-34) at 100 nM-0.01 nM for 24-96 hours and pooled, concentrated media were analyzed by functional assay for collagenase (3H-methyl collagen) and gelatinase (3H-methyl gelatin). Collagenase activity significantly decreased (P less than 0.01) in the PTH conditioned media in a dose-dependent manner before (98-64%) and after (91-39%) reduction and alkylation. SDS-PAGE and fluorography apparently showed the most degradation to alpha A chains in collagen with controls, whereas this substrate remained intact with PTH (100 nM). PTH (100 nM) media also showed neutral gelatinase activity approximately 2% compared to control before and after reduction and alkylation (P less than 0.01). Significant amounts of an inhibitor to collagenase and gelatinase might have been secreted at 1 nM and 0.01 nM PTH, since collagenase and gelatinase activities were greater after reduction and alkylation. Reduction and alkylation likely destroyed these significant amounts of inhibitor. Polymorphonuclear leukocyte collagenase activity was also inhibited 80% by PTH conditioned media, but not by control. However, upon reduction and alkylation which destroyed inhibitor, the PTH treated media showed only a 14% inhibition against polymorphonuclear leukocyte collagenase (P less than 0.01). PTH appeared to downregulate neutral metalloproteinase activities through its effects on an inhibitor. This downregulation may represent a specific phenotypic response to PTH in ROS 17/2.8 cells.

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.

Osteopenia is a recognized complication of diabetes mellitus in humans and experimental animals. We recently found that tetracyclines prevent osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990). The present study extends these initial ultrastructural observations by assessing osteoblast function in the untreated and tetracycline-treated diabetic rats. After a 3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline analog (CMT), were perfusion-fixed with an aldehyde mixture; the humeri were dissected and processed for ultracytochemical localization of alkaline phosphatase (ALPase) and Ca-ATPase activities. Some rats from each experimental group received an intravenous injection of 3H-proline as a radioprecursor of procollagen, and the humeri were processed for light microscopic autoradiography. In addition, the osteoid volume in each experimental group was quantitatively examined by morphometric analysis of electron micrographs. During the diabetic state, active cuboidal osteoblasts in the endosteum of control rats were replaced by flattened bone-lining cells that contained few cytoplasmic organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared to "restore" osteoblast structure. During diabetes, bone-lining cells incorporated little 3H-proline or secreted little labeled protein and produced only a very thin osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)