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We previously demonstrated that subantimicrobial-dose-doxycycline (SDD) treatment of post-menopausal osteopenic women significantly reduced periodontal disease progression, and biomarkers of collagen destruction and bone resorption locally in periodontal pockets, in a double-blind placebo-controlled clinical trial. We now hypothesize that SDD may also improve biomarkers of bone loss systemically in the same women, consistent with previous studies on tetracyclines (e.g., doxycycline) in organ culture and animal models of bone-deficiency disease. 128 post-menopausal osteopenic women with chronic periodontitis randomly received SDD or placebo tablets daily for 2 years adjunctive to periodontal maintenance therapy every 3-4 months. Blood was collected at baseline and at one- and two-year appointments, and sera were analyzed for bone resorption and bone formation/turnover biomarkers. In subsets of the study population, adjunctive SDD significantly reduced serum biomarkers of bone resorption (biomarkers of bone formation were unaffected), consistent with reduced risk of future systemic bone loss in these post-menopausal women not yet on anti-osteoporotic drugs. [ABSTRACT FROM AUTHOR]

We previously demonstrated that subantimicrobialdose- doxycycline (SDD) treatment of post-menopausal osteopenic women significantly reduced periodontal disease progression, and biomarkers of collagen destruction and bone resorption locally in periodontal pockets, in a double-blind placebo-controlled clinical trial. We now hypothesize that SDD may also improve biomarkers of bone loss systemically in the same women, consistent with previous studies on tetracyclines (e.g., doxycycline) in organ culture and animal models of bone-deficiency disease. 128 post-menopausal osteopenic women with chronic periodontitis randomly received SDD or placebo tablets daily for 2 years adjunctive to periodontal maintenance therapy every 3-4 months. Blood was collected at baseline and at one- and two-year appointments, and sera were analyzed for bone resorption and bone formation/turnover biomarkers. In subsets of the study population, adjunctive SDD significantly reduced serum biomarkers of bone resorption (biomarkers of bone formation were unaffected), consistent with reduced risk of future systemic bone loss in these post-menopausal women not yet on anti-osteoporotic drugs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lacloferrin and TIMP-1 of saliva (n =10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment. [ABSTRACT FROM AUTHOR]

Lysyl oxidase activity and the extractability of recently synthesized collagen were measured in gingiva and skin, two tissues which exhibit altered pathophysiologic responses during diabetes. Both tissues showed a reduced content and extractability of newly synthesized, 14C-Hydroxyproline labeled, collagen after induction of streptozotocin-induced diabetes. Diabetes increased lysyl oxidase activity in skin and gingiva but the effect on gingiva was much less dramatic. The altered characteristics of interstitial collagen in the diabetic rat, including aging-like changes, appear to reflect multiple alterations in collagen metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The degradation of recently synthesized collagen (probably procollagen) in rat incisor gingiva was three times greater than that in skin. Concomitantly, the formation of undegraded (intact) collagen molecules in gingiva was slower than that in skin. This high basal rate of degradation in gingiva was just slightly increased in streptozotocin-induced diabetic rats, whereas the low basal rate in skin was dramatically increased by the diabetic state. The degradation of recently synthesized collagen was measured by the relative amounts(%o) of [³H]hydroxyproline-containing material in the TCA-soluble fraction of a tissue, compared with the total amount (TCA-soluble + TCA-insoluble) of [³H]Ahydroxyproline-containing material. Separation of the TCA fractions allowed the formation of collagen degradation products (TCA-soluble) to be viewed separately from the formation of undegraded collagen molecules (TCA-insoluble). The [³H]hydroxyproline-containing material in the TCA-soluble fraction was greatest in amount and in specific activity, 30 min after [³H]proline injection, supporting the origin of this material as being procollagen. At this time period, the relative amounts of TCA-soluble [³H]hydroxyproline-containing material were 40.3% (gingiva) and 12.7% (skin). For diabetic rats, the values were 55% and 48.8%o, respectively. For the [³H]hydroxyproline-containing material in the TCA-insoluble fraction, at 30 min, the specific activity of[³H]hydroxyproline was 4.3 for gingiva and 7.4 for skin. At all other time periods, the values were also greater for skin than for gingiva, making it unlikely that the formation of intact collagen molecules occurred faster in gingiva than in skin. Overall, the prevailing concept of rapid collagen turnover in oral tissues seems applicable to procollagen, but not to collagen of rat incisor gingiva. [ABSTRACT FROM AUTHOR]

The serum protem, α1-proteinase inhibitor (α1-PI), defends the host against serine proteinases, e.g PMN elastase. Using a rabbit anti-serum against human α1-PI, this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCI/CaCl2 buffer, pH 7.6. α1-PI concentration increased with G and was highest in AP subjects. H sites only showed intact α1-PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, α1-PI degradation fragments were seen 17% and 71% of GCF samples, respectively. Both collagenase and α1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow) Moreover, the α1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. We propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting α1-PI regulation of elastase and other serineproteinases, thereby favouring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double-blind study using the techniques described above for α1-PI analysis, that low-dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation. [ABSTRACT FROM AUTHOR]

Tetracylines 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 alvcolar 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; eact 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. Significantly greater bone loss was observed in the untreated P. gingivalis-infected group compared to the controls based on both morphometric and radiographic measurements. oral administration of doxycycline or CMT-1 to the P. gingivalis-infected rats completely inhibited this bone loss. As expected, CMT-1 did not exhibit an antimicrobial effect against P. gingivalis. Therefore, this study indicates that tetracyclines can inhibit alveolar bone loss in vivo by a mechanism which is independent of the antimicrobial efficacy of these drugs. [ABSTRACT FROM AUTHOR]

Recently, a new mechanism was proposed to explain the therapeutic efficacy of tetracyclines in periodontal disease - that specifically these antibiotics directly inhibit the activity of collagenase (and possibly other collagenolytic enzymes) produced by the host tissues resulting in a reduced rate of collagen breakdown (Golub et al. 1983). In the current studies, the effect of tetracyclines (minocycline, doxycydline, tetracycline) on collagenolytic enzyme activity was examined in human periodontal pockets and in several animal systems. In the clinical studies, gingival crevicular fluid (GCF) was collected on filter paper strips from individual periodontal pockets, the volume measured with the Periotron 6000. and the Gingival Index and pocket depth scored immediately thereafter at the same sites. The strips were incubated with [³H-methyl] collagen maintained in solution (or with 14C-glycine labeled collagen fibrils), and the radiolabeled collagen degradation products measured in a liquid scintillation spectrometer. Using the techniques of SDS-PAGE and fluorography, the GCF was observed to generate collagen degradation products characteristic of those produced by mammalian collagenase, a finding consistent with previous studies. Both minocycline and tetracycline therapy reduced GCF collagenolytic activity about 70% during the initial weeks of treatment; the clinical parameters also showed improvements at the same time periods. This reduction in collagenolytic activity had largely regressed by S weeks after terminating tetracycline therapy and by 19 weeks after minocyclne therapy, in the absence of other forms of periodontal treatment. In a preliminary study, 50 days of doxycycline treatment also reduced GCF collagenase activity, an effect which persisted for at least 2 months (but less than 1 year) after terminating its administration. In several studies involving experimental animals, the tetracyclines (1) in vivo, were found to reduce the pathologically excessive collagenase activity in gingiva and skin of diabetic rats; and (2) in vitro, directly inhibited the activity of collagenases from rat PMNL's and rabbit chondrocytes. The data in the current and our previous studies (Golub et al. 1983, Gomcs, Golub & Ramamurthy 1984) continue to support the hypothesis that tetracyclines, by directly inhibiting collageno/ytic enzyme(s) activity, may be therapeutically useful in treating diseases, such as periodorititis, characterized by excessive collagen breakdown. [ABSTRACT FROM AUTHOR]

Minocycline, a semi-synthetic tetracycline has the ability to directly inhibit the activity of collagenolytic enzymes including, mammalian collagenase. This property of tetracycline could be clinically useful in treating diseases characterized by collagen breakdown. The paper examines the therapeutic effect of different tetracyclines on collagenase activity within periodontal pockets of humans, confirming that tetracyclines can inhibit the activity of collagenase from several mammalian sources and demonstrating that tetracyclines can inhibit the breakdown of different connective tissues mediated by excessive collagenolytic activity.

Diabetes increases gingival collagenase activity, an effect that may be mediated by endogenous tissue changes and exacerbated by an overgrowth of Gram-negative organisms in the gingival crevice (see Ramamurthy & Golub 1983, McNamara et al. 1982). In an attempt to reverse this collagenolytic abnormality, we administered an appropriate antibiotic, minocycline (a semi-synthetic tetracycline), to diabetic rats and humans. Adult male conventional or germfree rats were made diabetic with streptozotocin, and half of these animals were administered minocycline (20 mg per day) by tube feeding for 3-4 weeks prior to sacrifice. The buccal gingival, entire skins, and mandibles were dissected and tested for collagenolytic enzyme activity, collagen content, and alveolar bone loss, respectively. Ina preliminary study, inocycline (200 mg per day) was administered for 7 days to an insulin-dependent diabetic adolescent human and an adult non-diabetic human; and twin brother of the diabetic was treated with penicillin. Gingival fluid collagenase activity was measured (using [³H-methyl] collagen as substrate ina new microassay) in 8 periodontal pockets in each subject before and after antibiotic therapy. Examination of collagenase digestion products by SDS-polycrylamide gel electrophoresis and fluorography was also carried out. In rats , minocycline treatment: (1) suppressed the abnormally elevated collagenolytic enzyme activity in gingival of diabetic rats, even under germfree conditions; (2) inhibited PMN leukocyte collagenase activity in vitro, an effect that was reversed by the addition of calcium ions (pencillin-streptomycin had no effect on the activity of this enzyme); and (3) retarded the abnormal loss of skin collagen and alveolar bone in diabetic rats. In a preliminary study on humans, minocycline therapy reduced the collagenase activity of gingival crevicular fluid, an effect not produced by penicillin. Our data suggests that (1) tetracycline therapy inhibits tissue collagenolytic enzyme activity by a mechanism at least in part unrelated to its antibacterial efficacy, and (2) this mechanism may provide a new therapeutic approach for suppressing excessive collagen resorption which occurs during periodontal disease and which can occur during other pathologic conditions. [ABSTRACT FROM AUTHOR]

The connective tissues of diabetic humans and experimental animals exhibit abnormalities in collagen metabolism including an increased production of collagenolytic enzymes by gingival explants in tissue culture. In the current study, gingival tissue and skin were obtained from control and diabetic rats, the latter on the 5th, 10th, 15th, and 22nd day after inducing the disease with streptozotocin. Extracts of the tissues were assessed for collagenase activity using either 14C-labeled collagen fibrils or Peptide-P as substrate and by detecting collagenase digestion products by SDS-polyacrylamide gel electrophoresis. Elastase was measured using the specific substrate, succinyl-(L-Alanyl)3-p-nitroanilide. Diabetes increased the activity of collagenase in extracts of gingiva and skin; elastase, however, was increased dramatically only in the latter tissue. These findings (1) indicate that diabetes-induced enhanced gingival collagenase activity occurs in vivo as well as in vitro and (2) explain, at least in part, the greater loss of collagen in skin than in gingiva of the diabetic rat and other complications such as unusually severe periodontal breakdown. [ABSTRACT FROM AUTHOR]

We monitored the in vivo response of human and rat crevicular leucocytes (CL) and fluid (CE) to a chemical challenge in an attempt to develop an in vivo chemotaxis assay. The mesial crevices of #21 and #22 ot male dental students (mean Gl = 0; crevice depth = 2.3 mm) were washed and air dried. The baseline values for CL (#21) and CF (#22) were recorded. CL were collected in 10 μl washes of the crevice and an aliquot immediately counted in a hemacytometer. CF was collected on filter paper strips and measured with a fluid meter. Casein. a nutrient and a standard in vitro chemo-attractant was applied with a calibrated wire loop into each crevice. CL and CF were monitored 15 mm later and at 5 mm intervals up to 45 min CL and CF peaked approximately 30 min after the casein challenge; both parameters returned to baseline rapidly thereafter. In rats, a dose-response was obtained when concentrations of 0.01-4.0 mg casein/ml were applied to the gingival crevice. The maximum response of CL and CF occurred at 2.0 mg/ml. The application of buffer alone or Fab protein, which lacks chemotactic activity. produced no rise in CL and CF. We suggest that this crevicular technique can be used to monitor the in vivo response of subjects with defects in leucocyte chemotaxis. [ABSTRACT FROM AUTHOR]