Thomas F Mcnamara

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

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

Tetracyclines Inhibit Tissue Collagenases: Effects of Ingested Low-Dose and Local Delivery Systems*

In a series of experiments, Golub et al. demonstrated that tetracyclines, but not other antibiotics, can inhibit mammalian collagenases and proposed that this property could be useful in treating diseases, such as periodontal disease (but also included certain medical conditions, e.g., corneal ulcers) characterized by excessive collagen degradation {JPeriodont Res 1983, 1984 and 1985; Experientia 1984; Cornea 1984). One effect was the dramatic reduction of tissue collagenase activity within the gingival crevicular fluid (GCF) of periodontal pockets after administering a standard regimen of a tetracycline (e.g., 200 mg minocycline or 1000 mg tetracycline/day). The preliminary studies described below determined the effect of (1) low-dose (LD; 40-80 mg/day) orally administered minocycline on GCF collagenase activity and on the subgingival microflora (Exp. I), and (2) tetracyclineloaded monolithic fibers (TF) on collagenase activity in vitro (Exp. II). In Exp. I, GCF collagenase activity was reduced by 45 to 80% 2 weeks after initiating LD minocycline therapy, an effect that lasted for at least several weeks after stopping drug treatment. No consistent change in the relative proportions of G(+), G(-) and motile subgingival microorganisms was detected as a result of LD treatment suggesting that the reduction in GCF collagenase activity was a direct inhibition of the enzyme by the drug. In Exp. II, 3- and 6mm lengths of TF in vitro established tetracycline concentrations in 250 μl of 132 μg/ml, from 3-mm lengths, and 265 μg/ml, from 6-mm lengths, after an 18-hour incubation. Although these concentrations were slightly lower than those established in the GCF of individual TF-treated periodontal pockets in vivo (Goodson et al. 1983, J Periodontol), the released tetracycline from 3- and 6-mm TFs inhibited rat polymorphonuclear leukocyte collagenase activity in vitro by 21 % and 45%, respectively (bacterial collagenase activity was also inhibited in vitro). These results indicate that TF placed into individual periodontal pockets could maintain a level of tetracycline in GCF that would effectively inhibit tissue (and perhaps also bacterial) collagenases at the lesion site.

Effect of doxycycline hyclate on corneal epithelial wound healing in the rabbit alkali-burn model. Preliminary observations.

We examined the effects of doxycycline hyclate on epithelial healing in vivo in the rabbit alkali-burn model. Twelve 2–3-kg Dutch belted rabbits were divided into three groups and received standard bilateral alkali burns (1 N sodium hydroxide for 30 s in an 11-mm circular plastic well). In group 1, two rabbits (four eyes) served as untreated controls. In group 2, five rabbits (10 eyes) received doxycycline hyclate (1.5 mg/kg) orally daily for 14 days. In group 3, five rabbits (10 eyes) received doxycycline hyclate (5 mg/kg) orally daily for 14 days. The epithelial defects were drawn and photographed on alternate days, after fluorescein staining. At conclusion, extracts of the corneas were evaluated for collagenase activity. At 14 days, the mean percentage of epithelial defects results in groups 1–3 were 50.0, 50.7, and 7.1%, respectively. Using the Wilcoxon rank sum test (two tailed), the differences were found to be statistically significant (p=0.0015). Preliminary data indicated that oral doxycycline administration also decreased the collagenase activity in corneas obtained from these animals. Our preliminary findings indicated that systematically administered doxycycline hyclate, 5 mg/kg/day, promotes corneal reepithelialization in the rabbit alkali-burn model, a result, perhaps, of the drugs ability to inhibit excessive collagenase activity.

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

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

HPLC determination of a chemically modified nonantimicrobial tetracycline: Biological implications

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

Topically Applied Cmt-2 Enhances Wound Healing in Streptozotocin Diabetic Rat Skin

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.

Root-Surface Caries in Rats and Humans: Inhibition by a Non-Antimicrobial Property of Tetracyclines

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.

Fluorescence Quantitation of S-IgA in Parotid Saliva and of S-IgA Bound to Oral Microorganisms

Quantitation of secretory IgA (s-IgA) in parotid saliva (1), whole saliva (2,3) and in other exocrine secretions (4) is generally performed by radial immunodiffusion. Although simple and quantitative, this method does not readily lend itself to the determination of s-IgA bound to oral microorganisms. Two techniques quantitating the binding of parotid salivary s-IgA to Streptococcus mutans, a modified ELISA assay (5) and an agglutinin assay (6), have recently been published. The aim of the present work was to develop fluorescence methods to quantitate the concentration of s-IgA in parotid saliva and the percentage of parotid s-IgA bound to a specific oral microorganism. Fluorescence assays have the advantage of high sensitivity and moreover permit direct visualization of binding by fluorescence microscopy.