tetracycline and 4-des-dimethylaminotetracycline

A new therapeutic approach involves the discovery by the "Stony Brook group," that tetracyclines, but not other antibiotics, can inhibit host-derived collagen-destructive enzymes. This newly discovered property of tetracyclines is unrelated to the antimicrobial activity of these drugs. Examples support the hypothesis that this unexpected property of tetracyclines provides a new approach to treating periodontal diseases as well as a variety of medical disorders.

Topics: Gingival Crevicular Fluid; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Periodontal Diseases; Periodontal Pocket; Tetracycline; Tetracyclines

Tetracyclines have recently been shown to inhibit the activity of some but not all mammalian matrix metalloproteinases believed to mediate periodontal destruction. However, the specificity of this effect, which could have significant therapeutic implications for different periodontal diseases, has not been examined in detail. Doxycycline and 4-de-dimethylaminotetracycline (CMT-1) have been tested in vitro for their ability to inhibit human neutrophil and fibroblast interstitial collagenases and collagenase in human gingival crevicular fluid (GCF). The GCF samples were obtained from systemically healthy and insulin-dependent diabetic adult periodontitis patients and from localized juvenile periodontitis (LJP) patients. The concentrations of these 2 tetracyclines required to inhibit 50% of the collagenase activity (IC50) were found to be 15 to 30 microM for human neutrophil collagenase and for collagenase in GCF of systemically healthy and diabetic adult periodontitis patients. These concentrations approximate the tetracycline levels observed in vivo during treatment with these drugs. In contrast, human fibroblast collagenase and GCF collagenase from LJP patients were both relatively resistant to tetracycline inhibition; the IC50 for doxycycline and CMT-1 for these 2 sources of collagenase were 280 and 500 microM, respectively. Based on these and other findings, we propose the following: 1) that systemic levels of tetracycline may inhibit connective tissue breakdown by inhibiting neutrophil collagenase; 2) that tetracyclines do not inhibit fibroblast-type collagenase, which may help explain their lack of effect on normal connective tissue remodeling; 3) that tetracycline inhibition of collagenases may serve to identify the cellular origin of the enzyme; and 4) that tetracyclines can also prevent the oxidative activation of latent human procollagenases.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Collagenases; Doxycycline; Fibroblasts; Gingival Crevicular Fluid; Humans; Inflammation; Matrix Metalloproteinase Inhibitors; Neutrophils; Periodontal Diseases; Periodontitis; Tetracycline; Tetracyclines

The bacterial tetracycline transcription regulation system mediated by the tetracycline repressor (TetR) is widely used to study gene expression in prokaryotes and eukaryotes. To study multiple genes in parallel, a triple mutant TetR(K(64)L(135)I(138)) has been engineered that is selectively induced by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type TetR. In the present study, we report the crystal structure of TetR(K(64)L(135)I(138)) in the absence and in complex with 4-ddma-atc at resolutions of 2.1 A. Analysis of the structures in light of the available binding data and previously reported TetR complexes allows for a dissection of the origins of selectivity and specificity. In all crystal structures solved to date, the ligand-binding position, as well as the positioning of the residues lining the binding site, is extremely well conserved, irrespective of the chemical nature of the ligand. Selective recognition of 4-ddma-atc is achieved through fine-tuned hydrogen-bonding constraints introduced by the His64-->Lys substitution, as well as a combination of hydrophobic effect and the removal of unfavorable electrostatic interactions through the introduction of Leu135 and Ile138.

Topics: Amino Acid Substitution; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Hydrogen Bonding; Ligands; Models, Molecular; Mutagenesis, Site-Directed; Protein Engineering; Recombinant Proteins; Repressor Proteins; Static Electricity; Tetracycline; Tetracycline Resistance

Here, we have studied the effects of chemically modified tetracyclines (CMTs) on apoptosis both at the level of the cytoplasmic proteolytic caspase cascade, and on Bcl-2 and c-myc mRNA expression in the J774 macrophage cell line. The results indicate that CMTs induce morphological changes consistent with apoptotic events, as clearly demonstrated both by the acridine orange and ethidium bromide staining, and by TUNEL and fragmentation ELISA assays. Furthermore, the analysis of the cell cycle by flow cytometry shows an evident apoptotic sub-G0G1 peak, without important modifications in the cell cycle distribution. CMTs induce programmed cell death (PCD) in a dose-dependent manner and CMT-8 is the strongest among them. CMT-1 and CMT-8 activate mainly caspase-8 as attested by the inhibitory effects of Z-VAD-fmk and Z-IEDT-fmk on CMT-induced apoptosis. Part of CMT-induced PCD is due to the activation of caspase-9, since it is reduced by the specific caspase-9 inhibitor, Z-LEHD-fmk. Besides, CMTs increase Bcl-2 and c-myc mRNA expression. Collectively, these data indicate that CMTs are potentially anti-tumour agents, since they strongly trigger apoptosis both activating the proteolytic system of the caspase family and modulating genes involved in PCD regulation.

Topics: Animals; Apoptosis; Caspases; Cell Survival; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Macrophages, Peritoneal; Mice; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Messenger; Tetracycline; Tetracyclines; Tumor Cells, Cultured

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.. 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.. 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.. 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.

Topics: Adhesins, Bacterial; alpha 1-Antitrypsin; Anti-Bacterial Agents; Blotting, Western; Chromogenic Compounds; Chymotrypsin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Doxycycline; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Enzyme Precursors; Gelatinases; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Neutrophils; Porphyromonas gingivalis; Protease Inhibitors; Serine Proteinase Inhibitors; Serpins; Serum Albumin; Tetracycline; Tetracyclines; Treponema; Trypsin Inhibitors

Clinical, electrophysiological, and neuropathological features are reported associated with a novel heterozygote point mutation in the extracellular domain of the MPZ gene, where a transversion at codon 71 in exon 3 leads to a codon stop: Glu71stop (ie GAA-->TAA). A 36 year old woman developed a mild recurrent neuropathy after intensive manual work. The motor nerve conduction velocities were slow without conduction blocks and the nerve biopsy showed signs of demyelination-remyelination, axonal loss, and regular uncompacted myelin lamellae. She inherited the mutation from her father who displayed the same mutation with a normal phenotype. This nonsense mutation may cause a dosage difference of normal P0, and is probably underrepresented in the current mutation data bases. This report further extends the phenotype of MPZ mutations and also emphasises that mild phenotype of CMT1B may be more frequent than has been appreciated.

Topics: Adult; Codon, Nonsense; Female; Humans; Nervous System Diseases; Neural Conduction; Pedigree; Recurrence; Tetracycline

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.

Topics: Animals; Collagen; Collagenases; Diabetes Mellitus, Experimental; Gelatinases; Gingiva; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Molecular Structure; Rats; Rats, Sprague-Dawley; Skin; Structure-Activity Relationship; Tetracycline; Tetracyclines

Charcot-Marie-Tooth 1A (CMT1A) neuropathy is caused by duplication of the peripheral myelin protein 22 (PMP22) gene, leading to protein overexpression. Although this protein has a role in regulating Schwann cell growth and peripheral myelin compaction, how altered concentrations of PMP22 impair myelination is unknown. We established dorsal root ganglia (DRG) cultures from a transgenic rat overexpressing PMP22 (PMP22tg) to study the behavior of PMP22tg Schwann cells in early stages of development and myelination. We used reverse transcriptase-polymerase chain reaction and light and electron microscopy to study PMP22 expression and myelin formation. Myelin ultrastructure was evaluated in sural nerves from CMT1A patients to compare experimental and human findings. PMP22tg DRG cultures contained a greater number of internodes devoid of myelin, in the absence of remyelination, and increased periodicity of myelin lamellae compared with normal cultures. Widening of myelin lamellae was also observed in CMT1A biopsy specimens. Our results suggest that both functions of PMP22, in regulating Schwann cell differentiation and contributing to peripheral myelin compaction, are affected by its overexpression. The presence of similar myelin abnormalities in PMP22tg cultures and human nerves emphasizes the importance of developing in vitro models of hereditary neuropathies to study their underlying pathomechanisms.

Topics: Animals; Animals, Genetically Modified; Cell Culture Techniques; Ganglia, Spinal; Humans; Male; Microscopy, Electron; Myelin Proteins; Myelin Sheath; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sural Nerve; Tetracycline; Ultrasonography

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.

Topics: Affinity Labels; Animals; Anti-Bacterial Agents; Bacteroidaceae Infections; Collagen; Connective Tissue; Digoxigenin; Disease Models, Animal; DNA Probes; DNA, Complementary; Doxycycline; Extracellular Matrix; Gene Expression Regulation; Gingiva; In Situ Hybridization; Male; Matrix Metalloproteinase Inhibitors; Periodontal Ligament; Periodontitis; Porphyromonas gingivalis; Protease Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tetracycline

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.

Topics: Animals; Blood Glucose; Body Weight; Collagen; Diabetes Mellitus, Experimental; Intubation, Gastrointestinal; Male; Procollagen; Rats; Rats, Sprague-Dawley; RNA, Messenger; Skin; Tetracycline

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.

Topics: Adult; Analysis of Variance; Animals; Anti-Bacterial Agents; Collagenases; Doxycycline; Gingiva; Gingival Recession; Humans; Male; Matrix Metalloproteinase Inhibitors; Periodontal Diseases; Plants, Toxic; Porphyromonas gingivalis; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Root Caries; Tetracycline; Tobacco, Smokeless

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.. 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.. 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.. 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.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Collagenases; Drug Synergism; Drug Therapy, Combination; Flurbiprofen; Gelatinases; Indoles; Male; Oxindoles; Protease Inhibitors; Rats; Rats, Inbred Lew; Tetracycline

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)

Topics: Alveolar Bone Loss; Analysis of Variance; Animals; Antibodies, Bacterial; Doxycycline; Intubation, Gastrointestinal; Male; Matrix Metalloproteinase Inhibitors; Microbial Sensitivity Tests; Porphyromonas gingivalis; Rats; Rats, Sprague-Dawley; Tetracycline

The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.

Topics: Animals; Blotting, Northern; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Doxycycline; Epithelial Cells; Epithelium; Gelatinases; Gene Expression; Humans; Keratinocytes; Kinetics; Ligaments; Male; Matrix Metalloproteinase 2; Metalloendopeptidases; Microscopy, Electron, Scanning; Mouth; RNA, Messenger; Skin; Swine; Tetracycline; Tetracyclines

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Gelatinases; Indoles; Male; Matrix Metalloproteinase Inhibitors; Oxindoles; Radiography; Rats; Rats, Inbred Lew; Temporomandibular Joint; Temporomandibular Joint Disorders; Tetracycline

Topics: Animals; Bacteroidaceae Infections; Bone Resorption; Collagenases; Doxycycline; Enzyme Activation; Gelatinases; Gingiva; Male; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Pancreatic Elastase; Periodontitis; Porphyromonas gingivalis; Rats; Rats, Sprague-Dawley; Tetracycline

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bone and Bones; Bone Resorption; Drug Therapy, Combination; Indoles; Male; Matrix Metalloproteinase Inhibitors; Oxindoles; Rats; Rats, Inbred Lew; Tetracycline

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)

Topics: Administration, Oral; Analysis of Variance; Animals; Collagen; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gingiva; Hydroxyproline; Male; Matrix Metalloproteinase Inhibitors; Rats; Rats, Sprague-Dawley; Skin; Solubility; Structure-Activity Relationship; Tetracycline

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

Topics: Animals; Calcium; Cytosol; Doxycycline; Minocycline; Nickel; Osteoclasts; Rats; Receptors, Cytoplasmic and Nuclear; Tetracycline

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

Topics: Animals; Calcium; Image Processing, Computer-Assisted; Linear Models; Microscopy, Phase-Contrast; Minocycline; Osteoclasts; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Tetracycline

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.

Topics: Animals; Chromatography, High Pressure Liquid; Half-Life; Hydrogen-Ion Concentration; Male; Microbial Collagenase; Neutrophils; Rats; Rats, Inbred Strains; Tetracycline

Tetracyclines (Tc's) have the ability to inhibit neutrophil (PMN) functions which may be of value in the treatment of neutrophil-driven pathologic processes. However, long term use of Tc's frequently lead to emergence of antibiotic resistant strains of bacteria. A chemically modified analogue of Tc, 4DTc, having minimal antibiotic activity was compared to Dc, a member of the Tc family, for its ability to inhibit neutrophil functions. 4DTc was significantly less effective at inhibiting PMN superoxide synthesis, PMA-induced degranulation and PMN-mediated RBC lysis than was Dc. 4DTc and Dc were equally as effective inhibiting monocyte, but not PMN, adherence to gelatin-coated surfaces. When incubated in media containing varying 4DTc or Dc concentrations, Dc accumulated in RBC's and PMN's at levels 3 times greater than that found for similar media levels of 4DTc. The data suggest that the lesser ability of 4DTc to inhibit several PMN functions as compared to Dc may be related to its reduced intracellular accumulation. It also suggest that Tc inhibition of monocyte adherence may be more influenced by extracellular than intracellular processes.

Topics: Cell Adhesion; Cell Degranulation; Doxycycline; Erythrocytes; Humans; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides; Tetracycline; Tetradecanoylphorbol Acetate

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.

Topics: Administration, Oral; Animals; Arthritis, Experimental; Bone and Bones; Cartilage, Articular; Collagenases; Dose-Response Relationship, Drug; Doxycycline; Drug Therapy, Combination; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Flurbiprofen; Matrix Metalloproteinase 9; Metalloendopeptidases; Minocycline; Rats; Tetracycline; Tetracyclines

Tetracyclines have recently been shown to inhibit the activity of mammalian matrix metalloproteinases, i.e. type I collagenase (MMP-1) and type IV collagenase/gelatinase (MMP-2). The specificity of this effect, however, has not been examined in detail. In the present study, doxycycline (a clinically widely used commercial tetracycline) and 4-de-dimethylaminotetracycline (CMT-1, a chemically modified non-antimicrobial tetracycline) were tested, at a wide range of concentrations, for their ability to inhibit human neutrophil and fibroblast interstitial collagenases, which are distinct gene products, as well as collagenase in human gingival crevicular fluid (an inflammatory exudate in periodontal lesions) obtained from adult, juvenile and diabetic adult periodontitis patients. The concentrations of these two tetracyclines, required to inhibit 50% of the collagenase activity (IC50), were found to be 15-30 microM for purified human neutrophil collagenase as well as collagenase in gingival crevicular fluid of adult periodontitis patients and diabetic adult periodontitis patients, thus approximating in vivo therapeutic tetracycline levels. In contrast, the fibroblast collagenase and collagenase in gingival crevicular fluid of patients with juvenile periodontitis were relatively resistant to tetracycline inhibition: the IC50 for doxycycline and CMT-1 were 280 and 500 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Child; Doxycycline; Fibroblasts; Gingival Crevicular Fluid; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 8; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Neutrophils; Periodontal Diseases; Tetracycline

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.

Topics: Animals; Carbon Radioisotopes; Cell Line; Doxycycline; Insulin; Kinetics; Minocycline; Muscles; Proteins; Radioisotope Dilution Technique; Recombinant Proteins; Tetracycline; Tetracyclines; Tyrosine

Diabetes induces osteopenia, which is characterized by a deficiency of osteoid and decreased activity of osteoblasts. We recently found that tetracyclines prevent the loss of osteoid and bone matrix and the degeneration of osteoblasts in diabetic rats by a mechanism independent of their antimicrobial efficacy. However, bone remodeling requires the activity of osteoclasts as well as osteoblasts. To determine the in vivo effects of tetracycline on osteoclasts in long bones, either a tetracycline (minocycline, TC) or its chemically modified non-antibiotic analogue (CMT), 4-de-dimethylaminotetracycline, was administrated daily to streptozotocin-induced diabetic rats by oral intubation. After 21 days, the rats were perfusion-fixed with a mixture of formaldehyde and glutaraldehyde, and the humeri were dissected and processed for ultracytochemical demonstration of acid trimetaphosphatase (ACPase) activity. In untreated non-diabetic (control) rats, the osteoclasts at the zone of provisional ossification exhibited abundant mitochondria and cisterns of rough endoplasmic reticulum (RER) throughout the cytoplasm, prominent stacks of Golgi membranes, and lysosomes in the perinuclear cytoplasm, and numerous various pale vacuoles in the cytoplasmic area adjacent to well-developed ruffled border. Intense ACPase activity was observed in the Golgi saccules, lysosomes, pale vacuoles, and the extracellular canals of ruffled border. The reaction products were also noted along the resorbing bone surfaces associated with the osteoclast ruffled border. The osteoclasts in the untreated diabetic rats showed a cytoplasmic organization similar to that of the non-diabetic control rats, but showed little or no ruffled border which was replaced by a broad clear zone in some of these cells. However, most of the osteoclasts on bone matrix in the diabetics were devoid of both a ruffled border and a clear zone. ACPase activity was detected in the osteoclast cytoplasm of diabetic rat, as in the controls, but to a much lesser extent along the broad clear zone facing the resorbing bone surfaces. The osteoclasts in TC-treated diabetic rats possessed both a clear zone and a small ruffled border. However, in some cases, they lacked both structures reminiscent of the untreated diabetic cells. The osteoclasts of CMT-treated diabetic rats exhibited structural and enzymatic features essentially identical to those of the non-diabetic control rats. These results suggest that the diabetes-induced osteo

Topics: Acid Phosphatase; Administration, Oral; Animals; Diabetes Mellitus, Experimental; Male; Microbial Collagenase; Microscopy, Electron; Minocycline; Osteoclasts; Rats; Rats, Inbred Strains; Streptozocin; Tetracycline

Effects of metal ion-tetracycline (TC) interactions on both gastrointestinal absorption and pharmacological activity of these drugs are well documented. In particular, recent simulation studies based on newly determined complex stability constants have drawn attention to the potential influence of Ca2+ and Mg2+ ions on the bioavailability of various TC derivatives in blood plasma. Contrary to previous thoughts, it was demonstrated in these studies that the fraction of antibiotic not bound to proteins almost exclusively occurs as calcium and magnesium complexes. Among this fraction, predominant binuclear species are electrically charged, and as such cannot passively diffuse through cell membranes. It was thus postulated that the partial blocking of one of the potential coordination sites of the TC molecule, which would favor the formation of neutral mononuclear complexes, should result in a better tissue penetration of the drug. Such correlations were recently established for specific derivatives. Before possible modifications of the TC molecule can be envisaged, it is necessary that all the chelating sites involved in the relevant complexes be properly assigned. As tetracyclines are very complex ligands, the present paper first deals with the coordination of calcium and magnesium with two simpler parent substances, i.e., 4-dedimethylamino-tetracycline (DTC) and 6-desoxy-6-demethyl-tetracycline (DSC). After the quantitative investigation of the proton and metal complex equilibria involved, UV and circular dichroism spectroscopies are used to study the corresponding structural aspects. In DTC complexes, the BCD ring system acts as the exclusive coordination site for both metals. For DSC, however, the N4 atom plays a leading role in the metal binding and would be the only donor involved in 1:1 species; in ML2 complexes, the second ligand is thought to bind through the BCD ring system.

Topics: Calcium; Circular Dichroism; Humans; Magnesium; Potentiometry; Spectrophotometry; Tetracycline; Tetracyclines

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.

Topics: Animals; Arthritis, Rheumatoid; Cartilage, Articular; Diabetes Mellitus, Experimental; Humans; Male; Metronidazole; Microbial Collagenase; Minocycline; Rats; Rats, Inbred Strains; Rickets; Skin; Streptozocin; Tetracycline; Tetracyclines