Demeclocycline attenuates hyponatremia by reducing aquaporin-2 expression in the renal inner medulla.

Binding of vasopressin to its type 2 receptor in renal collecting ducts induces cAMP signaling, transcription and translocation of aquaporin (AQP)2 water channels to the plasma membrane, and water reabsorption from the prourine. Demeclocycline is currently used to treat hyponatremia in patients with the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Demeclocycline's mechanism of action, which is poorly understood, is studied here. In mouse cortical collecting duct (mpkCCD) cells, which exhibit deamino-8-D-arginine vasopressin (dDAVP)-dependent expression of endogenous AQP2, demeclocycline decreased AQP2 abundance and gene transcription but not its protein stability. Demeclocycline did not affect vasopressin type 2 receptor localization but decreased dDAVP-induced cAMP generation and the abundance of adenylate cyclase 3 and 5/6. The addition of exogenous cAMP partially corrected the demeclocycline effect. As in patients, demeclocycline increased urine volume, decreased urine osmolality, and reverted hyponatremia in an SIADH rat model. AQP2 and adenylate cyclase 5/6 abundances were reduced in the inner medulla but increased in the cortex and outer medulla, in the absence of any sign of toxicity. In conclusion, our in vitro and in vivo data indicate that demeclocycline mainly attenuates hyponatremia in SIADH by reducing adenylate cyclase 5/6 expression and, consequently, cAMP generation, AQP2 gene transcription, and AQP2 abundance in the renal inner medulla, coinciding with a reduced vasopressin escape response in other collecting duct segments.

In vitro study of the inactivation by dentine of some endodontic medicaments and their bases.

BACKGROUND: The aim of this study was to investigate the antimicrobial effect of endodontic medicaments and their bases in the presence of dentine powder. METHODS: The medicaments tested were Ledermix paste, Pulpdent paste, a 50:50 combination of the Pulpdent:Ledermix and their bases. The test organism was Enterococcus faecalis ATCC 29212. The presence or absence of dentine was examined as well as the effect of autoclaving dentine. Serial dilutions of samples at 1 hour, 1 day and 3 days were used for colony counting. The effects of dentine powder on pH for saturated Ca(OH)2 solution and Pulpdent paste at 1 hour and 24 hours were also measured. RESULTS: Pulpdent and the 50:50 combination of Pulpdent:Ledermix completely inhibited the growth of E. faecalis from 1 hour onwards, and these results were not affected by the presence/absence of dentine powder, pre-incubation period, timing of autoclaving, or exposure time. Saturated solutions of Ca(OH)2 are prone to inactivation by dentine powder unlike Pulpdent paste. Ledermix paste took 3 days to exert a significant effect on the growth of E. faecalis. CONCLUSIONS: In this laboratory study, both Pulpdent and the 50:50 mixture of Pulpdent with Ledermix were effective medicaments against E. faecalis in the presence of dentine powder.

An in vitro study of the antimicrobial activity of some endodontic medicaments against Enteroccus faecalis biofilms.

BACKGROUND: The in vitro antimicrobial activity of a series of endodontic medicaments and their bases against biofilms of Enterococcus faecalis was investigated. METHODS: The medicaments tested were Pulpdent paste, Ledermix paste, a 50:50 Ledermix and Pulpdent mixture, and a replica of Ledermix paste. Bases included methyl cellulose with water, polyethylene glycol (PEG), and PEG with zinc oxide, calcium chloride and the other components (inactives) that make up the Ledermix paste base. Biofilms grown on cellulose nitrate membrane filters were exposed to the medicaments for up to 5 days. The number of surviving colony forming units (CFU) was determined at days 1, 3 and 5. The results were expressed as a bacterial survival index (BSI) when compared to the unexposed control. RESULTS: Pulpdent produced the greatest reduction of BSI, followed by the 50:50 mixture of Pulpdent and Ledermix pastes. Ledermix paste, its replica and the individual bases showed no significant reductions in the BSI for E. faecalis. CONCLUSIONS: Within the limitations of this laboratory study, calcium hydroxide containing preparations had greater potential for reducing the survival of E. faecalis in a biofilm environment.

An in vitro study of the antimicrobial activity of some endodontic medicaments and their bases using an agar well diffusion assay.

BACKGROUND: The aim of this study was to determine the in vitro antimicrobial activities of various endodontic medicaments and their bases against selected organisms using an agar diffusion assay. METHODS: An agar well diffusion assay was used to test the antimicrobial action of some commonly used endodontic medicaments (Ledermix paste, Pulpdent paste, Ultracal paste, and a 50:50 mix of Ledermix and Pulpdent pastes) and their bases. Three bacterial species (E. faecalis, P. micros, P. intermedia) and one yeast (C. albicans) were selected. The diameters of growth inhibition zones and pH were assessed. RESULTS: P. micros demonstrated the highest level of in vitro resistance. Pulpdent and Ultracal pastes had the highest pH (12.64 and 12.53, respectively). The addition of Pulpdent to Ledermix did not increase the zone sizes significantly. CONCLUSIONS: All the commercial products showed some in vitro antimicrobial activity. Ledermix paste and the 50:50 Ledermix/Pulpdent mixture being the most effective in this model. The known anti-inflammatory/analgesic properties of Ledermix and the results from this agar model suggest that the 50:50 Ledermix/Pulpdent combination would be the preferred medicament for clinical use in symptomatic cases, even though the addition of calcium hydroxide to Ledermix did not appear to be synergistic in terms of enhancing the antimicrobial action.

Identification of cultivable microorganisms from root canals with apical periodontitis following two-visit endodontic treatment with antibiotics/steroid or calcium hydroxide dressings.

The study was aimed at comparing the efficacy of disinfection of root canals with periapical radiolucencies when treated with either antibiotics/steroid medicaments (Ledermix or Septomixine) or a calcium hydroxide paste (Calasept). Microbiological samples were taken before and after two-visit endodontic treatment from 88 canals with apical periodontitis. All of the canals but one (87 of 88) had cultivable growth before treatment. After dressing with Ledermix, Septomixine, or Calasept, the percentages of canals remained with positive growth were 48% (13 of 27), 31% (8 of 26), and 31% (11 of 35), respectively. The chi(2) tests showed there were no significant differences in the number of canals with positive growth or mean colony forming units counts after instrumentation, irrigation and dressing. In the Ledermix group, 38 strains of bacteria were recovered. The Septomixine group had 25 strains, and the Calasept group had 25 strains. Gram-positive facultative anaerobic cocci (including staphylococci and streptococci) were more prevalent than the Gram-negative obligate anaerobic rods after treatment in all three groups. Similarities in the reduced number of canals with residual growth, and the prevalence of Gram-positive facultative anaerobic cocci suggest that the use of different inter-appointment dressings produced similar microbiological outcomes. However, factors other than the antimicrobial effectiveness of intracanal medicaments may also be responsible for the results observed.

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

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The effect of intracanal Ledermix on root resorption of delayed-replanted monkey teeth.

Progressive root resorption is one of the common sequelae in replanted teeth, which is detrimental to their long-term prognosis. Ledermix paste, with its composition of triamcinolone acetonide and demethylclortetracycline, has been shown to inhibit inflammation and the associated hard tissue resorption. This study evaluated the effect of immediate intracanal Ledermix on root resorption of delayed-replanted monkey teeth. A total of 36 roots were root canal treated. For the experimental group, intracanal Ledermix was placed prior to extraction and replantation after 1-h bench dry. The positive control group was root filled and replanted after 1 h while the negative control group was root filled and replanted immediately. The monkeys were sacrificed after 12 weeks. The H&E histological tissue specimens were prepared and evaluated using a method modified from that of Andreasen's morphometric analysis, as favorable or complete healing and unfavorable healing, comprising inflammatory root resorption and replacement resorption. The results were analyzed using Kruskal-Wallis and Mann-Whitney U-tests as well as Wilcoxon signed ranks test. The negative control group produced highly significant favorable healing and unfavorable healing as compared to the Ledermix group (P = 0.000). The Ledermix group only showed significantly higher occurrence of complete healing (35.46%) compared to the positive control group (16.58%) (P = 0.037) but there were no significant differences in the inflammatory root resorption and replacement resorption. Nevertheless, when the latter two unfavorable healing patterns were combined, there was a significantly lower overall unfavorable healing in the Ledermix group (64.54%) when compared to the positive control group (83.43%) (P = 0.037). This unfavorable healing outcome in the Ledermix group, however, was not significantly different from the favorable healing outcome with the same treatment modality (P = 0.110).

Effect of immediate intracanal placement of Ledermix Paste(R) on healing of replanted dog teeth after extended dry times.

Ledermix Paste is a paste containing triamcinolone and demeclocycline with demonstrated anti-inflammatory activity that may slow down resorptive processes after severe traumatic injuries to the dentition. A total of 29 premolar roots of six mongrel dogs were extracted and instrumented with rotary nickel titanium files. Fifteen of these roots were then filled with a calcium hydroxide (Ca(OH)2) slurry and 14 roots were filled with Ledermix Paste paste. All accesses were sealed with glass ionomer and the roots replanted after an extraoral dry time of 60 min. After 4 months, the dogs were killed and the roots prepared for histological evaluation. Five-micrometer thick cross-sections of the root and surrounding tissue taken every 90 micro m were evaluated for healing. In addition, residual root mass was also measured to determine the extent of root structure loss for each treatment method. The Ledermix Paste-treated roots had statistically significantly more healing and less resorption than the roots treated with Ca(OH)2. Root filling with Ledermix Paste also resulted in significantly less loss in root mass due to resorption compared to those roots filled with Ca(OH)2. Immediate intracanal placement of Ledermix Paste at the emergency visit after an avulsion injury appears to decrease resorption and increase favorable healing.

Tetracyclines reduce Na+/K+ pump capacity in Calu-3 human airway cells.

We studied the effect of tetracyclines on the Na+/K+ pump activity in Calu-3, a human airway cell line. To estimate Na+/K+ pump capacity on the basolateral membrane, an ouabain-sensitive component of the short-circuit current (Isc) was measured in the presence of nystatin, an ionophore of Na+. The application of ouabain (1 mM) to the basolateral solution completely inhibited the Isc generated by adding nystatin (50 microM) to the apical solution. Tetracycline (TC), minocycline (MC), or demethylchlortetracycline (DC) at 0.5 mM applied to the apical but not to the basolateral solution also decreased the nystatin-induced Isc. Neither phlorizin- nor diphenylamine-2-carboxylic acid-sensitive Isc was affected by TC, MC, or DC. These results indicate that tetracyclines may permeate only through the apical membrane with the result that the Na+/K+ pump's capacity for Na+ extrusion should be suppressed without a decrease in Cl- transport.

[Effective demeclocycline therapy in a patient with over-secretion of antidiuretic hormone following head trauma]. / Hatásos demeclocyclin-terápia koponyatraumát követö antidiuretikus hormontúltermelés szindróma esetében.

The syndrome of inappropriate secretion of antidiuretic hormone (SIADH) is a common cause of hyponatremia. In this study a case of SIADH caused by head trauma is reported, in which severe hyponatraemia, escorted by life-threatening neurological symptoms was observed that could only be managed by parenteral sodium chloride infusions. Severe hyponatraemia was accompanied by elevated urinary sodium excretion, a characteristic sign of SIADH. After introducing the therapy with demeclocycline, a tetracycline type antibiotic that inhibits the renal action of antidiuretic hormone, serum sodium levels began to rise gradually, and the urinary sodium excretion slowly decreased. These observations show the effectiveness of demeclocycline in the treatment of SIADH.

Perioperative vasopressin secretion treated by demeclocycline.

The purpose of this study was to evaluate the perioperative effects of demeclocycline on vasopressin (VP) in patients undergoing surgery, specifically coronary artery bypass grafting (CABG). This was a prospective, double-blind placebo-controlled clinical study using human subjects in a 575-bed tertiary care teaching community hospital. Thirty patients (20 males and 10 females) undergoing elective CABG over a 6-month period were randomized preoperatively to receive either demeclocycline or a placebo. Each patient received either a total of 1200 mg daily of demeclocycline or a placebo beginning 5 days preoperatively and continuing through postoperative day 2. Urine and serum osmolality, electrolytes, and VP levels were measured daily. Perioperative VP levels were significantly higher (P = 0.05) in the demeclocycline group despite decreased VP activity. The postoperative serum sodium and osmolality remained normal in the demeclocycline group and significantly decreased in the placebo group (P < 0.01). The urine osmolality increased significantly in the placebo group (P = 0.04) on postoperative day 1. We conclude that perioperative administration of demeclocycline reliably inhibits the effects of increased VP secretion commonly seen in patients undergoing CABG procedures. Applying these findings to surgical patients who are at increased risk of complicated fluid and electrolyte problems requires further study.

Effect of calcium hydroxide and combinations of Ledermix and calcium hydroxide on inflamed pulp in dog teeth.

The effects of Ledermix+calcium hydroxide (Ca(OH)2) or Ca(OH)2 alone on inflamed pulp tissues of dogs were studied. Fifty-nine upper incisor teeth of 10 dogs were used. Class V cavities were prepared and filled with amalgam after placement of decayed dentin particles. After 7 days, the decayed dentin and alloy were removed, and the pulps of the teeth were exposed. Ledermix and Ca(OH)2 mixtures or Ca(OH)2 alone were applied to the cavities. At the end of 7, 30, and 90 days, the teeth were extracted and examined histopathologically. Inflammation was found to be more prevalent in the 7- and 30-day groups that were treated with the Ledermix+Ca(OH)2 combination, whereas fibrosis and necrosis were nearly similar in both groups. In the 90-day groups, no inflammation was seen. No difference between the two 90-day groups with regard to reparative dentin was found.

The quantification of tertiary dentine formation in response to materials commonly placed in deep cavities in general practice in the UK.

PURPOSE OF STUDY: To determine the rate of formation of tertiary dentine in dogs' teeth, in response to five commonly used lining materials. Also, to record the resulting differences in quality of the formed dentine. MATERIALS AND METHODS: Representatives of five types of materials commonly used in general practice in the UK were placed in deep standardised cavities with a remaining dentine thickness of 400-500 microns, and sealed to the cavo-surface with zinc oxide-eugenol. The daily rate of tertiary dentine formed in response to each material was measured over a period of 119 days using a tetracycline staining method. A concomitant study to show the quality of the formed dentine was undertaken using the same materials and experimental conditions. RESULTS: There was a significant difference between the rates of tertiary dentine formation for all materials except zinc oxide-eugenol and Ledermix paste. For each material there was a increased rate of tertiary dentine formation in the second period (29-49 days) compared to the first period (0-28 days). This is considered to be due, in part, to a lag period when damaged odontoblasts are differentiating from mesenchymal elements. The rate of tertiary dentine formation in response to all the test materials was always greater than that of normal physiological dentine formation in unprepared teeth. Qualitatively, there was difference in the formed tertiary dentine beneath each material. All the materials caused odontoblast destruction, altered ground substance production and alterations in the calcification process. CONCLUSION: None of the materials routinely used at present can be considered to be 'ideal' as odontoblast destruction, altered ground substance production and altered calcification occur beneath zinc oxide-eugenol preparations, calcium hydroxide preparations as well as in response to corticosteroid-containing materials such as Ledermix paste and cement.

A comparative study on the effects of tetracyclines and lithium on the cyclic AMP second messenger system in rat brain.

1. This study was aimed at investigating the effects of demeclocycline (DMC), minocycline (MC), and lithium (Li) in vitro on cyclic AMP (cAMP) accumulation in rat cerebral cortex stimulated by noradrenaline, forskolin, and ouabain. 2. DMC, MC, and Li dose-dependently reduced noradrenaline-stimulated cAMP formation in cortical slices, but only Li inhibited the cAMP formation induced by forskolin. 3. In contrast to Li, DMC and MC did not affect noradrenaline-stimulated adenylate cyclase activity in cortical membranes. 4. In cortical slices, ouabain stimulated the cAMP production (required the presence of extracellular Ca2+ and was blocked by verapamil). Ouabain-stimulated cAMP accumulation in cortical slices was inhibited by DMC, MC, and Li. 5. DMC and MC do not seem to interact directly with the adenylate cyclase as reported for Li. It is concluded that the tetracyclines, DMC and MC, affect the cAMP signaling system in rat brain by mechanisms that differ from that of Li. The decreased receptor agonist-stimulated cAMP production in cortical slices in the presence of DMC and MC may be due to the Ca(2+)-chelating ability of these tetracyclines.

Morphology of the mineralizing front and observations of reparative dentine following induction and inhibition of dentinogenesis in the rat incisor.

An extra oral approach was made to 26 pairs of Wistar rat incisors and a small bur used to expose the pulps which were then dressed with either calcium hydroxide paste (Pulpdent) zinc oxide-eugenol cement (Kalzinol) or a triamocinolone acetonide/tetracycline containing cement (Ledermix). Animals were sacrificed at 7 days, incisors removed, a window cut to the pulp opposite the site of exposure, soft tissues removed with 7% NaOCl for 30 minutes before teeth were dehydrated in graded concentrations of acetone and gold coated for examination under SEM. Calcium hydroxide produced rapid complete repair with a remarkably regular formation of calcospherites, except for a zone of relative inhibition perforated by nutrient canals at the periphery of each lesion. Both zinc oxide-eugenol and triamcinolone acetonide cements inhibited both bridging of the defect created by the exposure and dentine formation in the surrounding pulpal wall. Data was grouped according to the degree of hard tissue repair. The results were statistically significant (Chi-square p < or = 0.001). This method demonstrated qualitative and quantitative differences in the repair process resulting from chemical variations in the dressings applied.

Efficacy of Ledermix paste in eliminating Staphylococcus aureus from infected dentinal tubules in vitro.

The efficacy of Ledermix paste in disinfection of dentinal tubules was studied in a model developed by Haapasalo and Orstavik with some modifications. Ledermix and 3% Tetracycline in a hydrous base were effective in reducing the amount of Staphylococcus aureus in dentinal tubules after 7 days of incubation and also after recontamination. They were not effective after 24 h.

Transepithelial water movement in response to carbamazepine, chlorpropamide and demeclocycline in toad urinary bladder.

1. Osmotic water movement across toad isolated hemibladders was measured by a gravimetric method. 2. The influence of carbamazepine, chlorpropamide and demeclocycline on the antidiuretic hormone (ADH)-induced water flow rate was examined. 3. No antidiuretic activity due to carbamazepine alone was observed but a slight inhibition due to ADH-induced water flow was observed in the presence of carbamazepine over a selected dose-range. This was unexpected and is inconsistent with data from in vivo studies in man. 4. Chlorpropamide potentiated ADH-induced water flow, in keeping with the hypothesis that chlorpropamide sensitizes the renal tubules to ADH-induced water flow. 5. Demeclocycline inhibited ADH-induced water flow. The mechanism of action remains unclear.

Inhibition by antibiotic tetracyclines of rat cortical noradrenergic adenylate cyclase and amphetamine-induced hyperactivity.

Two antibiotic tetracyclines, demeclocycline (DMC) and minocycline, share several biochemical and behavioral properties with lithium (Li). DMC inhibited both noradrenaline- and chloradenosine-sensitive cyclic AMP accumulation in rat cerebral cortical slices both in vitro and ex vivo following two weeks of chronic dietary treatment. Minocycline, a lipophilic tetracycline, produced similar results in vitro. Both DMC and minocycline reduced open-field activity levels in rats following acute treatment, four hours prior to testing. Moreover, both drugs inhibited amphetamine-induced hyperactivity in the open field. Chronic treatment with 0.4% and 0.8% dietary DMC for two weeks attenuated amphetamine hyperactivity without affecting baseline activity levels in the open field. Neither DMC nor minocycline attenuated apomorphine-induced stereotypy at doses that attenuated amphetamine hyperactivity, a profile which is similar to that of lithium. Unlike lithium, however, DMC did not reverse reserpine-induced hypoactivity.