In vitro and in vivo antitumoral activity of a ternary copper (II) complex.

Recently, a high number of copper derivatives has been evaluated as DNA-targeting metallodrugs, due to the lower toxicity and its potential to cleave DNA. Several strategies have been testing to develop metal compounds effective against tumour cells. In this work, the ternary copper (doxycycline)-(1,10-phenanthroline) complex [Cu(dox)(phen)]2+ was especially designed as an antitumoral drug, previously showing high cytotoxicity and DNA cleavage activity. We aimed to further investigate the in vitro cytotoxic activity in both tumoral and non-tumoral cells, in vitro genotoxic potential, and in vivo antitumor activity using BALB/C mouse injected with sarcoma S180 and Ehrlich cell lines. Our results indicated that this compound exhibits a moderate genotoxic potential, with selective growth inhibition of tumor cells, especially the murine melanoma B16F10. Its main mechanism of action seems to be through ROS generation. We have further shown a significant reduction of the implanted tumor size in the animal model, suggesting that this compound has great antitumoral potential against many tumor types. [Cu(dox)(phen)]2+ is selectively cytotoxic for melanoma B16F10 and showed high chemotherapeutic potential in vivo against implanted sarcoma S180 and Ehrlich ascites tumours.

Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression.

We report the synthesis and photolytic properties of caged 9-aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9-aminodoxycycline showed complex photochemical reactions but did release 10 % of 9-aminodoxycycline. However, 9-(PEG7-DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells.

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most difficult Gram-negative bacteria to treat and eradicate. In a cell-based screening of pleuromutilin derivatives against a drug sensitive A. baumannii strain, new molecules (2-4) exhibit bacteriostatic activity with 3.13 µg/mL concentration and 1 shows bactericidal activity with an MBC of 6.25 µg/mL. The pleuromutilin derivative 1 displays strong synergistic effects with doxycycline in a wide range of concentrations. A 35/1 ratio of 1 and doxycycline (1-Dox 35/1) kills drug susceptible A. baumannii with the MBC of 2.0 µg/mL and an MDR A. baumannii with the MBC of 3.13 µg/mL. In vitro anti-Acinetobacter activity of 1-Dox 35/1 is superior to that of clinical drugs such as tobramycin, tigecycline, and colistin. The efficacy of 1-Dox 35/1 is evaluated in a mouse septicemia model; treatment of the infected C57BL/6 mice with 1-Dox 35/1 protects from lethal infection of A. baumannii with an ED50 value of <2.0 mg/kg.

Synthesis and antibacterial activity of doxycycline neoglycosides.

A set of 37 doxycycline neoglycosides were prepared, mediated via a C-9 alkoxyamino-glycyl-based spacer reminiscent of that of tigecycline. Subsequent in vitro antibacterial assays against representative drug-resistant Gram negative and Gram positive strains revealed a sugar-dependent activity profile and one doxycycline neoglycoside, the 2'-amino-α-D-glucoside conjugate, to rival that of the parent pharmacophore. In contrast, the representative tetracycline-susceptible strain E. coli 25922 was found to be relatively responsive to a range of doxycycline neoglycosides. This study also extends the use of aminosugars in the context of neoglycosylation via a simple two-step strategy anticipated to be broadly applicable for neoglycorandomization.

Pharmacokinetic behavior of doxycycline after intramuscular injection in sheep.

OBJECTIVE: To determine the pharmacokinetics of a commercial formulation of doxycycline hyclate after IM administration of a single dose to sheep. ANIMALS: 11 healthy domestic sheep. PROCEDURES: For each sheep, doxycycline was administered as a single dose of 20 mg/kg, IM. Blood samples were obtained prior to and for 84 hours after doxycycline administration. Plasma concentrations of doxycycline were determined via high-performance liquid chromatography with UV detection. Pharmacokinetic data were analyzed with noncompartmental methods. RESULTS: Mean ± SD values for pharmacokinetic parameters included maximum plasma concentration (2.792 ± 0.791 µg/mL), time to reach maximum plasma concentration (0.856 ± 0.472 hours), mean residence time (91.1 ± 40.78 hours), elimination half-life (77.88 ± 28.45 hours), and area under the curve (65.67 ± 9.877 µg•h/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that doxycycline had prolonged absorption and elimination in sheep after IM administration. A daily dose of 20 mg/kg would be sufficient to reach effective plasma concentrations against Chlamydia spp (minimum inhibitory concentration, 0.008 to 0.031 µg/mL) and Staphylococcus aureus (minimum inhibitory concentration, 0.12 µg/mL). Doxycycline administered IM could be an option for therapeutic use in sheep, although further studies are needed.

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes.

Doxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 µg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 µg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.

Mechanical and biological characterization of resin-modified glass-ionomer cement containing doxycycline hyclate.

OBJECTIVES: To characterize the mechanical and biological properties of a resin-modified glass ionomer cement (RMGIC) containing doxycycline hyclate. METHODS: The antibacterial effect of RMGIC containing 1.5, 3.0 and 4.5% doxycycline hyclate was assessed using two experiments - agar diffusion test for 24h and biofilm assay for 24h and 7 days - against some cariogenic bacteria. Briefly, base layers of BHI agar and 300µL of each inoculum were prepared in Petri dishes with 6 wells that were completely filled with materials. After 24h incubation, zones of bacterial growth inhibition were measured using a digital caliper. Biofilm assays were conducted using RMGIC specimens immersed in 24-well plates containing the inoculum in BHI broth. After 24h and 7 days, each specimen were removed, vortexed and the suspension diluted and inoculated in BHI plates for subsequent bacterial counting. Cytotoxicity tests used 50 specimens made in sterilized metal molds, including Vitrebond as positive control. Extracts from every specimen were applied on the MDPC-23 odontoblast-like cells for 24h. The MTT assay and SEM evaluation determined cell metabolism and morphology, respectively. 80 cylindrical specimens were made from the previously cited groups, and were submitted to testing with a universal testing machine (Instron 4411) using a crosshead speed of 1.0mm/min for compressive strength and 0.5mm/min for diametral tensile strength, respectively. Data from antibacterial and cytotoxic effects, and mechanical properties were submitted to appropriated statistical tests. RESULTS: All tested groups showed growth inhibition of all tested strains (p<0.05) in 24h for both microbiological tests, but only 4.5% doxycycline have antibacterial effect after 7 days. None of doxycycline concentrations caused toxic effect to the MDPC-23 cells or presenting alterations to mechanical properties. CONCLUSION: The incorporation of up to 4.5% doxycycline hyclate into RMGIC inhibits important oral microorganisms, without modifying biological and mechanical characteristics of the dental material, suggesting a new alternative for the treatment of dental caries.

Formulation optimization and characterization of spray dried microparticles for inhalation delivery of doxycycline hyclate.

The local delivery of antibiotics in the treatment of infectious respiratory diseases is an attractive alternative to deliver high concentration of antimicrobials directly to the lungs and minimize systemic side effects. In this study, inhalable microparticles containing doxycycline hyclate, sodium carboxymethylcellulose, leucine and lactose were prepared by spray drying of aqueous ethanol formulations. Box-Behnken design was used to study the influence of various independent variables such as polymer concentration, leucine concentration, ethanol concentration and inlet temperature of the spray dryer on microparticle characteristics. The microparticles were characterized in terms of particle morphology, drug excipient interaction, yield, entrapment efficiency, Carr's index, moisture content, thermal properties, X-ray powder diffraction, aerosolization performance and in vitro drug release. The effect of independent variables on spray dryer outlet temperature was also studied. The overall shape of the particles was found to be spherical like doughnuts in the size range of 1.16-5.2 µm. The optimized formulation (sodium carboxymethylcellulose concentration 14% w/v, leucine concentration 33% w/v, ethanol concentration 36% v/v, inlet temperature of 140°C) exhibited the following properties: yield 56.69%, moisture content 3.86%, encapsulation efficiency 61.74%, theoretical aerodynamic diameter 3.11 µm and Carr's index 23.5% at an outlet temperature 77°C. The powders generated were of a suitable mass median aerodynamic diameter (4.89 µm) with 49.3% fine particle fraction and exhibited a sustained drug release profile in vitro.

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility.

BACKGROUND: Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). METHODS: Stable cell lines with TFPI (both α and ß) and only TFPIß downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. RESULTS: Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIß was downregulated, revealing a novel function of this isoform in cancer metastasis. CONCLUSIONS: Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

Comparative evaluation of the efficacy of 10% doxycycline hyclate in the periodontal treatment of smokers - a clinical and microbiological study.

PURPOSE: The aim of this present clinical and microbiological study was to evaluate the efficacy of the association of locally delivered 10% doxycycline hyclate (Atridox®) with scaling and root planing in the periodontal treatment of smokers. MATERIALS AND METHODS: Forty-five patients with chronic periodontitis having a minimum of two periodontal pockets (>5 mm) and satisfying the inclusion and exclusion criteria were selected. Sites were randomly assigned to scaling and root planing (SRP) or scaling and root planing followed by local application of 10% doxycycline hyclate (SRP-D). Plaque index (PI), gingival index (GI), periodontal pocket depth (PD), clinical attachment level (CAL) and total anaerobic colony count (TACC) were recorded at baseline, one month and three months respectively. Differences between baseline and each period were considered for analysis. RESULTS: There was a significant reduction in plaque score, gingival score, periodontal pocket depth and total anaerobic colony count from baseline in both groups at all time intervals. Clinical attachment level showed a significant gain in both groups. However, PD reduction (P < 0.001) and CAL gain (P < 0.001) were significant in the test group as compared to control at the end of 3 months. Reduction in total anaerobic colony count from baseline was significant (P = 0.02) in the test group compared to control at the end of 3 months. CONCLUSIONS: The use of locally delivered doxycycline may constitute an important adjunct for the treatment of chronic periodontitis in smokers.

Radiation crosslinking polymerization of sterculia polysaccharide-PVA-PVP for making hydrogel wound dressings.

The present study deals with the modification of sterculia gum by PVA-PVP through radiation crosslinking, to develop the hydrogels meant for the delivery of antimicrobial agent to the wounds. The hydrogels were characterized by SEM, FTIR, TGA and swelling studies. For the evaluation of swelling and drug release mechanism, the swelling kinetics and in vitro release dynamics of model drug from this matrix have been studied respectively in the solution of different pHs and simulated wound fluid. After 24h swelling per gram of the hydrogel has taken (17.03±0.19)g of simulated wound fluid and has released (0.230±0.01)mg of drug in the simulated fluid. The release of drug in simulated fluids occurred through non-Fickian diffusion mechanism.

Sensitive and selective spectrophotometric assay of doxycycline hyclate in pharmaceuticals using Folin-Ciocalteu reagent.

A spectrophotometric method for the determination of doxycycline (DOX) is described. The method is based on the formation of blue colored chromogen due to reduction of tungstate and/or molybdate in Folin-Ciocalteu (F-C) reagent by DOX in alkaline medium. The colored species has an absorption maximum at 770 nm and the system obeys Beer's law over the concentration range 0.75-12.0 µg mL-1 DOX. The apparent molar absorptivity is 2.78 × 104 L mol-1 cm-1. The limit of quantification and detection values are reported to be 0.20 and 0.08 µg mL-1, respectively. Over the linear range applicable, the accuracy and precision of the method were evaluated on intra-day and inter-day basis. The reported mean accuracy value was 101.0 ± 1.7 %, the relative error was ≤ 2.7 % and the relative standard deviation was ≤ 2.5 %. Application of the proposed method to bulk powder and commercial pharmaceutical tablets is also presented. No significant difference was obtained between the results of the proposed method and the official BP method. The procedure described in this paper is simple, rapid, accurate and precise.

Tetracycline sensing using novel doxycycline derivatives immobilized on different surface plasmon resonance biosensor surfaces.

BACKGROUND: This article aims to explore novel doxycycline derivatives for analyzing low concentrations of tetracyclines in biological matrices and food in competitive assays. RESULTS: Surface plasmon resonance (SPR) was employed in an indirect competitive format using a bacterial tetracycline-dependent regulatory protein as receptor. Three doxycycline derivatives were synthesized and covalently bound to the surface of four different sensor chips. Parameters that influence the immobilization of the doxycycline derivatives and subsequent binding of the receptor protein were studied. CONCLUSION: The novel doxycycline derivatives were successfully used as competitors in an indirect SPR assay.

Effective and safe combination therapy for severe acne vulgaris: a randomized, vehicle-controlled, double-blind study of adapalene 0.1%-benzoyl peroxide 2.5% fixed-dose combination gel with doxycycline hyclate 100 mg.

There is a paucity of treatment options for severe acne vulgaris aside from oral isotretinoin. This randomized, vehicle-controlled, multicenter, double-blind study evaluated the efficacy and safety of combination therapy using adapalene 0.1%-benzoyl peroxide 2.5% (A/BPO) fixed-dose combination gel with doxycycline hyclate 100 mg in the treatment of severe acne vulgaris. A total of 459 participants were randomized in a 1:1 ratio to receive oral doxycycline hyclate 100 mg once daily and either A/BPO or vehicle once daily for 12 weeks. Efficacy in the A/BPO with doxycycline group was demonstrated as early as week 2 compared with the vehicle arm for total, inflammatory, and noninflammatory lesions (all P < .005). At week 12, this combination was superior to vehicle with doxycycline in reducing total, inflammatory, and noninflammatory lesion counts (an added incremental benefit of 23%, 24%, and 21%, respectively), as well as for global success and overall participant satisfaction (all P < .001). Digital UV fluorescence photography demonstrated a rapid reduction in Propionibacterium acnes in the A/BPO with doxycycline group, particularly within the first 4 weeks. These findings provide evidence on the efficacy of combining A/BPO and the oral antibiotic doxycycline in the treatment of severe acne vulgaris.

Click-chemistry-derived tetracycline-amino acid conjugates exhibiting exceptional potency and exclusive recognition of the reverse tet repressor.

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). The final products were tested in a variety of TetR and revTetR systems, and the C-terminally linked phenylalanine conjugate 12 c exhibited high selectivity for revTetR over TetR. Besides the unique property of the specific effector 12 c to effectively differentiate TetR and its reverse phenotype, the test compound proved to be almost devoid of any antibacterial activity; this will be highly beneficial for future applications to control gene expression in bacterial systems.

Biowaiver monographs for immediate release solid oral dosage forms: Doxycycline hyclate.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing doxycycline hyclate are reviewed. According to the Biopharmaceutics Classification System (BCS), doxycycline hyclate can be assigned to BCS Class I. No problems with BE of IR doxycycline formulations containing different excipients and produced by different manufacturing methods have been reported and hence the risk of bioinequivalence caused by these factors appears to be low. Doxycycline has a wide therapeutic index. Further, BCS-based dissolution methods have been shown to be capable of identifying formulations which may dissolve too slowly to generate therapeutic levels. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing doxycycline hyclate as the single Active Pharmaceutical Ingredient (API) provided that (a) the test product contains only excipients present in doxycycline hyclate IR solid oral drug products approved in the International Conference on Harmonization (ICH) or associated countries; and (b) the comparator and the test products comply with the BCS criteria for "very rapidly dissolving" or, alternatively, when similarity of the dissolution profiles can be demonstrated and the two products are "rapidly dissolving.".

Doxycycline-mediated inhibition of matrix metalloproteinases improves healing after rotator cuff repair.

BACKGROUND: Recent studies demonstrate a potentially critical role of matrix metalloproteinases (MMPs) and their inhibitors in the pathophysiology of rotator cuff tears. HYPOTHESIS: Doxycycline-mediated MMP inhibition after rotator cuff repair will improve tendon-to-bone healing. STUDY DESIGN: Controlled laboratory study. METHODS: Rats (n = 183) underwent acute detachment and repair of the supraspinatus tendon and the animals were divided into 4 groups: In controls (n = 66), the supraspinatus was repaired to its anatomical footprint. In experimental groups, an identical surgery was performed with doxycycline (130 mg/kg/d) administered orally at (1) preoperative day 1 (n = 66), (2) postoperative day (POD) 5 (n = 28), or (3) POD 14 (n = 23). Animals were sacrificed at 5 days, 8 days, 2 weeks, and 4 weeks. Tendon-bone interface was evaluated with histomorphometry. Enzyme-linked immunosorbent assay for local MMP-13 activity was performed at 8 days and 4 weeks. Biomechanical testing of the healing enthesis was performed at 8 days, 2 weeks, and 4 weeks. Serum doxycycline levels were measured at sacrifice. Statistical analysis was performed using unpaired t tests and 2-way analysis of variance (P < .05). RESULTS: Serum doxycycline levels were significantly higher in all treated groups compared with controls (1830 +/- 835 vs 3 +/- 3 ng/mL, respectively; P < .001). Doxycycline-treated animals demonstrated greater metachromasia and improved collagen organization at the healing enthesis at POD 5 (P < .06), POD 8 (P < .03), and 2 weeks (P < .04). The MMP-13 activity was significantly reduced in doxycycline-treated compared with control animals at POD 8 (6740 +/- 2770 vs 10400 +/- 2930 relative fluorescent units [RFU], respectively; P < .02) but not at 4 weeks (3600 +/- 3280 vs 4530 +/- 2720 RFU, respectively). The healing enthesis of animals started on doxycycline preoperatively or at POD 5 had an increased load to failure compared to controls at 2 weeks (13.6 +/- 1.8 and 13.2 +/- 1.94 N vs 9.1 +/- 2.5 N, respectively; P < .01). CONCLUSION/CLINICAL RELEVANCE: Modulation of MMP-13 activity after rotator cuff repair may offer a novel biological pathway to augment tendon-to-bone healing.

Loading and release of doxycycline hyclate from strontium-substituted calcium phosphate cement.

Novel Sr-substituted calcium phosphate cement (CPC) loaded with doxycycline hyclate (DOXY-h) was employed to elucidate the effect of strontium substitution on antibiotic delivery. The cement was prepared using as reactants Sr-substituted beta-tricalcium phosphate (Sr-beta-TCP) and acidic monocalcium phosphate monohydrate. Two different methods were used to load DOXY-h: (i) the adsorption on CPC by incubating the set cement in drug-containing solutions; and (ii) the use of antibiotic solution as the cement liquid phase. The results revealed that the Sr-substituted cement efficiently adsorbs the antibiotic, which is attributed to an enhanced accessibility to the drug-binding sites within this CPC. DOXY-h desorption is influenced by the initial adsorbed amount and the cement matrix type. Furthermore, the fraction of drug released from CPCs set with DOXY-h solution was higher, and the release rate was faster for the CPC prepared with 26.7% Sr-beta-TCP. The analysis of releasing profiles points to Fickian diffusion as the mechanism responsible for antibiotic delivery. We can conclude that Sr substitution in secondary calcium phosphate cements improves their efficiency for DOXY-h adsorption and release. The antibiotic loading method provides a way to switch from rapid and complete to slower and prolonged drug release.