Synthesis, in vitro and in vivo antimycobacterial activities of diclofenac acid hydrazones and amides

Various diclofenac acid hydrazones and amides were synthesized and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis. Preliminary results indicated that most of the compounds demonstrated better in vitro antimycobacterial activity (MIC: 0.0383-7.53 microM) than diclofenac (MIC: 21.10 microM) and ciprofloxacin (MIC: 9.41 microM). Among the synthesized compounds, 1-cyclopropyl-6-fluoro-8-methoxy-7-[[N4-(2-(2-(2,6-dichlorophenylamino)phenyl)acetyl)-3-methyl]-N1-piperazinyl]-4-oxo-1,4-dihydro-3-quinoline carboxylic acid (5d) was found to be the most active compound in vitro with MIC of 0.0383 microM and was more potent than first line antitubercular drug isoniazid (MIC: 0.1822 microM). In the in vivo animal model 5d decreased the bacterial load in lung and spleen tissues with 2.42- and 3.66-log10 protections, respectively, at 25 mg/kg body weight.     

Synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones

The purpose of this study was to prepare various isoniazid derivatives by introducing the isoniazid pharmacophore into several molecules and screening for antimycobacterial activity. Ortho-hydroxy acetophenone reacts with isoniazid to form acid hydrazones. The C-Mannich bases of the above acid hydrazones were prepared by reacting them with formaldehyde and various secondary amines. The synthesized compounds were screened against M. tuberculosis H(37)R(v) using the alamar blue susceptibility test. The synthesized compounds inhibit Mycobacterium tuberculosis strain H(37)R(v) with minimum inhibitory concentrations ranging from 0.56 to 4.61 microM. Compound N'-{1-[2-hydroxy-3-(piperazin-1-ylmethyl)phenyl]ethylidene}isonicotinohydrazide 8 was found to be the most active compound with an MIC of 0.56 microM, and was more potent than isoniazid (MIC of 2.04 microM). After 10 days of treatment, compound 8 decreased the bacterial load in murine lung tissue by 3.7-log10 as compared to controls, which was equipotent to isoniazid. The results demonstrate the potential and importance of developing new isoniazid derivatives against mycobacterial infections.     

In vivo and in vitro sodium pump activity in subjects with thyrotoxic periodic paralysis.

OBJECTIVE--To examine whether sodium pump activity plays a part in the pathogenesis of thyrotoxic periodic paralysis. DESIGN--Measurement of platelet sodium-potassium ATPase and in vivo sodium pump activities in healthy subjects and thyrotoxic subjects with and without paralysis. SETTING--University hospital in Hong Kong. SUBJECTS--21 healthy subjects, 23 untreated thyrotoxic subjects, 13 untreated men with periodic paralysis, seven treated thyrotoxic subjects, and six treated men with periodic paralysis. MAIN OUTCOME MEASURES--Platelet Na+, K(+)-ATPase activity and plasma rubidium concentration after oral loading. RESULTS--Median (range) platelet Na+, K(+)-ATPase activity in thyrotoxic subjects was 253 (169-821) mumol inorganic phosphate/h/g protein--significantly higher than that in healthy subjects (134 (81-180) mumol/h/g protein; p less than 0.001). Na+, K(+)-ATPase activity in those with periodic paralysis was 374 (195-1196) mumol/h/g protein, again significantly higher than that in healthy subjects (p less than 0.001) and that in other thyrotoxic subjects (p less than 0.01) despite similar degrees of hyperthyroidism. Activities in treated thyrotoxic subjects with and without periodic paralysis were 148 (110-234) and 131 (86-173) mumol/h/g protein respectively. Mean (95% confidence interval) plasma rubidium concentration five hours after oral administration in thyrotoxic subjects (7.0 (6.6 to 7.5) mumol/l) was significantly lower than in healthy subjects (10.2 (9.5 to 10.9) mumol/l; p less than 0.001) and higher than in those with periodic paralysis (6.0 (5.7 to 6.3) mumol/l; p less than 0.01). CONCLUSIONS--Sodium pump activity in untreated subjects with periodic paralysis is higher than in other thyrotoxic subjects, and this may be responsible for the hypokalaemia.     

In vitro and in vivo assessment of herb drug interactions

Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions.     

Mefloquine, an antimalaria drug with antiprion activity in vitro, lacks activity in vivo

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.     

In vivo and in vitro cytogenetic damage induced by sodium nitrite

The mutagenicity of sodium nitrite was assayed by in vivo and in vitro experiments. The in vivo experiments were carried out in male rats and mice intragastrically treated twice, with an interval of 24 h, with nitrite in doses of 1.72, 5.18, 15.55 and 46.66 mg/kg body weight and in male rabbits treated with the same doses of nitrite administered daily in drinking water for 3 months. Chromosomal aberration analysis was conducted in all 3 species of animals and micronucleus induction was only evaluated in mice. Nitrite induced increases in aberrant metaphases in all 3 species of animals. Likewise, in mice it induced increases of the numbers of micronucleated polychromatic erythrocytes and a light bone marrow depression. Neither in the increases of the numbers of chromosomal aberrations nor in that of micronuclei, were dose-related responses observed. The in vitro experiments were carried out on BSC-1 and HeLa cells grown in cultures with nitrite in doses of 0.265 and 0.530 mg/ml for 24 h. Both doses produced significant increases of the percentage of chromosomal aberrations but also without demonstration of positive dose-effect relationships.     

In vitro and ex vivo antimycobacterial potential of azole drugs against Mycobacterium tuberculosis H37Rv

The antimycobacterial activity of two clinically approved antifungal azole drugs, clotrimazole and econazole, was evaluated against Mycobacterium tuberculosis H₃₇Rv under in vitro and ex vivo conditions. The minimum inhibitory concentration (MIC₉₀) was 0.120 μg ml⁻¹, whereas the minimum bactericidal concentration and effective concentration was 0.125 μg ml⁻¹ for both the drugs demonstrating their potent antimycobacterial activity. Further, the azole drugs exhibited a synergistic activity with either rifampicin or isoniazid as evaluated on the basis of reduction of colony forming units. The results suggest that azole compounds bear the potential to enhance the efficacy of currently prescribed antitubercular drugs.     

Comparative in vitro and in vivo evaluation of matrix,osmotic matrix,and osmotic pump tablets for controlled delivery of diclofenac sodium

The aim of this investigation was preparation and comparative evaluation of fabricated matrix (FM), osmotic matrix (OM), and osmotic pump (OP) tablets for controlled delivery of diclofenac sodium (DS). All formulations were evaluated for various physical parameters, and in vitro studies were performed on USP 24 dissolution apparatus II in pH 7.4 buffer and distilled water. In vivo studies were performed in 6 healthy human volunteers; the drug was assayed in plasma using HPLC, and results were compared with the performance of 2 commercial tablets of DS. Various pharmacokinetic parameters (ie, C_max, T_max, area under the curve [AUC_0–24], and mean residence time) and relative bioavailability were compared. All fabricated formulations showed more prolonged and controlled DS release compared with commercial tablets studied. The OM and OP tablets, however, performed better than the matrix tablets. The rate and extent of drug release from FM1 matrix tablets (single polymer) was significantly different from that of FM2 (admixed polymers). Type of porosigenic agents and osmogens also influenced the drug release. Analysis of in vitro data by regression coefficient analysis revealed zero-order release kinetics for OM and OP tablets, while FM tablets exhibited Higuchi kinetics. In vivo results indicated prolonged blood levels with delayed peak and improved bioavailability for fabricated tablets compared to commercial tablets. It was concluded that the osmotic matrix and osmotic pump tablets could provide more prolonged, controlled, and gastrointestinal environmental-independent DS release that may result in an improved therapeutic efficacy and patient compliance.     

Nitroquinolones with broad-spectrum antimycobacterial activity in vitro.

During search on quinolonecarboxylic acids we used a facile, convenient two- or three-step procedure to synthesize new quinolone analogs, bearing at the C-7 position alkylamino substituents, and at the C-6 position a fluorine or alternatively a nitro group. The new derivatives were tested against both Gram-positive and Gram-negative bacteria and against a number of different mycobacteria. In vitro assays showed 1-tert-butyl-7-tert-butylamino-6-nitro-1,4-dihydro-4-quinolone-3-carboxy lic acid to be a potent inhibitor of Streptococcus and Staphylococcus with potencies superior to those of ofloxacin and ciprofloxacin, used as reference drugs. Some 6-nitroquinolones were found to exert good inhibiting activities against Mycobacterium tuberculosis and various atypical mycobacteria, whereas the 6-fluoro counterparts showed poor or no activity against this bacterium.     

In vitro and in vivo osteogenic activity of licochalcone A

We investigated the in vitro and in vivo osteogenic activity of licochalcone A. At low concentrations, licochalcone A stimulated the differentiation of mouse pre-osteoblastic MC3T3-E1 subclone 4 (MC4) cells and enhanced the bone morphogenetic protein (BMP)-2-induced stimulation of mouse bi-potential mesenchymal precursor C2C12 cells to commit to the osteoblast differentiation pathway. This osteogenic activity of licochalcone A was accompanied by the activation of extracellular-signal regulated kinase (ERK). The involvement of ERK was confirmed in a pharmacologic inhibition study. Additionally, noggin (a BMP antagonist) inhibited the osteogenic activity of licochalcone A in C2C12 cells. Licochalcone A also enhanced the BMP-2-stimulated expression of various BMP mRNAs. This suggested that the osteogenic action of licochalcone A in C2C12 cells could be dependent on BMP signaling and/or expression. We then tested the in vivo osteogenic activity of licochalcone A in two independent animal models. Licochalcone A accelerated the rate of skeletal development in zebrafish and enhanced woven bone formation over the periosteum of mouse calvarial bones. In summary, licochalcone A induced osteoblast differentiation with ERK activation in both MC4 and C2C12 cells and it exhibited in vivo osteogenic activity in zebrafish skeletal development and mouse calvarial bone formation. The dual action of licochalcone A in stimulating bone formation and inhibiting bone resorption, as described in a previous study, might be beneficial in treating bone-related disorders.     

Heterocyclic benzazole derivatives with antimycobacterial in vitro activity

The series of 2-benzylsulfanyl derivatives of benzoxazole and benzothiazole were synthesized, evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria, and the activity expressed as the minimum inhibitory concentration (MIC) in micromol/L. The substances bearing two nitro groups (4e, 4f, 5e, 5f) or a thioamide group (4i, 4j, 5i, 5j) exhibited appreciable activity particularly against non-tuberculous strains. The most active compounds were subjected to the toxicity assay and were evaluated as moderately cytotoxic.     

In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms

Staphylococcus aureus is now amongst the most important pathogenic bacteria responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics, partly attributed to its ability to form biofilms, is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of a coral associated actinomycete (CAA) - 3 on S. aureus biofilms both in vitro and in vivo. Methanolic extracts of CAA-3 showed a reduction in in vitro biofilm formation by S. aureus ATCC 11632, methicillin resistant S. aureus ATCC 33591 and clinical isolates of S. aureus at the biofilm inhibitory concentration (BIC) of 0.1 mg ml^?1. Furthermore, confocal laser scanning microscope (CLSM) studies provide evidence of CAA-3 inhibiting intestinal colonisation of S. aureus in the nematode Caenorhabditis elegans. To conclude, this study for the first time, reports CAA as a promising source of anti-biofilm compounds, for developing novel drugs against highly resistant staphylococcal biofilms.     

In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic chalcones

The in vitro screening of 43 polysubstituted chalcones against Leishmania amazonensis axenic amastigotes, led to the evaluation of 9 of them in a macrophage-infected model with the two other most infectious Leishmania species prevalent in Peru (L. braziliensis and L. peruviana). The five most active and selective chalcones were studied in vivo, resulting on the identification of two chalcones with high reduction parasite burden percentages.     

In vitro antimycobacterial activities of Physalis angulata L.

The HIV-tuberculosis co-infection has caused an impact on tuberculosis epidemiology all over the world and the efficacies of the therapeutic schemes traditionally prescribed in the treatment of tuberculosis, such as isoniazid, rifampicin and pyrazinamide, have decreased due to the appearance of multidrug-resistant M. tuberculosis strains (MDR). This work is part of research on natural antimicrobial agents from plant extracts through bioassay-guided fractionation, by in vitro determination of the minimum inhibitory concentration (MIC) using the microdilution method with Alamar blue oxidation-reduction dye. Crude CHCl3 Physalis angulata extracts and physalin-containing fractions displayed antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium malmoense and Mycobacterium intracellulare.     

In vitro and in vivo ACTH-releasing activity of ovine CRF,sauvagine and urotensin I

The ability of three homologous peptides, ovine corticotropin-releasing factor (CRF), sauvagine and urotensin I, to release ACTH was examined in vitro and in vivo. All three peptides exhibit statistically equivalent potencies to stimulate ACTH secretion both by cultured pituitary cells and in pharmacology blocked rats. These results suggest that all three peptides evolved from a common mammalian precursor that possessed high hypophysiotropic potency.     

In vitrouptake and antimycobacterial activity of liposomal usnic acid formulation

The cellular uptake and antimycobacterial activity of usnic acid (UA) and usnic acid-loaded liposomes (UA-LIPOs) were assessed on J774 macrophages. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of UA and UA-LIPO against Mycobacterium tuberculosis were determined. Concentrations required to inhibit 50% of cell proliferation (IC₅₀) were 22.5 (±0.60) and 12.5 (±0.26) μg/ml, for UA and UA-LIPO, respectively. The MICs of UA and UA-LIPO were 6.5 and 5.8 μg/mL, respectively. The MBC of UA-LIPO was twice as low (16 μg/mL) as that of UA (32 μg/mL). An improvement in the intracellular uptake of UA-LIPO was found (21.6 × 10⁴ ± 28.3 × 10² c.p.s), in comparison with UA (9.5 × 10⁴ ± 11.4 × 10² c.p.s). In addition, UA-LIPO remains much longer inside macrophages (30 hours). All data obtained from the encapsulation of usnic acid into liposomes as a drug delivery system (DDS) indicate a strong interaction between UA-liposomes and J774 macrophages, thereby facilitating UA penetration into cells. Considering such a process as ruling the Mycobacterium-transfection by magrophages, we could state that associating UA with this DDS leads to an improvement in its antimycobacterial activity.