Unprecedented Epimerization of an Azithromycin Analogue: Synthesis, Structure and Biological Activity of 2'-Dehydroxy-5″-Epi-Azithromycin.

Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton-McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities.

Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening.

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Design, synthesis, antitrypanosomal activity, DNA/RNA binding and in vitro ADME profiling of novel imidazoline-substituted 2-arylbenzimidazoles.

Novel imidazoline benzimidazole derivatives containing diversely substituted phenoxy moieties were synthesized with the aim of evaluating their antitrypanosomal activity, DNA/RNA binding affinity and in vitro ADME properties. The presence of the diethylaminoethyl subunit in 18a-18c led to enhanced antitrypanosomal potency, particularly for 18a and 18c, which contain unsubstituted and methoxy-substituted phenoxy moieties. They were found to be > 2-fold more potent against African trypanosomes than nifurtimox. Fluorescence and CD spectroscopy, thermal denaturation assays and computational analysis indicated a preference of 18a-18c toward AT-rich DNA and their minor groove binding mode. Replacement of the amidine group with less basic and ionisable nitrogen-containing moieties failed to improve membrane permeability of the investigated compounds. Due to structural diversification, the compounds displayed a range of physico-chemical features resulting in variable in vitro ADME properties, leaving space for further optimization of the biological profiles.

Rational design, synthesis and biological profiling of new KDM4C inhibitors.

The human histone demethylases of the KDM4 family have been related to diseases such as prostate and breast cancer. Majority of currently known inhibitors suffer from the low permeability and low selectivity between the enzyme isoforms. In this study, toxoflavin motif was used to design and synthesize new KDM4C inhibitors with improved biological activity and in vitro ADME properties. Inhibitors displayed good passive cellular permeability and metabolic stability. However, diminishing of redox liability and consequently non-specific influence on cell viability still remains a challenge.

Lipophilicity and bio-mimetic properties determination of phytoestrogens using ultra-high-performance liquid chromatography.

This paper presents lipophilicity and bio-mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein-4,7-dimethylether, prunetin, 3,4,7-trihydroxyisoflavon, 4,6,7-trihydroxyisoflavon, 4,6,7-trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High-performance liquid chromatography with fast gradient elution and Caco-2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl-sylane stationary phase using pH 2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α-1-acid-glycoprotein (AGP) stationary phases. Caco-2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco-2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco-2 permeability.

A comparison of in vitro ADME properties and pharmacokinetics of azithromycin and selected 15-membered ring macrolides in rodents.

The purpose of this study was to evaluate the impact of structural modifications on the 15-membered macrolactone ring and/or substituents on the in vitro ADME properties and in vivo pharmacokinetic (PK) profile for selected derivatives in rodents in comparison to azithromycin. Azithromycin and seven selected 15-membered macrolide derivatives, modified either by removal of the sugar moieties, replacement of the amine with a lactam, or addition of lipophilic substituents, were screened in several in vitro ADME assays and in vivo PK studies in rodents. In vitro ADME profiling included assessment of passive permeability and P-gp substrate, metabolic stability in liver microsomes and hepatocytes, as well as CYP direct inhibition measurements. In vivo PK studies were performed in rats (Sprague-Dawley), mice (Balb/c), and P-gp wild-type and deficient mice (CF-1™). Different structural modifications on the azithromycin scaffold resulted in substantial changes in disposition kinetics and oral bioavailability in both rodent species. However, these differences in vivo cannot be predicted based on in vitro results since most of these molecules are classified in the same category. Therefore, in the case of 15-membered ring macrolides, the in vitro ADME screens presented here seem to have low predictive value for in vivo prediction, making their use as routine in vitro screens prior to PK assessments questionable.

The effect of apigenin on cyclophosphamide and doxorubicin genotoxicity in vitro and in vivo.

The aim of this study was to investigate the mutagenic and antigenotoxic effects of different doses of the flavonoid, apigenin, alone and in combination with the antitumor drugs, cyclophosphamide and doxorubicin, in vitro and in vivo. Using bacterial reverse mutation inhibition in vitro, with and without metabolic activation, the effect of apigenin (10-400 µg/plate) was studied on genotoxicity induced by cyclophosphamide (800 µg/plate) and by doxorubicin (0.2 µg/plate). Subsequent to a dose-finding study in vivo, CD1 mice were treated with either cyclophosphamide (40 mg/kg, i.p.) or doxorubicin (5 mg/kg, i.p.) with or without co-administration of apigenin (1-100 mg/kg, p.o.). Micronuclei were determined microscopically in blood smears and glutathione peroxidase (GPX), superoxide dismutase (SOD) and total antioxidative status (TAS) in whole blood, erythrocytes and plasma, respectively. Apigenin decreased doxorubicin-induced, but not cyclophosphamide-induced mutagenicity in vitro. In vivo, apigenin caused a statistically significant decrease in micronucleus frequency in response to cyclophosphamide, possibly due to active flavonoid metabolite formation or inhibition of cyclophosphamide metabolic activation. In animals treated with apigenin and doxorubicin, a significant decrease in micronucleus frequency was not observed, probably due to interindividual variability. No changes in GPX, SOD or TAS were observed in response to either cytotoxic agents or the flavonoid, possibly due to limited metabolic transformation of the drugs at the doses used. The results of the present study provide further evidence for the chemo-preventative properties of apigenin.

Enhancement by PL 14736 of granulation and collagen organization in healing wounds and the potential role of egr-1 expression.

Apart from becaplermin (recombinant human platelet-derived growth factor homodimer of B chains, PDGF-BB), for the treatment of lower extremity diabetic ulcers, few agents are available for pharmacological stimulation of wound healing. We have compared the mechanism of action of the potential wound healing agent, PL 14736 (G E P P P G K P A D D A G L V), with that of PDGF-BB on granulation tissue formation following sponge implantation in the normoglycemic rat and in healing full-thickness excisional wounds in db/db genetically diabetic mice. Expression of the immediate response gene, early growth response gene-1 (egr-1) was studied in Caco-2 cells in vitro. While PDGF-BB and PL 14736 had similar selectivity for stimulation of granulation tissue in both sponge granuloma and in healing wounds in db/db mice, PL 14736 was more active in stimulating early collagen organization. It also stimulated expression of egr-1 and its repressor nerve growth factor 1-A binding protein-2 (nab2) in non-differentiated Caco-2 cells more rapidly than PDGF-BB. EGR-1 induces cytokine and growth factor generation and early extracellular matrix (collagen) formation, offering an explanation for the beneficial effects of PL 14736 on wound healing.