Assessment of Macrolide Transport Using PAMPA, Caco-2 and MDCKII-hMDR1 Assays

The transport of commercially available macrolide antibiotics erythromycin, clarithromycin, roxithromycin, azithromycin, and telithromycin was studied by PAMPA, a model of passive diffusion, and by two models of active transport, Caco-2 and MDCKII-hMDR1. An involvement of efflux pump P-glycoprotein (P-gp) in macrolide transport was also examined. Generally, in Caco-2 and MDCKII-hMDR1 assays without P-gp inhibition, a significantly lower apparent permeability (Papp) in apical-to-basolateral (AB) than basolateral-to-apical (BA) direction was observed [efflux ratio, Pappratio(BA/AB) > 4.2]. Upon P-gp inhibition, Papp(AB) was increased and Papp(BA) was decreased. The observed Papp values, their efflux ratios, as well as a significant decrease in efflux ratios caused by P-gp inhibition (> 50 %) qualify all tested macrolides as P-gp substrates. Results obtained with PAMPA suggest that this assay is not a method of choice for screening of macrolide permeability due to the lack of P-gp efflux pump and/or other transporters potentially involved in macrolide transport

Polyphenol bioavailability and modulatory potential on brain antioxidative markers in C57BL/6 mouse

Background and purpose: Prunus spinose L. is a traditionally consumed, recently scientifically reexamined plant. Brain bioavailability and functionality of polyphenols (PPH) of blackthorn flower extract (PSE) was investigated.Materials and methods: C57BL/6 mice received oral daily repeated doses of 25 mg/kg body weight of total PSE polyphenols for 28 days. Brain concentrations of individual polyphenols from PSE were determined by UPLC/MS on 1st,7th, 14th, 21st, and 28th day. Brain antioxidative defense markers were examined as indicators of functionality after bioaccumulation.Results: A total of 68.7% PPH present in PSE were detected in the brain. Higher (p≤0.05) Cmax/AUClast in the PSE treatment vs. control group was recorded for 59.1% of brain detected compounds, indicating relatively good bioaccumulation in the brain. The highest present compounds in PSE were not necessarily the ones mostly bioapsorbed in the brain. Kaempherols were not significantly distributed, opposite to phenolic acids, quercetins or epicgaloatechin-3-gallate. The compounds with the highest concentrations on 28th day were 4-p-coumaroylquinonic acid, (-)-epicatechin, quercetin-3-O-rutinoside, quercetin–rhamnoside, kaempherol-3-rutinoside and quercetin-3-gucoside. Brain lipid peroxidation (MDA) decreased (p<0.05) on the 21st and 28th day in the PSE group. Increase (p<0.05) in GSH concentration was observed on the 21st and 28th and SOD activity on the 28th day. Catalase activity was unchanged. It could be hypothesized that highest PPH concentration-ratios, caused reduction of lipid peroxidation by radical scavenging and simultaneous induction of glutathione and SOD pathways.Conclusions: Screened compounds could be candidates for examining or creation of brain targeted “neuro-nutriceuticals” polyphenol mixtures

Isolation of MDCK cells with low expression of mdr1 gene and their use inmembrane 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

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

Macrolactonolides: a novel class of anti-inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described

Synthesis, structure-activity relationship, and antimalarial activity of ureas and thioureas of 15-membered azalides

Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class. Ureas and thioureas of 15-membered azalides, N''-substituted 9a-(N'-carbamoyl-β-aminoethyl), 9a-(N'-thiocarbamoyl-β-aminoethyl), 9a-[N'-(β-cyanoethyl)-N'-(carbamoyl-β-aminoethyl)], 9a-[N'-(β-cyanoethyl)-N'-(thiocarbamoyl-β-aminoethyl)], 9a-{N'-[β-(ethoxycarbonyl)ethyl]-N'(carbamoyl-β-aminoethyl)}, and 9a-[N'-(β-amidoethyl)-N'-(carbamoyl-β-aminoethyl)] of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin), particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a-position. The improved in vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of molecules represents a good starting platform for the design of novel antimalarial azalides

Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis