Allylbenzenes (apiol, dillapiol, myristicin and allyltetramethoxybenzene) are individual components of plant essential oils that demonstrate antitumor activity and can enhance the antitumor activity of cytotoxic drugs, such as paclitaxel, doxorubicin, cisplatin, etc. Triphenylphosphine (PPh3) derivatives of allylbenzenes are two to three orders of magnitude more potent than original allylbenzenes in terms of IC50. The inhibition of efflux pumps has been reported for allylbenzenes, and the PPh3 moiety is deemed to be responsible for preferential mitochondrial accumulation and the depolarization of mitochondrial membranes. However, due to poor solubility, the practical use of these substances has never been an option. Here, we show that this problem can be solved by using a complex formation with cyclodextrin (CD-based molecular containers) and polyanionic heparin, stabilizing the positive charge of the PPh3 cation. Such containers can solubilize both allylbenzenes and their PPh3 derivatives up to 0.4 mM concentration. Furthermore, we have observed that solubilized PPh3 derivatives indeed work as adjuvants, increasing the antitumor activity of paclitaxel against adenocarcinomic human alveolar basal epithelial cells (A549) by an order of magnitude (in terms of IC50) in addition to being quite powerful cytostatics themselves (IC50 in the range 1-10 µM). Even more importantly, CD-solubilized PPh3 derivatives show pronounced selectivity, being highly toxic for the A549 tumor cell line and minimally toxic for HEK293T non-tumor cells, red blood cells and sea urchin embryos. Indeed, in many cancers, the mitochondrial membrane is more prone to depolarization compared to normal cells, which probably explains the observed selectivity of our compounds, since PPh3 derivatives are known to act as mitochondria-targeting agents. According to the MTT test, 100 µM solution of PPh3 derivatives of allylbenzenes causes the death of up to 85% of A549 cancer cells, while for HEK293T non-cancer cells, only 15-20% of the cells died. The hemolytic index of the studied substances did not exceed 1%, and the thrombogenicity index was < 1.5%. Thus, this study outlines the experimental foundation for developing combined cytostatic medications, where effectiveness and selectivity are achieved through decreased concentration of the primary ingredient and the inclusion of adjuvants, which are safe or practically harmless substances.
The drug resistance of pathogenic bacteria is often due efflux pumps-specific proteins that remove foreign compounds from bacterial cells. To overcome drug resistance, adjuvants are often used that can inhibit efflux pumps or other systems that ensure the resistance of bacteria to the action of antibiotics. We assumed that a new level of effectiveness with the use of an antibiotic + an adjuvant pair could be achieved by their joint delivery into the pathogen. To test this hypothesis, we constructed a series of molecular carriers based on poly-(olygo-, dendry)mers based on cyclodextrin-grafted PEI or mannan, as well as glycol chitosan, covalently bound to antibiotic, adjuvant, and the oligosaccharide ligand to the macrophage mannose receptor (CD206), which we studied earlier and showed high efficiency and selectivity of delivery of a therapeutic "cargo" to macrophages. Moxifloxacin was used as an antibiotic, and terpenoid and allylbenzene compounds were used as adjuvants, for which we previously discovered the ability to inhibit bacterial efflux pumps. We show that: (a) the resulting structures were stable in vitro for a long time (up to 10 days); (b) they were adsorbed on bacterial cells, providing a local increase in the concentration of the antibiotic and adjuvant in pathogen cells; (c) they were internalized by bacterial cells, ensuring the accumulation of both antibiotic and adjuvant inside bacterial cells; (d) the adjuvant, after entering the bacterial cell, provided inhibition of the efflux pumps; (e) due to this action of the adjuvant, combined with the targeted delivery by the carrier, the antibiotic's half-life in rats increased by more than 2 times, the effective concentration of the drug in the blood plasma (AUC) increased up to 8-10 times; (f) a significant increase in the effectiveness of the antibacterial action against Gram+ and Gram- cells was achieved (up to 3 times). Potentially, such an approach would significantly increase the effectiveness of therapies for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing bacterial resistance. Moreover, the use of a polymer carrier with covalently bound organic molecules of different structures will avoid problems linked to different (suboptimal) solubility and bio-distribution of the administered molecules, which would be almost inevitable when using the same compounds separately. It would be very difficult to find antibiotic/adjuvant pairs that simultaneously achieve optimal concentrations in the same target cells. In our case, terpenoids and alkylbenzenes used as adjuvants are practically insoluble as individual compounds, and their unacceptable pharmacological properties would not allow them to be used as efflux pump inhibitors.
Combretastatin derivatives is a promising class of antitumor agents, tubulin assembly inhibitors. However, due to poor solubility and insufficient selectivity to tumor cells, we believe, their therapeutic potential has not been fully realized yet. This paper describes polymeric micelles based on chitosan (a polycation that causes pH and thermosensitivity of micelles) and fatty acids (stearic, lipoic, oleic and mercaptoundecanoic), which were used as a carrier for a range of combretastatin derivatives and reference organic compounds, demonstrating otherwise impossible delivery to tumor cells, at the same time substantially reduced penetration into normal cells. Polymers containing sulfur atoms in hydrophobic tails form micelles with a zeta potential of about 30 mV, which increases to 40-45 mV when cytostatics are loaded. Polymers with tails of oleic and stearic acids form poorly charged micelles. The use of polymeric 400 nm micelles provides the dissolution of hydrophobic potential drug molecules. Micelles could significantly increase the selectivity of cytostatics against tumors, which has been shown using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Fourier transform infrared (FTIR) spectroscopy, flow cytometry and fluorescence microscopy. Atomic force microscopy presented the difference between the unloaded micelles and those loaded with the drug: the size of the former was 30 nm on average, while the latter had a "disc-like" shape and a size of about 450 nm. The loading of drugs into the core of micelles was confirmed by UV and fluorescence spectroscopy methods; shifts of absorption and emission maxima into the long-wavelength region by tens of nm was observed. With FTIR spectroscopy, a high interaction efficiency of micelles with the drug on cells was demonstrated, but at the same time, selective absorption was observed: micellar cytostatics penetrate into A549 cancer cells 1.5-2 times better than the simple form of the drugs. Moreover, in normal HEK293T, the penetration of the drug is reduced. The proposed mechanism for reducing the accumulation of drugs in normal cells is the adsorption of micelles on the cell surface and the preservation of cytostatics to penetrate inside the cells. At the same time, in cancer cells, due to the structural features of the micelles, they penetrate inside, merging with the membrane and releasing the drug by pH- and glutathione-sensitive mechanisms. From a methodological point of view, we have proposed a powerful approach to the observation of micelles using a flow cytometer, which, in addition, allows us to quantify the cells that have absorbed/adsorbed cytostatic fluorophore and distinguish between specific and non-specific binding. Thus, we present polymeric micelles as drug delivery systems in tumors using the example of combretastatin derivatives and model fluorophore-cytostatic rhodamine 6G.
Inhibition of biosynthetic pathways of compounds essential for Trypanosoma cruzi is considered as one of the possible action mechanisms of drugs against Chagas disease. Here, we investigated the inhibition of galactonolactone oxidase from T. cruzi (TcGAL), which catalyzes the final step in the synthesis of vitamin C, an antioxidant that T. cruzi is unable to assimilate from outside and must synthesize itself, and identified allylbenzenes from plant sources as a new class of TcGAL inhibitors. Natural APABs (apiol, dillapiol, etc.) inhibited TcGAL with IC50 = 20-130 µM. The non-competitive mechanism of TcGAL inhibition by apiol was established. Conjugation of APABs with triphenylphosphonium, which ensures selective delivery of biologically active substances to the mitochondria, increased the efficiency and/or the maximum percentage of TcGAL inhibition compared to nonmodified APABs.
Chagas Disease , Trypanosoma cruzi , Humans , Trypanosoma cruzi/metabolism , Oxidoreductases/metabolism , Sugar Acids/metabolism
Allylpolyalkoxybenzenes (APABs) and terpenoids from plant essential oils exhibit a range of remarkable biological effects, including analgesic, antibacterial, anti-inflammatory, antioxidant, and others. Synergistic activity with antibiotics of different classes has been reported, with inhibition of P-glycoprotein and impairment of bacterial cell membrane claimed as probable mechanisms. Clearly, a more detailed understanding of APABs' biological activity could help in the development of improved therapeutic options for a range of diseases. However, APABs' poor solubility in water solutions has been a limiting factor for such research. Here, we found that complex formation with ß-cyclodextrins (CD) is an efficient way to transform the APABs into a water-soluble form. Using a combination of spectroscopic (FTIR, NMR, UV) methods, we have estimated the binding constants, loading capacity, and the functional groups of both APABs and monoterpenes involved in complex formation with CD: ethylene, aromatic, methoxy and hydroxy groups. In the presence of a molar excess of CD (up to 5 fold) it was possible to achieve the complete dissolution of APABs and terpenoids in an aqueous medium (at 90-98% encapsulation) higher by 10-1000 times. Further, we have demonstrated that CD-APABs, if used in combination with levofloxacin (Lev), can be antagonistic, indifferent, additive, or synergistic, mostly depending on the concentration ratio: at high Lev concentration with the addition of APAB is typically neutral or even antagonistic; while at a Lev concentration below MIC, the addition of CD-APAB is either additive or synergistic (according to FICI criteria). An over three-fold increase in Lev antibacterial activity was observed in combination with eugenol (EG), as per the growth inhibition diameter measurement in agar. Interestingly, a synergistic effect could be observed with both Gram-positive and Gram-negative bacteria. So, obviously, the APAB-CD and terpenoid-CD mechanism of action is not limited to their interaction with the bacterial membrane, which has been shown earlier for CDs. Further research may open new prospects for the development of adjuvants to improve the therapeutic regimens with existing, as well as with new anti-infective drugs.
The molecular and crystal structure of the widely used antiseptic benzyldimethyl{3-[(1-oxotetradecyl)amino]propyl}ammonium chloride monohydrate (Miramistin, MR), C26H47N2O+·Cl-·H2O, was determined by a single-crystal X-ray diffraction study and analyzed in the framework of the QTAIM (quantum theory of atoms in molecules) approach using both periodic and molecular DFT (density functional theory) calculations. The various noncovalent intermolecular interactions of different strengths were found to be realized in the hydrophilic parts of the crystal packing (i.e. O-H...Cl, N-H...Cl, C-H...Cl, C-H...O and C-H...π). The hydrophobic parts are built up exclusively by van der Waals H...H contacts. Quantification of the interaction energies using calculated electron-density distribution revealed that the total energy of the contacts within the hydrophilic and hydrophobic regions are comparable in value. The organic MR cation adopts the bent conformation with the head group tilted back to the long-chain alkyl tail in both the crystalline and the isolated state due to stabilization of this geometry by several intramolecular C-H...π, C-H...N and H...H interactions. This conformation preference is hypothesized to play an important role in the interaction of MR with biomembranes.
