Azithromycin-liposomes as a novel approach for localized therapy of cervicovaginal bacterial infections.

BACKGROUND: Efficient localized cervicovaginal antibacterial therapy, enabling the delivery of antibiotic to the site of action at lower doses while escaping systemic drug effects and reducing the risk of developing microbial resistance, is attracting considerable attention. Liposomes have been shown to allow sustained drug release into vaginal mucosa and improve delivery of antibiotics to bacterial cells and biofilms. Azithromycin (AZI), a potent broad-spectrum macrolide antibiotic, has not yet been investigated for localized therapy of cervicovaginal infections, although it is administered orally for the treatment of sexually transmitted diseases. Encapsulation of AZI in liposomes could improve its solubility, antibacterial activity, and allow the prolonged drug release in the cervicovaginal tissue, while avoiding systemic side effects. PURPOSE: The objective of this study was to develop AZI-liposomes and explore their potentials for treating cervicovaginal infections. METHODS: AZI-liposomes that differed in bilayer elasticity/rigidity and surface charge were prepared and evaluated under simulated cervicovaginal conditions to yield optimized liposomes, which were assessed for antibacterial activity against several planktonic and biofilm-forming Escherichia coli strains and intracellular Chlamydia trachomatis, ex vivo AZI vaginal deposition/penetration, and in vitro cytotoxicity toward cervical cells. RESULTS: Negatively charged liposomes with rigid bilayers (CL-3), propylene glycol liposomes (PGL-2) and deformable propylene glycol liposomes (DPGL-2) were efficient against planktonic E. coli ATCC 700928 and K-12. CL-3 was superior for preventing the formation of E. coli ATCC 700928 and K-12 biofilms, with IC50 values (concentrations that inhibit biofilm viability by 50%) up to 8-fold lower than those of the control (free AZI). DPGL-2 was the most promising for eradication of already formed E. coli biofilms and for treating C. trachomatis infections. All AZI-liposomes were biocompatible with cervical cells and improved localization of the drug inside vaginal tissue compared with the control. CONCLUSION: The performed studies confirm the potentials of AZI-liposomes for localized cervicovaginal therapy.

Selective photocytotoxicity of anthrols on cancer stem-like cells: The effect of quinone methides or reactive oxygen species.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that share properties of embryonic stem cells like pluripotency and self-renewal and show increased resistance to chemo- and radiotherapy. Targeting CSC, rather than cancer cells in general, is a novel and promising strategy for cancer treatment. Novel therapeutic approaches, such as photodynamic therapy (PDT) have been investigated. A promising group of phototherapeutic agents are reactive intermediates - quinone methides (QMs). This study describes preparation of QM precursor, 2-hydroxy-3-hydroxymethylanthracene (2) and a detailed photochemical and photobiological investigation on similar anthracene derivatives 3 and 4. Upon photoexcitation with near visible light at λ > 400 nm 1 and 2 give QMs, that were detected by laser flash photolysis and their reactivity with nucleophiles has been demonstrated in the preparative irradiation experiments where the corresponding adducts were isolated and characterized. 3 and 4 cannot undergo photodehydration and deliver QM, but lead to the formation of phenoxyl radical and singlet oxygen, respectively. The activity of 1-4 was tested on a panel of human tumor cell lines, while special attention was devoted to demonstrate their potential selectivity towards the cells with CSC-like properties (HMLEshEcad). Upon the irradiation of cell lines treated with 1-4, an enhancement of antiproliferative activity was demonstrated, but the DNA was not the target molecule. Confocal microscopy revealed that these compounds entered the cell and, upon irradiation, reacted with cellular membranes. Our experiments demonstrated moderate selectivity of 2 and 4 towards CSC-like cells, while necrosis was shown to be a dominant cell death mechanism. Especially interesting was the selectivity of 4 that produced higher levels of ROS in CSC-like cells, which forms the basis for further research on cancer phototherapy, as well as for the elucidation of the underlying mechanism of the observed CSC selectivity based on oxidative stress activation.

Novel 1-acyl-4-substituted semicarbazide derivatives of primaquine - synthesis, cytostatic, antiviral and antioxidative studies.

A series of novel 1,4-substituted semicarbazides 5a-g with a primaquine moiety bridged by a carbonyl group at position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically evaluated. The synthetic pathways applied for preparation of the title compounds involved benzotriazole as synthetic auxiliary. Primaquine semicarbazides 5a-g and their synthetic precursors benzotriazolecarbonyl semicarbazides 4 were evaluated for cytostatic, antiviral and antioxidative activities. All compounds of the series 5 showed high selectivity towards MCF-7 cells (breast carcinoma) with IC(50) values in the low micromolar range and the most active was benzyl derivative 5c (IC(50) 1 ± 0.2 µM). The benzhydryl derivative 5e showed significant cytostatic activities towards all the tested cell lines (IC(50) 4-18 µM). The same compound was the strongest lipoxygenase inhibitor as well (51%). The highest antioxidant activity was demonstrated for the hydroxy derivative 5g and benzotriazolecarbonyl semicarbazides 4b,c (61.2-68.5%). No antiviral activity was observed against a wide variety of DNA and RNA viruses.

Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

PURPOSE: p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. METHODS: p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. RESULTS: In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. CONCLUSIONS: This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.

The phytohormone auxin induces G1 cell-cycle arrest of human tumor cells.

The plant hormone auxin is the key regulator of plant growth and development. Auxin regulates transcription of plant genes by targeting degradation of transcriptional repressor proteins Aux/IAA. While there are many reports describing its potential to modulate human cell functions, the majority are based on auxin action following enzymatic activation. A study focused on auxin alone and its antiproliferative potential, with emphasis on modulation of the cell cycle, has not been performed. Therefore, we analyzed tumor growth inhibitory effects and the cell-cycle perturbations of natural (IAA, IBA) and synthetic (NAA, 2,4-D) auxins. All derivatives showed cytostatic effects on selected human tumor cell lines. The cell-cycle analysis revealed that IAA and 2,4-D induce strong G1 arrest, along with a drastic decrease in the percentage of S-phase cells in MCF-7 cell line. This phenomenon demonstrates that auxins may have novel, unexploited antitumor potential and should be further investigated.

Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis.

We have previously shown that N-1-sulfonylpyrimidine derivatives have strong antiproliferative activity on human tumor cell lines, whereby 1-(p-toluenesulfonyl)cytosine showed good selectivity with regard to normal cells and was easily synthesized on a large scale. In the present work we have used an interdisciplinary approach to elucidate the compounds' mechanistic class. An augmented number of cell lines (11) has allowed a computational search for compounds with similar activity profiles and/or mechanistic class by integrating our data with the comprehensive DTP-NCI database. We applied supervised machine learning methodology (Random Forest classifier), which offers information complementary to unsupervised algorithms commonly used for analysis of cytostatic activity profiles, such as self-organizing maps. The computational results taken together with cell cycle perturbation and apoptosis analysis of the cell lines point to an unusual mechanism of cytostatic action, possibly a combination of nucleic acid antimetabolite activity and a novel molecular mechanism.

The novel C-5 aryl, alkenyl, and alkynyl substituted uracil derivatives of L-ascorbic acid: synthesis, cytostatic, and antiviral activity evaluations.

The novel C-5 substituted uracil derivatives of l-ascorbic acid were synthesized by coupling of 5-iodouracil-4,5-didehydro-5,6-dideoxy-l-ascorbic acid with unsaturated stannanes under Stille reaction conditions. The new compounds were evaluated for their antitumoral and antiviral activities. Among all compounds evaluated the 5-propynyl substituted uracil derivative of l-ascorbic acid (7) exhibited the most pronounced cytostatic activities against all examined tumor cell lines (IC(50): 0.2-0.78 microM). However, this compound was also cytotoxic to human normal fibroblasts WI 38. The 5-(phenylethynyl)uracil-2,3-di-O-benzylated l-ascorbic acid derivative (4) exhibited an albeit slight (IC(50): 55-108 microM), but selective inhibitory effect toward all tumor cell lines except for cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), laryngeal carcinoma (Hep-2), and colon carcinoma (SW 620), and no cytotoxicity to normal human fibroblast (WI 38). Compound 7 showed some, not highly specific, inhibitory potential against vesicular stomatitis virus, Coxsackie B4 virus, and Sindbis viruses (EC(50): 1.6 microM).

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation.

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).