FACTORS OF THE ACUTE PHASE OF INFLAMMATION AS MODULATORS OF THE INTERACTION OF MAST CELLS AND FIBROBLASTS

We investigated the influence of factors of the acute phase of inflammation (purified anaphylatoxins C3a and C5a, pentraxin C-reactive protein (CRP), and aggregated. IgG (aIgG) — as an analogue of circulating immune complexes) on the adhesive interaction of human mast cells (MC) with human fibroblasts (FB). HMC-1 MC line and primary human foreskin FB were used. MC or FB were treated with each of the studied inflammatory proteins within 24 h and then the changes in their adhesive properties were examined. Cell adhesion was assessed by flow cytometry. The results showed that all studied proteins increased MC adhesiveness, whereas FB adhesiveness was increased only by anaphylatoxins. Stimulation of the adhesive properties of the MC was associated with increased expression of CD11b and ICAM-1 molecules

Apoptosis/necrosis ratio in THP-1 cell line (human monocytic leukemia cells) exposed to esters of 2,3-dihydroazete series.

Objective: The failure of many anti-cancer chemotherapeutics is due to their inability to induce apoptosis at the cellular level. Many cancers can develop pro-survival adaptations and anti-apoptotic signaling pathways, leading to drug resistance. Development of prodrugs that induce targeted necrosis is one strategy to circumvent apoptosis-resistance. However, we hold the opinion that necrosis should be the natural outcome of primary and fully developed apoptosis, in order to maximally avoid side effects with healthy tissues at the stage of preclinical and clinical studies. Recently we developed two two-step procedures for the synthesis of thermally and hydrolytically stable 2,3-dihydroazete-2,3-di-/2,2,3-tricarboxylates (2,3-dihydroazetes) [in press]. The objective of the present research was to assess their pro-apoptotic and/or pro-necrotic abilities with cell suspension culture of human monocytic leukemia cells (THP-1), along with their general cytotoxic effect. Methods: Cultivation of the THP-1 cell line was in supplemented RPMI-1640 medium. For assessment of cell viability, two DNA binding dyes - YO-PRO-1 and propidium iodide (PI) - were used (‘normal’cells, not stained; cells at early stages of apoptosis, YO-PRO-1 positive; cells at later stages, YO-PRO-1 and PI positive). Results: 2,3-Dihydroazetes tested over the range 10-5 to 2×10-4 M showed significant differences in their ability not only to induce the cell death, but also in the way they do so by inducing predominantly apoptosis or necrosis. To adequately describe and quantitatively assess their apoptotic and/or necrotic potential, on one hand, and their general cytotoxic potential, on the other hand, we took the difference between the two areas under the curves, apoptotic and necrotic, over the concentration range. Dihydroazete 2a exhibited the maximal apoptotic/necrotic difference with the THP-1 cell line (Figs. 1, 2); 2m,n,k showed a lower and non-significant difference due to higher necrotic potential at nearly the same cytotoxicity as 2a; dihydroazetes 2b,e and 8a showed a lower though significant difference due to higher necrotic potential coupled with a lower cytotoxicity; 2h had both a very low difference and a very low cytotoxicity, and dihydroazetes 8g,c exhibited a negative difference due to predominantly necrotic cell death at a moderate to high level of cytotoxicity. Figure 1. The quantitative estimates of the apoptotic/necrotic difference (AND) of the dihydroazetes 2a,b,e,h,k,m,n and 8a,c,g exhibited with the THP-1 cell line. *) Р<0.05 Figure 2. Concentration dependency (C = mole × L-1) of apoptosis and necrosis induction. Percentage of dead cells as determined by DNA binding dyes YO-PRO-1 and PI via flow cytometry after treatment of THP-1 cells for 24 h with different concentrations of 2a. Conclusion: The newly synthesized thermally stable 2,3-dihydroazetes greatly differ in their ability to induce apoptosis and/or necrosis, and could be good candidates for further studies of their anti-cancer activities

Synthesis and Characterization of Novel Succinyl Chitosan-Dexamethasone Conjugates for Potential Intravitreal Dexamethasone Delivery

The development of intravitreal glucocorticoid delivery systems is a current global challenge for the treatment of inflammatory diseases of the posterior segment of the eye. The main advantages of these systems are that they can overcome anatomical and physiological ophthalmic barriers and increase local bioavailability while prolonging and controlling drug release over several months to improve the safety and effectiveness of glucocorticoid therapy. One approach to the development of optimal delivery systems for intravitreal injections is the conjugation of low-molecular-weight drugs with natural polymers to prevent their rapid elimination and provide targeted and controlled release. This study focuses on the development of a procedure for a two-step synthesis of dexamethasone (DEX) conjugates based on the natural polysaccharide chitosan (CS). We first used carbodiimide chemistry to conjugate DEX to CS via a succinyl linker, and we then modified the obtained systems with succinic anhydride to impart a negative ζ-potential to the polymer particle surface. The resulting polysaccharide carriers had a degree of substitution with DEX moieties of 2–4%, a DEX content of 50–85 μg/mg, and a degree of succinylation of 64–68%. The size of the obtained particles was 400–1100 nm, and the ζ-potential was −30 to −33 mV. In vitro release studies at pH 7.4 showed slow hydrolysis of the amide and ester bonds in the synthesized systems, with a total release of 8–10% for both DEX and succinyl dexamethasone (SucDEX) after 1 month. The developed conjugates showed a significant anti-inflammatory effect in TNFα-induced and LPS-induced inflammation models, suppressing CD54 expression in THP-1 cells by 2- and 4-fold, respectively. Thus, these novel succinyl chitosan-dexamethasone (SucCS-DEX) conjugates are promising ophthalmic carriers for intravitreal delivery

Succinyl Chitosan-Colistin Conjugates as Promising Drug Delivery Systems

The growth of microbial multidrug resistance is a problem in modern clinical medicine. Chemical modification of active pharmaceutical ingredients is an attractive strategy to improve their biopharmaceutical properties by increasing bioavailability and reducing drug toxicity. Conjugation of antimicrobial drugs with natural polysaccharides provides high efficiency of these systems due to targeted delivery, controlled drug release and reduced toxicity. This paper reports a two-step synthesis of colistin conjugates (CT) with succinyl chitosan (SucCS); first, we modified chitosan with succinyl anhydride to introduce a carboxyl function into the polymer molecule, which was then used for chemical grafting with amino groups of the peptide antibiotic CT using carbodiimide chemistry. The resulting polymeric delivery systems had a degree of substitution (DS) by CT of 3–8%, with conjugation efficiencies ranging from 54 to 100% and CT contents ranging from 130–318 μg/mg. The size of the obtained particles was 100–200 nm, and the ζ-potential varied from −22 to −28 mV. In vitro release studies at pH 7.4 demonstrated ultra-slow hydrolysis of amide bonds, with a CT release of 0.1–0.5% after 12 h; at pH 5.2, the hydrolysis rate slightly increased; however, it remained extremely low (1.5% of CT was released after 12 h). The antimicrobial activity of the conjugates depended on the DS. At DS 8%, the minimum inhibitory concentration (MIC) of the conjugate was equal to the MIC of native CT (1 µg/mL); at DS of 3 and 5%, the MIC increased 8-fold. In addition, the developed systems reduced CT nephrotoxicity by 20–60%; they also demonstrated the ability to reduce bacterial lipopolysaccharide-induced inflammation in vitro. Thus, these promising CT-SucCS conjugates are prospective for developing safe and effective nanoantibiotics

Succinyl Chitosan-Colistin Conjugates as Promising Drug Delivery Systems

The growth of microbial multidrug resistance is a problem in modern clinical medicine. Chemical modification of active pharmaceutical ingredients is an attractive strategy to improve their biopharmaceutical properties by increasing bioavailability and reducing drug toxicity. Conjugation of antimicrobial drugs with natural polysaccharides provides high efficiency of these systems due to targeted delivery, controlled drug release and reduced toxicity. This paper reports a two-step synthesis of colistin conjugates (CT) with succinyl chitosan (SucCS); first, we modified chitosan with succinyl anhydride to introduce a carboxyl function into the polymer molecule, which was then used for chemical grafting with amino groups of the peptide antibiotic CT using carbodiimide chemistry. The resulting polymeric delivery systems had a degree of substitution (DS) by CT of 3&ndash;8%, with conjugation efficiencies ranging from 54 to 100% and CT contents ranging from 130&ndash;318 &mu;g/mg. The size of the obtained particles was 100&ndash;200 nm, and the &zeta;-potential varied from &minus;22 to &minus;28 mV. In vitro release studies at pH 7.4 demonstrated ultra-slow hydrolysis of amide bonds, with a CT release of 0.1&ndash;0.5% after 12 h; at pH 5.2, the hydrolysis rate slightly increased; however, it remained extremely low (1.5% of CT was released after 12 h). The antimicrobial activity of the conjugates depended on the DS. At DS 8%, the minimum inhibitory concentration (MIC) of the conjugate was equal to the MIC of native CT (1 &micro;g/mL); at DS of 3 and 5%, the MIC increased 8-fold. In addition, the developed systems reduced CT nephrotoxicity by 20&ndash;60%; they also demonstrated the ability to reduce bacterial lipopolysaccharide-induced inflammation in vitro. Thus, these promising CT-SucCS conjugates are prospective for developing safe and effective nanoantibiotics

Combined Use of Antimicrobial Peptides with Antiseptics against Multidrug-Resistant Bacteria: Pros and Cons

Antimicrobial peptides (AMPs) are acknowledged as a promising template for designing new antimicrobials. At the same time, existing toxicity issues and limitations in their pharmacokinetics make topical application one of the less complicated routes to put AMPs-based therapeutics into actual medical practice. Antiseptics are one of the common components for topical treatment potent against antibiotic-resistant pathogens but often with toxicity limitations of their own. Thus, the interaction of AMPs and antiseptics is an interesting topic that is also less explored than combined action of AMPs and antibiotics. Herein, we analyzed antibacterial, antibiofilm, and cytotoxic activity of combinations of both membranolytic and non-membranolytic AMPs with a number of antiseptic agents. Fractional concentration indices were used as a measure of possible effective concentration reduction achievable due to combined application. Cases of both synergistic and antagonistic interaction with certain antiseptics and surfactants were identified, and trends in the occurrence of these types of interaction were discussed. The data may be of use for AMP-based drug development and suggest that the topic requires further attention for successfully integrating AMPs-based products in the context of complex treatment. AMP/antiseptic combinations show promise for creating topical formulations with improved activity, lowered toxicity, and, presumably, decreased chances of inducing bacterial resistance. However, careful assessment is required to avoid AMP neutralization by certain antiseptic classes in either complex drug design or AMP application alongside other therapeutics/care products