Design, Synthesis, and Evaluation of Biological Activity of Ferrocene-Ispinesib Hybrids: Impact of a Ferrocenyl Group on the Antiproliferative and Kinesin Spindle Protein Inhibitory Activity.

With the aim to combine more than one biologically-active component in a single molecule, derivatives of ispinesib and its (S) analogue were prepared that featured ferrocenyl moieties or bulky organic substituents. Inspired by the strong kinesin spindle protein (KSP) inhibitory activity of ispinesib, the compounds were investigated for their antiproliferative activity. Among these compounds, several derivatives demonstrated significantly higher antiproliferative activity than ispinesib with nanomolar IC50 values against cell lines. Further evaluation indicated that the antiproliferative activity is not directly correlated with their KSP inhibitory activity while docking suggested that several of the derivatives may bind in a manner similar to ispinesib. In order to investigate the mode of action further, cell cycle analysis and reactive oxygen species formation were investigated. The improved antiproliferative activity of the most active compounds may be assigned to synergic effects of various factors such as KSP inhibitory activity due to the ispinesib core and ability to generate ROS and induce mitotic arrest.

Synthesis and Biological Activity of Ferrocenyl and Ruthenocenyl Analogues of Etoposide: Discovery of a Novel Dual Inhibitor of Topoisomerase II Activity and Tubulin Polymerization.

Two series of the ferrocenyl and ruthenocenyl analogues of etoposide bearing 1,2,3-triazolyl or aminoalkyl linker were synthesized and evaluated for their cytotoxic properties, influence on the cell cycle, ability to induce tubulin polymerization, and inhibition of topoisomerase II activity. We found that the replacement of the etoposide carbohydrate moiety with a metallocenyl group led to organometallic conjugates exhibiting differentiated antiproliferative activity. Biological studies demonstrated that two ferrocenylalkylamino conjugates were notably more active than etoposide, with submicromolar or low-micromolar IC50 values towards SW620, etoposide-resistant SW620E, and methotrexate-resistant SW620M cancer cell lines. Moreover, the simplest ferrocenylmethylamino conjugate exerted dual inhibitory action against tubulin polymerization and topoisomerase II activity while other studied compounds affected only topoisomerase II activity.

Upper-airway dysbiosis related to frequent sweets consumption increases the risk of asthma in children with chronic rhinosinusitis.

BACKGROUND: Innate immunity response to local dysbiosis seems to be one of the most important immunologic backgrounds of chronic rhinosinusitis (CRS) and concomitant asthma. We aimed to assess clinical determinants of upper-airway dysbiosis and its effect on nasal inflammatory profile and asthma risk in young children with CRS. METHODS: We recruited one hundred and thirty-three children, aged 4-8 years with doctor-diagnosed CRS with or without asthma. The following procedures were performed in all participants: face-to-face standardized Sinus and Nasal Quality of Life questionnaire, skin prick test, taste perception testing, nasopharynx swab, and sampling of the nasal mucosa. Upper-airway dysbiosis was defined separately by asthma-specific microbiome composition and reduced biodiversity. Multivariate methods were used to define the risk factors for asthma and upper-airway dysbiosis and their specific inflammatory profile of nasal mucosa. RESULTS: The asthma-specific upper-airway microbiome composition reflected by the decreased ratio of Patescibacteria/Actinobacteria independently of atopy increased the risk of asthma (OR:8.32; 95%CI: 2.93-23.6). This asthma-specific microbiome composition was associated with ≥ 7/week sweet consumption (OR:2.64; 95%C:1.11-6.28), reduced biodiversity (OR:3.83; 95%CI:1.65-8.87), the presence of Staphylococcus strains in the nasopharynx (OR:4.25; 95%CI:1.12-16.1), and lower expression of beta-defensin 2, IL-5, and IL-13 in the nasal mucosa. The reduced biodiversity was associated with frequent antibiotic use and with a higher nasal expression of IL-17 and T1R3 (sweet taste receptor). In asthmatic children, reduced sweet taste perception was observed. CONCLUSIONS: Specific upper-airway dysbiosis related to frequent sweet consumption, frequent antibiotic courses, and altered nasal immune function increases the risk of asthma in young children with CRS.

Metal-Dependent Cytotoxic and Kinesin Spindle Protein Inhibitory Activity of Ru, Os, Rh, and Ir Half-Sandwich Complexes of Ispinesib-Derived Ligands.

Ispinesib is a potent inhibitor of kinesin spindle protein (KSP), which has been identified as a promising target for antimitotic anticancer drugs. Herein, we report the synthesis of half-sandwich complexes of Ru, Os, Rh, and Ir bearing the ispinesib-derived N,N-bidentate ligands (R)- and (S)-2-(1-amino-2-methylpropyl)-3-benzyl-7-chloroquinazolin-4(3H)-one and studies on their chemical and biological properties. Using the enantiomerically pure (R)- and (S)-forms of the ligand, depending on the organometallic moiety, either the SM,R or RM,S diastereomers, respectively, were observed in the molecular structures of the Ru- and Os(cym) (cym = η6-p-cymene) compounds, whereas the RM,R or SM,S diastereomers were found for the Rh- and Ir(Cp*) (Cp* = η5-pentamethylcyclopentadienyl) derivatives. However, density functional theory (DFT) calculations suggest that the energy difference between the diastereomers is very small, and therefore a mixture of both will be present in solution. The organometallics exhibited varying antiproliferative activity in a series of human cancer cell lines, with the complexes featuring the (R)-enantiomer of the ligand being more potent than the (S)-configured counterparts. Notably, the Rh and Ir complexes demonstrated high KSP inhibitory activity, even at 1 nM concentration, which was independent of the chirality of the ligand, whereas the Ru and especially the Os derivatives were much less active.

Ferrocenyl Paclitaxel and Docetaxel Derivatives: Impact of an Organometallic Moiety on the Mode of Action of Taxanes.

A series of ferrocenyl analogues and derivatives of paclitaxel and docetaxel were synthesised and assayed for their antiproliferative/cytotoxic effects, impact on the cell cycle distribution and ability to induce tubulin polymerisation. The replacement of the 3'-N-benzoyl group of paclitaxel with a ferrocenoyl moiety, in particular, led to formation of an analogue that was at least one order of magnitude more potent in terms of antiproliferative activity than the parent compound (IC50 values of 0.11 versus 1.11 µm, respectively), but still preserved the classical taxane mode of action, that is, microtubule stabilisation leading to mitotic arrest. Molecular docking studies revealed an unexpected binding pocket in the tubulin structure for the ferrocenoyl group introduced in the paclitaxel backbone.

The clinical effect of vitamin D supplementation combined with grass-specific sublingual immunotherapy in children with allergic rhinitis.

BACKGROUND: An important issue in sublingual immunotherapy (SLIT) is how to improve efficacy. OBJECTIVE: To compare the clinical efficacy of SLIT with vitamin D supplementation with placebo in children with allergic rhinitis. Secondary end points included lung function, exhaled nitric oxide concentration, methacholine bronchial provocation test, and serum level of calcifediol (25[OH]D). METHODS: Fifty children, ages 5-12 years, sensitive to grass pollen, with allergic rhinitis (eight patients had concomitant asthma) participated in a 5-month prospective, randomized, double-blind, placebo-controlled trial. Children received a 5-grass pollen sublingual 300 IR tablet with either vitamin D 1000 IU daily supplementation or placebo. RESULTS: When compared with the placebo group, SLIT plus vitamin D group therapy was more effective in the reduction of nasal symptoms (p = 0.04), asthma symptoms (p = 0.001), and the combined symptom-medication score (p = 0.001); there was no significant difference between the groups in medication and ocular scores. We observed a significant improvement of forced expiratory volume in 1 second (vitamin D group, p = 0.014; placebo group, p = 0.015) and the proportion of a person's vital capacity expired in the first second of forced expiration levels (vitamin D group, p = 0.004; placebo group, p < 0.001), within both groups, between visits. Fractional exhaled nitric oxide and provocative dose producing a 20% fall in forced expiratory volume in 1 second results did not statistically significantly differentiate the study participants in terms of receiving SLIT along with vitamin D or placebo. We showed a significant increase in calcifediol in the SLIT plus vitamin D group as well as in SLIT plus placebo group. CONCLUSIONS: Vitamin D supplementation combined with grass-specific SLIT was more effective in the reduction of nasal and asthma symptoms. Vitamin D supplementation combined with SLIT provides an effective and well-tolerated new immunotherapy modality for treating children with allergic rhinitis. A 5-grass pollen sublingual 300 IR tablet was effective in both studied groups and also in children with comorbid mild asthma.

Effect of Lactobacillus rhamnosus GG and vitamin D supplementation on the immunologic effectiveness of grass-specific sublingual immunotherapy in children with allergy.

BACKGROUND: An important issue in sublingual immunotherapy (SLIT) is how to improve efficacy. OBJECTIVE: To compare the clinical and immunologic efficacy of SLIT given alone and, to enhance clinical efficacy, given with probiotic or vitamin D supplementation. METHODS: One hundred children, ages 5-12 years, sensitive to grass pollen, with allergic rhinitis participated in a 5-month prospective, randomized, double-blind, placebo-controlled trial. Children received 5-grass SLIT 300 IR tablets with either vitamin D 1000 IU daily supplementation, probiotic, or placebo. The control group included children with allergy who did not qualify for immunotherapy. Primary end points included a symptom-medication score, lung function, and exhaled nitric oxide concentration. The secondary end point was the immunologic efficacy measured by the following: CD4(+)CD25(+)Foxp3(+) (forkhead box P3) cells, Toll-like receptor (TLR) 4, interleukin (IL) 1, IL-6, tumor necrosis factor, IL-10, and transforming growth factor ß-1 levels in cell culture supernatants. RESULTS: Reduction in the symptom-medication score and improvement in lung function as well as a significant increase in the percentage of CD4(+)CD25(+)Foxp3(+) in children who received SLIT in all the groups were observed compared with control group. In the SLIT-probiotic group, between-group analysis showed significantly higher CD4(+)CD25(+)Foxp3(+) induction compared with the SLIT group and higher reduction in the percentage of TLR-positive cell group compared with the SLIT-vitamin D group (Fig. 1). An increase in CD4(+)CD25(+)Foxp3(+) induction, reduction in TLR-positive cells recruitment and an increase in transforming growth factor ß-1 production were independently associated with a better clinical effect of SLIT in children. CONCLUSIONS: We demonstrated the clinical and immunologic effect of probiotic and vitamin D supplementation on SLIT. Probiotic supplementation showed better clinical and immunologic response in children with allergic rhinitis.

Half-sandwich ruthenium(II) biotin conjugates as biological vectors to cancer cells.

Ruthenium(II)-arene complexes with biotin-containing ligands were prepared so that a novel drug delivery system based on tumor-specific vitamin-receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru(II) -biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012-2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half-sandwich ruthenium(II)-biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed.

The connection between the toxicity of anthracyclines and their ability to modulate the P-glycoprotein-mediated transport in A549, HepG2, and MCF-7 cells.

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of solid tumors. We compared the resistance of the most popular solid tumors, breast adenocarcinoma (MCF-7 cell line) and nonsmall cell lung (A549 cell line) hepatocellular liver carcinoma (HepG2 cells), to aclarubicin (ACL) and doxorubicin (DOX). This research aimed at determining the relation between the toxicity of ACL and DOX, their cell accumulation, and then effect on P-glycoprotein functionality. ACL is more cytotoxic for tumor cells compared to DOX. The intracellular concentration of drugs in cancer cells was dependent on the dose of the drugs and the time of incubation. The P-gp inhibitor Verapamil (V) increased DOX accumulation in all tested cell lines. By contrast, the intracellular level of ACL was not affected by this modifying agent. The assessment of the uptake of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) or Rhodamine 123 (R123) allows the evaluation of the different influence of drugs on P-gp activity which is in agreement with the estimation of expression measured by MDR-1 shift assay. These data suggest that ACL is less P-gp dependent than DOX and consequently may be used in a clinical setting to increase treatment efficacy in resistant human tumors.

Oncostatin M reverses ABCG2-mediated mitoxantrone resistance.

Mitoxantrone resistant variant of SW620 line was developed, characterized and subsequently used as a model system to determine oncostatin M ability to modulate MDR phenomenon. The selection regimen allowed for overexpression of ABCG2 and ABCB1 both at the RNA and protein level, which was further confirmed by functional assays. Oncostatin M supplementation resulted in partial reversal of MDR phenotype by decreasing overexpression of ABCG2 demonstrating for the first time the ability of this cytokine for selective down-regulation of one of MDR proteins.

Development of Half-Sandwich Ru, Os, Rh, and Ir Complexes Bearing the Pyridine-2-ylmethanimine Bidentate Ligand Derived from 7-Chloroquinazolin-4(3H)-one with Enhanced Antiproliferative Activity.

Kinesin spindle protein (KSP) inhibitors are one of the most promising anticancer agents developed in recent years. Herein, we report the synthesis of ispinesib-core pyridine derivative conjugates, which are potent KSP inhibitors, with half-sandwich complexes of ruthenium, osmium, rhodium, and iridium. Conjugation of 7-chloroquinazolin-4(3H)-one with the pyridine-2-ylmethylimine group and the organometallic moiety resulted in up to a 36-fold increased cytotoxicity with IC50 values in the micromolar and nanomolar range also toward drug-resistant cells. All studied conjugates increased the percentage of cells in the G2/M phase, simultaneously decreasing the number of cells in the G1/G0 phase, suggesting mitotic arrest. Additionally, ruthenium derivatives were able to generate reactive oxygen species (ROS); however, no significant influence of the organometallic moiety on KSP inhibition was observed, which suggests that conjugation of a KSP inhibitor with the organometallic moiety modulates its mechanism of action.

Synthesis and Biological Properties of Ferrocenyl and Organic Methotrexate Derivatives.

Synthesis and biological activity of two series of modified side chain methotrexate (MTX) derivatives are presented, one with a ferrocenyl moiety inserted between the pteroyl and glutamate portions of the molecule and the other with glutamate substituted for short chain amino acids. Ferrocenyl derivatives of MTX turned out to be rather moderate inhibitors of dihydrofolate reductase (DHFR) although molecular modeling suggested more effective interactions between these compounds and the target enzyme. More interestingly, ferrocene-decorated MTX derivatives were able to impede the proliferation of four murine and human cell lines as well as their methotrexate-resistant counterparts, overcoming the multidrug resistance (MDR) barrier. They were also able to directly interact with Abcc1, an MDR protein. Of the amino acid pteroyl conjugates, the γ-aminobutyric acid derivative was an efficient inhibitor of DHFR but had no effect on cell proliferation in the concentration range studied while a taurine conjugate was a poor DHFR inhibitor but able to affect cell viability. We postulate that modification of the methotrexate side chain may be an efficient strategy to overcome efflux-dependent methotrexate resistance.

Organometallic Ru, Os, Rh and Ir half-sandwich conjugates of ispinesib - impact of the organometallic group on the antimitotic activity.

Antimitotic agents are among the most important drugs used in anticancer therapy. Kinesin spindle protein (KSP) was proposed as a promising target for new antimitotic drugs. Herein, we report the synthesis of Ru, Os, Rh, and Ir half-sandwich complexes with the KSP inhibitor ispinesib and its (S)-enantiomer. Conjugation of the organometallic moiety with ispinesib and its (S)-enantiomer resulted in a significantly increased cytotoxicity of up to 5.6-fold compared to the parent compounds, with IC50 values in the nanomolar range. The most active derivatives were the ispinesib Ru and Rh conjugates which were able to generate reactive oxygen species (ROS), which may at least partially explain their high cytotoxicity. At the same time, the Os and Ir derivatives acted as KSP inhibitors with no effects on ROS generation.

Effect of an Intranasal Corticosteroid on Quality of Life and Local Microbiome in Young Children With Chronic Rhinosinusitis: A Randomized Clinical Trial.

Importance: Intranasal corticosteroids (INCs) remain the first-line treatment of chronic rhinosinusitis (CRS) in both adults and children, despite the lack of evidence regarding their efficacy in the pediatric population. Similarly, their effect on the sinonasal microbiome has not been well documented. Objective: To assess the clinical, immunological, and microbiological effects of 12 weeks of an INC in young children with CRS. Design, Setting, and Participants: This open-label randomized clinical trial was performed in a pediatric allergy outpatient clinic in 2017 and 2018. Children aged 4 to 8 years with CRS diagnosed by a specialist were included. Data were analyzed from January 2022 to June 2022. Interventions: Patients were randomized to receive intranasal mometasone in an atomizer for 12 weeks (1 application per nostril, once per day) and supplemental 3-mL sodium chloride (NaCl), 0.9%, solution in a nasal nebulizer once a day for 12 weeks (INC group) or 3-mL NaCl, 0.9%, solution in a nasal nebulizer once a day for 12 weeks (control group). Main Outcomes and Measures: Measures taken both before and after treatment included the Sinus and Nasal Quality of Life Survey (SN-5), a nasopharynx swab for microbiome analysis by next-generation sequencing methods, and nasal mucosa sampling for occurrence of innate lymphoid cells (ILCs). Results: Of the 66 children enrolled, 63 completed the study. The mean (SD) age of the cohort was 6.1 (1.3) years; 38 participants (60.3%) were male and 25 (39.7%) were female. The clinical improvement reflected by reduction in SN-5 score was significantly higher in the INC group compared with the control group (INC group score before and after treatment, 3.6 and 3.1, respectively; control group score before and after treatment, 3.4 and 3.8, respectively; mean between-group difference, -0.58; 95% CI, -1.31 to -0.19; P = .009). The INC group had a greater increase in nasopharyngeal microbiome richness and larger decrease in nasal ILC3 abundance compared with the control group. A significant interaction was observed between change in microbiome richness and the INC intervention on the prediction of significant clinical improvement (odds ratio, 1.09; 95% CI, 1.01-1.19; P = .03). Conclusions and Relevance: This randomized clinical trial demonstrated that treatment with an INC improved the quality of life of children with CRS and had a significant effect on increasing sinonasal biodiversity. Although further investigation is needed of the long-term efficacy and safety of INCs, these data may reinforce the recommendation of using INCs as a first-line treatment of CRS in children. Trial Registration: ClinicalTrials.gov Identifier: NCT03011632.

Impact of the ferrocenyl group on cytotoxicity and KSP inhibitory activity of ferrocenyl monastrol conjugates.

The incorporation of the ferrocenyl moiety into a bioactive molecule may significantly alter the activity of the resulting conjugate. By applying this strategy, we designed ferrocenyl analogs of monastrol - the first low molecular weight kinesin spindle protein (KSP) inhibitor. The obtained compounds showed low micromolar antiproliferative activity towards a panel of sensitive and ABC-overexpressing cancer cells. Most cytotoxic compounds exhibited also higher KSP modulatory activity and ability for ROS generation compared to monastrol. The increased bioactivity of the studied compounds can be attributed to the presence of the ferrocenyl group.

Montelukast treatment may alter the early efficacy of immunotherapy in children with asthma.

BACKGROUND: Allergen-specific immunotherapy (SIT) is the only available potentially curative approach in the management of allergic diseases. Therapies that boost regulatory T cell induction during SIT might further enhance its effectiveness. OBJECTIVE: The purpose of this study was to assess the effect of montelukast treatment on early clinical and immunologic effects of allergen-specific immunotherapy in children with asthma. METHODS: It was a randomized, double-blind, placebo-controlled trial conducted in 36 children with asthma and allergy to house dust mites who required from 400 to 800 microg of inhaled budesonide per day during the 7-month run-in period. Patients were randomly allocated to receive 5 mg montelukast daily (n = 18) or placebo (n = 18) as an addition to inhaled corticosteroid (ICS) treatment during the 3-month build-up phase of SIT, when modification of ICS doses was not allowed. During the 7 months of the maintenance phase of SIT, ICS doses were adjusted to control the asthma symptoms. RESULTS: After 12 months of SIT, a reduction of the median daily ICS dose, necessary to control asthma symptoms, was 16.7% grater in patients from the placebo group than in patients from the montelukast group. Intervention with montelukast significantly impaired the induction of regulatory T lymphocytes. During the build-up phase of SIT, patients in the placebo group frequently experienced an increase in asthma symptoms leading to exclusions from the per protocol population. CONCLUSION: Our study failed to show a beneficial effect of montelukast on SIT. In fact, quite the opposite occurred: compared with placebo, montelukast intervention led to less effectiveness of SIT.

The effects of superoxide dismutase knockout on the oxidative stress parameters and survival of mouse erythrocytes.

The erythrocytes of 12-month old Sod1 (-/-) mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 (-/-) mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Heinz bodies and the reticulocyte count were indistinguishable in Sod1 (+/+) and Sod1 (+/-) mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.

Microtubule-Targeting 7-Deazahypoxanthines Derived from Marine Alkaloid Rigidins: Exploration of the N3 and N9 Positions and Interaction with Multidrug-Resistance Proteins.

Our laboratories have been investigating synthetic analogues of marine alkaloid rigidins that possess promising anticancer activities. These analogues, based on the 7-deazahypoxanthine skeleton, are available in one- or two-step synthetic sequences and exert cytotoxicity by disrupting microtubule dynamics in cancer cells. In the present work we extended the available structure-activity relationship (SAR) data to N3- and N9-substituted derivatives. Although N3 substitution results in loss of activity, the N9-substituted compounds retain nanomolar antiproliferative activities and the anti-tubulin mode of action of the original unsubstituted compounds. Furthermore, our results also demonstrate that multidrug-resistance (MDR) proteins do not confer resistance to both N9-unsubstituted and -substituted compounds. It was found that sublines overexpressing ABCG2, ABCC1, and ABCB1 proteins are as responsive to the rigidin analogues as their parental cell lines. Thus, the study reported herein provides further impetus to investigate the rigidin-inspired 7-deazahypoxanthines as promising anticancer agents.

Phenoxyherbicides induce production of free radicals in human erythrocytes: oxidation of dichlorodihydrofluorescine and dihydrorhodamine 123 by 2,4-D-Na and MCPA-Na.

Although it is known that phenoxyacetic herbicides significantly affect the oxidative status of human erythrocytes, there is no direct evidence of their ability to induce free radical production. To demonstrate this phenomenon we investigated the effect of two commonly used phenoxyherbicides-sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D-Na) and sodium salt of 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) on oxidation of dihydrorhodamine 123 and H(2)DCFDA as well as on carbonyl group content in cellular proteins. Moreover, haemoglobin denaturation was also measured. The rate of fluorescent probe oxidation was significantly higher for 2,4-D-Na, while both compounds exerted similar effects on protein carbonyl group (an increase in their content) and on denaturation of haemoglobin (no changes were observed). These results and the previous data led us to a conclusion that pro-oxidative action of phenoxyherbicides is strongly dependent on the localization of the substituent in the phenol ring. We also proposed a metabolic reaction chain that explains the mechanism of action of 2,4-D-Na in vivo.