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Background & aims: In CML, Leukemic Stem Cells (LSCs) that are insensitive to Tyrosine Kinase Inhibitors are responsible for leukemia maintenance and relapses upon TKI treatment arrest. We previously showed that downregulation of the BMI1 polycomb protein that is crucial for stem/progenitor cells self-renewal induced a CCNG2/dependent proliferation arrest leading to elimination of Chronic Myeloid Leukemia (CML) cells. Unfortunately, as of today, pharmacological inhibition of BMI1 has not made its way to the clinic. Methods: We used the Connectivity Map bioinformatic database to identify pharmacological molecules that could mimick BMI1 silencing, to induce CML cell death. We selected the bis-biguanide Alexidin (ALX) that produced a transcriptomic profile positively correlating with the one obtained after BMI silencing in K562 CML cells. We then evaluated the efficiency of ALX in combination with TKI on CML cells. Results: Here we report that cell growth and clonogenic activity of K562 and LAMA-84 CML cell lines were strongly inhibited by ALX. ALX didn’t modify BCR::ABL1 phosphorylation and didn’t affect BMI1 expression but was able to increase CCNG2 expression leading to autophagic processes that preceed cell death. Besides, ALX could enhance the apoptotic response induced by any Tyrosine Kinase Inhibitors (TKI) of the three generations. We also noted a strong synergism between ALX and TKIs to increase expression of caspase-9 and caspase-3 and induce PARP cleavage, Bad expression and significantly decreased Bcl-xL family member expression. We also observed that the blockage of the mitochondrial respiratory chain by ALX can be associated with inhibition of glycolysis by 2-DG to achieve an enhanced inhibition of K562 proliferation and clonogenicity. ALX specifically affected the differentiation of BCR::ABL1-transduced healthy CD34+ cells but not of mock-infected healthy CD34+ control cells. Importantly, ALX strongly synergized with TKIs to inhibit clonogenicity of primary CML CD34+ cells from diagnosed patients. Long Term Culture of Initiating Cell (LTC-IC) and dilution of the fluorescent marker CFSE allowed us to observe that ALX and Imatinib (IM) partially reduced the number of LSCs by themselves but that the ALX/IM combination drastically reduced this cell compartment. Using an in vivo model of NSG mice intravenously injected with K562-Luciferase transduced CML cells, we showed that ALX combined with IM improved mice survival. Conclusions: Collectively, our results validate the use of ALX bis-biguanide to potentiate the action of conventional TKI treatment as a potential new therapeutic solution to eradicate CML LSCs

Objectives The incidence of severe filamentous fungal infections has increased over the past decade. Some of these filamentous fungi are resistant to available antifungals; it is thus urgent to find new compounds that are active against such life-threatening pathogens. Methods In this study, 1280 drugs (Prestwick Chemical Library) were tested against six multidrug-resistant (MDR) filamentous fungi, includingAspergillus, Fusarium, Scedosporium/Lomentospora, Rhizopus and Lichtheimia species. Results Several hits were identified that induced fungal growth inhibition ≥70%. Among the non-antifungal compounds that were effective against the clinical moulds tested in this study, clioquinol, alexidine dihydrochloride, hexachlorophene and thonzonium bromide displayed a broad activity against all strains tested. Conclusion This study enriches the potential antifungal options that can be used against MDR invasive fungal diseases.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Objective During use, contact lens disinfecting solutions are exposed to tears and clinical microbial isolates. The current study was designed to test the performance of several disinfecting solution in the presence of organic soils or clinical isolates. Methods Standard and clinical isolates were exposed to the disinfecting solutions in the presence or absence of different organic soils. The number of microbial cells killed during disinfection was established by growing cells after disinfection on agar plates. Results The disinfecting activity of the povidone-iodine or hydrogen peroxide solutions was not affected by the organic soils or clinical isolates. The presence of yeast organic soil did not affect the performance of the disinfecting solutions when tested with standard microbial strains, but the addition of a model tear organic soil significantly reduced the disinfecting activity of the solutions containing various combinations of polyhexamethylene biguanide, polyquaternium-1, alexidine, and myristamindopropyl dimethylamine especially when tested against the standard fungal strains (reducing the effectiveness by between 0.5-4 log10) or the clinical bacterial isolates (reducing the effectiveness by between 0.5-3.5 log10). One disinfecting solution that contained polyquaternium-1 and myristamindopropyl dimethylamine had very poor activity against the clinical bacterial isolates in the absence or presence of either organic soil. Conclusions Povidone-iodine or hydrogen peroxide disinfecting solutions are not affected by organic soils and are very active against clinical bacterial isolates. Disinfecting solutions containing combinations of polyhexamethylene biguanide, polyquaternium-1, alexidine, and myristamindopropyl dimethylamine are affected by model tear organic soil and may have poor activity against clinical isolates.

Purpose To evaluate the antiviral potential of five multipurpose disinfecting solutions against coronavirus (mouse hepatitis virus, a surrogate for SARS-CoV-2 human corona virus). Methods Test solutions (Biotrue, renu Advanced [Bausch and Lomb], ACUVUE RevitaLens [Johnson and Johnson Vision], cleadew [Ophtecs corp.] or AOSept Plus [Alcon]) were mixed with the coronavirus mouse hepatitis virus at 104 plaque forming units (PFU)/mL as the final concentration and incubated at room temperature for the specified disinfection time. Surviving virus from each sample was then quantified by standard plaque forming unit assay and the reduction of PFU for each disinfectant was compared to the phosphate buffer saline (PBS) treated negative control. A regimen test was also conducted using Biotrue. Results The three multipurpose disinfecting solutions Biotrue (containing PHMB and polyquaternium-1), renu Advanced (PHMB, polyquaternium-1 and alexidine) and ACUVUE RevitaLens (polyquaternium-1 and alexidine) did not kill the coronavirus at the manufacturers recommended disinfection time in the stand alone test. After treatment, the virus’s titer (3.8 ± 0.2 log10 for Biotrue, 3.7 ± 0.1 log10 for renu and 3.7 ± 0.2 log10 for RevitaLens) was similar to the negative control (3.7 ± 0.1 log10; p ≥ 0.996). AOSept Plus (hydrogen peroxide) and cleadew (povidone iodine) significantly (p

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The defined lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, mitochondrial stress responses that help maintain the integrity of mitochondrial membranes against internal or external insults are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen originally aimed at identifying inhibitors of the inner mitochondrial membrane (IMM)-resident protease OMA1, we found that two bis-biguanide compounds, Chlorhexidine and Alexidine, modified OMA1 activity by altering the integrity of the IMM. Interestingly, these compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found Alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that Alexidine severely perturbated the IMM, especially the cristae structure. Along with this, we observed the altered localization of IMM-resident membrane-shaping proteins, including Mic60. Notably, Alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of Alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that can induce acute and selective perturbation of the IMM integrity, which should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

AIM The aim of the study was to assess the antibacterial efficiency of a combination of 1% alexidine (ALX) and 5.25% sodium hypochlorite (NaOCl) against E. faecalis biofilm using a confocal scanning electron microscopy. MATERIALS AND METHODS An estimated 120 human root dentin disks were prepared, sterilized, and inoculated with E. faecalis strain (ATCC 29212) to develop a 3-weeks-old biofilm. The dentin discs were exposed to group I-control group: 5.25% sodium hypochlorite (NaOCl) (n = 20); group II-1% ALX + 5.25% NaOCl (n = 40); group III-1% alexidine (ALX) (n = 40) (Sigma-Aldrich, Mumbai, India); group IV-negative control: saline (n = 20). After exposure, the dentin disks were stained with the fluorescent live/dead dye and evaluated with a confocal scanning electron microscope to calculate the proportion of dead cells. Statistical analysis was done using the Kruskal-Wallis and Mann-Whitney U test (p < 0.05). RESULTS The maximum proportion of dead cells were seen in the groups treated with the combination of 1% ALX + 5.25% NaOCl (94.89%) and in the control group 5.25% NaOCl (93.14%). The proportion of dead cells presented in the 1% ALX group (51.79%) and negative control group saline (15.10%) were comparatively less. CONCLUSION The antibacterial efficiency of a combination of 1% ALX and 5.25% NaOCl was more effective when compared with 1% ALX alone. CLINICAL SIGNIFICANCE Alexidine at 1% could be used as an alternative endodontic irrigant to chlorhexidine, as alexidine does not form any toxic precipitates with sodium hypochlorite. The disinfection regimen comprising a combination of 1% ALX and 5.25% NaOCl is effective in eliminating E. faecalis biofilms.

Background: Chlorhexidine (CHX) is a widely utilized disinfectant that can cause IgE-mediated urticaria/anaphylaxis. The cross-reactivity of patients with IgE-mediated CHX allergy with other disinfectants, which share structural similarities with CHX like polyhexanide (polyhexamethylene biguanide; PHMB), alexidine (ALX), or octenidine (OCT), is unknown. Methods: Forty-four patients with anaphylaxis or urticaria upon CHX exposure and positive skin prick test (SPT) and/or positive CHX ImmunoCAP test (Phadia TFS, Uppsala, Sweden) were recruited. IgE to the biguanide and/or hexamethylene structure was investigated with PHMB ImmunoCAP (n = 32) and by basophil activation tests (BAT) with CHX and ALX (n = 37). Inhibition tests of CHX and PHMB ImmunoCAPs by CHX, ALX, PHMB, and OCT were performed. Results: IgE reactivity to PHMB as surrogate marker for biguanide/hexamethylene reactivity was detected in 5/32 sera. Seven of 37 patients showed a positive BAT with ALX, but only under optimized conditions. Binding to CHX ImmunoCAP was inhibited by ALX in 1/32 sera, and binding to PHMB was blocked by ALX (1/5) and by OCT in another (1/5). In SPT, 9/10 patients were positive for CHX and 3 of them with ALX (only at highest concentration at 5 mg/mL). A further patient reacted primarily with OCT and showed IgE cross-reactivity with CHX, ALX, and PHMB. Conclusion: The IgE response to CHX seems polyclonal. The chloroguanide ending of CHX is the main epitope for the IgE and is suitable as screening assay to detect CHX reactivity. IgE-reactivities with the biguanide or hexamethylene components of other disinfectants (ALX, PHMB) can be detected by SPT, PHMB ImmunoCAP, and ALX-BAT in 15%-33% of CHX-allergic patients.

The lack of effective therapies remains one of the main challenges for malignant pleural mesothelioma (MPM). In this perspective, drug repositioning could accelerate the identification of novel treatments. We screened 1170 FDA-approved drugs on a SV40-immortalized mesothelial (MeT-5A) and five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) cell lines. Biological assays were carried out for 41 drugs, showing the highest cytotoxicity and for whom there were a complete lack of published literature in MPM. Cytotoxicity and caspase activation were evaluated with commercially available kits and cell proliferation was assayed using MTT assay and by clonogenic activity with standard protocols. Moreover, the five most effective drugs were further evaluated on patient-derived primary MPM cell lines. The most active molecules were cephalomannine, ouabain, alexidine, thonzonium bromide, and emetine. Except for alexidine, these drugs inhibited the clonogenic ability and caspase activation in all cancer lines tested. The proliferation was inhibited also on an extended panel of cell lines, including primary MPM cells. Thus, we suggest that cephalomannine, ouabain, thonzonium bromide, and emetine could represent novel candidates to be repurposed for improving the arsenal of therapeutic weapons in the fight against MPM.

Objective: The objective of this study was to chemically evaluate precipitate formation on irrigation by different concentrations of chlorhexidine (CHX) and alexidine (ALX) with sodium hypochlorite (NaOCl). Materials and Methods: Six test tubes were prepared with 1 ml of 4% NaOCl. One milliliter of 2%, 1%, 0.5%, and 0.25% ALX was added to the first four, and in the last two, 1 ml of 2% CHX and 0.2% CHX was added, respectively. Samples were observed for color changes or precipitates at multiple time intervals. All solutions were then centrifuged at 1000 rpm for 10 min and re-examined for precipitates. This process was repeated twice. Fifty freshly extracted premolars were biomechanically prepared, dried, divided into two groups, and irrigated with 10 ml of 4% NaOCl and 10 ml of 2% ALX (Group 1) and 10 ml of 4% NaOCl and 10 ml of 2% CHX (Group 2). These samples were sectioned and observed for precipitates on the dentinal surfaces by scanning electron microscopy (SEM). Results: The color of the solution of ALX and NaOCl stayed transparent and no precipitate was observed. A color change was noted immediately on mixing CHX and NaOCl which did not change with time. Precipitates were only observed in the solutions of CHX with NaOCl and after centrifuging them. SEM views also showed dense precipitates covering the dentinal surface and occluding the dentinal tubules in Group 2. Conclusion: The interaction of ALX and NaOCl does not produce precipitates which together with its better antimicrobial action make ALX a more effective and safer replacement for CHX as an adjunctive endodontic irrigant.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disease caused primarily by mutations in NPC1. NPC1 encodes the lysosomal cholesterol transport protein NPC1. The most common NPC1 mutation is a missense mutation (NPC1(I1061T)) that causes misfolding and rapid degradation of mutant protein in the endoplasmic reticulum. Cholesterol accumulates in enlarged lysosomes as a result of decreased levels of lysosomal NPC1(I1061T) protein in patient cells. There is currently no cure or FDA-approved treatment for patients. We sought to identify novel compounds that decrease lysosomal cholesterol storage in NPC1(I1061T/I1061T) patient fibroblasts using a high-content screen with the cholesterol dye, filipin and the lysosomal marker, LAMP1. A total of 3532 compounds were screened, including 2013 FDA-approved drugs, 327 kinase inhibitors and 760 serum metabolites. Twenty-three hits were identified that decreased both filipin and LAMP1 signals. The majority of hits (16/21) were histone deacetylase (HDAC) inhibitors, a previously described class of modifiers of NPC cholesterol storage. Of the remaining hits, the antimicrobial compound, alexidine dihydrochloride had the most potent lysosomal cholesterol-reducing activity. Subsequent analyses showed that alexidine specifically increased levels of NPC1 transcript and mature protein in both control and NPC patient cells. Although unsuitable for systemic therapy, alexidine represents a unique tool compound for further NPC studies and as a potent inducer of NPC1. Together, these findings confirm the utility of high-content image-based compound screens of NPC1 patient cells and support extending the approach into larger compound collections.

Introduction: Genotoxicity testing is necessary to address potential mutagenic characteristics of pharmaceutical candidates and medical devices depending on their intended use. Adapting such tests to contact lens multipurpose solutions (MPS) may be required due to their antimicrobial nature or to fulfill specific regulatory requirements. We describe a novel methodology for genotoxicity and biocompatibility evaluation of MPS in the context of traditional testing. Materials and Methods: Blink RevitaLens® Multipurpose Disinfecting Solution (MPDS) or contact lens (Acuvue® Advance and Acuvue® 2 brand) extracts were tested by Ames assay and Chromosome Aberration (ChrAb) Assays. A novel procedure for cycling contact lenses in MPDS was developed for testing. Before assays with the Blink RevitaLens MPDS disinfectant ingredient, Alexidine Dihydrochloride (ALX) was diluted for dose-range tests identifying TA100 or Chinese Hamster Ovary cytotoxicity levels to determine the amount of ALX appropriate for each ...

The aim of this study was to use antimicrobial efficacy endpoint methodology to determine compatibility of multipurpose disinfecting solutions (MPSs), lens cases, and hydrogel lenses for disinfection (AEEMC) against International Organization for Standardization (ISO)–specified microorganisms and clinical ocular isolates of Stenotrophomonas maltophilia. Six MPSs (PQ/Aldox 1, 2, and 3; PQ/Alexidine; PQ/PHMB; and PHMB) were challenged against ISO-specified microorganisms and S. maltophilia using the AEEMC test. AEEMC tests were performed with and without balafilcon A, etafilcon A, and senofilcon A lenses in lens cases with organic soil. Exposure times included disinfection time (DT) and 24 hr. Additionally, all six MPSs were challenged with two strains of S. maltophilia, based on the ISO Stand-alone test. The efficacy against bacteria for PQ/Aldox and PQ/Alexidine MPSs was not diminished by the presence of lenses. The efficacy of PQ/PHMB and PHMB MPSs against Serratia marcescens was significantly reduced compared with the no-lens control at DT for at least one lens type. The PHMB MPS with lenses present also demonstrated reduced efficacy against Staphylococcus aureus at DT versus the control. PQ/Aldox MPSs retained activity against Fusarium solani with lenses present; however, all other test MPSs demonstrated reduced F. solani efficacy at DT with lenses present. With lenses, all MPSs showed reduced efficacy against Candida albicans. AEEMC antimicrobial efficacy test results vary based on challenge microorganism, contact lenses, and MPS biocide systems. This study highlights the importance of evaluating MPSs for compatibility with lenses and lens cases. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.

Background Because corneal infiltrative events (CIEs) may result from bacterial components on contact lenses, which can come from contaminated lens cases, we evaluated the biocidal efficacy of five multipurpose solutions against Gram-negative commonly isolated and CIE-associated organisms. Methods Of the multipurpose solutions tested, one contained polyhexamethylene biguanide (PHMB)/polyquaternium-1 (PQ-1; Bausch & Lomb Incorporated: Biotrue), one contained alexidine dihydrochloride (alexidine)/PQ-1 (AMO: RevitaLens OcuTec) and three contained PQ-1/myristamidopropyl dimethylamine (MAPD; Alcon: Opti-Free PureMoist, PQ-1/MAPD-1; Opti-Free RepleniSH, PQ-1/MAPD-2; Opti-Free Express, PQ-1/MAPD-3). Challenge organisms were CIE-associated Achromobacter xylosoxidans, Delftia acidovorans and Stenotrophomonas maltophilia at manufacturer-recommended durations (stand-alone), in lens cases without lenses (up to seven days) and in lens cases with etafilcon A lenses (up to 30 days). Results In stand-alone testing against CIE-associated organisms, PHMB/PQ-1 and alexidine/PQ-1 were significantly superior versus MAPD-based multipurpose solutions against A. xylosoxidans (all p ≤ 0.01), D. acidovorans (all p ≤ 0.001) and S. maltophilia (all p ≤ 0.05). In lens cases, PHMB/PQ-1 and alexidine/PQ-1 achieved greater than 3-log reductions against all challenge organisms at all times evaluated. PQ-1/MAPD-1 achieved a greater than 3-log reduction against D. acidovorans at 24 hours; PQ-1/MAPD-1 and PQ-1/MAPD-3 achieved greater than 3-log reductions at seven days against all organisms. In lens cases with lenses, PHMB/PQ-1 and alexidine/PQ-1 achieved greater than 3-log reductions against all organisms at all times. PQ-1/MAPD-1 and PQ-1/MAPD-3 achieved greater than 3-log reductions at seven or more days against all organisms. PQ-1/MAPD-2 did not achieve a greater than 3-log reduction at any time; some regrowth was observed. Conclusions PHMB- and alexidine-based multipurpose solutions demonstrated significantly greater biocidal activity compared with PQ-1/MAPD-based agents against Gram-negative organisms commonly isolated and CIE-associated pathogens.

The purpose of this study was to evaluate the antibacterial effectiveness of Octenisept (OCT; SchülkeMayr GmBH, Norderstedt, Germany), 1% alexidine (ALX) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and 2% chlorhexidine (CHX) against Enterococcus faecalis biofilm using confocal laser scanning microscopy.Root dentin discs were prepared from extracted human teeth, sterilized, and inoculated with E. faecalis strain (ATCC 29212) to establish 3-week-old biofilm model. Infected dentin discs were exposed to OCT (n = 20), 1% ALX (n = 20), and 2% CHX (n = 20) for 10 minutes. Dentin discs (n = 15) exposed to 5.25% sodium hypochlorite (NaOCl) were used as a positive control, whereas specimens exposed to saline (n = 15) were used as a negative control. After exposure, the dentin discs were stained with fluorescent LIVE/DEAD BacLight dye (Invitrogen Molecular Probes, Eugene, OR) and analyzed with confocal laser scanning microscopy to determine the proportion of dead cells in the biofilm. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests (P .05).The highest proportion of dead cells was found in the 5.25% NaOCl group (94.14%; range, 92.30%-98.20%) compared with the experimental groups (P .05). A significantly greater proportion of dead cells was found in the OCT group (74.14%; range, 70.03%-78.96%) compared with the 1% ALX and 2% CHX groups (P .05). The proportion of dead cells was 43.89% (range, 24.86%-55.63%) and 42.78% (range, 25.45%-55.06%) in the 1% ALX and 2% CHX groups, respectively, with no statistical significant difference between the 2 groups (P .05).NaOCl had significantly greater antimicrobial activity against E. faecalis biofilms compared with OCT, CHX, and ALX. OCT was more effective than CHX and ALX.

Purpose To compare the antimicrobial effect of topical anesthetics, antivirals, antibiotics, and biocides on the viability of Acanthamoeba cysts and trophozoites in vitro. Methods Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461). Test agents included topical ophthalmic preparations of common anesthetics, antivirals, antibiotics, and biocides. Organisms were exposed to serial two-fold dilutions of the test compounds in the wells of a microtiter plate to examine the effect on Acanthamoeba spp. In addition, the toxicity of each of the test compounds was determined against a mammalian cell line. Results Proxymetacaine, oxybuprocaine, and especially tetracaine were all toxic to the trophozoites and cysts of Acanthamoeba spp., but lidocaine was well tolerated. The presence of the benzalkonium chloride (BAC) preservative in levofloxacin caused a high level of toxicity to trophozoites and cysts. With the diamidines, the presence of BAC in the propamidine drops was responsible for the activity against Acanthamoeba spp. Hexamidine drops without BAC showed good activity against trophozoites, and the biguanides polyhexamethylene biguanide, chlorhexidine, alexidine, and octenidine all showed excellent activity against trophozoites and cysts of both species. Conclusions The antiamoebic effects of BAC, povidone iodine, and tetracaine are superior to the current diamidines and slightly inferior to the biguanides used in the treatment for Acanthamoeba keratitis. Translational Relevance Ophthalmologists should be aware that certain topical anesthetics and ophthalmic preparations containing BAC prior to specimen sampling may affect the viability of Acanthamoeba spp. in vivo, resulting in false-negative results in diagnostic tests.

Background: Highly aggressive and resistant to chemotherapy, pancreatic cancers are the fourth leading cause of cancer-related deaths in the western world. The absence of effective chemotherapeutics is leading researchers to develop novel drugs or repurpose existing chemicals. Alexidine Dihydrochloride (AD), an orally bioavailable bis-biguanide compound, is an apoptosis stimulating reagent. It induces mitochondrial damage by inhibiting a mitochondrial-specific protein tyrosine phosphatase, PTPMT1. The aim of this study was to test AD as a novel compound to induce apoptosis in a human pancreatic adenocarcinoma cell lines, Panc-1, MIA PaCa-2, AsPC-1, and Psn-1. Methods: After the IC50 value of the AD was determined by cytotoxicity assay, apoptosis was observed by a variety of methods, including the detection of early apoptosis marker Annexin V and the proteomic profile screening by apoptosis array. Multicaspase and mitochondrial depolarization were measured, and changes in the cell cycle were analyzed. Results: AD is found to initiate apoptosis by activating the intrinsic pathway and inhibit the cell cycle in pancreatic cancer cell lines. Conclusions: In conclusion, considering its anti-cancer properties and bioavailability, Alexidine dihydrochloride can be considered as a potential candidate against pancreatic adenocarcinomas.

Polymeric biguanides, as well as quaternary ammonium compounds, are ubiquitous antimicrobial agents in healthcare. Due to the highly cationic and polymeric nature of these compounds and the complex matrices in which they are found, the analytical characterization of products containing them remains challenging. In this work an efficient, sensitive, and high-resolution separation protocol was developed to perform quantitative measurements (sub-mg L −1 ) of alexidine dihydrochloride (ADH) and polyhexamethylene biguanide (PHMB) in commercial multipurpose contact lens solutions (MPS). Initially, contactless conductivity (C 4 D) detection was explored, but lacked adequate selectivity and sensitivity to quantify PHMB or ADH in commercial MPS. To overcome these limitations, an alternative approach using solid phase extraction (SPE) followed by separation with reversed phase ultra-performance liquid chromatography (RP-UPLC) was developed for both ADH and PHMB separation and detection. The most sensitive and reliable method investigated utilized standard additions to compensate for matrix effects. For ADH, concentration values measured with the presented method were consistent with data provided by the MPS manufacturer (1.6 mg L −1 ) within 0.10 mg L −1 . PHMB quantification in MPS products was successful at concentrations −1 with quantitative reproducibility better than 2% RSD. Comparison of blind sample testing using the RP-UPLC method showed strong correlation (R 2 = 0.939) of PHMB concentrations with results obtained by the United States Food and Drug Administration using a published HPLC-Evaporative light scattering detection (ELSD) assay. A significant advantage of this method is the ability to partially resolve PHMB polydispersity, which to date has been minimally studied and explained. By coupling with electrospray mass spectrometry (MS), a general trend was observed for increased retention as a function of PHMB chain length. The improved robustness and reproducibility of UV detection versus ELSD coupled with the superior resolving power of UPLC is an asset to the detection and characterization of PHMB and ADH. In addition to quality control of MPS, this method has potential application to the analyses skin wipes, wound dressings and other medical products where understanding how manufacturing processes lead to differences in polydispersity is important to maximize the antimicrobial properties while minimizing toxicologic effects.

Therapeutic success in endodontic treatment depends on successful infection control. Alexidine dihydrochloride (ALX) was recently proposed as a potential alternative to 2% chlorhexidine (CHX) as it possesses similar antimicrobial properties, expresses substantivity and does not produce p-chloroaniline (PCA) when mixed with sodium hypochlorite (NaOCl). However, the products released in this reaction have not been described to date. The aim of this study was to identify detected chemical compounds formed in the reaction of ALX and NaOCl with the ultra-high-performance liquid chromatography–mass spectrophotometry (UHPLC-MS) method and assess whether precipitates and PCA are formed in this reaction. Solutions of ALX were mixed with the equivalent volume of 2% and 5.25% (w/v) NaOCl solutions. As control, 2% (w/v) CHX was mixed with 2% and 5.25% (w/v) NaOCl. Samples were subjected to the UHPLC-MS analysis. The mixture of ALX and NaOCl resulted in a yellowish precipitate formation, the amount of which depended on NaOCl concentration. Interaction of ALX and NaOCl resulted in the production of aliphatic amines. No PCA was formed when NaOCl was mixed with ALX. However, for the first time, we identified the possible products of the interaction. The interaction between NaOCl and ALX results in the formation of aliphatic amines, therefore, these compounds should not be mixed during endodontic treatment.

AIM The aim of this systematic review and network meta-analysis (NMA) was to compare the efficacy of different anti-plaque chemical agents, in 6-month, home-use, randomized clinical trials (RCTs), in terms of plaque index (PlI) changes. MATERIAL AND METHODS RCTs assessing PlI were identified, screened, and evaluated for inclusion. Relevant information was extracted, and quality and risk of bias were assessed. Mean differences between baseline-end were calculated to obtain weighted mean differences and 95% confidence intervals. NMA protocols were applied to assess direct and indirect comparisons among products using Turesky PlI. RESULTS Eighty-three papers were included: 49 examined dentifrices, 32 mouthrinses and 2 both. The NMA analysed 51 studies including data from 4242 and 4180 subjects for dentifrices and mouthrinses respectively. For dentifrices, triclosan-copolymer and chlorhexidine showed the greatest effect, with significant differences when compared with stannous fluoride. For mouthrinses, essential oils and chlorhexidine showed the greatest effect, with significant differences when compared with delmopinol, alexidine and cetylpyridinium chloride. CONCLUSION Within the limitations of this study (including the severe imbalance in the amount of evidence), dentifrices containing triclosan-copolymer or chlorhexidine and mouthrinses containing essential oils or chlorhexidine showed the greatest effect on PlI scores as assessed with NMA.

Purpose: Highly aggressive and resistant to chemotherapy, pancreatic cancers are the fourth leading cause of cancer-related deaths in the western world. The absence of effective chemotherapeutics is leading researchers to develop novel drugs or repurpose existing chemicals. Alexidine dihydrochloride (AD), an orally bioavailable bis-biguanide compound, is an apoptosis stimulating reagent. It induces mitochondrial damage by inhibiting a mitochondrial-specific protein tyrosine phosphatase, PTPMT1. The aim of this study was to test AD as a novel compound to induce apoptosis in a human pancreatic adenocarcinoma cell lines, Panc-1, MIA PaCa-2, AsPC-1, and Psn-1. Methods: After IC50 value of the AD was determined by cytotoxicity assay, apoptosis was observed by a variety of methods including the detection of early apoptosis marker Annexin V and the proteomic profile screening by apoptosis array. Multicaspase and mitochondrial depolarization were measured and changes in cell cycle were analyzed. Results: AD is found to initiate apoptosis by activating the intrinsic pathway and inhibit cell cycle in pancreatic cancer cell lines. Conclusions: As a conclusion, considering its anti-cancer properties and bioavailability, Alexidine dihydrochloride can be considered as a potential candidate against pancreatic adenocarcinomas. Citation Format: Ezgi Kasikci, Esra Aydemir, Bekir Mustafa Yogurtcu, Fikrettin Sahin, Omer Faruk Bayrak. Repurposing of alexidine dihydrochloride as an apoptosis-initiator and cell cycle inhibitor in human pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-031.

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2-deoxy-d-glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG-6-phosphate (2DG6P), the anti-cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose-6-phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti-cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC50 = 0.53, 1.77, 8.1 and 165 μM, respectively). The anti-CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase.

Invasive fungal infections due to Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, constitute a substantial threat to hospitalized, immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices, and emergence of drug-resistant fungi such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms which increases the chance of infection relapses. Here, we screened a small molecule Prestwick Chemical Library, consisting of 1200 FDA approved off-patent drugs, against C. albicans, C. auris and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens, and their ability to kill preformed biofilms. Our studies identified the bis-biguanide Alexidine dihydrochloride (AXD), as a drug with the highest antifungal and anti-biofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mice central venous catheters in vivo, highlighting its potential as a pan-antifungal drug.ImportanceThe prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multi-drug resistant strains such as C. auris. High mortality related to fungal infections point to major limitations of current antifungal therapy, and an unmet need for new antifungal drugs. We screened a library of repurposed FDA approved inhibitors to identify compounds with activities against a diverse range of fungi, in varied phases of growth. The assays identified Alexidine dihydrochloride (AXD) to have pronounced antifungal activity including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro, and prevented biofilm growth in vivo. Thus AXD has the potential to be developed as a pan-antifungal, anti-biofilm drug.

The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as C. auris. High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro and prevented biofilm growth in vivo. Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug., Invasive fungal infections due to Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans constitute a substantial threat to hospitalized immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices and emergence of drug-resistant fungi, such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms, which increase the chance of infection relapses. Here, we screened a small-molecule New Prestwick Chemical Library, consisting of 1,200 FDA-approved off-patent drugs against C. albicans, C. auris, and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens and their ability to kill preformed biofilms. Our studies identified the bis-biguanide alexidine dihydrochloride (AXD) as a drug with the highest antifungal and antibiofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mouse central venous catheters in vivo, highlighting its potential as a pan-antifungal drug. IMPORTANCE The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as C. auris. High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro and prevented biofilm growth in vivo. Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug.

The increasing problem of multidrug resistance (MDR) in bacteria calls for discovery of new molecules and diagnostic methodologies that are effective against a wide range of microbial pathogens. We have studied the role of alexidine dihydrochloride (alex) as a bioaffinity ligand against lipopolysaccharide (LPS), a pathogen-associated surface marker universally present on all Gram-negative bacteria. While the activity of alex against bacteria is biologically known, little information exists on its mechanism of action or binding stoichiometry. We have used nuclear magnetic resonance (NMR), fluorescence, and surface plasmon resonance (SPR) spectroscopies to probe the binding characteristics of alex and LPS molecules. Our results indicate that LPS:alex stoichiometry lies between 1:2 and 1:4 and has a dissociation constant ( KD) of 38 μM that is mediated through electrostatic interactions between the negatively charged phosphate groups present on LPS and the positively charged guanidinium groups present in alex. Further, molecular dynamics (MD) simulations performed to determine the conformational interaction between the two molecules show good agreement with the experimental results, which substantiate the potential of alex molecule for LPS neutralization and hence, development of efficient in vitro diagnostic assays.

Aseptic loosening and periprosthetic infection leading to inflammatory osteolysis is a major complication associated with total joint arthroplasty (TJA). The liberation of bacterial products and/or implant-derived wear particles activates immune cells that produce pro-osteoclastogenic cytokines that enhance osteoclast recruitment and activity, leading to bone destruction and osteolysis. Therefore, agents that prevent the inflammatory response and/or attenuate excessive osteoclast (OC) formation and bone resorption offer therapeutic potential by prolonging the life of TJA implants. Alexidine dihydrochloride (AD) is a bisbiguanide compound commonly used as an oral disinfectant and in contact lens solutions. It possesses antimicrobial, anti-inflammatory and anticancer properties; however, its effects on OC biology are poorly described. Here, we demonstrate that AD inhibits OC formation and bone resorption in vitro and exert prophylatic protection against LPS-induced osteolysis in vivo. Biochemical analysis demonstrated that AD suppressed receptor activator of NF-κB ligand (RANKL)-induced activation of mitogen-activated protein kinases (ERK, p38, and JNK), leading to the downregulation of NFATc1. Furthermore, AD disrupted F-actin ring formation and attenuated the ability of mature OC to resorb bone. Collectively, our findings suggest that AD may be a promising prophylactic anti-osteoclastic/resorptive agent for the treatment of osteolytic diseases caused by excessive OC formation and function.

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba.

Introduction The root canal system must be mechanically instrumented and chemically cleaned using various antimicrobial irrigants in a sequential manner or in combination for the elimination of necrotic pulp tissue and reducing the number of root canal bacteria. For this reason, new methods and materials are continuously being developed to achieve the objectives of endodontic treatment. Materials and methods E. faecalis (ATCC 29212) and C. albicans (ATCC 90028) standard strains were used for this study. Colonies of E. faecalis and C. albicans were harvested from the agar plates and suspended in 4 mL of phosphate buffered saline (PBS). Microorganisms were diluted to obtain a suspension of approximately 108 colony-forming units/mL (CFU/mL) in sterile PBS using McFarland standard tubes no. 0.5. Results After a two-minute contact time, all alexidine (ALX) concentrations used in this study eradicated all E. faecalis strains, while chlorhexidine (CHX) didn't kill 100% of E. faecalis at 0.25% and lower concentrations even after a five-minute contact time. ALX also eradicated C. albicans at all concentrations even after a one-minute contact time. CHX showed antifungal activity against C. albicans at all concentrations higher than 0.031% after a one-minute contact time. Conclusion A 0.0156% concentration of ALX can be a good alternative to CHX as an irrigation solution in endodontic treatment when used for one minute against E. faecalis and C. albicans.

Bu çalışmanın amacı; kök kanallarının irrigasyonu sırasında kullanılan farklı yıkama solüsyonlarının epoksi rezin esaslı AH Plus kök kanal dolgu patının bağlanma dayanımına etkisinin değerlendirilmesidir. Çalışmada 90 adet insan üst santral kesici dişleri kullanıldı. Kök boyutlarını benzer tutmak amacıyla, dişlerin koronal kısımları kök boyları apeksten 13 mm uzaklıkta olacak şekilde uzaklaştırıldı. Kök kanalları, ProTaper Universal Ni-Ti döner eğe sistemi kullanılarak şekillendirildi. Daha sonra kök kanalları, kullanılacak farklı final irrigasyon solüsyonlarına göre her grupta 18 diş olacak şekilde rastgele 5 gruba ayrıldı: Grup 1: %5,25 NaOCl + %17 EDTA, Grup 2: %6 NaOCl + QMix, Grup 3: % 5,25 NaOCl + %17 EDTA + %2 CHX, Group 4: %5,25 NaOCl + %17 EDTA + %1 ALX, Grup 5: %5,25 NaOCl + Distile su. Kök kanallarını yıkama işlemi tamamlandıktan sonra, örnekler AH Plus kök kanal dolgu patı ve güta-perka kullanılarak dolduruldu. Dişler 37oC'de %100 nemli ortamda 1 hafta bekletildi. Daha sonra her örnekten apikal orta ve koronalden olmak üzere, 2 mm (± 0,1 mm) kalınlığında üçer adet yatay kesit elde edildi. Örnekler Universal Test cihazına yerleştirildi ve itme kuvveti, kök kanal dolgusu ve dentin arasındaki bağlantıda kopma gerçekleşene kadar 1mm/dk sabit hız ile uygulandı. Nexygen veri analiz programı (Lloyd LRX, Fareham, UK) kullanılarak kopma kuvvetleri Newton (N) cinsinden kaydedildi ve megapaskala (MPa) çevrilerek bağlanma dayanımı hesaplandı. Kullanılan yıkama solüsyonlarından bağımsız olarak en yüksek bağlanma dayanımı değerleri sırasıyla koronal, orta ve apikal üçlü bölgelerinden elde edildi ve tüm gruplar arasındaki farklılık anlamlı bulundu (p

SUMMARY Virtually all organisms seek to maximize fitness by matching fuel availability with energy expenditure. In vertebrates, glucose homeostasis is central to this process, with glucose levels finely tuned to match changing energy requirements. To discover new pathways regulating glucose levels in vivo, we performed a large-scale chemical screen in live zebrafish and identified the small molecule alexidine as a potent glucose-lowering agent. We found that alexidine inhibits the PTEN-like mitochondrial phosphatase PTPMT1 and that other pharmacological and genetic means of inactivating PTPMT1 also decrease glucose levels in zebrafish. Mutation of ptpmt1 eliminates the effect of alexidine, further confirming it as the glucose-lowering target of alexidine. We then identified succinate dehydrogenase (SDH) as a substrate of PTPMT1. Inactivation of PTPMT1 causes hyperphosphorylation and activation of SDH, providing a possible mechanism by which PTPMT1 coordinates glucose homeostasis. Therefore, PTPMT1 appears to be an important regulator of SDH phosphorylation status and glucose concentration.

Introduction Root canal irrigants play an important role in reducing intracanal microorganisms, which in turn helps in achieving a successful outcome for the root canal treatment. Objective To compare the antibacterial efficacy of alexidine and chlorhexidine against Enterococcus faecalis. Methods A total of 50 extracted single-rooted teeth were randomly divided into five groups after being infected with Enterococcus faecalis. The groups were based on irrigants used: Group I - 0.4% alexidine; Group II - 1% alexidine; Group III - 1.5% percent alexidine; Group IV - 2% alexidine; Group V - 2% chlorhexidine. Following irrigation, colony-forming units were determined from the dentinal shavings collected at 400 µm depth. Results Use of 2% alexidine reduced the bacteria effectively when compared to 0.4%, 1%, and 1.5% alexidine. A statistically significant difference was not observed between 2% alexidine and 2% chlorhexidine. Discussion Alexidine, due to its higher virulence factors for bacteria and better bacterial penetrability at 400 µm depth of dentin showed better eradication of Enterococcus faecalis in comparison to chlorhexidine. Conclusion The use of 2% alexidine against Enterococcus faecalis at 400 µm depth of dentin has efficacy comparable to chlorhexidine. Hence, alexidine can be used as an alternative irrigant for chlorhexidine during endodontic procedures.

Lung cancer is the leading cause of cancer-related deaths in the US with an overall 5-year survival of only 15-20%. Metastatic disease is a major contributor to poor survival and novel therapeutics that can selectively target invasive tumor cell populations are lacking. Metabolic reprogramming contributes to tumor progression with effective therapies targeting cancer metabolism available. However, it remains unclear whether targeting cancer metabolism will prove valuable in inhibiting invasive cancer populations. Recent studies have demonstrated anticancer activity by the mitochondria-targeting bioactive small molecule alexidine dihydrochloride. Our group has previously found that alexidine specifically inhibits the a highly invasive subpopulation of H1299 previous identified in our lab by decoupling and decreasing collective invasion. Here we report the structure activity relationship (SAR) between analogs of alexidine on the inhibition of collective invasion in NSCLC cell lines. For these studies, we utilized an in vitro 3D invasion model where NSCLC cell lines were allowed to form spheroids, and then were embedded into Matrigel and allowed to invade over 48h in the presence or absence of the analogs. Using this technique, we identified two novel alexidine analogs with improved efficacy at inhibiting the invasion of H1299 NSCLC cells. Additionally, we examined the phosphorylation of pyruvate dehydrogenase (PDH), the gatekeeper for the entrance of pyruvate into the TCA cycle, following treatment with the alexidine analogs. We observed a significant correlation between PDH phosphorylation and inhibition of invasion following treatment with the analogs. In conclusion, we have identified two novel alexidine analogs with improved inhibition of invasion in NSCLC cell lines in an in vitro model. Citation Format: Jamie Arnst, Rachel Commander, Adam Marcus. Structure activity relationship of alexidine analogs for the inhibition of collective invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1239.

OBJECTIVE To compare the short-term clinical performance of two dual-disinfectant multipurpose disinfecting solutions (MPDS) when used in conjunction with two silicone hydrogel contact lenses. METHODS Participants wore galyfilcon A and balafilcon A lenses, randomly allocated one type to each eye. Multipurpose disinfecting solutions were double-masked and allocated using a randomized crossover design. Lenses were presoaked overnight in the allocated MPDS, containing either polyquaternium-1/alexidine dihydrochloride (PQ-alexidine) or polyquaternium-1/myristamidopropyl dimethylamine (PQ-MAPDA). Lenses were worn daily for 4 days and disinfected with the same MPDS in which they had been soaked. Clinical assessments occurred immediately after insertion, 2 hours after insertion, and after 2 hours of wear on day 4. Subjective ratings were collected at each assessment and after 6 hours of wear on days 1 to 3. Linear mixed models were used for statistical analysis. RESULTS Solution-induced corneal staining (SICS) incidence was significantly different between MPDS lens combinations (P=0.01) with PQ-MAPDA-balafilcon A showing the highest incidence (27.8%). The incidence of SICS was significantly higher when balafilcon A lenses were worn compared with galyfilcon A (19.4% vs. 2.8%; P=0.04). There was no significant difference in SICS incidence between MPDS (PQ-alexidine=5.6%, PQ-MAPDA=16.7%; P=0.08). Polyquaternium-1/alexidine dihydrochloride was associated with significantly fewer symptoms of burning/stinging (9.7±0.6 vs. 9.1±2.2; P=0.03) and self-assessed redness (9.8±0.5 vs. 9.2±1.9; P=0.03) on initial lens insertion. There were no significant differences between MPDS for comfort ratings (P>0.05). CONCLUSION These short-term clinical results provide an initial indication that PQ-alexidine is associated with fewer symptoms on insertion but no significant difference in ocular comfort compared with PQ-MAPDA. Longer studies are required to provide further evidence.

A previous study demonstrated that alexidine has greater affinity for the major virulence factors of bacteria than chlorhexidine. The aim of this study was to compare the antimicrobial activity of 1% alexidine with that of 2% chlorhexidine using Enterococcus faecalis-infected dentin blocks. Sixty bovine dentin blocks were prepared and randomly divided into six groups of 10 each. E. faecalis was inoculated on 60 dentin blocks using the Luppens apparatus for 24 h and then the dentin blocks were soaked in 2% chlorhexidine or 1% alexidine solutions for 5 and 10 min, respectively. Sterile saline was used as a control. The antimicrobial efficacy was assessed by counting the number of bacteria adhering to the dentin surface and observing the degradation of bacterial shape or membrane rupture under a scanning electron microscope. Significantly fewer bacteria were observed in the 2% chlorhexidine- or 1% alexidine-soaked groups than in the control group (P0.05). Ruptured or antiseptic-attached bacteria were more frequently observed in the 10-min-soaked chlorhexidine and alexidine groups than in the 5-min-soaked chlorhexidine and alexidine groups. In conclusion, 10-min soaking with 1% alexidine or 2% chlorhexidine can be effective against E. faecalis infection.

The host–guest complexations of a series of biguanidinium cations (metformin and phenformin) and bis(biguanidinium) dications (chlorhexidine and alexidine) by the host molecule cucurbit[7]uril (CB[7]) in aqueous solution have been investigated by 1H NMR spectroscopy and ESI mass spectrometry. Metformin forms both 1:2 and 1:1 host–guest complexes, whereas phenformin forms only a 1:1 complex. The two bis(biguanidinium) dications both form sequential 1:1, 2:1 and 3:1 host–guest complexes, however, the mechanism is different in each case. With chlorhexidine, the first CB[7] binds to the central hexamethylene chain, followed by sequential complexation of the terminal 4-chlorophenyl groups. With alexidine, the two terminal 2-ethylhexyl groups are bound first, with the third CB[7] adding only with a concomitant relocation of one CB[7] to the central hexamethylene binding site. The stability constants of the various host–guest complexes were determined by 1H NMR titrations and competitive binding experiments, as well as by the application of a statistical binding model.

Introduction: Chlorhexidine (CHX) is generally used as the final irrigating solution in root canal therapy. Recent studies have reported that, toxic precipitates containing parachloroaniline (PCA) are formed when CHX reacts with sodium hypochlorite (NaOCl). Whereas, Alexidine (ALX), a bisbiguanide disinfectant similar to CHX, has proven to form no precipitates with NaOCl. Aim: To compare antimicrobial activity of different concentrations of ALX with CHX individually and when combined with NaOCl against E. faecalis strains. Materials and Methods: Different concentrations of ALX and CHX (0.5%, 1%, and 2%) were tested individually and when mixed with 2.5% NaOCl (1:1 ratio) using disc diffusion method against E. faecalis. After 24 hours incubation at 37°C, zones of inhibition were measured for each solution. The results obtained were statistically analysed using one way ANOVA and Scheffe's post-hoc tests. The p-value

Objective ReNu with MoistureLoc (ReNuML), containing the antimicrobial agent alexidine 0.00045%, was associated with the Fusarium keratitis epidemic of 2004 to 2006. Although a single-point source contamination was ruled out, only Fusarium organisms were reported during the outbreak. This study investigated whether the reported loss of antimicrobial effectiveness toward Fusarium of ReNuML after exposure to heat in high-density polyethylene (HDPE) plastic containers could also be demonstrated with other common fungal and bacterial agents of keratitis. Methods A buffered solution of alexidine 0.00045% was incubated in glass and ReNu HDPE plastic containers at room temperature (RT) and 56°C for 4 weeks, serially diluted, and tested for its ability to inhibit the growth of 20 bacterial isolates, 12 non-Fusarium fungal isolates, and 7 Fusarium isolates originally involved in the keratitis epidemic. Results A statistically significant loss of antimicrobial capability was seen with all fungi, all gram-positive bacteria, and all isolates of Klebsiella when alexidine 0.00045% was incubated at 56°C in ReNu HDPE containers compared with RT or glass incubation (P≤0.0001). Conclusions Heating of an alexidine solution in ReNu HDPE plastic (but not glass) containers results in the same loss of anti-Fusarium activity as reported when testing the original ReNuML solution. This loss of inhibitory activity is not specific to Fusarium and occurs with other fungi and bacteria that cause keratitis. The reasons for the lack of reports of bacterial and/or non-Fusarium fungal keratitis during the original Fusarium keratitis epidemic remain unclear at this time.

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Introduction Recent studies have reported the color change and formation of precipitates containing para-chloroaniline (PCA) after a reaction of sodium hypochlorite (NaOCl) and chlorhexidine (CHX). Alexidine (ALX), a biguanide disinfectant similar to CHX, has greater affinity for bacterial virulence factors than CHX. This study determined by electrospray ionization mass spectrometry (ESI-MS) and scanning electron microscopy (SEM) whether the chemical interaction between ALX and NaOCl results in PCA or precipitates. Methods ESI-MS was performed on 4 different concentrations of ALX (1%, 0.5%, 0.25%, and 0.125%) with 4% NaOCl to detect the presence of PCA. As control groups, 1% ALX, 0.5% PCA, and a mixture of 2% CHX and 4% NaOCl were analyzed. The formation of precipitates on the dentinal surfaces of premolar root canals treated with the solutions of ALX and NaOCl (AN) or CHX and NaOCl (CN) was observed by SEM and the color change in the reaction solutions was also analyzed. Results ESI-MS showed that the peak (mass/charge ratio = 128.026) in the PCA spectrum was not detected in any of the 4 AN solutions, whereas the peak was found in the CN solution. SEM revealed precipitates covering dentinal surfaces in the CN solution. The AN solutions produced no precipitate. The AN solutions changed in color from light yellow to transparent with decreasing ALX concentration, whereas peach-brown discoloration was observed in the CN solution. Conclusions The interaction of ALX and NaOCl did not produce PCA or precipitates, and the color of the reacted solution changed transparent with decreasing ALX concentration.

Purpose Non-compliance is a significant factor in contact lens related microbial keratitis and includes solution reuse and failure to recap the lens storage case resulting in evaporation effects. To address this, impact of partial evaporation on the antimicrobial efficacy of multipurpose contact lens care solutions was investigated. Methods Solutions were evaporated under a stream of air to 2× and 4× concentration and challenged with Fusarium solani (ATCC 36031), Candida albicans (ATCC 10231) and Acanthamoeba castellanii (ATCC 50370). The level of organism kill at 6 h was compared to the non-evaporated product. Results ReNu with MoistureLoc ® (RML) lost 90–100% of biocidal activity against C. albicans on evaporation, 75–99% for F. solani and 29–33% with A. castellanii at 2× or 4× concentration, respectively. OPTI-FREE ® RepleniSH ® lost 72–90% efficacy against C. albicans and F. solani , and 61% at 2× and 10% at 4× concentration with A. castellanii . ReNu ® MultiPlus, AQuify ® Multi-Purpose and Biotrue™ showed only loss in efficacy with C. albicans at 4× concentration giving 79%, 34.5% and 48% reduction, respectively. No loss in biocidal activity on evaporation was obtained with Complete ® Revitalens for all organisms. Conclusion Partial evaporation can affect biocidal efficacy of multi-purpose solutions and may have been a significant factor in an outbreak of Fusarium keratitis cases associated with RML. Evaporation results in increased binding of cationic disinfectants to counter-ions in the formulation, reducing ability to attach and rupture anionic microbial cell walls. Interaction may also occur between the biocidal ingredient and other components, such as surfactants, resulting in sequestration of activity through micelle formation.

Aims: To compare the antibacterial activity of alexidine (ALX) alone or as a final irrigant in combination with sodium hypochlorite (NaOCl), with the most common canal irrigants, NaOCl and chlorhexidine (CHX). Materials and methods: Ninety-four root fragments from extracted human teeth were infected with Enterococcus faecalis for 24 h and then distributed into 4 groups of 20 fragments each. The NaOCl, CHX and ALX groups were immersed in 1 ml of 2.5% NaOCl, 2% CHX, and 1% ALX for 10 min, respectively. The samples of the NaOCl+ALX group were immersed in 1 ml of 2.5% NaOCl for 10 min followed by 1% ALX for 10 min. Bacteriological samples were taken, cultured, and the colony-forming units were counted. Results: There was no significant differences among the experimental groups (P>0.05) except for the comparisons CHX versus ALX and NaOCl+ALX versus ALX (P=0.004). ALX alone was the worst irrigant. CHX and NaOCl+ALX eradicated all bacteria. All experimental groups were significantly more effective than the control group immersed in saline (P Conclusions: The antibacterial effect of ALX alone was inferior to 2% CHX and 2.5% NaOCl. However, the combination of NaOCl with ALX as a final irrigant eradicated the biofilms.

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 μM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic β-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.

Purpose. To compare the effects of four commercially available multipurpose solutions (MPSs) on the structure and barrier function of corneal epithelial tight junctions. Methods. Human corneal epithelial cells were cultured on collagen-coated slides and then exposed to MPS A (polyhexamethylene biguanide, macrogolglycerol hydroxystearate), MPS B (polyhexamethylene biguanide, poloxamine), MPS C (Alexidine, poloxamine), and MPS D (POLYQUAD, poloxamine) for 60 minutes. Tight junction integrity of the corneal epithelial cells was evaluated with ZO-1 (tight junction–related protein) labeling under laser confocal microscopy. To investigate the changes of ultrastructure in tight junctions of human corneal epithelial cells, an ultrathin cross-section of the cell on collagen membrane was also observed by transmission electron microscopy. For quantitative evaluation of barrier functions, transepithelial electrical resistance of the epithelial cell was measured 30, 60, and 120 minutes after MPS exposure by using a volt ohmmeter. Results. The control (i.e., without MPS treatment) and MPS A–treated epithelial cells showed a normal, continuous linear pattern in ZO-1 staining along with cell–cell junctions. However, epithelial cells treated with MPS B, MPS C, and MPS D showed discontinuous, disrupted line structures at cell–cell borders. This may correspond to a partial breakdown of epithelial tight junctions. Treatment of epithelial monolayers with MPS B, MPS C, and MPS D caused a time-dependent decrease in transepithelial electrical resistance, whereas there was no significant difference between the MPS A–treated group and the control group. Conclusions. These results suggest the possibility that frequent use of a MPS with high cytotoxicity may lead to the breakdown of epithelial barrier functions and increase the risk of associated microbial infections in hydrogel lens wearers.

The 2005–2006 outbreak of Fusarium keratitis associated with soft hydrophilic contact lens wear was both unprecedented and unexpected. More than 250 cases have been reported worldwide that have primarily been associated with Bausch & Lomb ReNu with MoistureLoc and, more recently, with Bausch and Lomb ReNu MultiPlus multipurpose contact lens disinfecting solutions. This article documents the outbreak, presenting the time line and the historical and scientific basis for its occurrence. Underlying causes are explored including likely mechanisms of contamination and subsequent corneal infection. Thorough exploration of this unique occurrence affords the opportunity to examine contact lens and lens care actions and interactions and to develop greater understanding of possible associated risks and ways to minimize them.

Aim To evaluate the antibiofilm activity of 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 2% alexidine (ALX) and 0.2% cetrimide (CTR) alone and in combination on mature polymicrobial root canal biofilms on human dentine using confocal laser scanning microscopy (CLSM). Methodology Twenty-eight human dentine specimens were infected for 21 days with microbial samples collected from infected root canals of three volunteers. Antibiofilm activity of the irrigating solutions was evaluated after 3 min of contact time under CLSM. For quantification purposes, bioimage_L software was used. The variables evaluated were the log10 of total biovolume (μm3 ) and percentage of live cells (green) population. Statistical analysis of both variables was performed using an anova test and a post hoc Duncan test to determine significant clusters amongst groups. The variable green population percentage was previously subjected to the normalized Anscombe transformation. Results The NaOCl group had a total biovolume and percentage of live cells significantly lower than the other groups (P ˂ 0.001). The addition of 0.2% CTR significantly increased the antimicrobial effect of 2% CHX (P ˂ 0.001). There were no significant differences between 0.2% CTR, 2% ALX and the combination of both (P ˂ 0.05). Conclusions Overall, 2.5% NaOCl dissolved and killed bacteria significantly more efficiently when used against polymicrobial mature biofilm on human dentine. Cetrimide improved the antimicrobial activity of chlorhexidine and alexidine.

Purposes The purposes of this study were to determine whether the contact lens solution RevitaLens Ocutec (containing the antimicrobial agents alexidine and polyquaternium-1) would inhibit Fusarium organisms when heated in ReNu plastic bottles; whether alexidine would inhibit Fusarium organisms when heated in non-ReNu plastic bottles; and whether an alexidine-neutralizing compound leaches from heated ReNu bottles. Methods RevitaLens and an alexidine solution (0.00045%), previously stored in ReNu bottles at room temperature (RT) and 56°C, were incubated with 7 different Fusarium organisms. The alexidine solution was similarly stored in seven non-ReNu plastic bottles and incubated with these same organisms. To determine if an alexidine-neutralizing compound might be leaching from heated ReNu bottles, phosphate-buffered saline (PBS) was incubated at RT and 56°C in ReNu bottles, combined with alexidine, and then tested for anti-Fusarium capability. Results After being heated in ReNu bottles, RevitaLens retained its anti-Fusarium capability, whereas the alexidine solution did not. The alexidine solution heated in seven non-ReNu plastic bottles retained its anti-Fusarium capability. The alexidine solution retained its anti-Fusarium capability when incubated with a PBS solution that had been heated in ReNu bottles, indicating, microbiologically, that an alexidine-neutralizing compound did not leach from the heated ReNu bottle. Conclusions Alexidine uniquely fails to inhibit Fusarium organisms when heated in a plastic ReNu bottle, but not in seven other plastic bottles, whereas the anti-Fusarium capability of RevitaLens (containing the antimicrobial agents alexidine and polyquaternium-1) is unaffected by heating in a ReNu bottle. There does not seem to be an alexidine-neutralizing compound leaching from heated ReNu bottles. An interaction between alexidine and its heated ReNu bottle may have been a critical factor in the worldwide ReNu with MoistureLoc-related Fusarium keratitis event of 2004 to 2006.

Introduction The use of irrigating solutions with long-lived activity helps to avoid failure of endodontic therapy. The aim of the present study was to evaluate and compare the antimicrobial substantivity against Enterococcus faecalis by using as carrier a dentin-volumetric unit exposed to chlorhexidine (CHX) and alexidine (ALX). Methods Standardized dentin blocks of human molars were treated for 1 minute with 0.5% CHX, 2% CHX, 1% ALX, and 2% ALX. They were exposed to E. faecalis in suspension to determine antimicrobial substantivity during a period of 80 days. Results were analyzed by means of Kaplan-Meier survival analysis (P Results ALX obtained the best results, with differences that were significantly different from CHX regardless of the concentration. Although all the samples treated with CHX were contaminated at the end of the study period, a direct relationship was seen between concentration and survival time. There was no statistically significant difference between 2% and 1% ALX. Conclusions The present study shows that 2% and 1% ALX used for 1 minute provide longer antimicrobial substantivity against E. faecalis than CHX when applied to 2% and 0.5%.

Endodontic irrigating solutions may interact chemically with one another. This is important, because even when solutions are not admixed, they will come into contact with one another during an alternating irrigation technique, forming unwanted by-products, which may be toxic or irritant. Mixing or alternating irrigants can also reduce their ability to clean and disinfect the root canal system of teeth by changing their chemical structure with subsequent loss of the active agent, or by inducing precipitate formation in the root canal system. Precipitates occlude dental tubules, resulting in less penetration of antimicrobials and a loss of disinfection efficacy. Sodium hypochlorite is not only a very reactive oxidizing agent, but is also the most commonly used endodontic irrigant. As such, many interactions occurring between it and other irrigants, chelators and other antimicrobials, may occur. Of particular interest is the interaction between sodium hypochlorite and the chelators EDTA, citric acid and etidronate and between sodium hypochlorite and the antimicrobials chlorhexidine, alexidine, MTAD and octenisept.