The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Pharmacokinetics and Pharmacology of Latanoprost 0.005% without Benzalkonium Chloride Vs Latanoprost 0.005% with Benzalkonium Chloride in New Zealand White Rabbits and Beagles.

Purpose: Many intraocular pressure (IOP)-lowering medications contain benzalkonium chloride (BAK), a preservative associated with unfavorable outcomes.A formulation of latanoprost 0.005% ophthalmic without BAK is approved by the FDA and indicated for reduction of IOP in patients with open-angle glaucoma or ocular hypertension. We present two preclinical studies of latanoprost 0.005% BAK-free vs latanoprost with BAK; one examining plasma and ocular tissue pharmacokinetics (PK) in New Zealand white rabbits, and one comparing in vivo IOP-lowering efficacy in healthy beagles.Methods: In the PK study, one drop of treatment (latanoprost BAK-free or latanoprost with BAK) was instilled into both eyes of rabbits in each treatment group (n = 18). At 0.25, 0.5, 1, 4, 6, and 24 hours postdose, three rabbits per study group underwent terminal blood and tissue collection.In the IOP study, in the first dosing period, both eyes of each beagle received either 1 drop latanoprost BAK-free or latanoprost with BAK, once daily for 10 days. After a 10-day washout period, a second 10-day dosing period was conducted and latanoprost BAK-free or latanoprost with BAK were dosed in the opposite eyes, respectively. IOP measurements were taken at 1, 6, and 12 hours postdose.Results: The maximum plasma concentration for latanoprost BAK-free and latanoprost with BAK occurred 0.25 hours after administration (174.1 vs 217.2 pg/mL, respectively). Area under the concentration time curve from zero to infinity was highest in aqueous humor for latanoprost BAK-free and latanoprost with BAK (133.1 vs 119.6 hr·ng/mL, respectively) and was not estimable in vitreous humor. In beagles, once-daily administration of latanoprost BAK-free or latanoprost with BAK led to a significant reduction in IOP vs baseline (P < .001); there was no difference between groups (P > .05).Conclusions: Latanoprost BAK-free showed comparable activity in reducing IOP, and comparable plasma and ocular PK parameters to latanoprost with BAK.

Nanoemulsion as an Effective Treatment against Human-Pathogenic Fungi.

Infections triggered by pathogenic fungi cause a serious threat to the public health care system. In particular, an increase of antifungal drug-resistant fungi has resulted in difficulty in treatment. A limited variety of antifungal drugs available to treat patients has left us in a situation where we need to develop new therapeutic approaches that are less prone to development of resistance by pathogenic fungi. In this study, we demonstrate the efficacy of the nanoemulsion NB-201, which utilizes the surfactant benzalkonium chloride, against human-pathogenic fungi. We found that NB-201 exhibited in vitro activity against Candidaalbicans, including both planktonic growth and biofilms. Furthermore, treatments with NB-201 significantly reduced the fungal burden at the infection site and presented an enhanced healing process after subcutaneous infections by multidrug-resistant C. albicans in a murine host system. NB-201 also exhibited in vitro growth inhibition activity against other fungal pathogens, including Cryptococcus spp., Aspergillus fumigatus, and Mucorales Due to the nature of the activity of this nanoemulsion, there is a minimized chance of drug resistance developing, presenting a novel treatment to control fungal wound or skin infections.IMPORTANCE Advances in medicine have resulted in the discovery and implementation of treatments for human disease. While these recent advances have been beneficial, procedures such as solid-organ transplants and cancer treatments have left many patients in an immunocompromised state. Furthermore, the emergence of immunocompromising diseases such as HIV/AIDS or other immunosuppressive medical conditions have opened an opportunity for fungal infections to afflict patients globally. The development of drug resistance in human-pathogenic fungi and the limited array of antifungal drugs has left us in a scenario where we need to develop new therapeutic approaches to treat fungal infections that are less prone to the development of resistance by pathogenic fungi. The significance of our work lies in utilizing a novel nanoemulsion formulation to treat topical fungal infections while minimizing risks of drug resistance development.

Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments.

Salmonellosis, caused by the consumption of contaminated foods, is a major health problem worldwide. The aims of this study were to assess the susceptibility of Salmonella spp. isolates to benzalkonium chloride (BC) disinfectant and the antimicrobial activity of Butia odorata Barb. Rodr. extract against the same isolates from food and food environments. Moreover, phenotypic and genotypic resistance profiles, the presence of virulence genes and biofilm forming ability were determined. The minimum inhibitory concentration (MIC) of B. odorata extract against Salmonella spp. ranged from 10 to >19 mg.mL-1. Resistance to ampicillin, streptomycin, nalidixic acid, sulfonamide, trimethoprim/sulfamethoxazole, trimethoprim, tetracycline, and chloramphenicol was observed. In addition, multidrug resistance was observed in seven isolates (26.92%). The MIC of BC ranged from 32 to 64 mg.L-1, higher concentrations in comparison with wild-type MICs, and therefore were considered tolerant. Several resistance genes were detected, of which the most common were aadA, qacEΔ1, blaTEM, int1, sul1, and tetA. All isolates carried at least one virulence gene and produced biofilms on stainless steel surfaces at 10 and 22 °C. On the other hand, the B. odorata extract showed activity against Salmonella spp., and it has the potential to be used as a natural antimicrobial to control this important foodborne pathogen, despite its virulence potential and antimicrobial resistance profile.

Nanoemulsion is an effective antimicrobial for methicillin-resistant Staphylococcus aureus in infected wounds.

AIM: To develop NB-201, a nanoemulsion compound, as a novel microbicidal agent against methicillin-resistant Staphylococcus aureus (MRSA) infection, which is a common threat to public health but with limited therapeutic options. MATERIALS & METHODS: NB-201 was tested in in vitro and in vivo murine and porcine models infected with MRSA. RESULTS: Topical treatment of MRSA-infected wounds with NB-201 significantly decreased bacterial load and had no toxic effects on healthy skin tissues. NB-201 attenuated neutrophil sequestration in MRSA-infected wounds and inhibited epidermal and deep dermal inflammation. The levels of proinflammatory cytokines were reduced in NB-201-treated MRSA-infected wounds. CONCLUSION: NB-201 can greatly reduce inflammation characteristic of infected wounds and has antimicrobial activity that effectively kills MRSA regardless of the genetic basis of antibiotic resistance.

Effects and Effectiveness of Cavity Disinfectants in Operative Dentistry: A Literature Review.

The degree of success in the elimination of bacteria during cavity preparation and prior to the insertion of a restoration may increase the longevity of the restoration and therefore the success of the restorative procedure. The complete eradication of bacteria in a caries-affected tooth, during cavity preparation, is considered a difficult clinical task. In addition to weakening the tooth structure, attempts to excavate extensive carious tissue completely, by only mechanical procedures, may affect the vitality of the pulp. Therefore, disinfection of the cavity preparation after caries excavation can aid in the elimination of bacterial remnants that can be responsible for recurrent caries, postoperative sensitivity, and failure of the restoration. However, the effects of disinfectants on the restorative treatment have been a major concern for dental clinicians and researchers. This review aims to explore existing literature and provide information about different materials and techniques that have been used for disinfecting cavity preparations and their effects and effectiveness in operative dentistry and, therefore, helps dental practitioners with clinical decision to use cavity disinfectants during restorative procedures. Antimicrobial effectiveness and effects on the pulp and dental restorations, in addition to possible side effects, were all reviewed in this paper.

Screening of Nanoemulsion Formulations and Identification of NB-201 as an Effective Topical Antimicrobial for Staphylococcus aureus in a Mouse Model of Infected Wounds.

Despite advances in antimicrobial therapies, wound infection remains a global public health concern. We aimed to formulate and assess various nanoemulsions (NEs) for potential effectiveness as stable antimicrobial agents suitable for topic application. A total of 106 NEs were developed that varied with respect to nonionic and cationic surfactants. Stability testing demonstrated that the NEs tested are broadly stable, with 97/106 formulations passing 2-week stability tests. Two NEs, NB-201 and NB-402, were selected to test antimicrobial activity in a wound model in mice. Skin abrasion wounds were infected with Staphylococcus aureus followed by NE treatment. Infected skin was then evaluated by measuring colony forming units. NB-201 reduced median bacterial counts by 4 to 5 log compared to animals treated with saline, whereas NB-402 reduced bacterial counts by 2 to 3 log. Additional stability tests on NB-201 demonstrated that NB-201 is stable in the presence of human serum, and is stable for at least 6 months at 5°C, 25°C, and 40°C. Finally, in in vitro studies, NB-201 was found to be effective against S. aureus at a higher dilution than the commercially available silver sulfadiazine. Altogether these results demonstrate that NB-201 is a stable and effective topical antimicrobial for the treatment of S. aureus.

Nanoemulsion Therapy for Burn Wounds Is Effective as a Topical Antimicrobial Against Gram-Negative and Gram-Positive Bacteria.

The aim of this study is to investigate the antimicrobial efficacy of two different nanoemulsion (NE) formulations against Gram-positive and Gram-negative bacteria in an in vivo rodent scald burn model. Male Sprague-Dawley rats were anesthetized and received a partial-thickness scald burn. Eight hours after burn injury, the wound was inoculated with 1 × 10(8) colony-forming units of Pseudomonas aeruginosa or Staphylococcus aureus. Treatment groups consisted of two different NE formulations (NB-201 and NB-402), NE vehicle, or saline. Topical application of the treatment was performed at 16 and 24 hours after burn injury. Animals were killed 32 hours after burn injury, and skin samples were obtained for quantitative wound culture and determination of dermal inflammation markers. In a separate set of experiments, burn wound progression was measured histologically after 72 hours of treatment. Both NE formulations (NB-201 and NB-402) significantly reduced burn wound infections with either P. aeruginosa or S. aureus and decreased median bacterial counts at least three logs when compared with animals with saline applications (p < .0001). NB-201 and NB-402 also decreased dermal neutrophil recruitment and sequestration into the wound as measured by myeloperoxidase (MPO) assay and histopathology (p < .05). In addition, there was a decrease in the proinflammatory dermal cytokines (interleukin 1-beta [IL-1ß], IL-6, and tumor necrosis factor alpha [TNF-α]) and the neutrophil chemoattractants CXCL1 and CXCL2. Using histologic examination, it was found that both NB-201 and NB-402 appeared to suppress burn wound progression 72 hours after injury. Topically applied NB-201 and NB-402 are effective in decreasing Gram-positive and Gram-negative bacteria growth in burn wounds, reducing inflammation, and abrogating burn wound progression.

Performance of stress resistant variants of Listeria monocytogenes in mixed species biofilms with Lactobacillus plantarum.

Population diversity and the ability to adapt to changing environments allow Listeria monocytogenes to grow and survive under a wide range of environmental conditions. In this study, we aimed to evaluate the performance of a set of acid resistant L. monocytogenes variants in mixed-species biofilms with Lactobacillus plantarum as well as their benzalkonium chloride (BAC) resistance in these biofilms. L. monocytogenes LO28 wild type and acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Homolactic fermentation of glucose by L. plantarum created an acidic environment with pH values below the growth boundary of L. monocytogenes. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, indicating differences in adaptive behaviour between the variants. Two variants that showed a high level of acid adaptation when grown in liquid cultures, showed also better performance in mixed species biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. It can be concluded that acid resistant variants of L. monocytogenes show diversity in their adaptation to acidic conditions and their capacity to survive in mixed cultures and biofilms with L. plantarum.

Horizontal intracorneal swirling water migration indicative of corneal endothelial function.

PURPOSE: To test our hypothesis about whether there is water migration in the horizontal corneal plane and investigate its developmental mechanism. METHODS: A fluorescein solution was intrastromally injected into normal and edematous corneas of rabbits, and the movement of the fluorescein solution was observed and recorded over time. RESULTS: In normal corneas, the water flow was characterized by a swirling movement from the center to the periphery in the stroma. The fluorescein solution ultimately spread and occupied the entire cornea, indicating horizontal intracorneal swirling of water. In contrast, when the corneal endothelia were injured by intracameral injection of a preservative to create corneal edema, no water migration occurred, suggesting that the integrity of the corneal endothelial function is essential for water migration. The water migration stopped with injection of a sodium-potassium pump inhibitor, indicating that the enzyme is necessary for physiologic water migration in the cornea. With recovery of corneal endothelial function, the water migration began, and focal edema remained in the periphery with no water migration in this edematous area. CONCLUSIONS: We report for the first time the presence of horizontal water migration in the cornea in a swirling pattern (i.e., intracorneal swirling migration of water, generated by the pump function in the corneal endothelial cells), which may supplement the conventional concept of development of corneal edema in the vertical plane. This dynamic water circulatory system may be involved in increasing the efficiency of the water transfer in the entire cornea.

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

Aliphatic ß-nitroalcohols for therapeutic corneoscleral cross-linking: corneal permeability considerations.

PURPOSE: Our recent tissue cross-linking studies have raised the possibility of using aliphatic ß-nitroalcohols (BNAs) for pharmacologic, therapeutic corneal cross-linking. The present study was performed to determine the permeability of BNAs and to explore the use of permeability-enhancing agents. METHODS: Ex vivo rabbit corneas were mounted in a typical Franz diffusion chamber. BNA permeability was determined by assaying the recipient chamber over time using a modification of the Griess nitrite colorimetric assay. The apparent permeability coefficient (Ptot) was determined for 2 mono-nitroalcohols [2-nitroethanol (2ne) and 2-nitro-1-propanol (2nprop)], a nitrodiol [2-methyl-2-nitro-1,3-propanediol (MNPD)], and a nitrotriol [2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD)]. Permeability-enhancing effects using benzalkonium chloride (BAC) (0.01% and 0.02%), ethylenediaminetetraacetic acid (0.05%), and a combination of 0.01% BAC + 0.5% tetracaine were also studied. RESULTS: The Ptot (±SE) values (in centimeters per second) were as follows: 4.33 × 10 (±9.82 × 10) for 2ne [molecular weight (MW) = 91 Da], 9.34 × 10 (±2.16 × 10) for 2nprop (MW = 105 Da), 4.37 × 10 (±1.86 × 10) for MNPD (MW = 135 Da), and 8.95 × 10 (±1.93 × 10) for HNPD (MW = 151 Da). Using the nitrodiol, permeability increased approximately 2-fold using 0.01% BAC, 5-fold using 0.02% BAC, and 5-fold using the combination of 0.01% BAC + 0.5% tetracaine. No effect was observed using 0.05% ethylenediaminetetraacetic acid. CONCLUSIONS: The results indicate that the corneal epithelium is permeable to BNAs, with the apparent permeability corresponding to MW. The findings are consistent with previous literature indicating that the small size of these compounds (<10Å) favors their passage through the corneal epithelium via the paracellular route. This information will help to guide dosing regimens for in vivo topical cross-linking studies.

Effectiveness of ophthalmic solution preservatives: a comparison of latanoprost with 0.02% benzalkonium chloride and travoprost with the sofZia preservative system.

BACKGROUND: Although in vitro and in vivo laboratory studies have suggested that benzalkonium chloride (BAK) in topical ophthalmic solutions may be detrimental to corneal epithelial cells, multiple short- and long-term clinical studies have provided evidence supporting the safety of BAK. Despite the conflicting evidence, BAK is the most commonly used preservative in ophthalmic products largely due to its proven antimicrobial efficacy. This study was designed to characterize the antimicrobial performance of two commonly used topical ocular hypotensive agents that employ different preservative systems: latanoprost 0.005% with 0.02% BAK and travoprost 0.004% with sofZia, a proprietary ionic buffer system. METHODS: Each product was tested for antimicrobial effectiveness by European Pharmacopoeia A (EP-A) standards, the most stringent standards of the three major compendia, which specify two early sampling time points (6 and 24 hours) not required by the United States Pharmacopeia or Japanese Pharmacopoeia. Aliquots were inoculated with between 10(5) and 10(6) colony-forming units of the test organisms: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis. Sampling and enumeration were conducted at protocol-defined time points through 28 days. RESULTS: BAK-containing latanoprost met EP-A criteria by immediately reducing all bacterial challenge organisms to the test sensitivity and fungal challenges within the first six hours while the preservative activity of travoprost with sofZia did not. Complete bacterial reduction by travoprost with sofZia was not shown until seven days into the test, and fungal reduction never exceeded the requisite 2 logs during the 28-day test. Travoprost with sofZia also did not meet EP-B criteria due to its limited effectiveness against Staphylococcus aureus. Both products satisfied United States and Japanese pharmacopoeial criteria. CONCLUSIONS: Latanoprost with 0.02% BAK exhibited more effective microbial protection than travoprost with sofZia using rates of microbial reduction, time to no recovery for all challenges and evaluation against EP-A criteria as measures. The rapid and complete reduction of all microbial challenges demonstrates that antimicrobial activity of latanoprost with 0.02% BAK exceeds that of travoprost with sofZia preservative system in these products and provides a more protective environment in the event of contamination and subsequent exposure to microorganisms during use.

Effect of chitosan, benzalkonium chloride and ethylenediaminetetraacetic acid on permeation of acyclovir across isolated rabbit cornea.

The overall objective of this study was to evaluate the effect of chitosan, benzalkonium chloride (BAK) and disodium ethylendiaminetetraacetic acid (EDTA), alone and in combination, on permeation of acyclovir (ACV) across excised rabbit cornea. Corneas of male New Zealand White rabbits were used in these studies. Transcorneal permeation studies were conducted at 34 degrees C using a side-bi-side diffusion apparatus. In the presence of 0.01% BAK, transcorneal permeability of ACV was observed to increase almost 10.5-fold, from 3.5x10(-6) to 37.4x10(-6)cm/s. At 0.005% BAK, permeability of ACV was almost 3-fold higher than control. Combination of BAK 0.005% and EDTA 0.01% increased transcorneal penetration of ACV by 2.5-fold. Chitosan 0.2 and 0.1% increased corneal permeability of ACV by 5.8- and 3.1-fold, respectively, whereas, at 0.02%, chitosan did not exhibit a statistically significant effect. BAK at 0.005%, in combination with 0.01% EDTA and 0.1% chitosan, increased transcorneal ACV permeation by 5.5-fold. This study suggests that a judicious combination of chitosan, BAK and EDTA can lead to a significant increase in ACV's transcorneal permeability and that chitosan can enhance diffusion of hydrophilic agents across the corneal membrane. Further in vivo evaluation is warranted.

[Estimation of activity of pharmakopeal disinfectants and antiseptics against Gram-negative and Gram-positive bacteria isolated from clinical specimens, drugs and environment]. / Wrazliwosc wybranych szczepów bakterii Gram-ujemnych i Gram-dodatnich izolowanych z materialu klinicznego, leków i srodowiska, na farmakopealne zwiazki dezynfekcyjne i antyseptyczne.

The MIC of nine different disinfectants and antiseptics were determined for the Gram-negative and Gram-positive bacteria. Strains originated from clinical specimens, drugs and environment. A sensitivity was determined against chlorhexidinum digluconate (Gram-negative: 0,625-80 mg/L, Gram-positive: 0,3-10 mg/L), benzalconium chloride (Gram-negative: 2,5-1280 mg/L, Gram-positive: 1,25-20 mg/L), salicilic acid (Gram-negative and Gram-positive: 400-1600 mg/L), benzoic acid (Gram-negative: 800-1600 mg/L, Gram-positive: 400-1 600 mg/L), boric acid (Gram-negative: 800-12 800 mg/L, Gram-positive: 1 600-6400 mg/L), chloramine B (Gram-negative: 1600-6400 mg/L, Gram-positive:800- 6400 mg/L), jodine (Gram-negative: 200-1600 mg/L, Gram-positive: 200-1600 mg/L), etacridine lactate (Gram-negative: 40 do > 20480 mg/L, Gram-positive: 40-1280 mg/L) and resorcine (Gram-negative: 1600-6400 mg/L, Gram-positive: 800-6400 mg/L). Diversified values of MIC for different strains were obtained, especially in the case of benzalconium chloride, etacridine lactate, chlorhexidinum digluconate, boric acid and iodine. Strains isolated from environment were usually more susceptible to examined compounds than clinical strains. The biggest diversification of sensitivity was observed among strains originated from drugs where besides sensitive appeared strains characterizing by very high MIC values of some substances, eg. boric acid.

[In vitro effects of antiallergic eyedrops on complement activation induced by particulate matter]. / Effets modulateurs de collyres anti-allergiques sur l'activation du complément induite in vitro par des polluants particulaires.

BACKGROUND: Recent decades have been marked by an increasing number of patients suffering from ocular allergic-like symptoms without being associated with an increase in IgE levels. These symptoms include heaviness of the lid, foreign body sensation, burning, stinging and photophobia. Both epidemiological studies and controlled human exposure clinical studies have shown cause-effect relationships between allergic-like symptoms and environmental factors such as outdoor air pollutants or poor indoor air quality. An ocular surface subclinical inflammation is thought to be responsible for pseudoallergic, pollution-related conjunctivitis. The complement system is considered as one of the major effector mechanisms involved in initiation of the subclinical inflammation that leads to IgE-independent eye irritation. PURPOSE: To study the capability of nine antiallergic eyedrops commonly used in the treatment of allergic conjunctivitis to inhibit complement activation induced in vitro by pollutants. METHODS: Normal human serum obtained from healthy individuals was used as a source of complement. Activation of complement was assessed using the complement hemolytic 50% (CH50) assay, in the absence or the presence of antiallergic eyedrops and in the absence or the presence of various stimuli, including sand, common house dust, eye mascara, and Dactylis glomerata pollen extract. Zymosan was used as a standardized complement activator. The following eyedrops were studied: Naabak (4.9% N-acetyl aspartic acid-glutamic acid, NAAGA, sodium salt), Almide (lodoxamide 0.1%), Levophta (0.05% levocabastine), Emadine (0.05% emedastine), Tilavist (2% nedocromil), Allergodil (0.05% azelastine), Patanol (olopatadine), and Zaditen (0.025% ketotifen). Effects of preservative-free lodoxamide and ketotifen were also assessed and compared to those of the preserved formulations. A solution of 0.01% benzalkonium chloride (BAC), the most widely used preservative in topical eyedrops, was also tested. RESULTS: Zymosan-induced activation of complement (30+/-6%) was significantly lowered by preincubation of serum with unpreserved NAAGA (16.6+/-4%, p=0.0026) or benzalkonium-preserved nedocromil (20+/-2%, p=0.022). Preserved levocabastine, emedastine, olopatadine and ketotifen did not interfere with zymosan-induced complement activation, whereas preserved azelastine, lodoxamide and benzalkonium chloride significantly aggravated complement activation induced by zymosan. Similar results were obtained when complement activation was triggered by sand, common house dust, mascara, or by an allergenic extract of Dactylis glomerata pollen. In the absence of complement activator, none of the antiallergic eyedrops induced a significant change in CH50 titer, indicating that the deleterious pro-inflammatory effect of preserved azelastine and lodoxamide may occur only once complement activation has been initiated, i.e., on an inflamed ocular surface. CONCLUSION: Among the antiallergic eyedrops tested in this study, only Naabak and Tilavist were found to significantly inhibit complement activation triggered by particulate matters or pollen allergenic extract. Such an anticomplement activity confers these two molecules a potential in the therapeutic management of pollution-related pseudoallergic conjunctivitis.

Human lens epithelial cell damage and stimulation of their secretion of chemical mediators by benzalkonium chloride rather than latanoprost and timolol.

OBJECTIVE: To investigate the effects of latanoprost, timolol maleate, and benzalkonium chloride on cell damage and induction of the secretion of chemical mediators of stress and wound healing by human lens epithelial cells in culture. METHODS: Cells from a human lens epithelial cell line (SRA01/04) were cultured in Dulbecco minimum essential medium supplemented with 5% fetal bovine serum. The amounts of latanoprost (50 micro g/mL), timolol maleate (5 mg/mL), or benzalkonium chloride (200 micro g/mL) used in eyedrops, and x10 to x1000 dilutions thereof, were added to the medium. After 7 days' culture, cell morphological changes were assessed using phase-contrast microscopy, and cell-free culture supernatants were collected for prostaglandin E2 (PGE2), interleukin 1alpha (IL-1alpha), and interleukin 6 (IL-6) iodine I 125 radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent enzyme immunoassay, respectively. RESULTS: All cells that were cultured with the concentrations of latanoprost, timolol, or benzalkonium chloride used in eyedrops detached from the culture dish and died within 3 days. At a x10 dilution of latanoprost or timolol or a x100 dilution of benzalkonium chloride, no proliferation or elongation of the cells was observed. Secretions of PGE2, IL-1alpha, and IL-6 at x10 dilutions of latanoprost or timolol were 3 to 77 times higher than in controls, whereas they were 190 to 305 times higher at a x180 dilution of benzalkonium chloride. The amounts of these soluble mediators in culture supernatants depended on the dose of latanoprost, timolol, or benzalkonium chloride added. CONCLUSION: Our results indicate that benzalkonium chloride, used as the preservative in eyedrops containing latanoprost or timolol, is the agent most damaging to lens epithelial cells and most strongly stimulates the expression of soluble chemical mediators in these cells.

Treatment of plastic and extracellular matrix components with chlorhexidine or benzalkonium chloride: effect on Candida albicans adherence capacity in vitro.

This study investigates the influence of treatment of plastic and extracellular matrix (ECM) proteins with chlorhexidine or benzalkonium chloride on subsequent adherence of Candida albicans. Three concentrations were tested for each antiseptic: (i) chlorhexidine, MIC (6.25-12.5 mg/L), 80 x MIC and 800 x MIC; and (ii) benzalkonium chloride, MIC (3.12 mg/L), 40 x MIC and 1600 x MIC. Chlorhexidine and benzalkonium chloride activities were correlated with the tested concentrations. Antiseptics used at MIC were unable to modify the adherence to plastic or ECM proteins. Chlorhexidine (80 x MIC) induced a decrease in plastic adherence of 31% of the 15 strains used and an increase in ECM protein adherence of 13% of strains. Benzalkonium chloride (40 x MIC) induced a decrease in adherence to ECM proteins or plastic of 13-27% of strains. Our results indicated that the treatment with 1600 x MIC benzalkonium chloride could induce the opposite effect on adherence, depending on the surface: 60% of the strains showed an increase in their adherence to ECM proteins, whereas 93% of the strains showed a decrease in their adherence to plastic. A similar phenomenon was observed after treatment with 800 x MIC chlorhexidine: 60% of the strains showed an increase in their adherence to ECM proteins, whereas 67% showed a decrease in adherence to plastic. Treatment of medical devices with at least 5000 mg/L of chlorhexidine or benzalkonium chloride could therefore reduce C. albicans adherence to plastic surfaces, but would be unable to prevent fungal adherence to ECM proteins.

Antimicrobial efficacy of biocides tested on skin using an ex-vivo test.

An ex-vivo test was used to evaluate the activity of antimicrobials against three microorganisms, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The ex-vivo test is a carrier test using freshly excised animal skin samples maintained in viable conditions for a short period of time. Skin samples came from a veterinary practice and were excised from either dogs or cats. The antimicrobial activity of povidone iodine, chlorhexidine diacetate, cetrimide and benzalkonium chloride was also evaluated with suspension and glass-carrier tests. Generally, the activity of the antimicrobials tested was reduced when applied to the skin surface. Apart from povidone iodine (2%) against S. aureus, the biocides investigated failed to achieve a 5 log10 reduction in bacterial titre when tested with the ex-vivo method. There was no significant difference in reduction of bacterial titres after treatment with antimicrobials between the glass-carrier and the suspension tests. Furthermore, the drying process of bacterial inoculum was less detrimental on skin than on glass surfaces. This study confirmed that the activity of a biocide tested in suspension or on an inanimate surface did not reflect its activity when tested on skin. Further development of the ex-vivo test may be useful, especially for testing the antimicrobial activity of formulations with antiseptic properties.

Factors influencing a suspension test method for antimicrobial activity of disinfectants.

Factors influencing the numbers of Escherichia coli DSM 682 and Staphylococcus aureus ATCC 6538 surviving exposure to disinfectants were evaluated by factorial design. Aerobic conditions during pre-cultivation rendered E. coli more resistant to the lethal activity of benzalkonium chloride (BC) and a disinfectant containing grape fruit extract (GSE), whereas Staph. aureus became more sensitive. The degree of shaking and the pre-growth medium (tryptone soy broth or Mueller-Hinton broth) did not influence the result of the bactericidal test. The number of E. coli surviving BC treatment was significantly lower if the neutralizing broth contained thiosulphate, plate pouring was used instead of plate spreading, or the plates were incubated at 37 instead of 30 degrees C. The negative effect of plate pouring was also found with Staph. aureus. The use of filtration without prior neutralization of the disinfectant decreased the numbers of chlorine-treated, but not BC-treated, E. coli. The results showed that rigorous standardization is necessary to obtain good reproducibility of bactericidal suspension tests.