Pharmaceutical Properties of a Tinted Formulation of a Biguanide Antiseptic Agent, Olanexidine Gluconate.

Olanexidine gluconate-containing preoperative antiseptic (OLG-C) is colorless, which makes it difficult to determine its area of application. To overcome this drawback, we realized a stable orange-tinted antiseptic (OLG-T) by adding new additives to OLG-C and investigated its pharmaceutical properties compared with OLG-C and povidone iodine (PVP-I). We evaluated the influence of the additives on the antimicrobial activity and adhesiveness of medical adhesives to OLG-T-applied skin by in vitro time-kill/ex vivo micropig skin assays and a peel test using excised micropig skin, respectively. In the in vitro time-kill assay, the bactericidal/fungicidal activity of OLG-T and OLG-C were equivalent. In the ex vivo micropig skin assay, their fast-acting and persistent bactericidal activities against vancomycin-resistant Enterococcus faecalis were higher than that of PVP-I. In the peel test, the adhesion force of the incise drape and the amount of stripped corneocytes on the peeled drape were comparable between OLG-T- and OLG-C-applied skin, but both were less than those of PVP-I-applied skin. The drapes for OLG-T- and OLG-C-applied skin had moderate adhesion force, and the drape-related injuries were expected to be weak. These results suggest that OLG-T performs no worse than OLG-C in terms of its antimicrobial activity and medical adhesive compatibility. Therefore, we expect OLG-T to lead to more convenient preoperative skin preparation and further contribute to lowering surgical site infection rates.

Evaluation of in Vitro Bactericidal Activity of 1.5% Olanexidine Gluconate, a Novel Biguanide Antiseptic Agent.

Recently, 1.5% olanexidine gluconate, a biguanide compounds, was launched as a new antiseptic agent in Japan. However, the comprehensive bactericidal spectrum of olanexidine gluconate is still unknown. In this study, we evaluated in vitro bactericidal activity of olanexidine gluconate using time-kill assay against various bacteria, mycobacteria, and fungi. With the exception of Burkholderia cepacia and Mycobacterium spp., 1.5% olanexidine gluconate exhibited fast-acting (≤60 s) bactericidal activity against all tested Gram-positive and Gram-negative bacteria, including vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended spectrum ß-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Pseudomonas aeruginosa. Furthermore, 1.5% olanexidine gluconate eradicated Candida albicans, Microsporum canis, and Malassezia furfur within 3 min. Our findings indicate that olanexidine gluconate has broad spectrum bactericidal activity; therefore, it may be useful for the prevention of a wide range of infectious diseases.

Bactericidal Effects and Mechanism of Action of Olanexidine Gluconate, a New Antiseptic.

Olanexidine gluconate [1-(3,4-dichlorobenzyl)-5-octylbiguanide gluconate] (development code OPB-2045G) is a new monobiguanide compound with bactericidal activity. In this study, we assessed its spectrum of bactericidal activity and mechanism of action. The minimal bactericidal concentrations of the compound for 30-, 60-, and 180-s exposures were determined with the microdilution method using a neutralizer against 320 bacterial strains from culture collections and clinical isolates. Based on the results, the estimated bactericidal olanexidine concentrations with 180-s exposures were 869 µg/ml for Gram-positive cocci (155 strains), 109 µg/ml for Gram-positive bacilli (29 strains), and 434 µg/ml for Gram-negative bacteria (136 strains). Olanexidine was active against a wide range of bacteria, especially Gram-positive cocci, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and had a spectrum of bactericidal activity comparable to that of commercial antiseptics, such as chlorhexidine and povidone-iodine. In vitro experiments exploring its mechanism of action indicated that olanexidine (i) interacts with the bacterial surface molecules, such as lipopolysaccharide and lipoteichoic acid, (ii) disrupts the cell membranes of liposomes, which are artificial bacterial membrane models, (iii) enhances the membrane permeability of Escherichia coli, (iv) disrupts the membrane integrity of S. aureus, and (v) denatures proteins at relatively high concentrations (≥160 µg/ml). These results indicate that olanexidine probably binds to the cell membrane, disrupts membrane integrity, and its bacteriostatic and bactericidal effects are caused by irreversible leakage of intracellular components. At relatively high concentrations, olanexidine aggregates cells by denaturing proteins. This mechanism differs slightly from that of a similar biguanide compound, chlorhexidine.

The effects of n-3 polyunsaturated fatty acid-rich total parenteral nutrition on neutrophil apoptosis in a rat endotoxemia.

Although recruited neutrophils function as first-line defense to remove bacteria, delayed apoptosis is implicated in persistent inflammation leading to organ injury. Leukotrien B4, n-6 polyunsaturated fatty acids (PUFAs) product, is one of the mediators that delay neutrophil apoptosis. The mechanism of the beneficial effects of supplementation of fish oil-based long-chain n-3 PUFAs in parenteral nutrition for critically ill patients has not been fully understood. One possible mechanism is the less inflammatory n-3 PUFAs products can compete with proinflammatory n-6 PUFAs products for access to the enzymes. The aim of this study was to determine whether n-3 PUFA rich parenteral nutrition may alter the composition of fatty acids in the neutrophil membrane and restore delay of neutrophil apoptosis during endotoxin-induced systemic inflammation in rats. The animals in group 1 were treated with 20% Hicaliq NC-N in Neoparen-2 for three days. The animals in group 2 (referred to as n-6 PUFA-rich parenteral nutrition) were given parenteral nutrition solutions containing 20% soybean oil in Neoparen-2 (n-6/n-3 = 10). The animals in group 3 (referred to as n-3 PUFA-rich parenteral nutrition) were administered parenteral nutrition consisting of 10% soybean oil and 10% fish oil emulsion (n-6/n-3 = 1.3). The n-3/n-6 ratio of the neutrophil membrane was significantly increased in group 3 and was associated with restored lipopolysaccharide-delayed-apoptosis of neutrophils in bone marrow cells and increased production of leukotriene B5 from peritoneal neutrophils stimulated by lipopolysaccharide. Our preliminary results showed that n-3 PUFA-rich parenteral nutrition regulated neutrophil apoptosis and prevented synthesis of pro-inflammatory eicosanoids, explaining the protective effects seen in the clinical setting.

Disinfection efficacy and mechanism of olanexidine gluconate against norovirus.

BACKGROUND: The purpose of this study was to evaluate the virucidal activity of a new olanexidine-containing formulation for hand hygiene (olanexidine gluconate hand rub; OLG-HR) against non-enveloped viruses and to understand its mechanism of action. METHODS: The virucidal activities of OLG-HR against two strains of caliciviruses and three adenovirus serotypes were evaluated through suspension tests. Also, virus-like particles were used to predict the effect of olanexidine gluconate on virus particle structure. RESULTS: The results of suspension tests under conditions with and without interfering substances (1.5% BSA) indicated that OLG-HR had a broad-spectrum effect against non-enveloped viruses, and the virucidal effect was unaffected by organic contaminants. Furthermore, olanexidine inhibited the binding ability of virus-like particles to the binding receptor of human norovirus and increased the aggregation of virus-like particles in a dose-dependent manner. Transmission electron microscopy showed that the morphology of the virus-like particles was affected by exposure to olanexidine, indicating that the protein-denaturing effect of olanexidine gluconate caused the loss of receptor-binding capability of the viral capsid protein. CONCLUSIONS: This study suggests that olanexidine gluconate is a potential biological and environmental disinfectant against norovirus and adenovirus.

Antiviral Activity of Olanexidine-Containing Hand Rub against Human Noroviruses.

Human norovirus (HuNoV) is the leading cause of epidemic and sporadic acute gastroenteritis worldwide. HuNoV transmission occurs predominantly by direct person-to-person contact, and its health burden is associated with poor hand hygiene and a lack of effective antiseptics and disinfectants. Specific therapies and methods to prevent and control HuNoV spread previously were difficult to evaluate because of the lack of a cell culture system to propagate infectious virus. This barrier has been overcome with the successful cultivation of HuNoV in nontransformed human intestinal enteroids (HIEs). Here, we report using the HIE cultivation system to evaluate the virucidal efficacy of an olanexidine gluconate-based hand rub (OLG-HR) and 70% ethanol (EtOH70%) against HuNoVs. OLG-HR exhibited fast-acting virucidal activity against a spectrum of HuNoVs including GII.4 Sydney[P31], GII.4 Den Haag[P4], GII.4 New Orleans[P4], GII.3[P21], GII.17[P13], and GI.1[P1] strains. Exposure of HuNoV to OLG-HR for 30 to 60 s resulted in complete loss of the ability of virus to bind to the cells and reduced in vitro binding to glycans in porcine gastric mucin. By contrast, the virucidal efficiency of EtOH70% on virus infectivity was strain specific. Dynamic light scattering (DLS) and electron microscopy of virus-like particles (VLPs) show that OLG-HR treatment causes partial disassembly and possibly conformational changes in VP1, interfering with histo-blood group antigen (HBGA) binding and infectivity, whereas EtOH70% treatment causes particle disassembly and clumping of the disassembled products, leading to loss of infectivity while retaining HBGA binding. The highly effective inactivation of HuNoV infectivity by OLG-HR suggests that this compound could reduce HuNoV transmission. IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause nonbacterial acute gastroenteritis in all age groups worldwide. Since the introduction of rotavirus vaccines, HuNoVs have become the leading cause of diarrheal illness in children. These viruses are very stable in the environment and resistant to common disinfectants. This study evaluated the virucidal efficacy of a new disinfectant, olanexidine-based hand rub (OLG-HR), against HuNoV strains in an ex vivo human intestinal stem cell-derived enteroid (HIE) cultivation system. Exposure of multiple HuNoV strains to OLG-HR for 30 to 60 s resulted in complete loss of infectivity and binding to HBGAs, possibly due to partial disassembly and conformational changes in the major virus capsid (VP1). By comparison, the virucidal efficiency of EtOH70% was strain specific, leading to loss of infectivity while retaining HBGA binding. These findings show the utility of the ex vivo HIE cultivation system to test the effectiveness of disinfectants and report a highly effective product.

Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus.

Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus infection yet, preventive healthcare interventions to block the infection routes play an important role in infection control. One of the possible infection routes of human noroviruses are through contaminated hands, but no hand antiseptics have been proven effective. Olanexidine gluconate is a new biguanide compound that has already been approved for sale as an antiseptic for the surgical field in Japan. A new hand antiseptic was developed using olanexidine gluconate in this study, and its virucidal efficacy against human noroviruses was evaluated using modified RT-qPCR that can account for genome derived from intact viruses using RNase A and photo-reactive intercalators. We tested the virucidal efficacy of five materials; two olanexidine gluconate antiseptics (hand rub formulation and surgical field formulation), two kinds of ethanol solutions at different pH (approx. 3 or 7), and a base component of olanexidine gluconate hand rub formulation against 11 human norovirus genotypes by culture-independent methods. The infectivity of murine norovirus (MNV), a surrogate for human norovirus, was significantly reduced after use of the antiseptics. The olanexidine gluconate hand rub demonstrated the strongest virucidal efficacy against human norovirus among the five tested materials. This study showed that olanexidine gluconate has the potential to become a strong tool for the prevention of human norovirus infection.

Evaluation of fast-acting bactericidal activity and substantivity of an antiseptic agent, olanexidine gluconate, using an ex vivo skin model.

PURPOSE: We assessed the fast-acting bactericidal activity and substantivity of olanexidine gluconate (OLG) to investigate its remaining bactericidal activity on the skin after rinsing and drying by using an ex vivo Yucatan micropig (YMP) skin model. METHODOLOGY: The fast-acting bactericidal activity was evaluated in pigskin models inoculated with methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, vancomycin-resistant Enterococcus faecalis (VRE), Acinetobacter baumannii, Corynebacterium minutissimum and Cutibacterium acnes. To evaluate substantivity, the YMP skin piece first had 1.5 % OLG, chlorhexidine gluconate (CHG) formulations or 10 % povidone-iodine (PVP-I) applied to it, and was then rinsed with distilled water, incubated for 4, 6, 8 or 12 h and inoculated with the test bacteria (MRSA, S. epidermidis and VRE). The viable bacteria remaining at 1 min of exposure of bacteria were counted to measure the quantity of antiseptic molecules retaining bactericidal activity. To determine the factors contributing to the substantivity, the stratum corneum (SC) of the YMP skin that had had OLG or CHG applied to it was exfoliated using a tape-stripping method and the amount of antiseptic was quantitated. RESULTS: OLG showed a fast-acting bactericidal activity that was similar to or stronger than that of CHG formulations up to a concentration of 1 % and PVP-I with a short exposure time of 30 s, and substantivity until 12 h after rinsing, whereas the other antiseptics hardly showed any substantivity. There was 2.8 times or more OLG in the SC than CHG. CONCLUSION: OLG has fast-acting activity and substantivity, which are required properties for an antiseptic, and is useful for preventing infections.

Effects of a fish oil-based emulsion on rat hepatoma cell invasion in culture.

OBJECTIVE: Total parenteral nutrition containing a lipid emulsion is often employed after surgical tumor resection. This study investigated the effects of a fish oil-based infusion on rat hepatoma cell invasion. METHODS: Rat ascites hepatoma cell line AH109A was precultured with a fish oil-based or safflower oil-based emulsion for 48 h. Changes in membranous fatty acid composition were evaluated by gas chromatography. The invasiveness of hepatoma cells was assessed by coculturing with mesentery-derived mesothelial cells. To examine ex vivo effects of the fish oil-based infusion on hepatoma invasion, sera were prepared from rats infused with fish oil- or safflower oil-based emulsion and the effects of these sera were assessed. To clarify the mechanism of inhibition of invasion by the fish oil-based emulsion, the effects of prostaglandin (PG) E(2) and PGE(3) on invasion were examined. RESULTS: Pretreatment with the fish oil-based emulsion reduced invasiveness without affecting growth compared with the safflower oil-based emulsion. Pretreatment with the sera from rats infused with the fish oil-based emulsion also reduced invasiveness compared with the sera from rats infused with the safflower oil-based emulsion. The addition of PGE(2) eliminated the inhibitory effect of the fish oil-based emulsion, and the addition of PGE(3) reduced the invasiveness of hepatoma cells pretreated with the safflower oil-based emulsion. CONCLUSION: These results suggest that the fish oil-based emulsion may have anti-invasive effects. Changes in the membranous fatty acid composition and consequent changes in the prostaglandins produced may be involved in this inhibitory effect.

Effects of olanexidine gluconate on preoperative skin preparation: an experimental study in cynomolgus monkeys.

PURPOSE: To determine the bactericidal efficacy of a new topical antiseptic for preoperative skin preparation, olanexidine gluconate (development code: OPB-2045G), against transient or resident bacterial flora on the skin of cynomolgus monkeys. METHODOLOGY: After measuring baseline bacterial counts on test sites marked on the abdomens, we applied olanexidine, chlorhexidine or povidone-iodine. After 10 min (fast-acting effect) and 6 h (long-lasting effect), bacterial counts were measured again and log10 reductions were calculated. In addition, we determined the bactericidal effects on the skin contaminated with blood before or after applying the antiseptics. RESULTS: In the non-blood-contaminated condition, the mean log10 reductions of olanexidine at doses of 1-2 % were significantly higher than those of saline (negative control), but did not significantly differ from those of 0.5 % chlorhexidine and 10 % povidone-iodine at either time point. But olanexidine was significantly more effective at both time points than chlorhexidine and povidone-iodine when applied after the site was contaminated with blood. Olanexidine was also significantly more effective than chlorhexidine and as effective as or more effective than povidone-iodine at both time points when skin was contaminated with blood after the antiseptics were applied. CONCLUSION: The bactericidal effects of olanexidine were comparable to those of commercial antiseptics such as chlorhexidine and povidone-iodine in non-blood-contaminated conditions. More importantly, the effect of olanexidine was hardly affected by blood unlike commercial antiseptics. Thus, it is considered that olanexidine has a favourable property for skin preparation in various types of surgical treatments.

Novel antiseptic compound OPB-2045G shows potent bactericidal activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus both in vitro and in vivo: a pilot study in animals.

There is a need for new compounds to effectively treat methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The novel monobiguanide compound 1-(3,4-dichlorobenzyl)-5-octylbiguanide gluconate (OPB-2045G) has potential bactericidal activity. We sought to elucidate the potency of OPB-2045G bactericidal activity against MRSA and VRE compared to those of chlorhexidine digluconate (CHG) and povidone iodine (PVP-I). In vitro bactericidal activity was analysed using minimum bactericidal concentration (MBC) as the index. The in vivo bactericidal efficacy of OPB-2045G was examined by determining MRSA and VRE contamination of the normal dorsal skin of mice following removal of hair. After a 3 min treatment period, the MBC of OPB-2045G was lower than that of CHG and PVP-I against standard strains and clinical isolates. Additionally, in our in vivo mouse model, the in vivo bactericidal activity of 1.5 % OPB-2045G (a clinically relevant dose) was higher than that of 0.5 % CHG and equivalent to that of 10 % PVP-I against MRSA. Similarly, the in vivo bactericidal activity of OPB-2045G was higher than that of 0.5 % CHG and 10 % PVP-I against VRE. OPB-2045G showed more potent bactericidal activity against MRSA and VRE both in vitro and in vivo compared to CHG and PVP-I, indicating that OPB-2045G may provide better protection against health care-associated infections caused by these pathogens.

Effects of the omega-6:omega-3 fatty acid ratio of fat emulsions on the fatty acid composition in cell membranes and the anti-inflammatory action.

BACKGROUND: This study investigated the effects of parenterally administered fish oil (FO) on the fatty acid composition in rats to determine the optimal omega-6:omega-3 polyunsaturated fatty acid (PUFA) ratio of fat emulsions to achieve an anti-inflammatory effect. METHODS: Male Sprague-Dawley rats were infused a parenteral nutrition (PN) solution containing fat emulsions with different omega-6:omega-3 PUFA ratios. The fatty acid content of phospholipids in the membranes of splenocytes was analyzed by gas chromatography (experiment 1). In addition, the amounts of leukotriene (LT) B(4) and LTB(5) released from peritoneal polymorphonuclear leukocytes (PMNs) were measured by high-performance liquid chromatography (experiment 2). RESULTS: In experiment 1, after infusion of the fat emulsion containing FO, the omega-3 PUFA content in cell membranes rose to 70% of the peak value on day 1 and nearly reached a plateau on day 3. The highest ratio of eicosapentaenoic acid (EPA) to arachidonic acid (AA) was achieved by administering a PN solution with the smallest omega-6:omega-3 PUFA ratio. In experiment 2, a larger amount of LTB(5) was released from Ca-ionophore-stimulated PMNs taken from rats given a larger quantity of FO. The ratio of LTB(5):LTB(4) released from PMNs correlated positively with the EPA:AA ratio in the membranous phospholipid and in serum. CONCLUSIONS: The omega-3 PUFAs were readily incorporated into the cell membrane within 3 days of infusion with the fat emulsion. The EPA:AA ratio in membranous phospholipid in PMNs was positively correlated with the LTB(5):LTB(4) production ratio and was a good indicator of anti-inflammatory effects.

Analysis of a bacterial lipopolysaccharide-activated serine kinase that phosphorylates p65/L-plastin in macrophages.

We previously identified p65/L-plastin as a phosphorylated protein in LPS-stimulated macrophages and determined its phosphorylation site. In vitro kinase assay using peptide substrates revealed that LPS-stimulated kinase activity selectively phosphorylated their serine-5 (Ser-5) residue. Kinase inhibitors for cAMP-dependent kinase such as H-89 inhibited the Ser-5 phosphorylation, but cAMP was not essential for the kinase activity. The LPS-stimulated kinase activity in cytosol fractions of macrophages was recovered as a sharp peak by anion exchange chromatography. These findings suggest that an as yet unknown H-89-sensitive serine kinase is rapidly activated by LPS stimulation and then phosphorylates p65/L-plastin, playing a vital role in macrophage activation.