Waldemar Gottardi

Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system.

ABSTRACT N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time.N-Chlorotaurine (12.5 to 50 μM) demonstrated a bactericidal effect i.e., a 2 to 4 log10 reduction in viable counts, after incubation at 37°C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5.0), with a 3 to 4 log10 reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated inN-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function ofN-chlorotaurine and other chloramines during inflammation.

Chemical properties of N-chlorotaurine sodium, a key compound in the human defence system.

N‐Chlorotaurine (NCT) is known to play an important role in the human defence system. The already proved utility of the sodium salt as a disinfectant in human medicine suggested a thorough investigation of its chemical properties. Chlorine transfer to N‐H groups (transhalogenation) and oxidation of thio and aromatic compounds represent its main reactions. Auto‐chlorination causes disproportionation forming N, N‐dichlorotaurine (NDCT) with Kd = [NDCT][taurine]/fa[NCT]2aH+ = (4.5 ± 0.8) × 106, while the reaction with ammonium releasing NH2Cl is characterised by KNHCl2 = [NH2Cl][taurine]/[NCT][NH  4+ ]f  a2 = 0.02 ± 0.004.The verified unique stability and lowlevel reactivity of NCT are considered essential for its function in the mammalian defence system and its practical applicability, which manifests itself in an optimal compromise between microbicidal activity and toxicity.

N-chlorotaurine, a natural antiseptic with outstanding tolerability

N-chlorotaurine, the N-chloro derivative of the amino acid taurine, is a long-lived oxidant produced by activated human granulocytes and monocytes. Supported by a high number of in vitro studies, it has mainly anti-inflammatory properties and seems to be involved in the termination of inflammation. The successful synthesis of the crystalline sodium salt (Cl-HN-CH(2)-CH(2)-SO(3)Na, NCT) facilitated its development as an endogenous antiseptic. NCT can be stored long-term at low temperatures, and it has killing activity against bacteria, fungi, viruses and parasites. Transfer of the active chlorine to amino groups of molecules of both the pathogens and the human body (transhalogenation) enhances rather than decreases its activity, mainly because of the formation of monochloramine. Furthermore, surface chlorination after sublethal incubation times in NCT leads to a post-antibiotic effect and loss of virulence of pathogens, as demonstrated for bacteria and yeasts. Being a mild oxidant, NCT proved to be very well tolerated by human tissue in Phase I and II clinical studies. A 1% aqueous solution can be applied to the eye, skin ulcerations, outer ear canal, nasal and paranasal sinuses, oral cavity and urinary bladder, and can probably be used for inhalation. Therapeutic efficacy in Phase II studies has been shown in external otitis, purulently coated crural ulcerations and keratoconjunctivitis, so far. Based upon all presently available data, NCT seems to be an antiseptic with a very good relation between tolerability and activity. Recently, C-methylated derivatives of NCT have been invented, which are of interest because of improved stability at room temperature.

Iodine and Disinfection: Theoretical Study on Mode of Action, Efficiency, Stability, and Analytical Aspects in the Aqueous System

Although they have been in use for nearly 170 years, the mode of action of iodine‐based disinfectants is not yet clearly understood, as is manifested, for example, in diverging judgements about the relevance of the individual iodine species. Although studies based on calculated equilibrium concentrations in pure iodine solutions have already been done, there is a lack of knowledge about iodine solutions in the presence of additional iodide which would be of intrinsic importance for disinfection practice. Therefore, a re‐calculation was undertaken considering variations of this parameter in the pH range 0—14. The presented calculations concern fresh iodine solutions not affected by disproportionation (iodate formation) and provide information about the equilibrium concentrations of the species I‐, I2, I3‐, I5‐, I62, HOI, OI‐, HI2O‐, IO2‐, and H2OI+. Additional iodide and the pH value have a very pronounced influence on the individual equilibrium concentrations (several powers of ten); hence, conditions can be indicated where the number of species of virtual importance is drastically reduced. In the most common case with iodine in the presence of additional iodide at pH < 6, only I‐, I2, and I3‐ play a role. In the absence of additional iodide, at pH 8‐9 and at high dilution (c(I2) < 10‐5M), on the other hand, HOI accounts for over 90% of the oxidation capacity. At high iodide concentration (e.g. Lugol′s solution) the species I5‐ and I62‐ make up 8.2% of the oxidation capacity. The iodine cation H2OI+, frequently quoted as an active agent in disinfection, is without any relevance under the conditions occurring in practice, as are IO‐ and HI2O‐ which become important only at pH > 10. The stability problem (i.e. rate of iodate formation) arising at pH > 6 can be reduced to hypoiodous acid, as manifested in the simple rate law d[IO3‐]/dt = 0.25[HOI]3/[H+] which allows an estimation of stability under weakly alkaline conditions. The results of this study allow us to deduce general qualities of aqueous iodine solutions, such as reactivity, stability, and analytical aspects, and to estimate major disinfection‐orientated properties such as microbicidal activity, irritation, and incorporation effects. Though the calculations consider primarily preparations devoid of polymeric organic compounds capable of complexing iodine species, the results can be largely transferred to iodophoric preparations.

Activity of N-chlorotaurine against herpes simplex- and adenoviruses

N-chlorotaurine, an essential weak oxidant produced by stimulated human leukocytes, is known to have bactericidal, fungicidal and vermicidal properties. This study for the first time demonstrates its virucidal activity. By viral suspension tests at incubation times between 5 and 60 min, virus titers of both Herpes simplex virus type 1 and 2 were reduced about 1.3-2.9 log10 and 2.8-4.2 log10 by 0.1 and 1%, (5.5 and 55 mM) N-chlorotaurine, respectively. Virus titer reduction of adenovirus type 5 between 15 and 60 min was 0.5-2.0 and 0.6-4.0 log10, respectively, by the same concentrations of N-chlorotaurine. These findings support a contribution of N-chlorotaurine in destruction of pathogens during inflammatory reactions and also the possibility of its application as an antiviral agent in human medicine.

Tolerability and efficacy of N‐chlorotaurine in comparison with chloramine T for the treatment of chronic leg ulcers with a purulent coating: a randomized phase II study

Background  The well‐known active chlorine compound chloramine T (CAT) with broad‐spectrum antimicrobial activity is in common therapeutic use for leg ulcers with purulent coatings; however, this treatment is painful. The tolerability of the less aggressive N‐chlorotaurine (NCT), an endogenous compound also produced in vivo by stimulated human granulocytes, could be superior.

Tolerance of N-Chlorotaurine, a New Antimicrobial Agent, in Infectious Conjunctivitis – A Phase II Pilot Study

N-Chlorotaurine (NCT) is an endogenous microbicidal oxidant. This open pilot study (phase IIa) with 9 patients was done to gain first knowledge on the tolerance of NCT in infectious conjunctivitis. By application of 1% NCT 5 times a day, no adverse effects could be observed. All 6 subjects with bacterial conjunctivitis were cured within 3–5 days. Two subjects with epidemic keratoconjunctivitis were treated for 7–10 days and 1 subject with herpes simplex blepharitis for 3 days with no rapid improvement but probable mitigation of inflammation. Therefore, NCT seems to be useful in the treatment of infectious conjunctivitis, and further investigation on its therapeutic efficacy is suggested.

N-Chloramines, a Promising Class of Well-Tolerated Topical Anti-Infectives

ABSTRACT Antibiotic resistance is a growing public health crisis. To address the development of bacterial resistance, the use of antibiotics has to be minimized for nonsystemic applications in humans, as well as in animals and plants. Possible substitutes with low potential for developing resistance are active chlorine compounds that have been in clinical use for over 180 years. These agents are characterized by pronounced differences in their chlorinating and/or oxidizing activity, with hypochlorous acid (HOCl) as the strongest and organic chloramines as the weakest members. Bacterial killing in clinical practice is often associated with unwanted side effects such as chlorine consumption, tissue irritation, and pain, increasing proportionally with the chlorinating/oxidizing potency. Since the chloramines are able to effectively kill pathogens (bacteria, fungi, viruses, protozoa), their application as anti-infectives is advisable, all the more so as they exhibit additional beneficial properties such as destruction of toxins, degradation of biofilms, and anticoagulative and anti-inflammatory activities. Within the ample field of chloramines, the stable N-chloro derivatives of ß-aminosulfonic acids are most therapeutically advanced. Being available as sodium salts, they distinguish themselves by good solubility and absence of smell. Important representatives are N-chlorotaurine, a natural compound occurring in the human immune system, and novel mono- and dichloro derivatives of dimethyltaurine, which feature improved stability.

Tolerance and bactericidal action of N-chlorotaurine in a urinary tract infection by an omniresistant Pseudomonas aeruginosa.

N-chlorotaurine, a weak antimicrobial oxidant produced by stimulated human leukocytes, was used to treat cystitis caused by an omniresistant Pseudomonas aeruginosa. A 21-year-old male patient was treated by repeated daily lavages of the urinary bladder with an aqueous solution of 1% N-chlorotaurine for one month. N-chlorotaurine was well tolerated; no local or systemic side effects could be detected. Despite killing of > 10(6) cfu/ml of bacteria within ten minutes in vitro and in vivo, it was not possible to eradicate the Pseudomonas infection obviously caused by inflammation of the upper urinary tract and perpetuated by intravesical concrements. Nevertheless, in actually localized infection, treatment with N-chlorotaurine might be successful because of its sufficient bactericidal action.

N-Chlorotaurine and ammonium chloride: An antiseptic preparation with strong bactericidal activity

The bactericidal activity of the endogenous antiseptic N-chlorotaurine (NCT) is significantly enhanced in the presence of ammonium chloride which induces the formation of monochloramine (NH(2)Cl) whose strong bactericidal activity is well known. In this study the properties of NCT plus ammonium chloride have been investigated. The reaction of active chlorine compounds like chloramine-T (N-chlorotoluene-sulfonamide sodium), chloroisocyanuric acid derivatives, hypochlorites (NaOCl, CaOCl(2)) with ammonium chloride did not stop at the stage of monochloramine, and the pungent smelling by-products di- and trichloramine, NHCl(2) and NCl(3), were also formed. This was not the case with NCT where only monochloramine was generated. The equilibrium constant of the reaction of NCT with ammonium was found to be [Formula: see text] , which allows to estimate the equilibrium concentration of monochloramine in aqueous solutions of NCT and ammonium chloride. At concentrations each ranging between 0.01% and 1.0% it comes to [NH(2)Cl]=3.5-254 ppm. As an unexpected result the monochloramine containing formulation turned out to be most stable in plain water without buffer additives. Quantitative killing assays revealed complete inactivation of 10(6) to 10(7)CFU/mL of seven bacterial strains by 0.1% NCT plus 0.1% ammonium chloride within 5 min, while with plain 0.1% NCT an incubation time of 2-4h was needed to achieve the same effect. The highly significant increase of bactericidal activity (200-300-fold) could be assigned to the presence of monochloramine which could be isolated by vacuum distillation. Aqueous solutions of NCT and ammonium chloride provide a highly effective and well tolerable antiseptic preparation appropriate to a treatment cycle of at least 1 month if stored in the refrigerator.