This invention relates to the use of N-butylbenzenesulphonamide as an antimicrobial agent, and in particular as a topical antimicrobial agent. PRIOR ART

The problem of treating bacterial and especially staphylococcal infections is rendered difficult by the current prevalence of strains with multiple resistance and the fact that many germs rapidly develop resistance to new antibiotics. In recent years, over 80% of staphylococci have proved resistant to penicillin. Strains of staphylococcus resistant to methicillin or to other penicillinase-resistant penicillins are also increasing, particularly in the hospital environment. Hence the urgent need to find and develop new molecules active against these and other bacteria.

N-butylbenzenesulphonamide (NBBS) is a compound produced by a particular strain of Ps eudomonas sp., called AB2 ¹ . The antifungal activity of NBBS towards some plant pathogens such as Pythium ultimum, Phytophthora capsici, Rhizoctonia solani and Botrytis cinerea is reported in the literature, but there are no studies dealing with its antibacterial activity. NBBS is manufactured industrially, and is widely used as a plasticiser in the curing of polyamide compounds.

It has now been discovered that NBBS has considerable antimicrobial activity.

This invention therefore relates to the use of N-butylbenzenesulphonamide as an antimicrobial agent in humans and animals. MATERIALS AND METHODS

The antimicrobial activity of N-butylbenzenesulphonamide (NBBS) has been tested on the following certified strains:

Staphylococcus aureus (strain ATCC 33862 - OXOID).

Bacillus cereus (strain BCR50/ATCC 11778 - IZS Rome);

Bacillus subtilis (strain BCS51 - IZS Rome);

Listeria monocytogenes (strain ATCC 7644 - OXOID); Micrococcus luteus (strain ML53/ATCC 9341 - IZS Rome);

Rhodococcus equi (strain ATCC 6939 - Biogenetics);

Salmonella typhimurium (strain STY12/ATCC 14028 - OXOID).

Preparation of medium

For each bacterial strain the corresponding solid selective medium was prepared in accordance with the manufacturer's instructions.

Preparation of dilutions

9 ml of absolute ethyl alcohol (diluent) was distributed under sterile conditions between 4 test tubes. 1 ml of pure NBBS was added to the first test tube; after agitation with a benchtop vortex (Continental Equipment, Lawrence, KS ₅ USA), 1 ml of solution was removed and transferred to the second test tube; after agitation, 1 ml of solution was removed and transferred to the third test tube, and this operation was repeated until the last test tube, from which 1 ml of solution was discarded. Double dilutions of NBBS were thus obtained. The following dilutions of NBBS were therefore used for all the bacterial strains tested: 1 :10; 1 :20; 1 :40; 1 :80.

Preparation of inoculum

The inoculum was standardised by comparing the turbidity of the bacterial suspension in saline solution with that of a barium sulphate standard

(0.5 McFarland). Inoculation of plates

After solidification of the corresponding agarised media, each bacterial strain was streaked with a swab on their surface, to ensure confluent development and a uniform growth patina. Immediately after inoculation,

15 μl of each dilution of NBBS was deposited on the surface of the plate. The plates were then incubated for 18-24 h in a thermostat at 37°C. A control plate was also incubated for each bacterial strain; 15 μl of pure solvent (absolute ethyl alcohol) was deposited on the surface of the control plate after inoculation. RESULTS

The results were read by establishing the minimum dilution of NBBS at which bacterial growth was totally absent. a) Staphylococcus aureus The genus Staphylococcus, represented by Gram-positive, facultative aerobic, immobile cocci, belongs to the Micrococcaceae family. Staphylococci, which are widely found in nature, are the most resistant of the sporeless bacteria: they can survive under a wide variety of adverse environmental conditions and are relatively resistant to heat, drying, disinfectants, and high concentrations of sodium chloride. Various species of Staphylococcus are part of the normal flora in humans and animals; they are found on the skin, on the mucous membranes of the upper respiratory tract, and in the intestinal tract. Many individuals are therefore asymptomatic carriers, who can represent sources of infection to themselves and others. ⁴ The skin is frequently colonised by staphylococci, and the skin, whether intact or damaged, represents a frequent entrance route for staphylococcal infection. Of the various species, S. aureus is considered to be a pathogen, or potential pathogen, of great importance, and is the species most frequently found in clinical samples. The main diseases caused by S. aureus are diseases of the skin and cutaneous adnexa, food poisoning, haematogenous infections, respiratory infections, toxic shock syndrome, and burnt skin syndrome.

The Staphylococcus aureus strain was streaked with a swab on the surface of Mueller Hinton Agar and Columbia Agar plates (Oxoid GmbH,

Wesel, Germany), and 15 μl of the various dilutions of NBBS (1 :10; 1 :20; 1 :40; 1:80) was then deposited on the same plates. After incubation in a thermostat at 37°C for 24 h, NBBS inhibited the growth of staphylococcus up to the dilution of 1:20 on Mueller Hinton Agar, whereas growth was only inhibited on Columbia Agar up to the dilution of 1: 10; at the other dilutions, the colonies grew normally on both media. b) Bacillus cereus

The genus Bacillus is represented by Gram-positive, spore-forming, aerobic bacilli. Most Bacillus species are ubiquitous in nature; some are part of the normal intestinal flora found in humans and various animals. Bacillus anthracis, the aetiological agent of anthrax, and Bacillus cereus, mainly responsible for food poisoning, are pathogenic for man. ⁴

The test was performed on plates of Mueller Hinton Agar, as described in a). No colonies of Bacillus cereus developed at the dilution of 1 :10, whereas the colonies grew normally at the other dilutions. c) Bacillus Subtilis

Bacillus Subtilis is weakly pathogenic: it is occasionally responsible for infections, especially respiratory and eye infections, and very rarely for septicaemia. It also causes food poisoning, and has been repeatedly isolated in stool samples. ⁴

The test for inhibiting substances was performed on plates of Agar according to the method described in a). No Bacillus subtilis colonies developed on the surface at the dilution of 1 :10, but some small colonies began to form inside the agar. The colonies grew normally at the other dilutions. d) Listeria monocytogenes

The genus Listeria is represented by Gram-positive, sporeless, facultative aerobic bacilli; it comprises eight species, only one of which

{Listeria monocytogenes) has been isolated in humans. Listeria is widely distributed in nature. ⁴ In humans, infection caused by Listeria monocytogenes can take various forms: infection of the central nervous system, infection during pregnancy, granulomatosis infantisepticum, septicaemia, and local infections. ⁴

The Listeria monocytogenes strain was streaked with a swab on the surface of plates of Palcam Medium and Oxford Listeria Agar (Oxoid GmbH, Wesel, Germany), and 15 μl of the various dilutions of NBBS (1 :10; 1 :20; 1:40; 1:80) was then deposited on the same plates. After incubation in a thermostat at 37 ⁰ C for 24 h, NBBS inhibited Listeria growth up to the dilution of 1 :20 on Palcam, whereas growth was only inhibited on Oxford agar up to the dilution of 1 :10. At the other dilutions, the colonies grew normally on both media. e) Micrococcus luteus Micrococcus luteus is a Gram-positive coccus belonging to the

Micrococcaceae family; it is found on human skin, and occasionally on the mucous membranes. This bacterium, which is usually apathogenic, can be transmitted by direct contact, and is found in all our environments. In immunocompromised patients it can cause septic shock, pneumonia or endocarditis.

The Micrococcus luteus strain was streaked with a swab on the surface of plates of Mueller Hinton Agar, and tested according to the method described in a). No colonies of Micrococcus luteus developed at the dilution of 1 : 10; a number of colonies were found at the dilution of 1 :20, and the colonies grew normally at the other dilutions. f) Rhodococcus equi

The genus Rhodococcus comprises Gram-positive bacteria which are widely found in nature, especially in the soil and in the faeces of herbivores.

This genus comprises 7 species, only one of which is pathogenic to animals. ³ Rhodococcus equi is the aetiological agent of purulent pneumonia, which has a high mortality rate in foals; it has been isolated from foals with arthritis, ulcerative lymphangitis and purulent pleurisy. In pigs and cattle it is responsible for cervical and mesenteric lymphadenitis. In sheep, goats and marine mammals, it can cause lung infections. ³

The test on the strain of Rhodococcus equi was performed on plates of Mueller Hinton Agar as described in a). No colonies of Rhodococcus equi developed up to the dilution of 1:12, whereas the colonies grew normally at the other dilutions. g) Salmonella typhimurium

Salmonella are Gram-negative bacilli belonging to the Enter obacteriaceae family. They are transmitted from human to human through faecal contamination of food and water. The majority of strains are ubiquitous. In humans, salmonella is the aetiological agent of salmonellosis. Salmonella typhimurium is the main serotype, which causes enteritis and septicaemia in cattle, sheep, pigs, horses, dogs, cats and birds; the source of contagion is represented by contaminated feed and forage, rodents and wild birds, fertigation, and contaminated bedding and water courses. As salmonellosis caused by Salmonella typhimurium is transmitted fairly frequently from animals to humans, it is considered a typical zoonosis.

The test on the strain of Salmonella typhimurium was performed on plates of Mueller Hinton Agar as described in a). Only one colony of Salmonella typhimurium developed at the dilution of 1 :10; a dozen colonies were found at the dilution of 1:20, and the colonies grew normally at the other dilutions. Toxicity

When administered intraperitoneally at high doses in the rat (300 mg/kg

every 6 hours), NBBS exhibited low neurotoxicity, with short-lived effects. ²

The invention therefore also relates to pharmaceutical compositions containing as active ingredient an effective dose of N-butylbenzenesulphonamide for the treatment of infections of the skin and cutaneous adnexa, especially following (staphylococcal) infections.

The compositions according to the invention will be administered topically.

The compositions to which this invention relates could be formulated suitably for topical administration, and will be prepared according to conventional methods well known in pharmaceutical technology, such as those described in Remington's Pharmaceutical Handbook, Mack Publishing Co., N.Y., USA, using excipients, diluents, fillers and anti-caking agents acceptable for their final use. Examples of formulations according to the invention include fatty ointments, ointments, suspensions and oily solutions.

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