A newly described 6d-amidinopenicillanic acid derivative, mecillinam (formerly called FL 1060), showed a high in vitro activity against Enterobacteriaceae. The effect on Escherichia coli was bactericidal and was due to lysis of the cells. The longer the culture grew under the influence of mecillinam or the lower the inoculum, the greater the bactericidal effect. In a previous paper from this laboratory a new group of penicillanic acid derivatives, 6,-amidinopenicillanic acids, with unusual in vitro antibacterial properties was described (4). One member of the group, mecillinam (proposed intemational nonproprietary name), 6,B-[(hexahydro-lH-azepin-1-yl)-methyleneamino ]-penicillanic acid (formerly called FL 1060), has been studied extensively, and it has been shown that this compound differs fundamentally from the hitherto known penicillanic acid derivatives both in regard to antibacterial spectrum (4, 8) and its mode of action (3, 5-8).The results presented in this paper deal with the bactericidal effect of mecillinam and its synergistic action with ampicillin in vitro.MATERIALS AND METHODS Antibiotics. Mecillinam was used as the hydrochloride dihydrate and ampicillin as the trihydrate. The concentrations of both compounds were calculated on the basis of the anhydrous zwitterion. Solutions of the compounds were freshly prepared for each experiment.Culture media. NIH agar medium and the corresponding liquid medium were used throughout the study. The medium contains 0.5% yeast extract (Difco), 1.5% casein hydrolysate (pancreatic), 0.1% dextrose, 0.25% sodium chloride, 0.005% L-cystine, and 1% agar (Oxoid no. 1) in distilled water, pH 7.1, after autoclaving. In some experiments NIH agar or liquid media without sodium chloride was used as a basic medium to which membrane-filtered solutions of different compounds were added. The osmolality of the liquid media was measured by the cryostatic method using a Knauer type M osmometer, and the specific conductivity was measured as millisiemens at 36 C using a conductivity meter, Radiometer type CDM 2c.Bactericidal activity. The bactericidal effect of mecillinam on a growing culture of Escherichia coli (Leo HA2) was determined by measuring the change in viable count and optical density (OD) during the first 6 h of incubation. Viable counts were performed in quadruplicate on NIH agar by plating 0.1 ml of serial 10-fold dilutions in NIH liquid medium. The colonies were counted after 18 h at 36 C. OD was measured by means of a Vitatron MPS photometer with a tungsten light without a filter. In some instances the total number of microbial cells was quantitatively determined by microscopy.In Fig. 1 to 4 the OD scale was adjusted to the viable count scale by measuring the OD of dilutions of an overnight culture of the test organism. Such a culture consists of separate minor cells, and the &adjustment is therefore not entirely valid for a culture in the log phase, where the cells are more voluminous and many pairs or short chains occur, each representing only one colony-forming unit...

Calcipotriol and betamethasone dipropionate are widely used effective treatments for psoriasis. Combined therapy is known to be superior to monotherapy, but current formulations do not permit simultaneous application as the drug substances will degrade when mixed. The purpose of the study was to develop a formulation which combines calcipotriol and betamethasone dipropionate in a single vehicle hereby achieving optimal delivery of both substances into the skin. As the two substances are incompatible in aqueous and alcoholic medias, different non-aqueous formulations were prepared. Skin permeation studies were investigated using Franz-type diffusion cells. Formulations based on isopropyl myristate were found to decrease the permeation rate (25-35%) as compared with marketed monotherapy products (set to 100%). Lanolin had no overall effect on skin permeability. However, polyoxypropylene-15 stearyl ether (PSE) had a marked effect. A 5% PSE formulation resulted in a permeation rate comparable to the marketed products. Thus, by using PSE as solvent, it was possible to combine calcipotriol and betamethasone dipropionate in a single formulation while optimal skin permeability was attained. Recently, the efficiency of this formulation (Daivobet) has been verified in clinical studies showing an improved efficacy in the treatment of psoriatic patients.

The ability of brobactam to inhibit beta-lactamases and the in vitro activity of ampicillin combined with brobactam was compared with another beta-lactamase inhibitor, clavulanic acid, and other beta-lactam antibiotics. Both inhibitors showed good and similar activity against staphylococcal penicillinase and most broad-spectrum beta-lactamases found in the Enterobacteriaceae, whether plasmid or chromosomally mediated. Both inhibitors were less active against chromosomally mediated cephalosporinases in Enterobacteriaceae, but brobactam was 8-50 times more potent than clavulanic acid. The amount and type of beta-lactamase produced by a particular bacterial strain was reflected in its sensitivity to a combination of ampicillin and brobactam, and correlated well with the sensitivity of extracted beta-lactamase to inhibition by brobactam. The in-vitro activity of ampicillin/brobactam in a 3:1 ratio was compared to amoxycillin/clavulanic acid (4:1 ratio), amoxycillin, cefaclor and cefuroxime. The two inhibitor combinations were generally of similar activity, but the brobactam combination had superior activity against Proteus vulgaris, Morganella morganii, Citrobacter freundii and Yersinia enterocolitica. The cephalosporins were less effective against the Bacteroides fragilis group, Prot. vulgaris and M. morganii, but had advantages in the case of Escherichia coli, Klebsiella spp. and C. diversus. The MBC of ampicillin/brobactam was similar to the MIC. No resistant sub-populations were observed amongst the staphylococcal strains investigated. Exposure of bacteria to sub-inhibitory levels of ampicillin/brobactam did not lead generally to the development of resistance.

The morphological response of E. coli to a new antibiotic, 6p-[ (hexahydro-IH-azepin-l-yl)methyleneamino] -penicillanic acid (FL 1060), has been investigated and compared with the response to benzylpenicillin and ampicillin. At high concentrations of FL 1060 (1000 rg/ml) in osmotically stabilized media, E. coli responds in the same way as to penicillins by forming "rabbit ears" and spheroplasts. At lower concentrations down to the IC50 value (0.02 pg/ml), the cells, even in unstabilized media, first become ellipsoidal and later spherical. After 2-3 hours, lysis of the cells occurs. This is rather late, as compared to the early lysis obtained with penicillins. Electron microscopical investigations show no characteristic changes in the subcellular pattern. During the second hour of treatment, the bacterial culture contains a considerable number of cells presenting asymmetrical cell divisions. During the same period, nuclear stainings show abnormal nuclear regions with impaired segregation, resulting in chromatine bridges and horseshoe-shaped chromatine regions. The results support the conception that, on cells in the pre-lytic stage, FL 1060 interferes with the balance between lengthwise growth and cell division through cross wall formation.A previous paper ( 10) described the synthesis and antimicrobial properties of a new group of penicillanic acid derivatives, viz. 6p-amidinopenicillanic acids. I t was shown that the antibacterial properties of this class of compounds differ from those of penicillins such as benzylpenicillin and ampicillin, which can be regarded as acyl derivatives of 6p-aminopenicillanic acid.Whereas the penicillins in general are much more active against gram-positive than Received 6.iv.73