A new cephalosporin with a highly reactive 3-lactam ring was found to give an immediate color change in the presence of 3-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomnonas. The reaction is confined to organisms producing ,B-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme in small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380-to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the 3-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only 3-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.There are several techniques available for the detection and estimation of ,3-lactamases (2). Some are normally used quantitatively, and others can also be applied as qualitative spot tests. None of these methods is entirely satisfactory and, for carrying out spot tests, there is a requirement for a new reliable and sensitive diagnostic agent. This report describes the properties of such a substance. In the course of a screening program, a new cephalosporin was made: 3-(2,4-dinitrostyryl)-(6R, 7R)-7-(2-thienylacetamido) -ceph-3-em-4-carboxylic acid, E-isomer. It will be referred to here under the code number 87/312, and has the structure shown in Fig. 1 Spectrophotometric method for determination of g-lactamase activity. The method used was adapted from one described previously using cephaloridine as a substrate (9), in which the rate of breakdown of this substrate in the presence of g-lactamase was determined by measuring the rate of change of the ultraviolet absorption associated with the ,3-lactam ring.In aqueous solution at pH 7, 87/312 has two main absorption peaks, one at 217 nm and one at 386 nm (Fig. 2). The peak at 217 nm is associated with the 7-acyl group, and no change in absorbance occurred after the enzymatic hydrolysis. (Fig. 2). There was also a change in spectrum and color when 87/312 was attacked by serum; in this case, the decrease at 386 nm was the same, but the new peak was formed at 510 nm instead of 482 nm. At this wavelength, the maximal change observed was from 0.12 to 1.74 for a solution of 10-4 M.The shift in wavelength is due to serum binding (Fig. 3).

GR 20263 is a new broad-spectrum injectable cephalosporin which is stable to most 8i-lactamases. Its in vitro activities were of the same order as those of cefotaxime against most gram-negative bacteria, were clearly inferior to cefotaxime against Staphylococcus aureus, but were significantly more active against Pseudomonas aeruginosa. Against the 25 strains used, GR 20263 was significantly more active than any of the other agents tested: piperacillin, azlocillin, gentamicin, amikacin, and carbenicillin. GR 20263 protected mice against experimental infections with P. aeruginosa more effectively than other f-lactam antibiotics; its general effectiveness in this test was comparable with gentamicin. Studies on human volunteers showed that it produces high, long-lasting blood levels, with much of the antibiotic being recovered in the urine. Intramuscular and intravenous injections were well tolerated by the volunteers, and there were no untoward side effects.Although the cephalosporins have been used in clinical medicine for many years, the usefulness of the currently available members of the group is impaired by limitations such as restricted antibacterial spectrum, susceptibility to ,8-lactamases from gram-negative organisms, and metabolic degradation in the body. Compounds such as cefuroxime (4), cefotiam (7), and cefotaxime (1,5) have overcome some of these problems, but further improvements were desirable. For example, cefuroxime has good resistance to ,8-lactamases from many gram-negative organisms and is stable to metabolic degradation, but its intrinsic antibacterial activity is not as high as that of cefotiam. The spectrum of cefotiam is rather restricted by a degree of susceptibility of the compound to many ,B-lactamases. Cefotaxime, with good enzyme resistance and very high intrinsic antibacterial activity, suffers from metabolic degradation which reduces its activity in the body. To a large extent the newly developed GR 20263

Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to f8-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinaseproducing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-,8-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many 1B-lactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range.

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A cephalosporin, (6R,7R)-7-[(2R)-2-hydroxy-2-phenylacetamido]-3-(pyrid-2-yl- N -oxide) thiomethylceph-3-em-4-carboxylic acid (MCO), that could lead to a novel approach to the problem of β-lactamase destruction is described. The compound is slightly more resistant to some β-lactamases than is cephalothin, but it is still hydrolyzed by many to a varying degree. Hydrolysis of the β-lactam bond of a cephalosporin releases the 3-substituent, which in MCO is itself an antibacterial agent, 2-mercaptopyridine- N -oxide. Thus, MCO has a dual mode of action, and bacteria that do not produce an effective amount of a β-lactamase are inhibited by the intact cephalosporin, whereas those that do hydrolyze it are inhibited by the released antibacterial compound.