Peter Angehrn

In Vitro and In Vivo Properties of Ro 63-9141, a Novel Broad-Spectrum Cephalosporin with Activity against Methicillin-Resistant Staphylococci

ABSTRACT Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of Enterococcus faecium. Its MIC at which 90% of the isolates tested were inhibited (MIC90) for methicillin-resistant Staphylococcus aureus (MRSA) was 4 μg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant Streptococcus pneumoniae(MIC90 = 2 μg/ml). It was active against ceftazidime-susceptible strains of Pseudomonas aeruginosaand against Enterobacteriaceae except Proteus vulgaris and some isolates producing extended-spectrum β-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2′ of MRSA, and its stability towards β-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.

Design, synthesis, and structure–activity relationship studies of ATP analogues as DNA gyrase inhibitors

We report herein the design and synthesis of ATP-analogues, namely 4-amino-pyrazolo[3,4-d]pyrimidines and 4-amino-pyrazolo[1,5-a][1,3,5]triazines, with DNA gyrase inhibitory activity. Among these series, some compounds exhibited promising antibacterial activity.

Ro 13-9904, a long-acting broad-spectrum cephalosporin: in vitro and in vivo studies.

Ro 13-9904, a new parenteral cephalosporin, was found to have high in vitro activity against Enterobacteriaceae and other gram-negative bacteria, including various isolates resistant to cefuroxime, cefamandole, cefoxitin, and cefazolin. It showed promising activity against Pseudomonas aeruginosa. Although inhibitory against Staphylococcus aureus at concentrations readily achievable in plasma, it was less potent against this pathogen than cefamandole, cefazolin, or cefuroxime. Isolates of Streptococcus faecalis were uniformly resistant to all the cephalosporins tested. Ro 13-9904 was more active than cefotaxime against Proteus mirabilis, Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae, but less active against S. aureus. Ro 13-9904 was stable to various types of beta-lactamases. Its therapeutic efficacy against experimental septicemias in mice was equal to or slightly superior to that of cefotaxime and SCE-1365 when the antibiotics were administered in repeated subcutaneous doses after bacterial challenge. Cefoperazone, and particularly cefamandole nafate, cefazolin, and mezlocillin were less effective. Although structurally related to cefotaxime and SCE-1365, Ro 13-9904 was found to differ from them in one important respect, namely, in having a long duration of action; this was observed with single-dose treatment given before bacterial challenge. Its broad spectrum of activity coupled with favorable pharmacokinetic properties make Ro 13-9904 a promising compound for clinical studies.

Low Trimethoprim Susceptibility of Anaerobic Bacteria Due to Insensitive Dihydrofolate Reductases

All the 28 Bacteroides fragilis strains investigated were susceptible to sulfamethoxazole (minimal inhibitory concentration < 16 μg/ml) and resistant to trimethoprim (TMP; minimal inhibitory concentration > 4 μg/ml). Synergism between sulfamethoxazole and TMP was present in all strains at a ratio of 1:1. The few clostridia investigated proved more resistant to both compounds. Dihydrofolate reductases from B. fragilis, C. perfringens, and some other anaerobic species were isolated. Inhibition profiles with six structurally different inhibitors revealed major differences in all enzymes. For 50% inhibition, the enzyme from B. fragilis and all clostridia required concentrations of TMP which were between several hundredfold and 1,000-fold higher than those required for the enzyme of Escherichia coli, whereas the enzyme from Propionibacterium acnes only needed a threefold higher concentration. In vitro activities of TMP were seen to correspond to the activity at the enzymatic level in B. fragilis and P. acnes, but correspond to a much lesser extent to the activity at the enzymatic level in clostridia, where a poor penetration is assumed to be involved. Dihydrofolate reductase inhibitors other than TMP were found to be as active as TMP both at the enzyme and in vitro. In B. fragilis, higher concentrations of exogenous thymidine were required for increasing the minimal inhibitory concentration of TMP than in E. coli and probably also in C. perfringens.

In vitro antibacterial properties of cefetamet and in vivo activity of its orally absorbable ester derivative, cefetamet pivoxil

The in vitro activity of cefetamet, the microbiologically active metabolite of the orally administered prodrug cefetamet pivoxil, was compared with that of cephalexin, cefaclor, cefuroxime and amoxicillin. Cefetamet was highly active againstEnterobacteriaceae, Neisseria spp.,Vibrio spp.,Haemophilus influenzae and streptococci other than enterococci. Cefetamet inhibited cefaclor-resistant species such asProteus vulgaris, Providencia stuartii, Providencia rettgeri andSerratia marcescens. Staphylococci,Pseudomonas aeruginosa and cephalosporinase-overproducing strains ofEnterobacter cloacae were resistant to cefetamet. The superior activity of cefetamet compared with older oral beta-lactam antibiotics against a large number of gram-negative pathogens correlated with enhanced stability towards betalactamases. In accordance with the in vitro findings, cefetamet pivoxil showed good activity in experimental infections in the mouse and rat, suggesting satisfactory bioavailability in these animals after oral administration.

The biochemical basis of the antimicrobial action of sulfonamides and trimethoprim in vivo--I. Action of sulfonamides and trimethoprim in blood and urine.

Abstract Trimethoprim (2 μg/ml), sulfamethoxazole (5 μg/ml) and a combination of these compounds reduced the viable cell count of Escherichia coli in human blood and urine in a bactericidal manner. This bactericidal action can be prevented by thymidine or thymine, the effectiveness of thymidine being considerably greater than that of thymine. Growth kinetics in the presence of trimethoprim, thymidine or thymine differed strikingly. Concentrations of thymidine below 0.05 μg/ml were detectable by their growth promoting effect, which started without a lag. Considerably higher concentrations of thymine, however, did not initially prevent the onset of cell inactivation, but resulted in a higher level of survivors. It is concluded that the concentrations of thymidine and thymine in blood and urine are too low to prevent a bactericidal action of trimethoprim, sulfonamides or a combination of these whereas other compounds, such as amino acids and purines, which support the bactericidal action, are present in sufficient quantities.

The biochemical basis of the antimicrobial action of sulfonamides and trimethoprim in vivo—II. Experiments on the limited thymine and thymidine availability in blood and urine

Abstract Escherichia coli 15T − , which undergoes “thymineless death” upon thymine deprivation, is not viable in human blood and urine. The addition of as little as 0·05 μg/ml thymidine results in a distinct growth promoting effect in both fluids whereas the thymine concentrations needed for growth are higher. Growth kinetics with thymine are different from those observed with thymidine. By comparison of the growth of E. coli 15T − in synthetic media that in blood and urine it is assumed that the concentrations of thymidine and thymine in the biological fluids are below 0·05 μg/ml and 0·2 μg/ml, respectively. These concentrations are not sufficient to antagonize the bactericidal action of trimethoprim, sulfonamides or their combinations. From the growth characteristics of E. coli 15 (TAU) − in blood it is concluded that other pyrimidines such as uracil (or uridine) are present and that there is a specific lack of thymine and thymidine. The in vivo conditions for the action of antifolates are discussed.

Antibacterial properties of Ro 40-6890, a broad-spectrum cephalosporin, and its novel orally absorbable ester, Ro 41-3399.

Ro 41-3399 is a novel orally active ester of Ro 40-6890, an aminothiazolyl cephalosporin with potent in vitro activities against commonly encountered aerobic gram-positive bacteria (streptococci and methicillin-susceptible staphylococci) and gram-negative bacteria (members of the family Enterobacteriaceae, haemophili, meningococci, and gonococci). In terms of the MICs determined by the methods recommended by the National Committee for Clinical Laboratory Standards, for 50 and 90% of gram-positive organisms, the water-soluble free carboxylic acid Ro 40-6890 proved to be at least as active as or two- to fourfold more active than cefpodoxime, cefuroxime, cefaclor, amoxicillin, amoxicillin-clavulanic acid, and ceftriaxone; against aerobic gram-negative organisms, Ro 40-6890 was usually two- to fourfold more active than cefpodoxime, the next most potent of the oral drugs under comparison, but remained usually two- to fourfold weaker than ceftriaxone. Ro 40-6890 showed a high affinity for the essential penicillin-binding proteins of susceptible bacteria and was resistant to hydrolysis by a broad array of beta-lactamases. Ro 41-3399 bopentil was well absorbed in mice when administered by oral gavage and proved effective in several experimental bacterial infections. Further studies with Ro 41-3399 and Ro 40-6890 are in progress.

Effects of trimethoprim and its antagonists on RNA synthesis in Escherichia coli.

Abstract The influence of trimethoprim on total RNA synthesis of Escherichia coli in minimal medium was investigated, as well as the reversing abilities of its antagonists thymidine, adenosine, guanosine, methionine and glycine. A purine source and the amino acids glycine and methionine are sufficient for reversing the inhibition of RNA synthesis completely. The experiments demonstrate that the inhibition of RNA synthesis upon addition of 5 μg/ml trimethoprim in minimal medium is caused by a lack of purines as well as by control mechanisms via the deprived amino acids, as is known from stringent strains. The addition of the single substances to cultures inhibited by trimethoprim leads to a short-time stimulation of RNA synthesis. The nature of this stimulation is not completely understood, especially the stimulation by thymidine, which is not necessary for the reversal of inhibited RNA synthesis. Whereas the lack of methionine results in a very strong inhibition of RNA synthesis, it seems that the synthesis of purines is most sensitive to a shortage of tetrahydrofolate, and which is responsible for the rapid stop of RNA synthesis.

From the DNA gyrase inhibitor Cyclothialidine to a new class of antibacterial agents

Cyclothialidine (Ro 09-1437) is a potent DNA gyrase inhibitor that was isolated from S. filipinensis NR0484. In this account we describe the identification of potent antibacterials structurally derived from this virtuallyneat enzyme inhibitor.