Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases.

Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5-4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known.

Staphylococcus aureus and Escherichia coli adhesion to human cells is reduced by sub-MICs of gemifloxacin.

This study was designed to investigate the capacity of subinhibitory concentrations of the newly developed fluoroquinolone antibiotic gemifloxacin to interfere with the mechanism of bacterial adhesion. Human buccal epithelial cells were incubated with Staphylococcus aureus and Escherichia coli, and grown in the presence of serial dilutions of gemifloxacin from 1/2 MIC to 1/128 MIC. A significant decrease in the adhesion of both S. aureus and E. coli was observed from 1/2 MIC to 1/32 MIC. Morphological changes including filamentous forms of E. coli and cluster formation and swelling of S. aureus were also observed, mainly from 1/2 MIC to 1/8 and 1/16 MIC. These findings are discussed in terms of dose-effect relationships and the interpolation of this pharmacodynamic data with the pharmacokinetics curve of gemifloxacin.

Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.

Comprehensive genomic analysis of the important human pathogen Staphylococcus aureus was achieved by a strategy involving antisense technology in a regulatable gene expression system. In addition to known essential genes, many genes of unknown or poorly defined biological function were identified. This methodology allowed gene function to be characterized in a comprehensive, defined set of conditionally growth-defective/lethal isogenic strains. Quantitative titration of the conditional growth effect was performed either in bacterial culture or in an animal model of infection. This genomic strategy offers an approach to the identification of staphylococcal gene products that could serve as targets for antibiotic discovery.

Pleuromutilins. Part 1. The identification of novel mutilin 14-carbamates.

A novel series of mutilin 14-carbamates has been discovered as a result of structure-activity studies on the naturally occurring antibiotic pleuromutilin (1). In particular, the 4-methoxybenzoylcarbamate, SB-222734 (15o) displays potent antibacterial activity against a number of bacterial pathogens which are resistant to currently used agents and shows enhanced metabolic stability when compared to earlier pleuromutilin derivatives. Such derivatives therefore have the potential to provide a new class of antibacterial agents for human therapy which address the threat of bacterial resistance.

Pharmacodynamics to combat resistance.

The ability to identify agents with the optimal combination of potency, pharmacokinetics and pharmacodynamics should help to maximize bacteriological cure and thus minimize the potential for selection and spread of resistance. Gemifloxacin demonstrated excellent correlation between efficacy and the AUC0-24h/MIC ratio whereas there was little correlation with time above MIC. Thus, gemifloxacin is similar to other quinolones in that it is the amount of drug present, not the frequency of administration, that determines antibacterial effect. In a neutropenic murine thigh model of infection, caused by Gram-negative bacilli, a AUC0-24h/MIC ratio of approximately 100 was necessary to protect >90% of the animals, which is similar to data reported previously for other quinolones. However, in order to achieve the same protection in an immunocompetent murine infection caused by Streptococcus pneumoniae, the AUC-24h/MIC ratio was approximately 25. The magnitude of this AUC0-24h/MIC ratio did not alter for strains exhibiting penicillin or macrolide resistance. Importantly, when gemifloxacin was examined against strains of S. pneumoniae with well-characterized ciprofloxacin resistance (including mutations in gyrase, parC and parE as well as efflux strains) there was little impact on the in vivo efficacy. Overall, the data showed a trend towards a decrease in the AUC0-24h/MIC ratio for these more resistant strains. The lower AUC0-24h/MIC ratio was especially noticeable for the efflux mutants suggesting that the quinolone efflux mechanism may be down-regulated in vivo and may be of minimal relevance to the clinical activity of gemifloxacin against S. pneumoniae. The efficacy of gemifloxacin, in comparison with other oral agents used to treat respiratory infections, has also been evaluated in a rat model using doses, and therefore AUC0-24h/MIC ratios, that approximate those in man. These data confirm the excellent activity of gemifloxacin against strains of Haemophilus influenzae and S. pneumoniae, including those demonstrating penicillin, macrolide and quinolone resistance.

Comparative in vivo activity of gemifloxacin in a rat model of respiratory tract infection.

The in vivo efficacy of the novel quinolone gemifloxacin (SB-265805) was examined in a rat respiratory tract infection (RTI) model against four strains of Streptococcus pneumoniae and two strains of Haemophilus influenzae with varying susceptibilities to standard antimicrobial agents. Animals were infected intrabronchially to produce pneumonia and therapy with oral gemifloxacin, amoxycillin-clavulanate, ciprofloxacin, cefuroxime, azithromycin, trovafloxacin, grepafloxacin or levofloxacin was started 24 h after infection. The doses administered were chosen to approximate in the rat the serum or tissue concentrations measured in humans following therapeutic dosing. Therapy continued once- or twice-daily for 3 days, and approximately 17 h after the end of therapy the lungs were excised for bacterial enumeration. Following infection with strains of S. pneumoniae, gemifloxacin produced a 3-5 log reduction in bacterial numbers compared with untreated animals. Gemifloxacin was as effective as amoxycillin- clavulanate, and was as potent or more potent than all other comparators. Notably, the quinolone agents trovafloxacin, ciprofloxacin, grepafloxacin and levofloxacin were significantly less effective (P < 0.01) than gemifloxacin: these agents reduced bacterial numbers by < or =3 log compared with untreated animals. Gemifloxacin produced a marked response against H. influenzae infection, reducing bacterial numbers significantly (P < 0.01) compared with untreated controls. Gemifloxacin was significantly more potent than cefuroxime and azithromycin. None of the other comparator agents was more potent than gemifloxacin. The excellent efficacy seen in these experimental models of RTI with S. pneumoniae and H. influenzae confirms the in vitro activity of gemifloxacin against these organisms. This indicates that gemifloxacin may be of significant benefit in the treatment of RTI.

Comparative efficacy of gemifloxacin in experimental models of pyelonephritis and wound infection.

Gemifloxacin (SB-265805) is a potent, novel fluoroquinolone with broad-spectrum antimicrobial activity. In this study, the efficacy of gemifloxacin was studied in experimental models of Gram-negative pyelonephritis (caused by Escherichia coli or Proteus mirabilis) and Gram-positive wound infection resulting from Streptococcus pyogenes, Staphylococcus epidermidis or Staphylococcus aureus. Gemifloxacin activity against these pathogens was compared with those of amoxycillin-clavulanate, ciprofloxacin, cefuroxime, azithromycin, trovafloxacin, grepafloxacin, levofloxacin and tosufloxacin. Oral treatment was initiated 1 h after infection and continued once or twice daily for 3 days. Around 17 h after the end of treatment, animals were killed and the infected kidneys or the skin around the wound site were excised for the enumeration of viable bacteria. In the pyelonephritis model (either microorganism), gemifloxacin reduced bacterial numbers significantly (P < 0.01) compared with no treatment. No comparator agent had a greater effect than gemifloxacin. Notably, grepafloxacin and azithromycin were significantly less effective (P < 0.01) than gemifloxacin against E. coli pyelonephritis, and amoxycillin-clavulanate, azithromycin and trovafloxacin were inferior (P < 0.01) against P. mirabilis infection. In the S. pyogenes wound infection model, gemifloxacin, amoxycillin-clavulanate, cefuroxime and azithromycin reduced bacterial numbers significantly compared with controls (P < 0.01). Results for the comparator quinolones were not significantly different from untreated controls (P > 0.05). Gemifloxacin was also effective against staphylococcal infection, as were grepafloxacin and levofloxacin, while ciprofloxacin, trovafloxacin and tosufloxacin were significantly less effective against these pathogens than gemifloxacin (P < 0.01). No comparator agent had greater activity than gemifloxacin against S. pyogenes or S. aureus infections. These data demonstrate the potential benefit of gemifloxacin in the treatment of Gram-negative urinary tract infection and Gram-positive skin and soft tissue infection.

In vitro activity of gemifloxacin (SB 265805; LB20304a) against human mycoplasmas.

The in vitro activity of gemifloxacin, a new broad-spectrum fluoroquinolone, was compared with those of ciprofloxacin, erythromycin, azithromycin and doxycycline against 29 human respiratory or urogenital tract mycoplasmas. Gemifloxacin was highly active against all of the mycoplasma and ureaplasma species tested (MIC range 0.001-0.25 mg/L) and was 5- to 100-fold more active than ciprofloxacin. Doxycycline was less active than gemifloxacin against the mycoplasmas (MIC range 0.01-1 mg/L) but had similar activity against Ureaplasma urealyticum (MIC ranges 0.025-0.25 mg/L and 0.1-0. 25 mg/L, respectively). The macrolides, particularly azithromycin, were more active than gemifloxacin against Mycoplasma pneumoniae (MIC range 0.001-0.0025 mg/L) and Mycoplasma genitalium (0.0005-0. 001 mg/L) isolates but were less active against Mycoplasma fermentans and U. urealyticum and inactive against Mycoplasma hominis. Gemifloxacin may therefore be useful in the treatment of respiratory, urogenital or systemic mycoplasma infections in humans.

Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection.

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted a tet regulatory expression system for use in Staphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the S. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisense hla RNA downregulated chromosomally derived hla gene expression in vitro approximately 14-fold. Similarly, induction of hla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureus infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the tet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.

Two pharmacodynamic models for assessing the efficacy of amoxicillin-clavulanate against experimental respiratory tract infections caused by strains of Streptococcus pneumoniae.

Two models of respiratory tract infection were used to investigate the pharmacodynamics of amoxicillin-clavulanate against Streptococcus pneumoniae. Eight strains of S. pneumoniae were used in a mouse model in which the animals were infected intranasally and were then treated with a range of doses and dose intervals. The time that the plasma amoxicillin concentration remained above the MIC (T>MIC) correlated well with bacterial killing, such that if T>MIC was below 20% there was no effect on bacterial numbers in the lungs. As T>MIC increased, the response, in terms of decreased bacterial load, improved and at T>MICs of greater than 35 to 40% of the dosing interval, bacteriological cure was maximal. On the basis of equivalent T>MICs, these data would suggest that in humans a dosage of 500 mg three times daily (t.i.d.) should have efficacy equal to that of a dosage of 875 mg twice daily (b.i.d.). This hypothesis was evaluated in a rat model in which amoxicillin-clavulanate was given by computer-controlled intravenous infusion to achieve concentrations that approximate the concentrations achieved in the plasma of humans following oral administration of 500/125 mg t.i.d. or 875/125 mg b.i.d. Infusions continued for 3 days and bacterial numbers in the lungs 2 h after the cessation of the infusion were significantly reduced (P < 0.01) by both treatments in strains of S. pneumoniae for which amoxicillin MICs were below 2 microg/ml. When tested against a strain of S. pneumoniae for which the amoxicillin MIC was 4 microg/ml, the simulated 500/125-mg dose was ineffective but the 875/125-mg dose demonstrated a small but significant (P < 0. 01) reduction in bacterial numbers. These data confirm the findings in the mouse and indicate that amoxicillin-clavulanate administered at 875/125 mg b.i.d. would be as effective clinically as amoxicillin-clavulanate administered at 500/125 mg t.i.d.

Efficacy of high-dose amoxicillin-clavulanate against experimental respiratory tract infections caused by strains of Streptococcus pneumoniae.

The purpose of the present investigation was to determine if the efficacy of amoxicillin-clavulanate against penicillin-resistant Streptococcus pneumoniae could be improved by increasing the pediatric amoxicillin unit dose (90 versus 45 mg/kg of body weight/day) while maintaining the clavulanate unit dose at 6.4 mg/kg/day. A rat pneumonia model was used. In that model approximately 6 log10 CFU of one of four strains of S. pneumoniae (amoxicillin MICs, 2 microg/ml [one strain], 4 microg/ml [two strains], and 8 microg/ml [one strain]) were instilled into the bronchi of rats. Amoxicillin-clavulanate was given by computer-controlled intravenous infusion to approximate the concentrations achieved in the plasma of children following the administration of oral doses of 45/6.4 mg/kg/day or 90/6.4 mg/kg/g/day divided every 12 h or saline as a control for a total of 3 days. Infusions continued for 3 days, and 2 h after the cessation of infusion, bacterial numbers in the lungs were significantly reduced by the 90/6.4-mg/kg/day equivalent dosage for strains for which amoxicillin MICs were 2 or 4 microg/ml. The 45/6.4-mg/kg/day equivalent dosage was fully effective only against the strain for which the amoxicillin MIC was 2 microg/ml and had marginal efficacy against one of the two strains for which amoxicillin MICs were 4 microg/ml. The bacterial load for the strain for which the amoxicillin MIC was 8 microg/ml was not reduced with either dosage. These data demonstrate that regimens which achieved concentrations in plasma above the MIC for at least 34% of a 24-h dosing period resulted in significant reductions in the number of viable bacteria, indicating that the efficacy of amoxicillin-clavulanate can be extended to include efficacy against less susceptible strains of S. pneumoniae by increasing the amoxicillin dose.

Bacteriological efficacies of three macrolides compared with those of amoxicillin-clavulanate against Streptococcus pneumoniae and Haemophilus influenzae.

Comparative antibacterial efficacies of erythromycin, clarithromycin, and azithromycin were examined against Streptococcus pneumoniae and Haemophilus influenzae, with amoxicillin-clavulanate used as the active control. In vitro, the macrolides at twice their MICs and at concentrations achieved in humans were bacteriostatic or reduced the numbers of viable S. pneumoniae slowly, whereas amoxicillin-clavulanate showed a rapid antibacterial effect. Against H. influenzae, erythromycin, clarithromycin, and clarithromycin plus 14-hydroxy clarithromycin at twice their MICs produced a slow reduction in bacterial numbers, whereas azithromycin was bactericidal. Azithromycin at the concentrations achieved in the serum of humans was bacteriostatic, whereas erythromycin and clarithromycin were ineffective. In experimental respiratory tract infections in rats, clarithromycin (equivalent to 250 mg twice daily [b.i.d.]) and amoxicillin-clavulanate (equivalent to 500 plus 125 mg b.i.d., respectively) were highly effective against S. pneumoniae, but azithromycin (equivalent to 500 and 250 mg once daily) was significantly less effective (P < 0.01). Against H. influenzae, clarithromycin treatment (equivalent to 250 or 500 mg b.i.d.) was similar to no treatment and was significantly less effective than amoxicillin-clavulanate treatment (P < 0.01). Azithromycin demonstrated significant in vivo activity (P < 0.05) but was significantly less effective than amoxicillin-clavulanate (P < 0.05). Overall, amoxicillin-clavulanate was effective in vitro and in vivo. Clarithromycin and erythromycin were ineffective in vitro and in vivo against H. influenzae, and azithromycin (at concentrations achieved in humans) showed unreliable activity against both pathogens. These results may have clinical implications for the utility of macrolides in the empiric therapy of respiratory tract infections.

Effect of protein binding on penetration of beta-lactams into rabbit peripheral lymph.

The relevance of protein binding to penetration of beta-lactams into body fluids was investigated by examining the distribution of amoxicillin, ceftriaxone, clavulanic acid, temocillin, and ticarcillin into rabbit peripheral lymph after intravenous administration. The elimination half-lives in rabbit plasma varied between 0.34 h (temocillin) and 1.80 h (ceftriaxone), and the half-lives measured in lymph were similar to those in plasma (0.37 to 1.76 h). The percent penetration (area under the concentration-time curve in lymph/area under the concentration-time curve in plasma x 100) was high for amoxicillin (97.6%), temocillin (89.4%), and clavulanic acid (90.8%) but was lower for ticarcillin (76.0%) and for ceftriaxone (67.3%). There was a direct correlation between plasma protein binding and percent penetration. Correction for plasma and tissue binding increased the percent penetration for all compounds, and figures approached 100%. The results presented demonstrate the use of this model to examine the relationships between plasma pharmacokinetics, protein binding, and distribution of antibiotics.

The influence of uptake from the gastrointestinal tract and first-pass effect on oral bioavailability of (Z)-alkyloxyimino penicillins.

We have investigated the contribution of uptake from the gastrointestinal tract and first-pass effect to the poor oral bioavailability of a series of (Z)-alkyloxyimino penicillins in mice. Investigative studies in gut sacs and perfused small intestine demonstrated that these penicillins were able to pass across the mucosal epithelium although to a lesser extent than amoxycillin and cyclacillin, both of which exhibit excellent oral bioavailability in man and animals. In the jejunal gut sacs the mucosal to serosal flux for BRL 44154 was approximately half that of amoxycillin and four times less than that of cyclacillin, and for all, uptake was pH dependent. The serosal to mucosal fluxes were however similar for these compounds and significantly lower than mucosal to serosal fluxes, suggesting involvement of carrier mechanisms in uptake from the mucosal surface. The order of results for the alkyloxyimino penicillins paralleled that observed for oral bioavailability in the mouse. For the alkyloxyimino penicillins, between 5.5 and 9.9% was taken up from the perfused intestine, values which were significantly less than those for amoxycillin (13.2%) and cyclacillin (33.3%). However, uptake was concentration-dependent for BRL 44154 as it was for amoxycillin, thus confirming the possible use of carrier mechanisms in absorption. These observations suggest that the poor peripheral blood concentrations of the alkyloxyimino penicillins achieved after oral dosing were not a consequence of the inability of the compounds to cross the mucosal epithelium. The biliary clearance of the alkyloxyimino penicillins was, however, considerably greater than for amoxycillin and cyclacillin, a finding which may well have been a contributory factor to the comparatively low peripheral concentrations of BRL 44154 and its analogues achieved after oral administration.

Bactericidal and morphological effects of amoxicillin on Helicobacter pylori.

The growth kinetics of Helicobacter pylori after it has been exposed to amoxicillin have been investigated in conjunction with studies of cell morphology. A potent bactericidal effect was observed at concentrations 10-fold higher than the MIC, but this was accompanied by an increase in the residual numbers of coccoid forms observed. In the presence of 10 micrograms of amoxicillin per ml, these forms could be detected as rapidly as 6 h after exposure to the antibiotic. Although the clinical relevance of coccoid forms remains unknown, such forms should be considered when potential anti-Helicobacter agents are tested in vitro.

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man.

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.

Simulation of human serum pharmacokinetics of cefazolin, piperacillin, and BRL 42715 in rats and efficacy against experimental intraperitoneal infections.

Studies were performed to determine the effects of BRL 42715, a potent beta-lactamase inhibitor, on the activity of cefazolin and piperacillin against experimental intraperitoneal infections caused by either Escherichia coli or Serratia marcescens in rats. Compounds were administered to rats as a continuous infusion of an exponentially diluted solution to simulate in rat plasma the concentration-versus-time curves obtained for humans following intravenous bolus administration. A simulated 1-g dose of cefazolin was ineffective in reducing the bacterial counts in blood and peritoneal fluid samples of animals infected with S. marcescens US20, which produced class Ia beta-lactamase, and as a result, mortality was similar to that of infected controls. Similarly, a simulated 2-g dose of piperacillin was ineffective in reducing bacterial numbers and mortality in animals infected with E. coli 41548, producing a TEM-1 beta-lactamase. However, when the antibiotics were coadministered with BRL 42715, bacterial numbers were reduced significantly and all animals survived at least 16 h after infection. These data demonstrate the ability of BRL 42715 to potentiate the activity of cefazolin and piperacillin against beta-lactamase-producing bacteria that would otherwise be resistant to these antibiotics and illustrate the application of a model to simulate human serum concentrations in conscious rats.

Penetration of amoxycillin, ticarcillin and clavulanic acid into lymph after intravenous infusion in rabbits to simulate human serum pharmacokinetics.

The distribution of amoxycillin, ticarcillin and clavulanic acid into lymph collected from the right lymphatic duct of rabbits was examined after intravenous administration. The compounds were administered to simulate, in the plasma of rabbits, the concentrations of amoxycillin, ticarcillin and clavulanic acid measured in human serum after the administration of either an iv bolus dose of amoxycillin 1.0 g plus clavulanic acid 200 mg, ticarcillin 3.0 g plus clavulanic acid 200 mg, or an iv infusion of amoxycillin 2.0 g plus clavulanic acid 200 mg or ticarcillin 3.0 g plus clavulanic acid 200 mg given over 30 min. Lymph concentrations of the compounds reached a peak rapidly after the simulation of a bolus dose (0-1 h) and the concentration-versus-time profiles in plasma and lymph were generally similar after 45 min. Following simulation of an iv infusion, peak concentrations of amoxycillin and clavulanic acid in lymph were reached at approximately the same time as for the bolus simulation, but that of ticarcillin occurred slightly later. The elimination half-lives of the compounds were similar in plasma and lymph. The percentage penetration values were high (greater than 80%) irrespective of the concentration-versus-time curve simulated. The penetration of clavulanic acid was compatible with that of the coadministered penicillin agent and was similar when given with either amoxycillin or ticarcillin.