Full text of DOI:10.1517/13543784.9.12.2945

Expert Opinion on Investigational Drugs
Ryan, Ho, Wu, Frosco, Dougherty & Barrett
40th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC)
40th Interscience Conference on
Antimicrobial Agents and Chemotherapy
(ICAAC)
Toronto, Canada, September 17 - 20, 2000
http://www.ashley-pub.com
Brenda Ryan, Hsu-Tso Ho, Ping Wu, Mary-Beth Frosco, Thomas
Dougherty & John F Barrett
Meeting Highlights
1. Carbapenems
Infectious Diseases, Bristol-Myers Squibb Pharm. Res. Institute, Wallingford,
CT 06492, USA
2. Quinolones
The 40th ICAAC Meeting provided one of the widest varieties of new
antibacterial and antifungal agents in years, with a range of improved ‘class-
ical’ agents (β-lactams, carbapenems), novel quinolone agents and totally
new ideas for novel therapeutic intervention. Among the compounds
reported were potent anti-MRSA carbapenems, novel des-fluoroquinolones
(BMS 284756/T-3811), non-fluorinated quinolones, quinolone-related
antibacterials, novel cephalosporins (cefditoren, RWJ-54428, MC 04546),
ketolides (telithromycin, ABT-773), novel streptogramins and novel
β-lactamase inhibitors, bacterial and fungal efflux pump inhibitors and
novel antifungals (azasordarins, echinocandins, azoles).
3. Quinolone-related agents
4. Ketolides
5. New
β-lactams/cephalosporin
6. β-Lactamases and
inhibitors
7. Streptogramins
8. Novel oxazolidinones
9. Antifungals
Keywords: β-lactamase, antibacterials, antifungals, cephalosporins, efflux
pump inhibitors, ketolides, quinolones
10. Efflux pump inhibitors
11. Exploratory research
12. Novel bacterial targets
Exp. Opin. Invest. Drugs (2000) 9(12):2945-2972
1. Carbapenems
1.1 CP5068
13. Summary and expert
opinion
CP5068 (Meiji Seika Kaisha Ltd., Yokohama, Japan) (Figure 1), a novel
carbapenem with potent anti-MRSA activity, possesses methicillin-resistant
Staphylococcus epidermidis (MRSE) and methicillin-resistant Staphylo-
coccus aureus (MRSA) activity nearly equivalent to vancomycin (E Shitara et
al., Meiji Seika Kaisha Ltd.) with an MIC90 of 4 µg/ml against MRSA and 4
µg/ml against MRSE (T Ida et al., Meiji Seika Kaisha Ltd.). CP5068 has a
6,7-disubstituted imidazo [S, 1-β] thiazolium-2-yl group on the C-2-position
of the carbapenem nucleus.
Specifically designed to target PBP2′ of MRSA, CP5068 exhibited 124-times
greater affinity than imipenem to S. aureus strain MF535HR (MRSA) with an
IC50 to PBP2′ binding of 1.7 µg/ml (vs. IC50, 210 µg/ml for imipenem) (T Ida
et al.). CP5068 is more bactericidal than cefotaxime or penicillin G against
highly penicillin-resistant Streptococcus pneumoniae (PRSP) (K Ubukata et
al., Inst. Microb. Chem., Tokyo, Japan). In direct comparison with other
penems, CP5068 was 16-times more active than imipenem, 32-times more
active than meropenem and four-times more active than panipenem and
CP5068 was 128-times more active than cefotaxime, 64-times more active
than levofloxacin and 16-times more active than vancomycin against PRSP
2945
2000 © Ashley Publications Ltd. ISSN 1354-3784

2946 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 1: Chemical structures of carbapenems.
CP5068
E-1010
Faropenem
OH
NH2
HO
HO
HO
H
H
H
S
N
H
N
N
S
NH
O
O
S
S
N
N
N
H
O
CO2Na
CO2-
COOH
O
O
MK0826
J-114,870
OH
H
NH2
S
*HCl
HO
N
H
CONH2
H
H
N
S
O
O
N
HO
N
H
N
H
O
COOH
O
O
OH
and penicillin-intermediate S. pn eumon ia e (K
Ubukata et al.). In a PRSP systemic infection model in
mice, CP5068 was 14-times more potent (ED50, 0.16
mg/ kg/ d ay) th an me ro p e n e m (ED50, 2.22
mg/ kg/ day), three-times more p otent than
panipenem (ED50, 0.44 mg/kg/day), 78-times more
potent than ceftriaxone, 142-times more potent than
cefotaxime (ED50, 22.8 mg/kg/day) and 23-times
mo re p o ten t th an van co mycin (ED50, 3.72
mg/kg/day) (K Ubukata et al.). CP5068 demonstrated
excellent potency in a PRSP experimental meningitis
model in rabbits, with a > 3 log reduction in CFU at 20
1.2 J-114,870
J-114,870 (Figure 1), a new 1-β-methycarbapenem
w ith a C-2-p o sitio n tran s-3,5-d isu b stitu te d
5-arylpyrrolidin-3-ylthio moiety, is a broad spectrum
carbapenem with excellent activity against MRSA
(MIC90, 4 µg/ml), PRSP (MIC90, 0.125 µg/ml) and
Pseudomonas aeruginosa (MIC90, 4 µg/ml) (K Shibata
et al., Tsukuba Res. Inst., Banyu Pharm. Co. Ltd.,
Tsukuba, Japan).
Efficacy of J-114,870 against MRSA and P. aeruginosa
mouse septicaemia models, was with an ED50 of 5.49
mg/kg, as compared with imipenem (ED50 74.35
mg/kg) or vancomycin (ED50, 4.25 mg/kg) against
MRSA and ED50 values for J-114,870, imipenem and
vancomycin of 0.16, 0.32 and > 100 mg/kg, respec-
tively, against P. aeruginosa (K Shibata et al.).
Superior in vivo potency by J-114,870 was also
demonstrated against PRSP, with ED50 values of 0.12,
0.15, 0.29 and 5.85 mg/ kg against J-114,870,
imipenem, meropenem and penicillin G, respectively
(K Shibata et al.). J-114,870 demonstrates equal
bactericidal activity to other penems (meropenem,
imipenem).
mg/kg. In the same model, the t was 0.29 h,
½
AUCO→∞ = 13.8 mg.h/l (in plasma) and AUCO→∞
=
4.6 mg.h/l (in CSF). Scaling predictions suggest a b.i.d.
dosing in man.
PK parameters of CP5068 in mice for a single dose, 44
mg/kg, resulted in a Cmax of 80.1 µg/ml, t of 12.3 min
½
and AUCO→∞ 38.7 mg.h/l in serum and a Cmax of 27
µg/ml, t of 32.3 min and AUCO→∞ of 14.6 µg.h/gm of
½
lung tissue (T Ida et al.). Protein binding to serum
protein for CP5068 was 19.8% in mice and 7.5% in
humans (E Shitana et al., Meiji Seika Kaisha Ltd.).
CP5068 exhibited the highest relative stability to renal
DHP-1, as compared with meropenem, panipenem
and imipenem (E Shitara et al.).
1.3 E-1010
E-1010 (Figure 1), a parenteral 1-β-methyl carbape-
nem with broad spectrum antibacterial activity in
development by Eisai Co. Ltd. (Tokyo, Japan). T
Hirawawa et al. (Eisai Co. Ltd.) reported on Phase I
double-blind, placebo-controlled, ascending, single-
dose study in healthy males, with doses ranging from
250 - 1000 mg. PK parameters for the 250 mg dose
CP5068 minimal lethal dose in mice was over 2000
mg/kg, with GABAA receptor binding in rats showed
CP5068 similar to panipenem, better than meropenem
and less safe than imipenem. No kidney nephrotox-
icity by CP5068 was observed in rats (E Shitara et al.).
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2947
were Cmax, 19.7 µg/ml, AUCO→∞ 37.4 mg.h/l, Vss, 0.18
β-lactamase-negative and β-lactamase-producing H.
influenzae, respectively, as compared with penicillin,
amoxicillin/ clavulanate and cefuroxime with
MIC50/90s of 0.25/1, 0.25/1 and 0.5/1 µg/ml, respec-
tively, versus β-lactamase-negative H. influenzae and
> 8/ > 8, 1/1 and 0.5/1 µg/ml, respectively, against
β-lactamase-producing H. influenzae (GG Zhanel et
al., Univ. of Manitoba, Winnipeg, Manitoba, Canada).
Likewise, MK0826 had excellent activity against
Moraxella catarrhalis, with MIC50/90s of ≤ 0.015/≤
0.015, 8/16, 0.25/0.5, 1/2 and 0.25/0.5 µg/ml for
MK0826, p enicillin, amoxicillin/ clavulanate,
cefuroxime and cefotaxime, respectively (GG Zhanel
et al.). Against a panel of 431 clinical isolates of
anaerobes including Bacteroides fragilis, Prevotella,
Porphyromonas and Fusobacterium, imipenem =
meropenem (MIC50/90s 0.12/1 µg/ml) > MK0826
(MIC50/ 90s, 0.25/ 2 µg/ ml) > clindamycin =
piperacillin/tazobactam (MIC50/90s, 0.5/16 µg/ml) >
metronidazole (MIC50/90s, 1/ > 16 µg/ml) > ceftria-
zone (MIC50/90s, 16/ > 64 µg/ml) (LM Kelly et al.,
Hershey Med. Ctr., Hershey, PA); 95.8% of anaerobes
tested were susceptible at a proposed breakpoint of ≤
4 µg/ml for MK0826. The MIC50/MIC90 value for
MK0826 against all strains tested was 0.25/2 µg/ml,
similar to those for imipenem and meropenem
(MIC50/90s, 0.12/1 and 0.12/1 µg/ml, respectively) and
two- to 32-fold lower than other comparators. In
time-kill experiments against one strain each of B.
fragilis, B. thetaiotaomicron, P. bivia and Clostridium
perfringens, MK0826 was bactericidal (≥ 3 log10
killing after 48 h) at and above the MIC (0.25, 0.5, 0.5,
0.06 µg/ml, respectively).
l/kg, t , 1.72 h and CL, 0.09 l/h/kg, while the 1000 mg
½
dose had Cmax, 66 µg/ml, AUCO→∞ 131.5 mg.h/l, Vss,
0.21 l/kg, t , 1.79 h and CL 0.10 l/h/kg (T Hirasama et
½
al.). All 41 volunteers initiated and completed this
Phase I trial. The plasma elimination t of E-1010 (1.7 -
½
1.8 h) was longer than meropenem or imipenem (~ 1
h). Urine was the major elimination route of E-1010,
with 60 - 76% of dose identified within 24 h of dosing.
Imipenem-susceptible P. aeruginosa (MIC values, 1.0
µg/ml) and imipenem-resistant P. aeruginosa (MIC
values, 16 µg/ml) were found to be susceptible to
E-1010, with MIC values of 0.25 and 1.0 µg/ml, respec-
tively (T Toyosawa and M Inoue, Kitasata Univ. Sch.
Med., Kanagawa, Japan). Using these strains in an in
vitro pharmacodynamic model simulating human
plasma levels at b.i.d. dosing with E-1010 or
imipenem, in vivo P. aeruginosa potency was
determined. E-1010 demonstrated high level killing in
5 h (99.9% killing), with a maximum 3.5 logs killing
overall. Imipenem, however, did not reach 99.9%
killing and overall had a maximum killing of 2.5 logs
against imipenem-resistant P. a eru gin osa (T
Toyosawa and M Inoue).
1.4 MK0826
MK0826 (ertapenem, L-749,345; Merck Pharm. Co.,
Rahway, NJ) (Figure 1) is a novel, parenteral,
long-acting 1-β-methyl carbapenem with broad
spectrum activity, including anti-pneumococcal and
anti-anaerobic activity (GA Pankoch et al., Hershey
Med. Center, Hershey, PA; KM Overbye et al., Merck
Res. Laboratories, Rahway, NJ). Against extended
spectrum β-lactamases (ESBLs) producing organisms,
MK0826 had an MIC90 of 0.125 µg/ml, as compared
1.5 Faropenem
The in vitro activity of faropenem (Bayer AG,
Wuppertal, Germany) (Figure 1) was reported by D
Felmingham et al. (GR Micro Ltd., London, UK), with
MIC90s of 0.008, 0.25 and 0.5 µg/ml, respectively,
against penicillin-sensitive, -intermediate and
-resistant S. pneumoniae. MIC90s against haemolytic
streptococci were 0.03 - 0.06 µg/ml, against MSSE
were 0.12 µg/ml and against MRSA were > 64 µg/ml.
Faropenem’s MIC90 for Gram-negative bacteria
ranged from 0.015 to 4 µg/ml, except for Serratia
marcescans (MIC90, 16 µg/ml), Stenotrophomonas
maltophilia (MIC90, > 64 µg/ml) and P. aeruginosa
(MIC90, > 64 µg/ml) (D Felmingham et al.).
w ith ce ftriaxo n e (MIC90
, 64 µg/ ml) an d
piperacillin/tazobactam (MIC90, 128 µg/ml) (KM
Overbye et a l., Merck Res. Laboratories). The
anti-pneumococcal activity (MIC50/90s) of MK0826,
amoxicillin, amoxicillin/clavulanate, cefuroxime,
cefp rozil, cefep ime, ceftriaxone, imip enem,
meropenem and clarithromycin were 0.5/1.0, 2/2,
2/2, 8/8, 16/16, 1/2, 1/2, 0.25/0.25, 0.5/1 and > 32/32
µg/ ml, respectively, against penicillin-resistant,
quinolone-sensitive strains of S. pneumoniae and
0.125/1, 0.25/2, 0.25/2, 0.5/8, 0.5/16, 0.25/1, 0.25/1,
0.03/0.25, 0.06/0.5 and ≤ 0.03/8 µg/ml, respectively,
for penicillin-resistant, quinolone-resistant strains of
S. pneumoniae (GA Pankuch et al.). MK0826 has
excellent anti-Haemophilus influenzae activity with
MIC50/90s of 0.03/0.06 and 0.03/0.06 µg/ml against
Faropenem exhibits excellent penicillin-resistant
anti-S. pneumoniae activity, with MIC90s of 1 µg/ml,
as compared with imipenem, penicillin, amoxicillin/
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2948 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
clavulanate, cefprozil, cefuroxime axetil, azithro-
mycin , clarith ro mycin an d trime th o p rim/
sulphamethoxazole, with MIC90s of 0.5, 8, 4, 16, 8, >
16, > 16 and > 4 µg/ml, respectively (JA Black et al.,
Creighton Univ. Sch. Med., Omaha, NE). Faropenem
exhibited excellent bactericidal activity against
common respiratory pathogens including M.
catarrhalis, H. influenzae and S. pneumoniae and
was more bactericidal than cephalosporins and
macrolides against a penicillin-resistant strain of S.
pneumoniae (JA Herrington et al., Bayer Corp, West
Haven, CT), making faropenem a good candidate for
treating respiratory tract bacterial infections.
Faropenem was reported to be resistant against a
panel of penicillinases and cephalosporinases from
Gram-positive and Gram-negative bacteria (A Dalhoff
et al., Bayer AG Pharma Res. Ctr., Wuppertal,
Germany). The MIC values of faropenem against S.
pneumoniae, Enterococcus faecalis and Escherichia
coli track with the extensive PBP1, PBP2 and PBP3
binding for S. pneumoniae, PBP5, PBP3, PBP1 and
PBP4 binding for E. faecalis and PBP2, PBP4, PBP1
PBP3 and PBP5 binding for E. coli (A Dalhoff et al.).
Morphological studies of faropenem binding affinity
for S. aureus PBP1/PBP3 and E. coli PBP3 confirmed
the primary target activity in whole bacteria (A Dalhoff
and K Okamoto, Bayer AG).
two dozen posters. RC Owens et al. (Maine Med.
Center, Portland, ME) presented data on a Monte
Carlo analysis of the probability of gatifloxacin,
moxifloxacin and levofloxacin achieving the target
AUC:MIC ratio of 30. Using in vitro susceptibility data,
free drug AUC:MIC ratios were calculated (using
standard PK numbers reported by each company for
their product) and the Monte Carlo analysis indicates a
98.8, 97.9 and 81.1% probability of achieving the
AUC:MIC ratio of 30 in man for gatifloxacin,
moxifloxacin and levofloxacin, respectively (RC
Owens et al.). The ‘dose-dependent total antimicro-
bial’ effect of gatifloxacin over ciprofloxacin was
confirmed by SH Zinner et al. (Mount Auburn Hosp.,
Cambridge, MA) in an in vitro pharmacodynamic
model of anti-pneumococcal activity. From this
model, a 1400 mg dose of ciprofloxacin would be
required to attain the same clinical effect of the
standard 400 mg gatifloxacin q.d. dosage (SH Zinner
et al.). Confirmation of gatifloxacin activity was
reported by PD Lister and JA Black (Creighton Univ.
Sch. Med., Omaha, NE) in an in vitro PD model, in
which killing by gatifloxacin was compared with
levofloxacin. Both gatifloxacin and levofloxacin
exhibited similar PK-based killing and lack of
emergence of resistant S. pneumoniae isolates in
vitro.
Pharmacokinetic (PK) profiles of a single 300 mg dose
of faropenem daloxate in human subjects provided
the following range of values:
2.2 BMS 284756 (T-3811)
BMS 284756 (T-3811) (Figure 2) is a novel gyrase
inhibitor without the C-6 fluorine typical of the older
generation fluoroquinolones. With a broad spectrum
of antibacterial activity, esp ecially against
Gram-positive organisms, as well as good anaerobic
activity, BMS 284756 may replace trovafloxacin as the
anaerobic, broad spectrum quinolone. E Gradelski et
al. (Bristol-Myers Squibb, Wallingford, CT) presented
on the broad spectrum activity of BMS 284756
comparing this new agent with five other quinolones
(trovafloxacin, moxifloxacin, levofloxacin, ofloxacin
and ciprofloxacin) against over 400 pathogens. BMS
284756 was of equal to or greater activity than all other
quinolones except against Enterococcus faecium,
with MIC90s of 0.03, 2, 0.03, 2, 0.25, 0.25, 0.12, 0.5, 0.5
and 8 µg/ml, respectively, against MSSA, MRSA, MSSE,
MRSE, β-haemolytic streptococci, viridans strepto-
cocci, ciprofloxacin-susceptible S. pneumoniae,
ciprofloxacin-resistant S. pneumoniae, E. faecalis and
E. faecium. The quinolones could be grouped into
‘more active’ (BMS 284756, trovafloxacin and
moxifloxacin), with MIC90s of BMS 284756 ranging
• Males: AUCO→∞ 26 - 32.8 mg.h/l; Cmax, 13.5 - 13.8
µg/ml; t , 0.88 - 1.32 h; and time over MIC, 4.6 - 5.5
½
h
• Females: AUCO→∞ 27.6 - 31.6 mg.h/l; Cmax, 12.9 -
13.3 µg/ml; t , 0.91 - 1.09 h; and time over MIC, 4.5
½
- 5.1 h (JT Lettieri et al., Bayer Corp., West Haven,
CT)
• Escalating dosing of faropenem daloxate from 300
to 1200 mg resulted in approximately linear
increases in all PK parameters (AUC, Cmax, time
over MIC), with similar t (1.14 - 1.39 h) (U
½
Schuehly et al., Bayer AG, Wuppertal, Germany)
2. Quinolones
2.1 Gatifloxacin
Gatifloxacin (Bristol-Myers Squibb, Princeton, NJ)
(Figure 2), a recently launched novel, 8-methoxy
quinolone, was again highlighted in approximately
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2949
Figure 2: Chemical structures of quinolones.
non-susceptible (resistant) and trovafloxacin and
gatifloxacin were 94.1% susceptible. Likewise, in a
SENTRY report of 24,961 isolates from Europe and the
USA from 1999, of which 11.3% of the isolates (955)
were S. pneumoniae, the MIC90s for BMS 284756,
ciprofloxacin, gatifloxacin and trovafloxacin were
0.12, 2, 0.5 and 0.25 µg/ml, respectively (RN Jones et
al.).
Gatifloxacin
O
F
COOH
N
N
HN
O
The anti-anaerobic activities of BMS 284756 were
reported by DW Hecht and JR Osmolski (Hines VA
Hosp., Loyola Univ. Med. Center, Maywood, IL). The
anti-anaerobic activity of BMS 284756 was equal to or
superior to other quinolones tested (trovafloxacin,
moxifloxacin) and superior to typical anti-anaerobic
agents such as clindamycin and cefoxitin. With the
exception of several anaerobes (i.e., Fusobacterium
mortiforum/varium, B. thetaiotaomicron, Porphyro-
monas anaerobius and Peptostreptococcus loeschii),
BMS 284756’s MIC90s ranged from 0.05 - 2 µg/ml,
providing broad spectrum anaerobic coverage,
whereas trovafloxacin’s MIC90s were generally equal
to BMS 284756 and moxifloxacin’s MIC90s were
generally four-times less active (DW Hecht and JR
Osmolski). Imipenem maintained the best overall
anti-anaerobic activity with MIC90s ranging from ≤
0.015 - 0.5 µg/ml; whereas clindamycin (MIC90s
BMS 284756 (T-3811)
O
COOH
N
HN
O
CHF2
from equal to, or up to eight-fold more active;
whereas the ‘less active’ quinolones (levofloxacin,
ofloxacin and ciprofloxacin) have MIC90s four- to
32-fold less active than BMS 284756 (E Gradelski et
al.). BMS 284756 had the best activity against
ciprofloxacin-resistant S. pneumoniae, with an MIC90
of 0.5 µg/ml, as compared with trovafloxacin,
moxifloxacin, levofloxacin, ofloxacin and ciprofloxa-
cin with MIC90s of 4, 2, 8, 16 and 16 µg/ml,
respectively. This activity was independently
confirmed in several other in vitro studies reported,
including the SENTRY data report by AC Gales et al.
(Univ. Iowa College of Medicine, Iowa City, IA), who
reported the same rank order of susceptibility (based
on MIC90s) with BMS 284756 > trovafloxacin >
gatifloxacin > levofloxacin > ciprofloxacin against all
S. pneumoniae isolates (regardless of ciprofloxacin
susceptibility). Among these 257 S. pneumoniae
isolates reported from Latin America medical centres,
72.4% were penicillin-susceptible and 13.2% had
high-level penicillin resistance. Breakpoint/MIC90s
for BMS 284756, trovafloxacin, gatifloxacin, levofloxa-
cin and ciprofloxacin were 16, 4, 2, 2 and ~ 0.5 µg/ml,
respectively, providing BMS 284756 with the highest
cushion of peak serum drug level to MIC ratio. In a
separate SENTRY report, RN Jones et al. (Univ. of
Io w a) re p o rte d o n cip ro flo xacin -re sistan t
Gram-positive cocci susceptibility to BMS 284756 and
two other quinolones (gatifloxacin and trovafloxa-
cin), as well as ampicillin, linezolid and vancomycin.
Against 197 ciprofloxacin-resistant S. pneumoniae
isolates, BMS 284756 was 100% susceptible at or
below the estimated breakpoint of 2 µg/ml (or
possibly 4 µg/ml), whereas ciprofloxacin was 100%
ran gin g fro m
≤
0.015
-
>
64 µg/ ml),
piperacillin/tazobactam (MIC90s ranging from < 0.03 -
16 µg/ml) and cefoxitin (MIC90s ranging from 0.125 -
128 µg/ml) continue to show resistance emergence
and reduced utility as the ‘gold standards’ of
anaerobic coverage.
An SAR study of BMS 284756 and analogues was
presented by P Wu et al. (Bristol-Myers Squibb,
Wallingford, CT). The specific mechanism of action of
this novel quinolone was shown to be a putative DNA
gyrase A inhibitor in a demonstration of both DNA
gyrase supercoiling inhibition and formation of the
cleavable complex (CC50; specific for GyrA inhibi-
tors). Target-based inhibition as measured by the
supercoiling inhibition assay ranks activity of BMS
284756 = ciprofloxacin > levofloxacin > moxifloxacin,
with IC50 values of 0.17, 0.18, 0.32 and 0.36 µg/ml,
respectively (P Wu et al.). Verification of the GyrA
target activity was provided in the CC50 assay, in
which novobiocin as a negative-control GyrB
inhibitor (CC50 > 3000 µg/ml), was compared with the
four quinolones, whose CC50s ranged from 1.23 - 6.55
µg/ml. Little difference was observed for microbio-
logical activity or target-based inhibition of closely
related analogues of BMS 284756, including the
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2950 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
S-isomer, the C-6 fluorine analogue and the -OCHF2
ether-minus analogue, however there were differ-
ences in selectivity and cytotoxicity (LE Lawrence et
al., Bristol-Myers Squibb; H Yamada et al., Toyama
Chem. Co, Toyama, Japan), with BMS 284756 being
among the quinolones with the greatest selectivity
over the human topoisomerase II inhibition. Microbial
physiology macromolecule precursor studies
demonstrated BMS 284756 to be the most selective
quinolone for targeting DNA synthesis over RNA or
protein synthesis, with AUC/IC50DNA ratios of 822,
640, 103 and 44 for BMS 284756, moxifloxacin,
levofloxacin and ciprofloxacin, respectively; as well
as Cmax/IC50DNA ratios of 78, 60, 12 and 10, for BMS
284756, moxifloxacin, levofloxacin and ciprofloxacin,
respectively, making BMS 284756 the most specific
DNA targeting quinolone tested (H Ho et al., Bristol-
Myers Squibb).
and characterisation of mutants of S. pneumoniae,
selected by either ciprofloxacin or BMS 284756.
Ciprofloxacin-selected mutants saw MIC values
change from 0.5 µg/ml (WT = wild type) to 2 µg/ml in
the first step and 16 µg/ml in the second step mutants
for ciprofloxacin, 0.5, 2 and 16 µg/ml, respectively for
levofloxacin and 0.125, 0.25 and 2 µg/ ml for
moxifloxacin, respectively, whereas BMS 284756 MIC
values were 0.015, 0.06 - 0.25 and 0.5 µg/ml, respec-
tively. This indicates that ciprofloxacin loses
susceptibility by the first step mutation, levofloxacin
by the second step mutation, moxifloxacin by the
second step mutation, but BMS 284756 maintains
susceptibility into the second step (and beyond) (SL
Hartman-Neumann et al.). In the converse experi-
me n t, BMS 284756 se le cte d mu tan ts o f S.
pneumoniae, MIC values rose from 0.015 µg/ml (WT)
to 0.06 - 0.125 µg/ml (first step) to 0.25 µg/ml (second
step) to 2 µg/ml (third step) to 8 - 16 µg/ml (fourth
step), whereas ciprofloxacin’s MIC values rose from
0.5 µg/ml (WT) to 1 µg/ml (first step) to 16 µg/ml
(second step) to 16 µg/ml (third step) to 32 µg/ml
(fourth step); levofloxacin’s MIC values rose from 0.5
µg/ml (WT) to 1 µg/ml (first step) to 16 µg/ml (second
step) to 16 µg/ml (third step) to 32 µg/ml (fourth
step); and moxifloxacin’s MIC values rose from 0.125
µg/ml (WT) to 0.25 µg/ml (first step) to 4 µg/ml
(second step) to 4 - 8 µg/ml (third step), to 64 µg/ml
(fourth step). Thus, ciprofloxacin-resistant laboratory
mutants with loss of ciprofloxacin susceptibility by the
first or second step and cross-resistant to levofloxacin
and moxifloxacin by the second step, are covered by
BMS 294756, suggesting the possibility of BMS 284756
being used to cover first and second step quinolone-
resistant mutants in the clinical setting.
The bactericidal activity of BMS 284756 was reported
by BM Ryan et al. (Bristol-Myers Squibb) for inhibition
of MSSA and MRSA. Matching human PK/PD parame-
ters to microbiological activity (killing and
post-antibiotic effect), Ryan et al. demonstrated
Cmax/MIC and AUC/MIC ratios of 188 and 1987,
respectively, for BMS 284756 against methicillin-
sensitive S. aureus (MSSA), whereas levofloxacin was
22.8 and 190, respectively and ciprofloxacin was 5.9
and 27.4, respectively. Likewise for MRSA, the
demonstrated Cmax/MIC and AUC/MIC ratios of 94
and 993, respectively for BMS 284756 against MSSA,
whereas levofloxacin was 22.8 and 190, respectively
and ciprofloxacin was 11.9 and 54.8, respectively (BM
Ryan et al.), demonstrating a predicted greater
potency in vivo. Quinolone-resistant strains of MSSA,
either GyrA or GyrA:GrlA, were compared for BMS
284756, ciprofloxacin and levofloxacin susceptibility
and BMS 284756 was shown to maintain the same
microbiological superiority with both mutants,
exhibiting four- to eight-fold and eight- to 16-fold
greater activity than levofloxacin and ciprofloxacin,
respectively, against the wild type MSSA strain, six- to
eight-fold and 12- to 16-fold greater activity against
the GyrA mutant for both levofloxacin and ciprofloxa-
cin, respectively and four- and ≥ eight-fold activity
over levofloxacin and ciprofloxacin, respectively
against the double GyrA:GrlA mutant (L Lawrence et
al., Bristol-Myers Squibb). In examining the rate of
resistance emergence frequency, site of mutation and
cross-resistance between BMS 284756, ciprofloxacin,
moxifloxacin and levofloxacin, SL Hartman-Neumann
et al. (Bristol-Myers Squibb) reported on the selection
2.3 Non-fluorinated quinolones
Researchers presented a series of posters on their
non-fluorinated quinolones (non-FQ) (JL Gray et al.,
TW Morris et al., S Roychoudhury et al., I Critchley et
al., Procter and Gamble). A series of non-FQ
compounds were described in a chemistry-based
p oster. The genesis of the non-fluorinated
compounds was the desire to move away from the
genotoxicity associated with the presence of the
fluorines. It was found that it was possible to synthe-
sise a series of compounds without the C6 fluorine
that could be as potent as the fluorinated congeners.
An interesting side finding was substitution of a
chlorine at C6, along with aminoethylpyrollidone at
C7 and O-methoxy at C8 yields a compound of
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2951
Figure 3: Chemical structures of quinolone-related agents.
non-FQs on clinical isolates from the USA and one on
Mycoplasma, Chlamydia and Legionella susceptibili-
ties to these agents. The conclusions of the clinical
isolate study were that non-FQs are particularly
potent against pneumococci and other streptococci
and equipotent against Gram-negatives and obligate
anaerobes. The non-FQs also had advantages against
ciprofloxacin-resistant staphylococci over the fluori-
nated quinolones. With regard to the atypical
pathogens, the non-FQs compounds were active
against Legionella pneumophila with activity similar
to clarithromycin and levofloxacin. With regard to
Chlamydia pneumoniae, the compounds were active,
with similar potency to clarithromycin and trovafloxa-
cin, but with eight- to 16-fold greater activity than
levofloxacin. The non-FQs were of similar activity to
levofloxacin and trovafloxacin when tested against
Mycoplasma pneumoniae. Clarithromycin had activity
against these organisms superior to all quinolones,
non-fluorinated or otherwise.
O
O
F
+
N
-
OH
O
N
N
N
N
F
RP60556A
O
F
COOH
F
N
H
N
H N
F
WQ-2944
NH
F
O
COOH
N
N
3. Quinolone-related agents
3.1 RP60556A
H N
F
F
D61-1113
RP60556A (RP) (Figure 3), the cholinate salt of a
benzo[b]-naphthyridone derivative, is structurally
related to quinolones. It is active against aerobic
Gram-positive cocci (staphylococci, streptococci and
enterococci) and anaerobes, with MIC values ranged
from 0.12 - 2 µg/ml (N Berthaud et al. Aventis Pharma,
Center de Recherche de Vitry-Alfortville, Vitry sur
Seine, France). The MIC values against Enterobacte-
riae and Pseudomonas were generally ≥ 64 µg/ml.
When tested against a panel of quinolone-sensitive
and quinolone-resistant S. aureus strains (with
mutations in GyrA, GrlA and/or NorA), RP60556A
demonstrated same level of activity (MIC values from
0.25 - 0.5 µg/ml) (N Berthaud et al., Aventis Pharma).
It was also equally active against quinolone-sensitive
and quinolone-resistant S. pneumoniae clinical
isolates, including the strains resistant to quinolone,
penicillin, macrolides, lincosamides and strepto-
gramin B. The MIC values ranged from 0.5 - 1 µg/ml.
Against S. aureus and S. epidermidis, RP60556A had
strong and quick bactericidal activity, generally
occurring at concentrations ranging from 2 - 4 times
MIC within a 3 - 6 h period (N Berthaud et al., Aventis
Pharma). Against streptococcus Group A, RP60556A
was generally bactericidal at concentrations ranging
from 2 - 4 times MIC within a 3 h period. The
hypothesis of transepidermal diffusion of RP was
exceptional potency that is equipotent with an
identical compound with C6 fluorine. Profiling of the
metabolic specificity of these compounds, by
monitoring DNA, RNA, protein and cell wall synthesis
in S. aureus, indicated that the DNA synthesis inhibi-
tion was in line with so-called third generation
quinolones. In this regard, the DNA synthesis inhibi-
tion was more specific than observed in earlier
compound generations. In general, the non-FQs are
rapidly bactericidal in vitro and are claimed to have
activity in mouse infection models. A series of S.
aureus mutants against the non-FQ compounds were
selected in vitro. These were interesting in that
non-FQs selected mutations at sites outside the
quinolone resistance determining region (QRDR)
w h e re FQ mu tatio n s are u su ally fo u n d .
‘Non-traditional’ mutations in GrlA include His103-Tyr
and SeR52-Arg; Glu472-Val in GrlB and Glu477-Val in
GyrB. Prior studies had indicated that the common
QRDR mutation Ser84-Leu led to only two- to
four-fold MIC increases for non-FQ compounds.
However, in the presence of these other mutations,
particularly the GrlA changes outside the QRDR, MIC
increases of 512-fold were observed. Two posters
rounded out the section, one on the activities of the
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2952 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
supported by the experiment by F-X Bernard et al.
(Aventis Pharma). These results suggest the potential
of RP as a topical anti-Gram-positive agent.
superior to those of levofloxacin. WQ-2944 appears to
be a new antibacterial agent with potent activity,
especially against quinolone-resistant Gram-positive
bacteria.
3.2 D61-1113
D61-1113 (Figure 3) is a leading compound from a
series of 5-amino-1-[2-(S)-fluoro-1-(R)-cyclopr-
op yl]-8-methylquinolones with the modified
3-(aminomethyl)pyrrolidin-1-yl substituents at the
C7-p osition. It has the 3,4-cis-oriented (3S,
4R)-4-(1-aminocyclopropyl)-3-fluoropyrrolidin-1-yl
C7 substituent. Its antibacterial activity, pharmacoki-
netics and preliminary safety profiles were described
by H Takahashi et al. (Daiichi Pharmaceutical Co. Ltd.,
Japan). D61-1113 showed a broad antibacterial
sp e ctru m again st b o th Gram-p o sitive an d
Gram-negative bacteria. It was more potent than
reference quinolones, vancomycin, teicoplanin and
linezolid especially against clinical isolates of
Gram-positive bacteria including ofloxacin-resistant
MRSA, MRCNS, PRSP and VRE. The activity of
D61-1113 against clinical isolates of Gram-negative
bacteria was comparable with those of aforemen-
tioned reference drugs. It achieved high C5min in the
serum and good tissue distribution pattern in liver,
kidney and lung (AUCtissue/AUCserum > 3.7) after iv.
administration to experimental animals. The serum
protein binding of D61-1113 in human was 75%.
D61-1113 also showed favourable safety profiles. It
did not prolong QT interval in monkeys under the
repeated-dose toxicity experimental conditions.
D61-1113 was selected for further evaluation as the
promising candidate for the treatment of serious
infections due to Gram-positive bacteria.
4. Ketolides
4.1 Telithromycin
Aventis researchers presented several posters on their
new ketolide, telithromycin, specifically developed
for respiratory tract infections. A poster presented by
B Leroy et al. (Aventis, Bridgewater, NJ and Aventis
Pharma, Hoechst Marion Roussell Romainville,
Romainville, France) reported the Phase III results of
the efficacy of telithromycin in treating bacteraemia
with community-acquired pneumonia (CAP). The
clinical trials included 755 patients ranging in age
from 18 - 65. A dose of 800 mg once daily for 7 - 10
days was evaluated in three multicentre, randomised,
double-blind comparator studies (comparators:
amoxicillin 100 mg three times a day for ten days,
clarithromycin 500 mg twice daily for ten days and
trovafloxacin 100 mg once daily for 7 - 10 days).
Clinical and bacteriological assessments were made
throughout the course of the study. The clinical and
bacteriological efficacy of telithromycin post-therapy
and test of cure (TOC) were excellent. The clinical
efficacy was 91.8% and bacteriological outcome was
90.0%. The clinical success rate for telithromycin for
bacteraemia was 90% (27/30) and 88.5% (23/26) for
documented pneumococcal bacteraemia. Telithro-
mycin at 800 mg once daily for 7 - 10 days was as
effective as the comparators in the treatment of CAP. B
Leroy et al. (Aventis and Aventis Pharma, Hoechst
Marion Roussell Romainville) presented on the in
vitro susceptibility of telithromycin against clinical
isolates of respiratory pathogens. This study obtained
clinical isolates from adult patients with community-
acquired respiratory tract infections (RTI) from a
multicentre clinical trial. The key respiratory tract
pathogens for this study were identified as S. pneumo-
n ia e, H. in flu en za e, H. pa ra in flu en za e, M.
catarrhalis, S. aureus and S. pyogenes. Patients were
treated with an oral dose of 800 mg of telithromycin
once daily: 7 - 10 days for CAP, five or ten days for
acute maxillary sinusitis (AMS) and five days for acute
exacerbation of chronic bronchitis (ACEB) or tonsilo-
pharyngitis. A single dose of 800 mg of telithromycin
provided excellent coverage for clinical isolates of key
respiratory tract pathogens. The clinical efficacy and
b acte rio lo gical cu re w e re e valu ate d at
3.3 WQ-2944/WQ-3402
Th e SAR o f n o ve l acid ic, 7-amin o o r
7-alkylamino-1-(5-amino-2,4-difluorophenyl)-8-meth
ylquinolones was studied by Y Kuramoto et al.
(Wakunaga Pharmaceutical Co. Ltd., Hiroshima,
Japan). The alkyl chain on the amino group at the
7-position is essential to enhance the antibacterial
activity against Gram-positive resistant strains.
Although not affecting the activity against
Gram-positive bacteria, the increase in the alkyl chain
length reduced the activity against Gram-negative
bacteria. WQ-2944 (Figure 3), with methylamino
group at the 7-position, was the most potent acidic
quinolone tested against clinical isolates including
quinolone-resistant strains. The therapeutic effects of
WQ-3402 (ethanolamine salt of WQ-2944) were
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2953
Figure 4: Chemical structure of ABT-773.
rat thigh infection model (ED50 for a 2 log reduction in
bacterial burden of 2.3 mg/kg/day). The agent was
bactericidal at 24 h to all pathogens tested (LM Edine
et al. and KL Credito et al., Hershey Medical Center,
Hershey, PA).
N
N
ABT-773 also showed both in vitro (MIC90 = 0.03 -
0.06 µg/ml) and cellular (HL-60 cells) activity against
L. pneumophila (K Sens et al., VA Medical Center,
Pittsburgh, PA). Susceptibility testing of clinical
isolates of S. pneumoniae obtained from upper
respiratory tract of children carrying erythromycin-
resistant genes (including erythromycin MIC > 256
µg/ml, strains), by C Johnson et al. (Univ. Alabama,
Birmingham, AL), showed minimal increase of the
MIC values of ABT-773 (MIC90 of 0.125, 0.032 and 0.5
µg/ml for mefE, ermB and both genes, respectively).
These isolates remained susceptible to ABT-773,
which was reported to have similar potency as the
newer quinolones and is two- to three-times more
active than telithromycin (Aventis). Posters by
Canadian researchers (BM Raily et al., Univ. Toronto
and K Weiss et al., Univ. Montreal, Montreal, Canada)
reported similar activities for ABT-773 against strepto-
cocci with one exception. A S. pneumoniae strain
with a MIC of 128 µg/ml was identified, raising
concern of existing resistance in the community. In
Japan, surveillance data reported similar potency of
ABT-773 for all S. pneumoniae, MSSA, S. pyogenes
tested and also observed one quinolone-resistant
MSSA showing MIC of ABT-773 at > 128 µg/ml (T
Fujikawa et al., Toho Univ., Tokyo, Japan).
O
O
O
HO
O
H
N
O
O
O
O
O
post-therapy/TOC at days 17 - 21, except for tonsilio-
pharyngitis evaluated at days 16 - 20. The clinical and
bacterial cure rates for telithromycin against the key
pathogens were: S. pn eu mon ia e (94.6%), H.
influenzae (79.3%), H. parainfluenzae (85.7%), M.
catarrhalis (92.3%), S. aureus (100.0%) and Strepto-
coccus pyogenes (88.3%).
4.2 ABT-773
ABT-773 (Figure 4) is a 14-membered ketolide
currently under clinical development by Abbott
Laboratories (Abbott Park, IL) for treatment of respira-
tory infections. It was reported to be active against
β-lactam and macrolide-resistant streptococci, H.
influenzae and M. catarrhalis. ABT-773 was reported
to be bactericidal against macrolide-sensitive and
resistant S. pneumoniae with faster time kill kinetics
than erythromycin (N Ramer et al. Abbott Labs.). At 8
times MIC, 2 h drug exp osure, the in vitro
post-antibiotic effect (PAE) for ABT-773 was 2 - 4 h
and 5 - 6 h for macrolide resistant (MICABT-773 0.004 -
0.008 µg/ml and MICerythromycin > 8 µg/ml) and
sensitive (MICABT-773 0.004 - 0.008 µg/ ml and
MICerythromycin 0.008 - 0.12 µg/ml) strains, respec-
tively. A 4 - 7 h in vitro PAE was shown for H.
influenzae (MIC 1 - 2 µg/ml) and M. catarrhalis (MIC
0.015 - 0.06 µg/ml). GA Pankuch et al. (Hershey
Medical Center, Hershey, PA) also reported similar
results. Dosing at 6 mg/kg resulted in in vivo PAE
(murine thigh infection) of 11 h with S. pneumoniae
and of 5 - 9 h with S. aureus (DR Andes et al., U
Wisconsin, Madison, WI). The 24 h AUC/MIC best
predicted the in vivo activity of ABT-773 for both S.
pneumoniae and S. aureus infections. The protein
binding was 90%. ABT-773 was active against
fluoroquinolone-resistant (parC and/or gyrA mutants)
S. pneumoniae (L Almer et al., Abbott Labs.) both in
vitro (MIC90 = 0.03 µg/ml) and in vivo in neutropoenic
In a dose-escalation study (RS Pradhan et al., Abbott
Labs.) ranging from 100 - 1200 mg in fasting human
subjects, AUC deviated from linearity primarily in the
100 - 400 mg dose range and once daily dosing of this
agent was suggested. Higher accumulation than the
dose proportion was observed in this range. The Cmax
increased proportionally with dose and mean t
½
ranged between 3.6 and 6.6 h. The bioavailability of
ABT-773 is unaffected by food (RS Pradhan et al.,
Abbott Labs.).
4.3 CP-654743
CP-654743, a fluoro ketolide, targeting respiratory
tract infections was reported (T Kaneko et al., Pfizer,
Inc., Groton, CT) with MIC values against key
pathogens (Table 1).
In murine peritonitis and pulmonary infection models
(D Girard et a l., Pfizer, Inc., Groton, CT) by
MLS-phenotype, penicillin-resistant S. pneumoniae,
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2954 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Table 1: MIC values of CP-654743 against key pathogens.
Pathogen
S. pneumoniae S. pneumoniae S. pneumoniae
H. influenzae
S. pyogenes S. pyogenes mefA
ermB/ermA
ermB
ermA
MIC
0.004 µg/ml
0.125 µg/ml
0.25 µg/ml
2.0 µg/ml
6.3 µg/ml
1.0 µg/ml
the mean oral ED50 values were 22.7 and 28 mg/kg,
respectively. In gerbil middle ear H. influenzae
infection model, the mean ED50 was 23 mg/kg.
penicillin-susceptible, 20.0% penicillin-intermediate
and 17.1% penicillin-resistant) collected in 2000.
Based on MIC90s, the order of activity for all S.
pneumoniae isolates was cefditoren (0.5 µg/ml) >
ceftriaxone = levofloxacin (1 µg/ml) > penicillin =
amoxicillin/clavulanate (2 µg/ml) > ampicillin =
cefuroxime (4 µg/ml) > azithromycin = SXT (> 4
µg/ml) > cefprozil (8 µg/ml). Cefditoren had higher
MIC90s against penicillin-resistant strains (PEN-R, 1
µg/ml) than penicillin-susceptible strains (PEN-S, 0.03
µg/ml), yet it was more active against PEN-S, PEN-I
(penicillin-intermediate) and PEN-R isolates than
other β-lactams tested. Cefditoren was also active
against strains resistant to other cephalosporins and
levofloxacin. Time-kill experiments were performed
by D Draghi et al. (MRL) against nine recent clinical
isolates of S. pneumoniae with different phenotypes.
Results showed that cefditoren had potent bactericidal
activity against strains of S. pneumoniae that were not
only resistant to penicillin, but also other antimicro-
bial classes including fluoroquinolones, macrolides
and SXT.
5. New β-lactams/cephalosporin
5.1 Cefditoren
Cefditoren, formerly ME-1206, is a novel oral cephalo-
sporin that has potent activity against the bacterial
species most commonly associated with respiratory
tract infection. A number of posters investigated the in
vitro activities of cefditoren against clinical isolates of
H. influenzae, M. catarrhalis, S. pneumoniae and
non-pneumococcal streptococci. DF Sahm et al. (MRL,
Herndon, VA and Brentwood, TN) tested the activities
of cefditoren against 1372 H. influenzae strains
(35.2% β-lactamase-positive) and 843 M. catarrhalis
strains (83.7% β-lactamase-positive) collected from US
hospitals during 1999 - 2000. Also tested were other
antimicrobials such as cefuroxime, cefprozil, ceftri-
axo n e , amo xicillin / clavu lan ate , amp icillin ,
erythromycin, clarithromycin, azithromycin,
trimethop rim/ sulp hamethoxazole (SXT) and
levofloxacin. Cefditoren has excellent activity against
H. influenzae (MIC values ranging from ≤ 0.008 to
0.25 µg/ml); in contrast, the MIC values of comparator
β-lactams ranged from ≤ 0.015 to 32 µg/ ml.
β-lactamase production in H. influenzae did not affect
the MIC distribution for cefditoren. The cefditoren
MIC90 was 0.015 µg/ml for both β-lactamase-positive
and β-lactamase-negative isolates, equivalent to
levofloxacin, similar to ceftriaxone and more active
than amoxicillin/clavulanate. Based on MIC90 values
the hierarchy of activity of tested β-lactams for all M.
catarrhalis isolates was as follows: amoxicillin/
clavulanate (0.25 µg/ml) > cefditoren = ceftriaxone
(0.5 µg/ml) > cefuroxime (2 µg/ml) > cefprozil (4
µg/ml) > ampicillin (8 µg/ml). The MIC90s for
cefditoren were higher against β-lactamase-positive
(0.5 µg/ml) than β-lactamase-negative (0.015 µg/ml)
isolates. The MIC90s for macrolides (0.03 - 0.25 µg/ml)
were lower than cefditoren.
Th e
activitie s
o f
ce fd ito re n
again st
non-pneumococcal streptococci were evaluated by
DF Sahm et al. (MRL). Isolates (n = 450) of viridans
streptococci, 917 S. pyogen es and 800 other
β-haemolytic streptococci were tested. Based on
MIC90s, the order of activity for viridans streptococci
isolates was cefditoren (0.5 µg/ml) > penicillin (2
µg/ml) > amoxicillin/clavulanate = ampicillin =
cefuroxime (4 µg/ml) > cefprozil (16 µg/ml). The
MIC90s for cefditoren against S. pyogenes and other
β-haemolytic streptococci were 0.015 and 0.06 µg/ml,
respectively. Cefditoren was the most active β-lactam
tested. All this data demonstrated cefditoren’s
potential as a treatment for respiratory tract infections.
5.2 RWJ-54428 (MC 02479)
RWJ-54428 (MC 02479) (Figure 5), a novel cephalo-
sporin, was previously shown to have excellent
activity against Gram-positive organisms. The
anaerobic activity of this compound was evaluated
against 363 anaerobes isolated from clinical
specimens by LM Kelly et al. (Hershey Med. Ctr,
Hershey, PA and Case Western Reserve Univ.,
A similar study was conducted by C Thornsberry et al.
(MRL) against 2597 S. pneumoniae isolates (62.9%
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2955
Figure 5: Chemical structures of new β-lactams/cephalo-
sporins.
were comparable with those of cefazolin. The
frequency of isolation of resistance to RWJ-333441 in
MRSA COL was very low (< 10-10 at 2x MIC). Besides
excellent anti-MRSA activity, RWJ-333441 was shown
to be potent against penicillin-resistant pneumococci,
viridans group streptococci and vancomycin-resistant
E. faecalis (J Blais et al., Microcide). Its MIC90 values (2
µg/ml) were similar to vancomycin against MRSA and
MRCNS (coagulase-negative staphylococci) and it was
active against MRSA with reduced susceptibility to
glycopeptides (MIC values 0.125 - 2 µg/ ml).
RWJ-333441 (MIC90, 1 µg/ml) was more active than
ampicillin (MIC90, 2 µg/ml) against vancomycin-,
gentamicin- and ciprofloxacin-resistant Enterococcus
faecalis and it was the most potent agent tested
against ampicillin susceptible E. faecium (MIC90, 4
µg/ml). RWJ-333441 showed good activity against H.
influenzae and M. catarrhalis (MIC90, 2 µg/ml), but
poor activity against other Gram-negative bacteria
(MIC90 > 128 µg/ml). It demonstrated excellent
bactericidal and significant post-antibiotic effects
against multi-resistant staphylococci (C Park et al.,
Microcide). The aqueous solubility of RWJ-333441 at
physiological pH (7) is 4.4 mg/ml, which is lower than
desired for iv. administration. A prodrug approach
was reported by SJ Hecker et al. (Microcide) to
improve its solubility. Several acyl derivatives of the
C(3) primary amino group of RWJ-333441 were
prepared and their aqueous solubility, cleavage in
vitro in serum and conversion to parent drug in vivo
w e re me asu re d . Th e asp artate d e rivative
(RWJ-333442) demonstrated desirable properties and
appeared to be suitable for parenteral administration.
The activity of RWJ-333441 (administrated as either
the active or the prodrug form RWJ-333442) in the
mouse sepsis and the neutropoenic mouse thigh
models was investigated by D Griffith et a l.
(Microcide). Both forms were shown to be efficacious
against MSSA, MRSA and GISA (glycopeptide-
intermediate S. aureus). The in vivo efficacy and
potency was similar or superior to current treatment
(e.g., vancomycin and Synercid™) for MRSA.
OH
N
H
N
N
S
S
H N
N
S
O
Cl
N
S
O
CO -
NH +
RWJ-54428 (MC 02479)
OH
H
N
N
S
N
S
H N
N
O
N
N
S
S
O
CO -
NH +
RWJ-333441 (MC 04546)
Cleveland, OH). The results showed it had low MIC
values only against β-lactamase-negative anaerobic
strains. The investigators suggested that if this
compound is to have additional activity against
β-lactamase-positive organisms, combination with a
β-lactamase inhibitor will be necessary.
5.3 RWJ-333441
Researchers from Microcide Pharmaceuticals, Inc.
(Mountain View, CA) presented a series of posters on
RWJ-333441 (MC 04546), a new cephalosporin active
against a variety of clinically important Gram-positive
bacteria, including MRSA. It is a lead compound from
a series of 3-(heteroarylthio)cephems. The discovery
of this compound was reported by T Glinka et al.
(Microcide). It has been found that in order to achieve
high in vitro activity against MRSA it is necessary to
append electron-withdrawing substituents at C-3 and
C-7 of the cephem core, which increase the reactivity
of the β-lactam ring. Optimisation of anti-MRSA
activity versus stability toward serum-mediated
degradation required a fine balance of substituent
effect; RWJ-333441 was the one with the superior
balance. It displays potent anti-MRSA activity (MIC90,
2 µg/ml), high rat serum stability (6% decomposition
over 60 min) and improved pharmacokinetics in rat
(total clearance 0.39 l/ h/ kg). The potency of
RWJ-333441 against MRSA was shown to be related to
its enhanced affinity for PBP2a by J Blais et al.
(Microcide). It had high activity against MRSA COL
(MIC, 1µg/ml) and high affinity for PBP2a (IC50 = 0.8
µg/ml). It was stable to staphylococcal β-lactamases
and its rates of hydrolysis by various β-lactamases
6. β-Lactamases and inhibitors
6.1 ESBLs (extended spectrum β-lactamases)
Reports covering β-lactamase (BLA)-mediated
bacterial resistance to β-lactams, especially the third
generation cephalosporins, were well represented at
the 2000 ICAAC. The patterns of resistance prevalence
are both dynamic and complex as well as regional and
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Exp. Opin. Invest. Drugs (2000) 9(12)

2956 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Table 2: New ESBLs and AmpC-like BLAs or new bacterial hosts for known BLAs as reported at the meeting.
Organism
P. mirabilis
Type
BLA name
Residing where?
Note
ESBL
TEM-67 (IRT)
Plasmid
T Naas et al., Hospital de Bicetre, le
Kremlin-Bicetre, France
P. mirabilis
ESBL
TEM-87
Plasmid
M Perilli et al., U L’aquila, Italy.
Tazobactam, IC50 55 nM, Clavulanate, IC50
130 nM
P. mirabilis
E. cloacae
AmpC-like
ESBL
ACC-1
Plasmid
Plasmid
A Ben Assen et al., Charles Nicolle Hospital,
Tunis, Tunisia
TEM-80
C Aprin et al., Univ. Bordeaux 2, Bordeaux,
France
First IRT reported in E. cloacae
E. coli
CTX-M-10
FOX-5
A Oliver et al., Hosp. Ramony Cajal, Madrid,
Spain
K. pneumoniae
AmpC-like
Plasmid
AM Queenan et al., R.W. Johnson Res. Inst.
Raritan, NJ, USA
Enterobacteriaceae
Norcardia asteroides
ESBL
A
Toho-3
AST-1
70-kb plasmid
Chromosome
Y Ishii, Toho Univ., Tokyo, Japan
F Laurent et al., Hospoces Civils de Lyon, Pierre
Benite, France
Ochrobactrum
anthropi
AmpC-like
Chromosome
CS Higgins et al., Univ. Bristol, Bristol, UK
Inducible, inhibited by BRL42715, not by
clavulanate
Chryseobacterium
indologenes
MBL IND-1, -2, -2a, -2b,
-3, -3a & -4
S Bellais et al., Hospital de Bicetre. Asist. Publ.
Paris, France
Shared 77 - 90% amino acid identity
Aeromonas sobria
MBL
ImiS
RM Mosi et al., AnorMED, Inc., BC, Canada
hospital sites dependent. Among the four Ambler-
typed BLAs, expression of ESBLs attributed to the
most frequently identified mechanism of β-lactam
(BL) resistance. Enterobacteriaceae such as E. coli and
Klebsiella pneumoniae are the mostly commonly
detected TEM and sulfhydryl variable (SHV) classes
ESBLs carrying pathogens. New enzymes with distinct
substrate profile and mutations continue to evolve. As
of July 2000, the TEM variant BLAs reported were up
to TEM-90 (many are clavulanate-resistant) and the
SHV variants were up to SHV-26. A website
monitoring the emergence of new ESBLs set up by G
Jacoby of the Lahey Clinic and K Bush of the RW
Johnson Pharmaceutical Research Institute can be
reached at http://www.lahey.org/studies/webt.htm.
Other pathogens harbouring ESBLs include Proteus
mira bilis, En teroba cter cloa ca e, Morga n ella
morganii, Enterobacter aerogenes and Klebsiella
oxytoca.
Klebsiella (9%) > Enterobacter spp. (7%) (D Mathai et
al., Univ. Iowa, Iowa City, IA). Characterisation of
ESBL phenotype in Europe (EU), North and Latin
America and Western Pacific indicated the high
prevalence of ESBL in K. pneumoniae: Latin America
(45%) > Western Pacific (24%) > EU (21%) > US (7%) >
Canada (4%). E. coli isolates showed similar order,
varying at 1 - 8%. Resistance due to ESBL continues to
evolve especially in EU and the Western Pacific
region. Significant inter-strain and -species dissemina-
tion was documented, but the rates of ESBL in US,
Latin America and Canada appears stable. RA
Bonomo (Veteran’s Affairs Medical Center, Cleveland
OH) also evaluated ESBLs prevalence in K. pneumo-
niae worldwide and reported that 58.9% of isolates
tested positive with TEM or SHV genes by PCR.
Among them, greater than 78% of strains harboured
multiple ESBLs that are located on plasmids.
RA Bonomo’s group (Veteran’s Affairs Medical Center,
Cle ve lan d , O H) co n d u cte d site -satu ratio n
mu tage n e sis at th e co n se rve d S130 an d
D104-positions of the SHV BLA. Except for the S130G
variant, all other E. coli variants containing individual
6.2 SENTRY
SENTRY worldwide surveillance databases from 1997
- 99 indicated the most prevailing Gram-negative
bacteria in ICU settings are Eschericha coli (9%) =
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Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2957
complicated protocols and separate confirmation at
biochemical levels.
Table 3: The identification of β-lactamase inhibitors.
New ESBLs and AmpC-like BLAs or new bacterial
N
O
hosts for known BLAs were reported (Table 2).
O
S
By ap p lyin g e le ctro sp ray io n isatio n / mass
spectrometry (ESI/MS) analysis to the tazobactam-
treated CMY-2 enzyme, RA Bonomo’s group
(Veteran’s Affairs Medical Center, Cleveland, OH)
demonstrated the covalent modification of SeR64 of
CMY-2 (a class C BLA) by a fragment of tazobactam.
No cross-linking to other amino acid residue was
observed.
N
R
O
CO
Enzyme (class) & IC50, µM
Compound, R P99 (C) TEM (A) PC1 (A) GC1 (C)
Tazobactam
49.8
0.5
0.32
0.3
2.8
2.6
0.1
0.72
0.3
1.4
75
3.4
NT
CH2-O-COCH3
CH = CHCONH2
CH = CHCN
JD Buynak (Southern Methodist Univ., Dallas, TX)
and JR Knox (Univ. Connecticut, Storrs, CT) reported
a crystal structure of E. cloa ca e GC1 class C
β-lactamase in complex with a 7-alkylidene-
cephalosporin sulphone inhibitor. Their goal was to
identify inhibitors with coverage over as many classes
of β-lactamase as possible (Table 3).
0.26
0.01
0.2
0.09
0.014
0.02
0.07
68
0.01
0.012
0.3
CH = CHCO2Me
CH = CHClCO2Me
CH = CH-CH = CH
CH = CHCO2But
0.9
0.18
NT
24
1.48
NT
240
NT
RA Powers et al. (North Western Univ., Chicago, IL)
reported the determination of β-lactamase inhibitory
activity of eight acylglycine boronic acid inhibitors
with the acyl group representing the R1 substitutions
of penicillin G, penicillin V, penethicillin, cloxacillin,
nafcillin, cephalothin, cephapirin and ceftazidime
(Figure 6). The Ki values of these inhibitors for AmpC
ranged between 0.02 - 0.7 µM, while R1 = CH3 gave a
Ki of 19 µM. The binding affinity of these inhibitors to
TEM-1 enzyme was significantly lower. Complexed
AmpC crystal structures with two acylglycine boronic
acid inhibitors (R1 = cloxacillin or cephalothin) were
determined. The inhibitors bound at the region where
the R1 side chain of β-lactam typically binds.
H-bonding and hydroxyl binding sites were identi-
fied. The key interacting residues are either conserved
or have equivalent counter part among classes A and
C enzymes. The information provides a map of
binding sites for future inhibitor design. Inhibitors
carrying the R1 chain of nafcillin (Ki = 0.03 µM) and
ceftazidime (Ki = 0.02 µM) were shown to potentiate
the antibacterial activity of ceftazidime against AmpC
expressing E. cloacae in disk diffusion plate assay.
The ceftazidime side chain containing inhibitor was
stated to be at least 6000-fold more selective for AmpC
than chymotripsin, trypsin and elastase.
Figure 6: Structure of acylglycine boronic acid inhibitors.
O
OH
OH
B
R
N
H
mutations at S130 were ampicillin and piperacillin-
susceptible (MIC < 8 µg/ml). This resistance is limited
to clavulanate only, not to sulbactam or tazobactam.
Except for D104C, mutations at D104 retained
resistance to ampicillin and piperacillin. D104H, 104R
and 104K variants showed increased resistance to
ceftazidime, as compared with SHV-1, suggesting the
positive charge interfering with ceftazidime binding.
6.3 AmpC-type β-lactamases (class C)
Testing and reporting of class C serine BLAs are not
standardised as those of ESBLs. These AmpC-like
enzymes are cephalosporinases that are not suscep-
tible to clavulanate inhibition and have pIs ranging
from 7 - 9. Although AmpC was initially identified as
chromosomally located and exhibited inducible
phenotype, more and more AmpC-like BLAs are
detected at plasmid locations and hyper-express
constitutively. A significant number of pathogens (E.
cloacae, K. pneumoniae, Citrobacter fruendii, etc.)
are now known to harbour multiple BLAs belonging
to different classes (A & C, A&B, etc.). Specific identi-
fication of AmpC-like BLAs and ESBLs in these
multiple BLAs-expressing organisms required more
D Tondi (Univ. Modena and Reggio Emilia, Modena,
Italy) presented a poster on carboxamide- and
sulphonamide boronic acid inhibitors of AmpC BLA.
One of the most potent compounds has a Ki of 83 nM
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Exp. Opin. Invest. Drugs (2000) 9(12)

2958 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 7: One of the most potent carboxamide- and sulphona-
mide boronic acid inhibitors of AmpC BLA (Ki = 83nm).
were synthesised and tested as MBL inhibitors using
nitrocefin as substrate. One of the Cys-7mers-Cys
identified showed mixed inhibition with Ki of 10 µM,
Km of 15 µM and Kcat of 24 s-1. Addition of Zn+2 was
able to rescue the peptide-mediated inhibition.
Binding of the peptide inhibitor resulted in no detect-
able structural change of L1 by circular dichroism.
S
H
S
N
O
S
O
O
O
B
OH
M Gilpin (SKB, Harlow, UK) reported on the SAR of
thiazolidine and proline mercaptocarboxylate MBL
inhibitors. Activities against four MBLs (L-1, IMP-1,
CfiA and Bc II) were evaluated. IC50 values of ~ 0.2 -
0.4 µM were achieved with the compound shown in
Figure 8. The D-stereochemistry at the C-terminal
amino acid conferred maximal inhibition of BcII and
CfiA while L-1 and IMP-1 were less discriminating.
ACE inhibition (captopril) was optimised on the
L-series compounds, indicating the selective differ-
ences that can be built in for MBL inhibitors.
HO
Figure 8: Chemical structure of a thiazolidine and proline
mercaptocarboxylate MBL inhibitor.
HS
O
N
S
O2C
JL Huber (Merck Research Laboratory, Rahway, NJ)
presented a poster on succinic acid derivatives as
IMP-1 MBL inhibitors (Figure 9).
(Figure 7). The crystal structure of AmpC in complex
with the inhibitor was determined to 1.9 Å resolution.
The inhibitors show little intrinsic antibacterial activity
alone. Reversal of amoxicillin resistance by this
inhibitor was observed in Gram-positive bacteria
only, but not in Gram-negative organisms.
These compounds have no intrinsic antibacterial
activity (MIC > 200 µM). A majority of the 18
imipenem-resistant strains of Pseudomonas and S.
marcescens (MIC values 8 - > 256 µg/ml, expressing
IMP-1 MBL) tested showed decreased MIC values of
imipenem to < 8 µg/ml when used in combination
with 12.5 µM of either inhibitors.
A Patera (North Western Univ., Chicago, IL), in
collaboration with Eli Lilly, reported complexed
crystal structures of loracarbef (substrate) and
cloxacillin (inhibitor) bound to deacylation-deficient
AmpC mutant (Q120L) and inhibitor moxalactam
bound to WT AmpC. Data obtained is consistent with
a β-phase catalytic water attack and both TyR150 and
the substrate ring nitrogen stabilised the hydrolytic
transition state while inhibitors can act by blocking
the activation.
T Walsh (Univ. Bristol, Bristol, UK) presented
bulgecin A as a weak L1-MBL inhibitor (IC50 = 150
µM). This group of compounds has antibacterial and
anticancer activities. A patent has been filed for the
use of these comp ounds in p harmaceutical
compositions.
6.4 Metallo β-lactamases (MBL, class B)
7. Streptogramins
CS Higgins (Univ. Bristol, Bristol, UK), in association
with SmithKline Beecham, reported a study that
showed the N-terminal 20 amino acid residue was not
essential for the tetramer formation of L1 MBL from S.
maltophilia, but significantly reduces the binding
affinity of substrates with large aromatic side chains.
Streptogramins belong to an antibiotic class consisting
of two structurally unrelated components, group A
and group B. The group A compounds are polyun-
saturated macrolactones and the group B compounds
are cyclic hexadep sip ep tides. The group A
compounds interfere with the elongation of the
peptide chain by preventing the binding of
aminoacyl-tRNA to ribosome. The group B
compounds stimulate the dissociation of peptidyl-
tRNA and may also interfere with the release of the
completed peptide. Combination of group A and B
compounds works synergistically in inhibiting protein
F Sanschagrin (Univ. Laval, Ste-Foy, PQ, Canada)
presented a poster on using the phage display
libraries to facilitate the identification of novel MBL
inhibitors. The L1 MBL from S. maltophilia was
cloned, overexpressed and purified. The purified
His-tagged L1 was used to screen phage peptide
libraries. Peptides containing consensus sequences
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Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2959
Figure 9: Chemical structures of some succinic acid deriva-
tives as IMP-1 MBL inhibitors.
activity included multi-drug resistant Gram-positive
cocci (MIC, 0.12 - 0.5 µg/ml) and some Gram-negative
bacteria (H. influenzae MIC, 1 µg/ml; not active
against enterobacteria) responsible for respiratory
tract infections. After a 3 h treatment at 8 times MIC (1 -
4 µg/ml), the agent caused a 2.16 - 1.24 log10 CFU/ml
d ro p o f in ge ste d S. a u r eu s (MSSA an d
MRSA-MLSB-CR) inside murine macrophage cells. In a
mo u se p n e u mo n ia mo d e l (S. pn eu m on ia e
MLSB-resistan t), d o sin g at 120 mg/ kg, p o .
RPR-20868/RPR-132552 resulted in a drop of bacterial
count of 2.93 log10 CFU/ml at 6 h post initial
treatment. In mouse lung, RPR-20868 accumulated
faster than RPR-132552 and was not in accordance
w ith th e in itial 30/ 70 ratio ad min iste re d .
RPR-20868/RPR-132552 demonstrated activities in
MRSA (MLSB-resistant) sepsis model with ED50 of 44
mg/kg (20 mg/kg for MSSA infection) and in the
mouse abscess model with ED50 of 60 mg/kg.
Clarithromycin was not active in these models at a
concentration of 300 mg/kg. When infected with
MLSB S. pneumoniae, the mouse sepsis model gave
ED50 values ranging 24 - 55 mg/kg and the mouse
pneumonia model with an ED50 at 50 mg/kg.
N
HO C
CO H
N
N
3 Cl
K = 0.13 nm
N
O
HO C
CO H
3 Cl
N
K = 0.15 nm
N
synthesis and are strongly bacteristatic, sometimes
bactericidal. These antibiotics are produced naturally,
but with very poor water solubility. Semisynthetic
analogues of these two groups of compounds have
been shown to possess improved solubility,
enhancing their therapeutic values.
7.1 Synercid
Synercid (quinupristin/dalfopristin; Aventis, Vitry-
sur-seine, France), an iv.-only agent, is the first
commercially available human antibiotic of the
streptogramin class. It was approved for the treatment
of serious or life-threatening infections associated
with vancomycin-resistant E. faecium (but not E.
faecalis) bacteraemia and for complicated skin and
skin structure infections caused by MSSA and S.
8. Novel oxazolidinones
Versicor is participating in a collaboration with
Pharmacia & Upjohn to identify, through combinato-
rial methodologies, and develop a second-generation
oxazolidinone with an improved spectrum of activity
and improved potency. Versicor (Z Nie et al., Versicor,
Fremont, CA) has synthesised VRC-322 (Figure 10), a
structurally novel C-5 cinnamide, that has an activity
profile against Gram-positive pathogens very similar
to that of linezolid. The evaluation of a C-5 linezolid
analogue library showed an unexpected tolerance for
extended C-5 lipophilic substitutions. The investiga-
tors hypothesised that there may be an as yet
unexplored lipophilic pocket at the ribosomal binding
site, which may offer even greater opportunity for
second-generation oxazolidinones. The four substitu-
ents at the 3-aryl site were also explored by Versicor.
pyogenes. Synercid
is bacteriostatic against E.
fa eciu m and bactericidal against strains of
methicillin-susceptible and methicillin-resistant
staphylococci. The mode of action differs from that of
other classes of antibacterial agents such as β-lactams,
aminoglycosides, glycopeptides, quinolones,
macrolides, lincosamides and tetracyclines. There was
no cross-resistance between Synercid
agents.
and these
7.2 RPR-20868/RPR-132552
The novel 3′-fluoro-4-thioether phenyloxazolidi-
nones had good activity against Gram-positive
pathogens (GW Luehr et al., Versicor, Fremont, CA).
VRC-3406, the most active compound in this series,
had MIC values of 2, 2 and 1 µg/ml against S. aureus,
VRE and S. pneumoniae, respectively. VRC-3406 also
had an MIC of 8 µg/ml against the linezolid-resistant S.
aureus mutant (linezolid MIC > 64 µg/ml). Versicor
Reported by J-C Barriere (Aventis, Vitry-sur-seine,
France) and several Aventis poster presentations (N
Berthaud et al., Aventis, Vitry-sur-seine, France), in
clinical development by Aventis was the oral strepto-
gramin RPR-20868/RPR-132552, a 30/70 mixture,
targeting community acquired respiratory tract
infections. The in vitro spectrum of bacteriostatic
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Exp. Opin. Invest. Drugs (2000) 9(12)

2960 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 10: Chemical structures of some novel oxazolidinones.
O
O
F
O
O
H
N
H
N
N
N
F
O
O
N
F
S
F
O
O
VRC-3816
VRC-322
O
O
N
O
N
F
O
H
N
H
N
F
H
N
O
O
S
S
F
N
O
F
O
VRC-3839
VRC-3054
O
N
O
H
N
O
N
O
H
N
O
O
S
F
R
HN
F
N
R1
O
R2
RO
RWJ-302377, R=Ch Ph
RWJ-306490, R=H
PNU: 2-aminomethyl-1,3,4-thiadiazole
phenyl oxazolidinones
has also identified novel 4-amido-3 fluorophenyloxa-
zolidinones (MF Gordeev et al., Versicor, Fremont,
CA). VRC-3125 was the most active in this series with
MIC values of 1 - 2, 1 and 2 µg/ml against S. aureus,
VRE and S. pneumoniae, respectively. No in vivo
efficacy was shown for VRC-3125, but VRC-3054
(Figure 10) and VRC-3055 were both found to be
orally efficacious with ED50 values of 8.5 and 7.5
mg/kg/day in a murine model of septicaemia. These
values are nearly identical to ED50 values resulting
from treatment with linezolid. The activities of novel
4-acylamino and 4-sulfonamido-3-fluorophenyl
oxazolidinones were also presented (MF Gordeev et
al., Versicor, Fremont, CA). VRC-3599 was the most
active compound in this series with MIC values of 0.25
- 0.5, 8, 0.5, 0.13 - 0.5 µg/ml, against S. aureus,
linezolid-resistant S. aureus, VRE and S. pneumoniae,
respectively and MIC values of 2 - 8 and 4 µg/ml
against the fastidious Gram-negative bacteria H.
influenzae and M. catarrhalis, respectively. Versicor
provided information on their synthesis and screening
of novel tetrahydo-4(2H)-thiopyranyl sulphoxide and
sulphone libraries. VRC-3816 and VRC-3839 (Figure
10) were active against the Gram-positive pathogens
(MIC values of 0.5 - 4 µg/ml), active against the
fastidious Gram-negative pathogens (MIC values of 4 -
8 µg/ml) and were also active in a murine model of
septicaemia (ED50 values of VRC-3816 and VRC-3839
were 3.75 and 6.52 mg/kg/day, respectively).
Investigators at AstraZeneca found that the acetami-
domethyl side chain could be replaced by limited 5-
and 6-membered oxygen-linked heteroaromatic
substituents and retain activity in vitro that was
comp arable with the amide analogues (MB
Gravestock, AstraZeneca, Macclesfield, UK). These
substitutions were made to oxazolidinones previously
modified by having an unsaturated piperidine or
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Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2961
pyran at the C-4-position on the phenyl ring. Hetero-
cycles containing sulphur were the most active. The
thiadiazolyl series yielded MIC values of 0.13 - 0.25
µg/ml against S. aureus and 0.25 µg/ml against E.
faecalis. In vivo efficacy was verified as a reduction in
bacterial numbers in a murine model of a localised S.
aureus infection. Investigators also found that the C-5
of the oxazolidinones (modified by having an unsatu-
rated piperidine or pyran at the C-4-position of the
phenyl ring) can be replaced by a wide variety of
heterocyclic aminomethyl groups (RG Wilson,
AstraZeneca, Macclesfield, Cheshire, England). The
unsubstituted 5-membered rings provided the
greatest amount of in vitro activity (MIC values of 0.13
- 0.5 µg/ml against the Gram-positive pathogens).
This in vitro activity was significantly reduced by the
increase in ring size to a 6-membered ring.
Compounds with an isoxazole at C-5 provided the
best combination of in vitro activity and in vivo
efficacy. In continuing the search for the second-
generation oxazolidinones, the investigators at
AstraZeneca showed that O-linked and N-linked
5-membered heterocycles at the C-5-position can be
combined with a wide range of N-linked heterocycles
at the C-4 of the phenyl ring. The most active
compounds in the 4-heteroaryl-1-piperazine series
had MIC values of 0.06 - 0.5 µg/ ml. In the
4-heteroaryl-1-piperazine series, the best compounds
had MIC values of 0.125 - 0.5 µg/ml against the
Gram-positive pathogens. The best compounds in the
N-linked heteroaromatic series had MIC values
ranging from 0.13 - 2 µg/ml against the Gram-positive
pathogens and the most active compounds in the
3-aminopyrrolidine series had MIC values of 0.13 - 1
µg/ml. The in vivo efficacy of these compounds were
verified in a murine model of a localised thigh
infection. The thiadiazolyloxymethyl and the isoxazo-
laminomethyl groups were the most active in vitro.
However, the most efficacious compounds in vivo
were those with the amino linkage.
20 mg/kg/day) when compared with linezolid (ED50
values of 2 - 9 mg/kg/day).
Investigations at Bayer on the imidazo-benzoxazinyl-
oxazolidinones (S Bartel et al., Bayer AG, Leverkusen,
Germany and Bayer AG, Wuppertal, Germany) have
shown that compounds in this series have very potent
in vitro activity against respiratory pathogens
including S. pneumoniae, H. influenzae and M.
pneumoniae. MIC values for these active compounds
were 0.125 - 4 µg/ml for S. pneumoniae and 0.5 - 8
µg/ml for H. influenzae. An N-acetyl derivative was
selected for further study (R Endermann et al., Bayer
AG, Wuppertal, Germany and Bayer AG, Leverkusen,
Germany). Although the pharmacokinetic parameters
were less favourable with this compound compared
with linezolid, the overall increase in in vitro activity
resulted in an in vivo efficacy that was as good as or
superior to linezolid. Both compounds significantly
reduced lung titres of S. pneumoniae (3 log10 CFU/g
tissue at 2 x 25 mg/kg/day, orally). The N-acetyl-
derivative caused only a slight reduction in bacterial
counts of H. influenzae in the murine respiratory
model. In the murine model of septicaemia, the
N-acetyl derivative was reportedly more efficacious
than linezolid.
Efforts at the RW Johnson Pharmaceutical Research
Institute (MA Weidner-Wells et al., Raritan, NJ)
focused on the modifications of the N-substituent of
piperidinyloxy-substituted oxazolidinones (Figure
10). These compounds were observed to have
moderate to weak activity against Gram-positive
pathogens, with the best compounds having MIC
values of 2 - 16 µg/ml.
9. Antifungals
9.1 Ravuconazole
Bristol-Myers Squibb presented a number of posters
on their new broad spectrum antifungal agent,
ravuconazole. SJ Olsen et al. (Bristol-Myers Squibb,
Princeton, NJ and Medeval Ltd., Manchester, UK)
described the clinical safety study of a single
ascending oral dose in healthy patients. This clinical
trial was a randomised, double-blind, placebo-dosed
and single-dosed study; the dose was escalated in
healthy male patients with the first dose of 50 mg oral
capsule of ravuconazole or placebo. Patients were
evaluated at days 4 and 7 for safety and tolerability.
Eight untreated patients were then given escalation
doses of 100, 200, 400, 600 and 800 mg. Clinical and
LM Thomasco et a l. (Pharmacia Corporation,
Kalamazoo, MI) provided in vitro and in vivo data on
the 2-aminomethyl-1,3,4-thiadiazole p henyl
oxazolidinone thioamides (Figu r e 10). Many
compounds in this series had excellent activity against
the Gram-positive pathogens (MIC values of 0.25 - 1
µg/ml) as well as activity against the fastidious
Gram-negative pathogens H. influenzae and M.
catarrhalis (MIC values of 0.5 - 41 µg/ml). However,
none of these very lipophilic compounds were effica-
cious in a murine model of septicaemia (ED50 values >
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Exp. Opin. Invest. Drugs (2000) 9(12)

2962 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
laboratory evaluations for the next four weeks were
followed. A second dose was provided to the patients
in the 100, 400 and 800 mg groups. The patients in the
200 mg groups received an oral solution of 10 ml of 20
mg/ml ravuconazole or placebo with clinical and
laboratory evaluations for the following four weeks.
The safety profile showed no serious adverse events
with headache and abdominal pain the most
frequently reported. Ravuconazole was 50% bioavail-
able at the 200 mg oral solution dose. A half-life
ranging from 3.5 - 7.5 days was reported supporting
once daily dosing regimen. DM Grasela et al. (Bristol-
Myers Squibb and Medeval Ltd.) reported on the
multiple ascending oral dose in healthy subjects. A
sequential ascending dose study of ravuconazole with
the following doses: 50, 100, 200 and 400 mg. Healthy
male patients were treated once a day for 14 consecu-
tive days, beginning with the lowest concentration
group. Safety and tolerability were monitored for up
to 28 days for each group. Each dose study began after
the results of the lowest study was available. The most
serious adverse event reported was headache.
Ravuconazole was safe and well-tolerated up to the
400 mg dose. The half-life ranged from 4.3 - 10 days
and continued to accumulate throughout the study.
Ravuconazole evaluation of the cytochrome P450
isoenzyme, CYP3A, showed no induction.
sub-inhibitory concentrations of amphotericin B,
5-flucytosine, fluconazole or GW 471558 and single
step selection of resistance on agar containing 10 or
100 times MIC of one of the antifungal agents. No
progressive increase in MIC values of GW 471558
occurred even after 40 serial passages. In single-step
studies, the frequency of GW 471558 mutants on 10
times MIC was in the order of 2 x 10-8 or lower and
similar to values obtained with amphotericin B. The
frequency of mutation with 5-flucytosine was about
three orders of magnitude greater than the frequency
found for GW 471558. The efficacies of GW 471552,
GW 471558 and GW 531920 were evaluated in two
different rat models of Pneumocystis carinii infections
(A Matinez et al., Glaxo Wellcome S A, Madrid, Spain).
In both models, doses of 0.25 - 5 mg/kg subcutane-
ously, twice a day for ten consecutive days,
significantly reduced the number of P. carinii cysts
and also strongly inhibited parasite development in
infected rats compared with the untreated controls.
Treatment of infected rats with trimethoprim/
sulphamethoxazole proved ineffective. The in vivo
efficacies of the azasordarins, GW 471552, GW 471558
and GW 531920 were also evaluated in rat models of
oral and vaginal candidiasis at doses of 1, 5 and 10
mg/kg, subcutaneously, three times a day, for seven
days or every 4 h for three days. In the rat model of
oral candidiasis, all the azasordarins eradicated the
infection at the 10 mg/kg dose. GW 471552 and GW
531920 at 5 mg/kg, markedly reduced the number of
recoverable organisms compared with the untreated
controls. In the rat model of vaginal candidiasis, GW
471558 and GW 521920 eradicated the organisms at 5
mg/kg. GW 471552 significantly reduced the number
of recoverable organisms at 5 mg/kg and eradicated
the organisms at 10 mg/kg. The therapeutic efficacy of
GW 531920 was tested in a murine model of systemic
candidiasis (P Aviles et al., GlaxoWellcome S A,
Madrid, Spain) at doses of 40, 20, 10 and 5 mg/kg,
subcutaneously, every 8 h, for seven consecutive
days. A pharmacokinetic analysis was performed as
well to obtain information on dosing proportionality.
Overall, there was a good correlation with the net
effect versus daily dose and the percent survivors
versus daily dose. Dose proportionality was also
demonstrated. For the 40, 20, 10 and 5 mg/kg dose,
the AUCs (µg.h/ml) were 63.4, 22.9, 12.2 and 5.6,
respectively and the Cmaxs (µg/ml) were 29.9, 11.3,
5.2 and 2.13, respectively, while the percent survivors
by day 18 were 90, 60, 20 and 0%, respectively. The
pharmacokinetic parameters of GW 471558 and GW
531920 were evaluated following iv. administration in
9.2 Azasordarins
There were several presentations that provided
information on the in vitro and in vivo activities of the
Glaxo azasordarins. Azasordarins are derivatives of
th e so rd arin s th at are ch aracte rise d b y a
6-methylmorpholi-2-yl group with N-4 substituents
different from the sugar moiety. According to the data
provided by E Herreros et al. (GlaxoWellcome S A,
Madrid, Spain), GW 471588 and GW 531920, respec-
tively, were found to have MIC90s of 0.015 and 0.004
µg/ml against Candida albicans, 0.25 and 0.25 µg/ml
against C. glabrata, 0.12 and 0.12 µg/ml against C.
tropicalis and > 16 and 0.25 µg/ml against C. parapsi-
losis. C. krusei was resistant to both azasordarins. GW
531920 was the more active agent against Aspergillus
and the dermatophytes, with MIC values of 16 µg/ml
against A. fumigatus, 4 µg/ml against A. flavus and
0.12 µg/ml against Trichophyton mentagrophytes. It
was also more active against the less common moulds,
with MIC values of 2 µg/ ml against Fusa rium
oxysporum. The development of resistance was also
investigated by E Herreros et al. Resistance emergence
was evaluated by sequential subculture in
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2963
the mouse, rat and dog. Based on the resulting AUCs
formerly LY-303366) (Figure 11), a semi-synthetic
antifungal agent under development for the iv.
treatment of serious systemic fungal infections due to
Candida and Aspergillus. The echinocandins are
fungicidal for Candida and act by inhibiting β-1,3
glucan synthase. The Phase I dose optimisation
studies (GL Brown et al., Versicor, Inc. and Charter-
house Clinical Research Unit, Royal Masonic Hospital,
London, England) showed that VEC could be dosed at
a higher concentration (loading/maintenance) than
tested in the earlier Phase I. Doses tested in this study
were 100/70 mg (cohort A) and 140/100 mg (cohort
B). VEC was well-tolerated in 5/6 patients given the
100/70 dose and in 3/6 patients given the 140/100
dose. Patients in both cohort A and B who did not
tolerate the drug well exhibited transient symptoms
consistent with a histamine release type of reaction. In
cohort B, 2/3 of these patients also exhibited a grade II
adverse event. Based on the PK and safety data
provided, the investigators believe that the 100/70
would be the maximum tolerated dose and that Phase
II/III trials can be conducted safely at doses higher
than the previously reported 70/35 mg regimen. The
Phase II, randomised, open-label study, consisted of
36 patients with oesophageal candidiasis given VEC in
either of two iv. doses, 50/25 mg (loading/mainte-
nance) or 70/35 mg, for a maximum of 14 - 21 days
(GL Brown et al., Versicor, Fremont, CA and Eli Lilly
and Company, Indianapolis, IN). At the termination of
the study, 29/36 patients were considered evaluable,
16 in the 50/25 mg group and 13 in the 70/35 mg
group. Endoscopy scores based on achieving a grade
0 or 1 improvement at the end of treatment gave an
efficacy of 81% for the 50/25 mg group and 85% for
the 70/35 mg group. In the 50/25 mg group, 68.8%
(11/16) had resolution of both dysphagia and retros-
ternal chest pain and in the 70/35 mg group, 81.8%
(9/11) had resolution of both dysphagia and retros-
ternal chest pain. Of all 36 patients enrolled, there
were a total of 28 adverse events in 16 patients (eight
patients/cohort) that were reported as possibly
related to the study drug, including injection site
reaction (three), headache (three), vasodilation (two),
hypotension (two) and nausea (two). There were
11/36 patients (31%) that experienced a serious
adverse event; three were reported as possibly related
to study drug and included aspartate amino transfe-
(µg.h/ ml), t and Vss (l/ kg), the investigators
½
concluded that the azasordarins have PK properties
that can be predicted across the different species.
Preliminary toxicology of the azasordarins was also
provided (E Herreros et al.) for GW 471552 and GW
471558. Cytotoxicity was tested against five different
cell lines and a primary hepatocyte cell line. In the
transformed cell lines from target organs including
brain, cervix, muscle, kidney, lung and liver, cytotox-
icity was evaluated by crystal violet exclusion and
protein synthesis inhibition at 48 and 72 h. For the
primary hepatocyte line, toxicity was evaluated by
intracellular lactic dehydrogenase. For GW 471552,
IC50 values ranged from 62 to > 128 µg/ml and for GW
471558 the IC50 values (µg/ml) were all > 128. In
considering the activity of the azasordarins against C.
albicans, this represents a therapeutic index of
~10,000. The LD50s were also evaluated against male
and female mice following a bolus (10 - 15 sec) iv.
administration. The LD50s (mg/kg) for GW 471552
were 196 (male) and 276 (female) and the LD50s for
GW 471558 were 222 (male) and 268 (female).
9.3 Echinocandins
CM Douglas et al. (Merck, West Point, PA and Thomas
Jefferson University, Philadelphia, PA) presented on
the activity of caspofungin against Aspergillus
fumigatus in vitro and in vivo in immunosuppressed
mice. The murine cyclophosphamide-induced
immunosuppression produced a chronic immuno-
sup p ression when mice were treated with
cyclophosphamide over the course of the experiment
(28 days) or a transient immunosuppression when
treatment was over 14 days. Mice in the chronic
immunosuppression murine model were treated once
daily with either caspofungin or amphotericin B for
seven days. The PD50s were 0.173 and 0.235 mg/kg
for caspofungin and amphotericin B, respectively.
Mice in the transient immunosuppression model were
treated once daily for 14 days, producing PD50s of
0.245 and 0.264 mg/ kg for caspofungin and
amphotericin B, respectively. The survival of mice in
both immunosuppressed murine models was 100%.
The effect of caspofungin at a dose of 0.3 µg/ml for 6 h
at 37°C against A. fumigatus in liquid culture showed
killing of the apical cells. The killing capacity of
caspofungin of the tips of A. fumigatus hyphae grown
in liquid cultures was 75%.
rase
(se ru m
glu tamic
o xalo ace tic
transaminase-SGOT) increase, alanine amino transfe-
rase (serum glutamic pyruvic transminase-SGPT)
increase and leukopoenia.
Versicor, Inc. (Fremont, CA) presented data from their
Phase I and Phase II trials of V-echinocandin (VEC,
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2964 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 11: Chemical structures of the echinocandins, VEC and FK-463.
O
OH
HO
HN
O
N
O
N
H
HO
H
O
HN
O
OH
H
O
O
N
H
HO
N
H
N
H
OH
H
O
HO
OH
VEC
(LY-303366)
OH
HO
OH
O
O
HO
N
H
N
O(CH2)4CH3
N O
N
H2N
O
OH
HN
O
O
O
HO
N
N
O
N
HO
OH
OH
O
FK-463
NaO3SO
HO
Several presentations also highlighted the develop-
me n t o f FK-463, th e e ch in o can d in u n d e r
development by Fujisawa Pharmaceutical Co. Ltd.,
Osaka, Japan (Figure 11). The in vitro activity of
FK-463 was evaluated against a spectrum of Candida
species (L Ostrosky-Zeichner et al., Univ. of Texas,
Houston, Medical School, Houston, TX). The values
were essentially identical after 24 or 48 h of incuba-
tion, whether evaluated visually or spectrophoto
metrically. The median MIC (range) was 0.015 µg/ml
(0.015 - 2 µg/ml) for C. albicans (272), 0.015 µg/ml
(0.015 - 2 µg/ml) for C. glabrata (94), 0.25 µg/ml
(0.015 - 0.5 µg/ml) for C. krusei (32), 0.062 µg/ml
(0.031 - 1 µg/ml) for C. lusitaniae (11), 1 µg/ml (0.015
- 4 µg/ml) for C. parapsilosis (104) and 0.031 µg/ml
(0.015 - 1 µg/ml) for C. tropicalis (92). V Petraitis et al.
(NCI, NIH, Bethesda, MD) presented a study that
involved the comparative antifungal activity of FK-463
against disseminated candidiasis and invasive
pulmonary aspergillosis in persistently neutropoenic
rabbits. Rabbits with candidiasis were treated
intravenously with FK-463 at 0.25, 0.5 and 1 mg/kg or
amphotericin B at 1 mg/kg 24 h following infection.
There was a dosage-dependent clearance of
organisms. In rabbits treated with 0.25 mg/kg FK-463,
there was clearance of C. albicans in all tissues except
the vena cava and lungs. In vitro time-kill studies also
showed a concentration-dependent killing of C.
albicans. Rabbits infected with A. fumigatus were
treated intravenously with FK-463 at 0.5, 1, or 2
mg/kg, amphotericin B or liposomal amphotericin B.
Although there was an increase in survival and a
decrease in infarcts, there was not a dosage-
dependent reduction in the CFU/g tissue of A.
fumigatus in this model. As expected, the rabbits
treated with amphotericin B had significant
reductions of C. albicans and A. fumigatus in both of
these models. In a murine model of aspergillosis (M
Nakajima et al., Kawasaki Medical School, Kurashiki,
Japan and Fujisawa Pharmaceutical Co. Ltd, Osaka,
Japan), mice were treated with FK-463 (0.5, 1, or 2
mg/kg), amphotericin B (0.25 or 0.5 mg/kg) or a
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2965
combination of FK-463 and amphotericin B at 1 and
0.25 mg/kg or 2 and 0.5 mg/kg. The investigators
concluded that pathological findings in the mice
treated with the combination of amphotericin B and
FK-463 were more favourable and correlated with
improved survival rates compared with the two
groups treated with FK-463 or amphotericin B alone.
Other investigators (S Kohno et al., Nagasaki Univ.
School of Medicine, Nagasaki, Japan and Saitama
Medical School, Saitama, Japan) who also used a
similar murine model of aspergillosis, found the
combination of FK-463 plus amphotericin B effective
(more effective than either agent alone) and the
combination significantly reduced the fungal burden
in the lungs of mice.
9.4 Novel azoles
S Takeda et al. (SSP Co. Ltd., Chiba, Japan) presented
the structure-activity relationship of SS750, a new
triazole containing a gem-difluormethylsulphonyl
moiety (Figure 12). Compounds were evaluated for
in vitro activity against various fungal pathogens
(yeast and moulds) and were tested for in vivo activity
against a murine model of systemic candidiasis. The
most active agent, SS750, contained an ethanol
substituent at R′. SS750 had MIC values of 0.063 µg/ml
for C. albicans, 0.5 µg/ml for C. krusei, 2 µg/ml for A.
fumigatus and 2 µg/ml for A. flavus. This compound
was also efficacious in vivo with 5 of 5 mice surviving
infection following treatment with oral SS750 at 1.25
mg/kg/day. Studies by M Matsumoto et al. (SSP Co.
Ltd., Chiba, Japan), were established to compare the
in vitro and in vivo activities of SS750 with the activi-
ties of fluconazole and itraconazole. In vitro, SS750
was more active than fluconazole, but less active than
itraconazole against a fluconazole-susceptible and
-resistant strain of C. albicans.
The pharmacodynamics of FK-463 were evaluated in
a neutropoenic mouse thigh infection induced by
injection of C. albicans. Mice were treated with 0.5, 1
and 2 mg/kg FK-463 and viable counts in the muscle
were evaluated 24 h after therapy. Mice were treated 1
h following infection. The reduction in viable CFU/g
showed that there was a dose-dependent response to
treatment with FK-463 and at 1 mg/kg there was a
significant reduction in CFU/ g compared with
untreated controls. The concentration of FK-463 in
muscle at 1 and 7 h following therapy with FK-463 at 1
mg/kg was 0.5 and 0.3 µg/ml, respectively. These
values are equivalent to 0.5 and 0.25 times the MIC in
90% mouse serum or 4% human serum, respectively
and were very much greater than the MIC values
determined in RPMI/MOPS. The plasma pharmacoki-
netics and tissue distribution of FK-463 were
evaluated in rabbits (AH Groll et al., NCI, Bethesda,
MD, NIH, Bethesda, MD, MDS, Harris, Lincoln, NE
and Fujisawa Healthcare, Deerfield, IL). FK-463 was
dosed at 0.5, 1 and 2 mg/kg iv. into normal rabbits
once a day for eight consecutive days. The Cmax
values were 10.3, 17.2 and 19.7 µg/ml and the AUC
values were 5.7, 13.4 and 22.9 µg.h/ml, respectively.
For all three doses, the VD was ~ 0.3 l/kg, the Cl was
When evaluated for in vivo efficacy (on day 7) against
the fluconazole-susceptible strain, SS750 given orally
was four- to five-fold more active than fluconazole
and 100- to 200-fold more active than itraconazole in a
murine model of systemic candidiasis (involving
either normal or immunosuppressed mice) as well as
in a murine model of pulmonary candidiasis. When
given intravenously, SS750 was four- to five-fold more
efficacious than fluconazole given intravenously.
Given orally, SS750 on day 7 was four-fold more
active than fluconazole and > 16-fold more active than
itraconazole against the fluconazole-resistant strain.
When given intravenously, SS750 was > ten-fold more
active than fluconazole given intravenously. The
therapeutic efficacy of SS750 was also evaluated in
murine models of systemic aspergillosis and crypto-
coccosis (K Yokoyama et al., Research Center for
Pathogenic Fungi and Microbial Toxicoses, Chiba,
Japan and SSP Co. Ltd., Chiba, Japan). The in vitro
activity against the strains used in these models
showed SS750 to be more active than fluconazole, but
less active than itraconazole. However, SS750 had
excellent efficacy in vivo, with equivalence shown for
iv. and oral administration, against systemic aspergil-
losis and cryptococcosis in immunosuppressed mice.
In both models, SS750 was significantly more effica-
cious than either fluconazole or itraconazole. T
Kogure et al. (SSP Co. Ltd., Chiba, Japan) investigated
the metabolic fate of SS750 in rats. Rats were dosed
with radiolabelled SS750, orally and intravenously, at
0.8 - 0.9 l/h/kg and the t values were ~ 3 h. The
½
plasma pharmacokinetics were best described by a
two-compartment pharmacokinetic model that
showed linear disp osition. FK-463 achieved
potentially therapeutic levels that exceeded the MIC
of susceptible fungi. FK-463 also penetrated into
tissues that are common sites of fungal infection.
Tissue concentrations near peak plasma concentra-
tions were observed in lung, liver, spleen and kidney.
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2966 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 12: Chemical structures of some novel azoles.
O
HO
N
O
O
HO
N
N
N
N
N
N
N
N
H
N
N
O
N
N
N
N
N
N
F
H
N
F
Cl
F
TAK-456
TAK-457
F
F
CN
O
O
F
OH
F
SO Et
F
HO
H
N
N
N
N
S
N
N
F
F
F
R-120758
SS750
4 mg/kg. A Cmax of ~ 2 µg/ml in plasma was achieved
in ~ 1 h. Plasma concentrations were reduced with a
found have an ED50 of 1.6 - 2.3 µM/kg in a murine
model of systemic candidiasis. However, TAK-456
had a water solubility of 5 µg/ml. Quaternary salts of
TAK-456 were synthesised to identify a water-soluble
prodrug for injectable formulation. TAK-457, which
contained an acetoxymethyl triazolium moiety, had
t
of 2 and 1.4 h for the iv. and oral administration,
½
respectively. Bioavailability achieved ~ 75% and
protein binding was 50 - 63%. At 1 and 4 h
post-administration, SS750 was found mainly
unchanged in the liver and plasma. The high bioavail-
ability and high absorption support the observed in
vivo efficacy. The effects of SS750 on cytochrome
P450 were studied by K Ishida et al. (SSP Co. Ltd.,
Chiba, Japan). The IC50 values of SS750 for inhibition
of [14C] mevalonic acid into ergosterol of C. albicans
and C. krusei were equivalent to the IC50 values of
itraconazole at 1.5- and 3.7-fold, respectively and less
than fluconazole. SS750 was a more potent inhibitor
of the fungal cytochrome P450 system than flucona-
zole, however, the affinity for the human cytochrome
P450 was equivalent for SS750 and fluconazole. These
data demonstrate a higher selectivity between the
fungal and human cytochrome P450 systems for SS750
compared with fluconazole.
water solubility (4 - 10 mg/ml) and stability (in vitro t
½
of 5 - 6.4 h in mouse, rat and human) sufficient for an
iv. formulation. In mouse, rat and human plasma in
vitro, TAK-457 was readily converted to TAK-456 and
it was also readily converted to TAK-456 in rats.
Further studies on the in vitro activity and in vivo
efficacy were provided by Y Iizawa et al. (Takeda
Chemical Industries Ltd, Osaka, Japan). Both TAK-456
and TAK-457 were found to be more active than
fluconazole, itraconazole and voriconazole in murine
models of systemic candidiasis in normal and neutro-
poenic mice, whether the infecting strain was
fluconazole-susceptible or fluconazole-resistant.
TAK-457 was also more active than amphotericin B in
a murine model of an A. fumigatus pulmonary
infection when survival rates, lung weights, reduction
of fungal burden in lungs and plasma β-glucan levels
were all compared. TAK-456 and TAK-457 may prove
useful in the treatment of fungal infections including
those caused by Aspergillus.
The in vitro and in vivo antifungal activities for
TAK-456 and the water soluble prodrug TAK-457
(Figure 12) were reported (T Kitazaki et al., Takeda
Chemical Industries Ltd, Osaka Japan). TAK-456 is a
new azole that contains an imidazolidinone nucleus.
TAK-456 has activity against C. albicans (MIC values,
0.016 - 0.03 µg/ml), azole-resistant C. albicans (MIC, 2
µg/ml), C. glabrata (MIC, 4 µg/ml), C. krusei (MIC, 1
µg/ml), Cryptococcus neoformans (MIC, 0.5 µg/ml),
A. fumigatus (MIC, 0.5 µg/ml) and A. flavus (MIC, 1
µg/ml). Following oral administration, TAK-456 was
The in vitro activity of another newer triazole,
R-120758 (Figure 12), against fungal clinical isolates
(50 strains of each species) was also presented (A
Sanchez et al., Harbor-UCLA Research and Education
Institute, Torrance, CA and Sankyo Co. Ltd, Tokyo,
Japan). R-120758 had MIC50/MIC90s of ≤ 0.004/0.016
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2967
µg/ml against C. albicans, 0.5/≥ 4 µg/ml against
fluconazole-resistant C. albicans, 0.5/2 µg/ml against
C. glabrata, 0.25/0.25 µg/ml against C. krusei,
0.016/0.016 µg/ml against C. parapsilosis, 0.016/0.5
µg/ml against C. tropicalis and ≤ 0.004/≤ 0.004 µg/ml
against C. neoformans. Continued development plans
were indicated for this triazole antifungal.
Acin etoba cter ba u ma n ii and P. a eru gin osa )
demonstrated an effect on the MIC90 values against
azithromycin, clarithromycin and erythromycin with
the efflux inhibitor MC 04124.
10.2 Fungal EPIs
M Warren et al. (Microcide Pharm. Co. and Inst. Of
Microbiology, Lausanne, Switzerland) presented a
poster on inhibitors of fungal efflux pumps. There
were two types of inhibitors, one broad spectrum
targeting ABC transporters, CDR1 and CDR2 and the
second narrow spectrum targeting the CDR1 pump.
Two broad spectrum inhibitors, milbemycin and
beauvericin (Figure 13), given in combination with
either fluconazole or terbinafine, inhibited CDR1 in
both C. albicans and C. glabrata, decreasing the MIC
16- to 128-fold. Beauvericin had no effect on potenti-
ating fluconazole against C. albicans with BEN
upregulated. BEN is an MDR pump of the major facili-
tator family and not effected by the inhibitors. In
addition, beauvericin had no effect against defined
strains of both C. albicans and C. glabrata with
deleted copies of CDR1 and CDR2. This showed
specificity for these pumps by its lack of effect on the
strains with the deleted pumps. The narrow spectrum
efflux pump inhibitors, MC 05,204 and MC 380,286,
treated at a concentration of 1 µg/ml with fluconazole,
ketoconazole, itraconazole or posaconazole resulted
in a decrease of MIC values ranging from 133- to
256-fold. Microcide’s inhibitors are effective against
ABC transporters, either broad spectrum, CDR1 and
CDR2 or narrow spectrum, CDR1 only and no effect
has been shown for the major facilitator family.
10. Efflux pump inhibitors
10.1 Bacterial
Microcide had an entire poster session devoted to its
work on efflux pump inhibitors (TE Renau et al., R
Leger et al., MS Warren et al., D Griffith et al., D Cho et
al.). The antibacterial efflux inhibitors were derived
from a dipeptide structure, which had been modified
with N-methyl groups to stabilise the derivative to
serum peptidases. The modified compounds were
found to be slightly less active in potentiation of
levofloxacin against efflux pump overexpressing
pseudomonads in vitro, but stable to human serum for
an extensive period of time. The resulting compound,
D-ornithine-D-homo-phenylalanine-3NHQ, was used
as a basis for further modification by substitutions,
which led to peptidomimetic versions of the inhibitor.
Classes made and evaluated included ether and
thioether analogues, which were very active in
potentiation. Benzoxazole derivatives also had good
potentiation, but benzimidazoles and benzthiazoles
were less active.
Another presentation focused on the inhibitor MC
207110 (Figure 13), identified from high throughput
screening of a library of efflux inhibitor candidates
based on the prototypes. When tested against the Mex
pumps of P. aeruginosa, the compound was found to
be an inhibitor of the RND family of efflux pumps and
antibiotics that are effluxed by the pump do not
compete with MC 207110. This compound also
permeabilises the outer membrane of P. aeruginosa.
Testing of the dipeptide efflux inhibitor MC 02595 in
mouse infection models with P. aeruginosa (MexCD,
OprJ overexpression strain) demonstrated an effect
on the ED50 values in the sepsis model, consistent
with the lowered in vitro MIC values. A similar result
was obtained in the reduction of bacteria isolated
from infected thigh muscle in the mouse by the
combination of efflux inhibitor and levofloxacin
versus levofloxacin alone. Another in vitro study with
a variety of Gram-negative organisms (E. coli,
Sa lm on ella typh im u r iu m , H. in flu en z a e,
Another poster presented by K Sorensen et al.
(Microcide Pharm. Co.) described the in vivo potentia-
tion of fluconazole by MC 510,011 (Figure 13). A
murine neutropoenic mouse model and two isogenic
pairs of C. albicans: YEM 14 fluconazole-susceptible
and YEM 15 overexpressing CDR1 and CDR2. The
compounds evaluated were MC 510,011 a broad
spectrum efflux pump inhibitor (CDR1 and CDR2),
treated at a dose of 2 and 200 mg/kg orally and
fluconazole treated at a dose of 3.1 and 100 mg/kg,
intraperitoneal. Against the strain YEM 14, MC 510,011
decreased the kidney fungal burden by 2.0 - 2.5 log10
CFU/g. In contrast to strain YEM 15, neither MC
510,011 or fluconazole (100 mg/kg) had an effect on
decreasing kidney fungal burden. Combination
treatment of fluconazole and MC 510,011 decreased
kidney fungal burden by 1 - 2 log10 CFU/g. Assess-
ment of kidney fungal burden showed that the
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)

2968 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
Figure 13: Chemical structures of some efflux pump inhibitors.
O
O
O
O
O
N
O
H
O
N
O
O
O
OH
N
O
HO
O
O
O
Milbemycin (MC-510,021)
Beauvericin (MC-510,065)
n = 0,1
H
O
O
O
H
N
H N
N
H
O
O
H
O
OH
NH
NH
N
H
O
H
N
HO
MC-207110
MC-510,011
fluconazole and MC 510,011 combination had a 26%
decrease for YEM 14 and 36% decrease for YEM 15
over fluconazole treatment alone. The pharmacoki-
netic profile of MC 510,011 showed no effect in
infected neutropoenic mice. The effect on fluconazole
MIC values by treatment with MC 510,011 at a dose
ranging 0 to 4 µg/ml was a 0- to 133-fold decrease in
MIC for YEM 14 and a 0- to 1032-fold decrease in MIC
for YEM 15. The poster suggested that combination
therapy of an azole and an efflux pump inhibitor
maybe a useful approach in overcoming azole
resistance.
to the lack of the cladinose moiety, including telithro-
mycin, ABT-773 and TE-802. Global structure of the
drug is important for induction of resistance, which
can be conveniently measured by a green fluorescent
reporter (GFP) on the methylase gene. As far as
mechanism of action, the prototype of this class,
erythromycin, interacts with the peptidyl transferase
and methylation of the ribosome leads to decreased
affinity of erythromycin. In the case of the ketolide
ABT-773, there is improved binding to methylated
ribosomes, albeit not enough to defeat resistance in
MLS constitutive strains. In streptococci, however,
MLS resistance is primarily inducible, therefore
ketolides are active.
11. Exploratory research
Other types of macrolide resistance include the efflux
pump, mefA. This pump uses proton motive force for
energy and telithromycin is a weak inducer and
substrate relative to erythromycin. Another efflux
pump is the msrA, which is an ABC transporter, is
inducible and can result in a 6 - 12 times MIC increase.
In pneumococci mutations in the stem-loop region
that controls the methylase expression can destabilise
the region, leading to low level expression of
methylase and low level resistance. Additional
11.1 Resistance to ketolides: role of ribosomal
modification and macrolide efflux
Ketolides are 14-membered ring macrolides, which
have a 3-keto group instead of an L-cladinose moiety
on the erythronolide A ring (R Leclercq, Hopital Cote
de Nacre, Caen, France). They are distinguished from
azalides, which have an N-modification of the rings.
Ketolides are weak inducers of the erm methylase due
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Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2969
pneumococcal mutations are in the L22 protein,
which lead to modest increases in the MIC of telithro-
mycin. Mutations in L4 can give similar types of
increases. These latter two, combined with the
stem-loop mutation can result in resistant
pneumococci.
norfloxacin, levofloxacin and trovafloxacin. Both
single and double mutants of FQ targets have been
detected in clinical isolates of pneumococci.
He then described work in which the four proteins
(GyrA, GyrB, ParC and ParE) of pneumococci were
his-tagged and overexpressed for in vitro assays.
There are three types of assays that can be performed
with these enzymes: supercoiling, decatenation and
cleavable complex. Fisher indicated that the cleavable
complex assay was the best indication of activity. He
also cautioned against using IC50 values, as
quinolones trap the enzyme on the DNA and induce a
double-stranded DNA break, therefore it is not similar
to a standard enzyme substrate reaction. In studies,
both ciprofloxacin and sparfloxacin were found to
trap cle avab le co mp le xe s o f gyrase an d
topoisomerase IV. The results from genetic (mutant
selection) and biochemical assays do not always
agree. For example, in pneumococcal topoisomerase
IV is more susceptible in cleavable complex assay to
all FQs compared with gyrase. Some quinolones have
enhanced affinity for their targets, for example, the
CC50 for ciprofloxacin against DNA gyrase is 80 and 1
µM against topoisomerase IV, whereas for clinafloxa-
cin the values are 2.5 and 0.1 µM. To conclude with,
Fisher presented a model for FQ action, in which DNA
gyrase and topoisomerase IV represent two arms that
can both lead to cleavable complexes. Subsequent to
the establishment of a cleavable complex, there are
double-stranded DNA breaks mediated by helicases
and DNA polymerases involved in DNA replication,
resulting in cell killing. There is competition between
the two enzyme arms in the cell and there are
downstream events that contribute to killing. When
one performs an in vitro assay, one is not measuring
these events, which explains the lack of correlation
observed between in vitro enzyme assays and in vivo
FQ killing.
11.2 Fluoroquinolone resistance in pneumococci
An overview of several aspects of fluoroquinolone
(FQ) interactions with S. pneumoniae was described
by LM Fisher (St. George’s Hospital, London,
England). These include efflux, with the pmrA
encoded pump and target changes; namely DNA
gyrase and topoisomerase IV gene mutations that lead
to resistance. The two enzymes that are FQ targets,
DNA gyrase and topoisomerase IV, both perform their
functions via double-stranded cleavages in the DNA.
Changes in the protein sequences that lead to
increased antibiotic resistance are found in regions,
originally identified in E. coli as QRDRs, which are hot
spots for resistance development. The QRDR regions
are highly similar in amino acid composition in both
gyrase and topoisomerase, with resistance changes
occurring largely in conserved amino acids. The
N-terminal portion of the gyrase of E. coli has been
crystallised and there is a helix-loop-helix domain that
recognises the DNA, with tyrosines at the active site
forming covalent bonds with the DNA during strand
cleavage. The enzyme has a central hole where the
active site resides, with ‘gates’ on either side for DNA
entry and exit. The resistance mutational hot spots lie
along an α-helix at the interface that interacts with
DNA.
There are three types of common inhibition
mechanisms for FQs, as distinguished by their in vivo
target affinities as defined by mutant selection and
genetic studies. The first, ciprofloxacin type, has
topoisomerase IV as its primary target. The second
type, sparfloxacin type, has DNA gyrase as a primary
target and clinafloxacin defines the third type with
approximately equal sensitivity of both DNA gyrase
and topoisomerase IV to the drug. When measured
with an in vitro biochemical assay, generally all FQs
are more active against pneumococcal topoisomerase
IV. With new compounds, one can quickly charac-
terise them by performing MIC values with a panel of
strains having mutations in DNA gyrase and
topoisomerase IV. Many of the newer FQs (e.g.,
gatifloxacin, grepafloxacin, moxifloxacin, sparfloxa-
cin) target GyrA. In contrast, topoisomerase IV
appears to be the primary target of ciprofloxacin,
11.3 Antibiotics and ribosomes: interaction and
resistance
J Hansen (Yale Univ., New Haven, CT) described the
3-D structure of the ribosome. The recent solution of
the 50S subunit at the 2.4 Å level by the Steitz labora-
tory has led to several major insights in peptide bond
formation. The major achievement was the
unequivocal demonstration that the peptidyl transfe-
rase is a ribozyme activity of the central loop in
domain V of 23S rRNA. Implicated in this mechanism
was the base of A 2451 in E. coli, which has an unusual
pKa, due to hydrogen bond interactions with G 2447
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Exp. Opin. Invest. Drugs (2000) 9(12)

2970 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
and the buried phosphate of A2450. This allows the A
2451 to exist in a rare imino form and act in a charge
relay that increases the negative charge density,
allowing it to function as a base to facilitate a nucleo-
philic attack by the α-amino acid of the A-site
substrate. Another feature of the ribosome is the
existence of a 15 Å wide and 100 Å long tunnel, which
leads from the peptidyl transferase site to an exit point
for the nascent polypeptide. The ribosomal proteins
were found to be largely globular, with long
extensions that threaded into the ribosome and
interacted with the ribosomal RNA to neutralise
charges, fill voids and presumably stabilise the
structure. It was found that none of the protein
sidechains that penetrate and interact with domain V
are closer than 18 Å to the peptidyl transferase site.
PGE-9739390 and PGE-6737410 was evaluated in a
mu rin e se p sis mo d e l o f in fe ctio n . Th e
3-dithiocarbamoyl(carba)cephalosporins protected
mice against infection by S. pneumoniae and S.
aureus, but did not protect mice against infection by
E. coli or methicillin-resistant, ciprofloxacin-resistant
S. aureus.
11.5 R-115685
R-115685 is a new parenteral carbapenem antibiotic
being developed by Sankyo Co. Ltd. (Tokyo, Japan).
This 1-β-methylcarbapenem possesses excellent
Gram-positive and Gram-negative antibacterial
activity in vitro, including activity against MRSA, PRSP
and imipenem-resistant P. aeruginosa (S Ohya et al.,
Sankyo Co. Ltd., Tokyo, Japan). R-115685 is stable to
dihydropeptidase I and appears to be less nephro-
toxic than meropenem (I Kawamoto et al., Sankyo Co.
Ltd.). R-115685’s balanced spectrum has the
meropenem-like Gram-positive activity (S Ohya et al.,
Sankyo Co. Ltd.). R-115685 has a 16-fold lower MIC
than meropenem or imipenem against resistant
mutants of P. aeruginosa, was stable against all but
the metallo-β-lactamases and is bactericidal (N
Masuda et al., Sankyo Co. Ltd.). In vivo efficacy
confirmed the activity of R-115685, with excellent
protection against systemic infections caused by
Staphylococcus epidermidis, S. aureus, S. pneumo-
niae and Enterobacteriaceae, with ED50 values of ≤ 8
mg/kg (N Masuda et al., Sankyo Co. Ltd.). PK results in
animals, extrapolated to humans, predicts less
frequent dosing that with other carbapenems (S Ohya
et al., Sankyo Co. Ltd.).
11.4 Novel 3-dithiocarbamoylcephalosporins
A number of novel 3-dithiocarbamoylcephalosporins
(PGE-9951357, PGE-856854, PGE-9882816,
PGE-9739390 and PGE-6737410) were presented by
researchers from Procter & Gamble Pharmaceuticals
(P&G, Mason, OH). Their compounds were among a
series of compounds synthesised to identify new
β-lactams with broad spectrum activity against multi-
resistant Gram-positive bacteria including MRSA and
PRSP, through the inhibition of multiple PBPs
including PBP2a and PBP2xR. The drug resistance in
MRSA and PRSP was attributed in part to the presence
of low affinity penicillin binding proteins (PBP2a and
PBP2xR, respectively). Synthesis and SAR study were
provided. PGE-9951357 and PGE-856854, both
possessing the 3-(isoindolinyl) dithiocarbamoyl
moiety, had activities against MRSA (MIC values, 8, 64
µg/ml) and PRSP (MIC values, 1, 0.5 µg/ml) (RE White
et al., P&G). Introduction of a 5-chloro substituent to
the aminothiazolyl(oximino)acetyl led to compounds
with enhanced anti-MRSA/PRSP and PBP2a/PBP2xR
activity (e.g., PGE-6737410 and PGE-9882816) (Z
Chen et al., P&G). The potency of these compounds
was reduced in the presence of fetal bovine serum
(FBS). Efforts toward changing the physical properties
by attaching an amino side chain to the isoindolinyl
moiety resulted in compounds (e.g., PGE-9739390)
with maintained in vitro potency, but reduced
solubility. MIC values (µg/ml) for PGE-9882816,
PGE-9739390 showed the following properties:
MRSA, 2, 2; PRSP, ≤ 0.125, 0.5; E. coli, 2, 0.5;
vancomycin-resistant E. faecium, 32, 8. IC50 values
(µM): PBP2a, 0.9, 1.5; and PBP2xR, 0.3, 0.15, respec-
tively. The in vivo efficacy of PGE-9951357,
12. Novel bacterial targets
12.1 Peptide deformylase
Peptide deformylase is an essential bacterial enzyme,
which is highly conserved. Versicor, in collaboration
with Novartis, has synthesised combinatorial libraries
of peptide deformylase (PDF) inhibitors. CJ
Hackbarth et al. (Versicor, Fremont, CA and Novartis
Pharma AG Summit, NJ) presented a poster on
compounds that were screened against the PDF zinc
metalloenzyme and a panel of Gram-positive and
Gram-negative organisms. Several compounds
showed improved antibacterial activity, while
maintaining excellent enzymatic activity. VRC-3324
(Figure 14) was identified as a promising lead, with
improved whole cell activity against S. pneumoniae,
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Exp. Opin. Invest. Drugs (2000) 9(12)

Ryan , Ho, Wu, Frosco, Dough erty & Barrett 2971
Figure 14: Compounds for novel bacterial targets.
Pharmacokinetics of VRC-3375 was determined in
mice at a dose of 100 mg/kg by iv., sc. and po. This
compound has 64% oral bioavailability, it is rapidly
absorbed from the gastrointestinal tract and distrib-
uted to various tissues and rapidly cleared from the
serum. A mouse septicaemia model with S. aureus
(Smith strain) was used to determine efficacy.
VRC-3375 has PD50 values of 32 mg/kg iv., 17 mg/kg
sc. and 21 mg/kg po.
O
O
H
N
HO
N
H
N
H
VRC-3324
British Biotech presented three posters on peptide
deformylase profiling the inhibitor, BB-3497 (Figure
14). The first poster by W Thomas et al. (British
Biotech, Oxford, UK) presented a method of
screening a library of metalloprotease inhibitors
against the E. coli PDF. A novel high-throughput assay
was developed using a fluorometric assay, utilising
flourescamine as the amine reactive reagent. BB-3497
has excellent enzymatic activity against PDF of several
organisms, E. coli 7 nM, S. aureus 100 nM and S.
pneumoniae 30 nM. BB-3497 was shown to be
selective for PDF when tested against a panel of
mammalian metalloproteases. JM Clements et al.,
(British Biotech) presented a poster on antibacterial
activity of BB-3497 against a panel of Gram-positive
and Gram-negative organisms showed moderate
activity ranging from 0.25 - 32 µg/ml. Mechanism of
action studies used an E. coli strain with the deletion
of fmt, removing the need for deformylation and an E.
coli strain with PDF on a plasmid for overexpression.
The whole cell activity of BB-3497 against the E. coli
parent strain and the mutant E. coli had MIC values of
4 and > 128 µg/ml, respectively, showing specificity of
the compound for deformylase by the increased MIC
in the mutant strain. The bactericidal effects were
evaluated for BB-3497 by in vitro time-kill studies
against S. aureus, E. coli and E. faecalis at concentra-
tions 4 times MIC. For all organisms, the decrease in
viable counts was less than one log, which showed
bacteriostatic activity. Rate of resistance emergence
was determined for BB-3497 at 4 times MIC against E.
coli, S. aureus, E. faecalis, S. pneumoniae and H.
influenzae. The rates were 10-7 for E. coli, S. aureus
and E. faecalis, while the rates were much lower at
10-9 for S. pneumoniae and H. influenzae. Mutations
in the resistant strains of E. coli, S. aureus and E.
fa eca lis were found to be either missense or
frameshifts in the fmt gene. Pharmacokinetics of
BB-3497 given iv. or po. at a dose of 100 mg/kg was
rapidly absorbed in mice which showed a Cmax of 23
mg/l and a AUC0-24 of 23 mg.h/l. In vivo efficacy was
tested in a murine systemic infection of S. aureus with
OH
N
O
H
N
H
NH
O
O
BB-3497
S. pyogenes, H. influenzae and M. catarrhalis with
MIC values ranging from 0.13 to 4 µg/ml.
Other lead compounds were VRC-3375, VRC-3488,
VRC-3896 and VRC-3997 with excellent enzymatic
activity, ranging from 1 - 8 nM and whole cell activity
against some Gram-positive organisms. Bactericidal
studies with these compounds determined them to be
bacteriostatic according to the results of in vitro
time-kill studies. Several other posters provided a
profile of VRC-3375 (IC50 = 2 nM against Ni-PDF E.
coli) a potent deformylase inhibitor. P Margolis et al.
(Versicor) presented mechanisms of resistance studies
against H. influenzae and S. pneumoniae. The rate of
resistance development to VRC-3375 for both
organisms was 10-8, which is 100-fold less than S.
aureus. A single functional deformylase gene is
present in H. influenzae, unlike S. pneumoniae and S.
aureus, which has two copies, with only one copy
functional (defB). Resistance to H. influenzae showed
frameshift mutations in formyltransferase (FMT),
which results in the bypass of the formyla-
tion/deformylation cycle. Identifying the resistance
mechanism for H. influenzae to be the same as S.
aureus. Resistance to S. pneumoniae was identified as
mis-sense mutations in defB. The resistant mutants to
H. influenzae and S. pneumoniae showed antibiotic
resistance to actinonin and VRC-3375, but a pattern of
antibiotic susceptibility to other classes of antibiotics.
This shows a resistance pattern specific to peptide
deformylase compounds. D Chen et al. (Versicor)
presented a poster on in vivo activity of VRC-3375.
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Exp. Opin. Invest. Drugs (2000) 9(12)

2972 40th In terscien ce Con feren ce on An tim icrobial Agen ts an d Ch em oth erapy (ICAAC)
a single iv. or po. dose of BB-3497 given 1 h following
infection. An ED50 of 7 mg/kg, iv. dose and 8 mg/kg,
po. dose, for S. aureus (Smith strain) and 14 mg/kg,
p.o. dose, for methicillin resistant S. aureus. P Baker et
a l. (British Biotech and Krebs Institute for
Biomolecular Research, University of Sheffield, UK)
presented data on x-ray crystal structures of actinonin
and BB-3497, which showed similar binding in the
complex, partially within the active site. The most
suitable sites for modification of compounds to
improve antibacterial activity appears to be P2′ and
P3′.
13. Summary and expert opinion
For the first time in many years, the ICAAC meeting
was rich, with a variety of late-stage development
antimicrobial compounds, filling multiple unmet
medical needs, with a high probability of reaching the
marketplace. Novel anti-MRSA carbapenems, novel
quinolones, new cephalosporins, the novel class
ketolides and novel antifungal agents top this list of
new agents. The innovative approaches to adjunct
therapy (novel β-lactamase inhibitors, efflux pump
inhibitor), as well as long-term genomics efforts, offer
great excitement and hope to combat emerging
resistance problems in the clinic.
Brenda Ryan, Hsu-Tso Ho, Ping Wu, Mary-Beth Frosco, Thomas
Dougherty & John F Barrett†
† Author for correspondence
Infectious Diseases, Bristol-Myers Squibb Pharm. Res. Institute,
Wallingford, CT 06492, USA
E-mail: John.Barrett@bms.com
© Ashley Publications Ltd. All rights reserved.
Exp. Opin. Invest. Drugs (2000) 9(12)