From antimalarial to antibacterial

1
From anti-malarial to antibacterial
1939 chloroquine
1958 7-chloroquinolone Anti-bacterial activity
noted in screening
1962 Nalidixic acid First new class of
synthetic antibiotic for 30 years
2
Mechanism of action
Inhibition of topoisomerases a family of
enzymes responsible for maintaining the topology
of DNA
relaxed
topo-isomerase
gyrase
supercoiled
3
Quinolones form a Quaternary Complex between
Protein, DNA and Neighbouring Quinolones
4
Accumulation of Covalent Complexes and Broken DNA
is Lethal
Target is gyrase (Gram-negative) and/or
topisomerase IV (Gram-positive) Killing is rapid
but reaches a plateau (60-120 for 104-fold
decrease in viable count) Killing antagonized by
inhibitors of translation and at high FQ
concentration Eagle effect) Anaerobic
conditions inhibit action
5
Concentration Dependence of Killing
Concentration dependence shows two phases of
inhibition, separated by a plateau. The cultures
in the plateau region give rise to first step
resistant mutants. Mutants are not recovered
above the onset of the second sharp drop. This
limit is defined as the mutant preventing
concentration (MPC).
MPC
6
Early Quinolone Antibiotics
Nalidixic Acid 1962 Sterling Winthrop
Pipemidic Acid 1974 Roger Bellon
Oxolinic Acid 1967 Werner Lambert
Cinoxacin 1974 Lilly
Flumequine 1973 Rikker Labs
Rosoxacin 1976 Sterling Winthrop
7
Potential of Early Quinolone Antibiotics
Nalidixic Acid Enterobacteriaceae Short
half-life (not P. aeruginosa) (1.5
hr) high protein binding Pipemidic
Acid Broader Gram-negative Longer
half-life spectrum (except Serratia) Oxolin
ic Acid Weak but broad lower protein
binding Gram-negative Flumequine Weak but
broad Gram-negative
8
The First Fluoroquinolone Antibiotics
Pipemidic Acid 1974 Roger Bellon
Flumequine 1973 Rikker Labs
Norfloxacin 1978 Kyorin
Broad Gram-negative spectrum (F, morpholino
grp) Less protein binding, 50 (quinolone not
naphthyridine) Longer half life, 3h (morpholino
group)
9
The First Generation of Fluoroquinolone
Antibiotics
Flumequine 1973 Rikker Labs
Norfloxacin 1978 Kyorin
Pefloxacin 1979 Dainippon
Ofloxacin 1979 Daiichi
Ciprofloxacin 1983 Bayer
Fleroxacin 1987 Roche
10
Potential of the First Generation Fluoroquinolone
Antibiotics
Gram-negative spectrum t1/2
(h) Flumequine Weak but broad
Norfloxacin Strong 3 - 4
Pefloxacin Weak 11 Ofloxacin Strong
6 Ciprofloxcin Very strong 3
4 Levofloxacin Extremely strong 6 Also
rans Amifloxacin 1984 Sterling
Winthrop Fleroxacin 1987 Roche Lomefloxacin 198
8 Searle
11
From Ofloxacin to Levofloxacin
Ofloxacin 1979 Daiichi
Levofloxacin 1987 Daiichi
Racemic mixture
Pure (-) S isomer Active component of
ofloxacin 2-fold improved activity Extended
patent life
12
The Later Fluoroquinolone Antibiotics
Difloxacin 1986 Abbott
Temafloxacin 1988 Abbott
Sparfloxacin 1991 Rhone-Poulenc-Rorer (Dainippon
1985)
Grepafloxacin 1995 Otskuda (1989)
Gatifloxacin 1993 Kyorin (1988)
Moxifloxacin 1994 Bayer
13
Later Fluoroquinolone Antibiotics

• Gram-negative spectrum retained
• MIC for E. coli
• Ciprofloxacin 0.125 - 0.5
• Grepafloxacin 0.06 - 2
• Gatifloxacin 0.06
• Increased potency or breadth of Gram-positive
  spectrum
• MIC for S. pneumoniae
• Ciprofloxacin 0.5 - 2
• Temafloxacin 0.5 1
• Sparfloxacin 0.125 0.5
• Moxifloxacin 0.01 0.5
• Improved activity against anaeobes
• MIC for B. fragilis
• Ciprofloxacin 2 128
• Gatifloxacin 0.25 - 8
• Moxifloxacin 0.25 - 8

14
Later Naphthyridines
Flumequine 1973 Rikker Labs
Pipemidic Acid 1974 Roger Bellon
Ciprofloxacin 1983 Bayer
Enoxacin 1984 Rhone-Poulenc Rorer
Temafloxacin 1988 Abbott
Trovafloxacin 1993 Pfizer
Gemifloxacin 1994 L G Chemicals
15
Later Naphthyridines
Gram ve Gram ve Anaerobes (E. coli)
(S. pneumoniae) (B. fragilis) Pipemidic Acid
0.025 16 4 gt64 gt64 Enoxacin 0.025
16 1.6 - 16 Trovafloxacin 0.008 - 1 0.007
0.25 0.128 - 8 Gemifloxacin 0.5 -
64 Ciprofloxacin 0.125 0.5 0.5 - 2 2 -
128
16
Current Uses of Fluoroquinolones
Ciprofloxacin wide range of infections
pneumonias, bone infections, diarrhea, skin
infections and urinary tract infections. Not
good for methicillin resistant Staphylococcus
aureus Norfloxacin better for UTI effective
against Gram-negative (including Pseudomonas
aeruginosa) and Gram-positive UTIs and
prostatitis, but not in systemic infections
Lomefloxacin and enoxacin -- UTIs and
bronchitis caused by Haemophilus influenzae or
Moraxella catarrhalis. Lomefloxacin not effective
against pseudomonal bacteremia. Enoxacin
recommended for STDs Moxifloxacin overcomes
the problems with S. pneumoniae Acute bacterial
sinusitis mild to moderate community-acquired
pneumonia
17
Frequent Side Effects of Quinolones
Chelation of cations (Fe3 , Al 3 , Mg2, Ca
2) incompatibility with antacids Phototoxicity
Drug interactions (inhibition of cytochrome
P450) Ciprofloxacin and enoxacin interfere with
hepatic biotransformation -- may cause toxicity
due to excess of e.g. theophylline, warfarin
CNS toxicity (GABA receptor antagonist) all
quinolones contraindicated in patients with
history of convulsions Gastro-intestinal
discomfort nausea or vomiting, abdominal or
stomach pain Cartilage and musculosqueletal
pathogenicity have caused arthralgias and joint
swelling in children Achilles tendinitis and
tendon rupture 2 - 42 days after start of therapy
Bacterial SOS response to DNA and other damage
caused by FQ may induce toxins. (E. coli, S.
typhimurium, B. anthracis)
18
Rare Side Effects of Quinolones
Nephrotoxicity crystalluria, hematuria,
interstitial nephritis, acute renal
failure Cardiac toxicity inhibition of hERG
channel leads to QT prolongation torsades de
pointe Hepatotoxicity temafloxacin syndrome,
trovafloxacin syndrome
19
Frequent Side Effects of Quinolones
GABA receptor binding CNS penetration
Cation complexation all quinolones
Inhibition of CyP450 ciprofloxacin, gemifloxacin
CyP450 inhibition
Photoxicity especially F fleroxacin,
lomefloxacin, sparfloxacin
20
Nephrotoxicity
crystalluria, hematuria, interstitial nephritis,
acute renal failure Poor solubility can lead to
crystallization of the FQ in concentrated urea.
Needle-like crystals form that can cause
mechanical damage
21
Cardiac Toxicity
Torsades de pointe paroxysm of ventricular
tachycardia in which the electrocardiogem shows a
steady undulation in the QRS axis in runs of 5 to
20 beats with progessive changes in direction.
It is a most severe type of arythmia and can
result in death. It is most often associated
with and preceeded by a prolongation of the QT
interval.
Population at risk QT greater than 20
ms Moxifloxacin 7 ms, grepafloxacin 10 ms,
sparfloxacin 15 ms
22
Hepatotoxicity
Temafloxacin syndrome Hemolytic uraemic
anemia discoloured urine fever jaundice nausea
, vomiting coagulopathy hepatic
dysfunction renal dysfunction 0.56 incidence 2
deaths Withdrawn June 1992
Trovafloxacin syndrome Serious hepatic
events laboratory abnormalities encephalopathies
nectrotic inflammation 0.0056 incidence 5
transplants 6 deaths Withdrawn/limited in June
1999
23
Resistance to Quinolones
Point mutations in the gyrase A gene cluster in
region around tyrosine 122, site of covalent
attachment of DNA Point mutations in the
regulatory regions of transport proteins porins
of Gram-negative bacteria efflux systems of P.
aeruginosa (mex systems) Enterobacteriaceae
(Acr system porin mutation) S. aureus (norA
system) S. pneumoniae (pmrA system, patAB
system) No transferable resistances recorded
until 1994 wild type susceptible genes tend to
be dominant plasmid conjugation is inhibited by
quinolones plasmids can be eliminated from
bacteria at sub-lethal quinolone concentrations T
he new plasmid pMG255 encodes a putative
quinolone binding protein.
24
Emergence of Quinolone Resistance During Therapy
Most often in difficult to treat infections and
long-term treatments
Pefloxacin bronchitis P. aeruginosa Ofloxacin p
ulmonary tuberculosis M. tuberculosis Ciprofloxa
cin cystic fibrosis ass. RTI P.
aeruginosa osteomyelitis P.
aeruginosa MRSA carriers S.
aureus Norfloxacin complicated UTI
P. aeruginosa

• Notably with infections caused by staphylococci,
  pneumococci, enterococci, and P. aeruginosa .
  Spontaneous mutation leading to single-step
  resistance is more common - 106-107 - than for
  other pathogens - 109-1011

25
Acr System of Enterobacteriaceae
Multi-drug resistance efflux system. System. Pumps
a broad range of antibiotics out of the cell.
Part of a multicomponent system that spans
cytoplasmic and outer membrane of Gram-negative
bacteria. Cytoplasmic membrane component acts as
energy transducer (pmf) as well As substrate
recognition module
26
Acr System of Enterobacteriaceae
27
Modelled Structure of Mex/OprM Efflux Pump
MexA,B-OprM exports quinolones, tetracycline,
chloramphenicol and other antibiotics MexC,D-OprJ
MexX,Y-OprM exports aminoglycoside antibiotics
(amikacin, tobramycin) and quinolones
Efflux pump inhibitors developed by Essential
Therapeutics are in clinical trials as
combination partner for levofloxacin
28

• Fluoroquinolone-resistance in Streptococci
• by the late 1980s
• FQ-resistant staphylococci established in
  clinics
• increased levels of resistance amongst
  streptococci reported from
• patients receiving FQ for Gram-negative urinary
  tract infections.
• Resistance in staphylococci
• point mutations in the genes encoding the
  molecular targets
• (gyrase subunit A and topoisomerase IV subunit
  A)
• point mutations in the promoter of an integral
  membrane protein
• leading to consitutive expression
• expression of the membrane protein alone is
  sufficient for clinical resistance

29
Efflux-mediated Resistance in Streptoocci
The NorA protein is an efflux transporter similar
to the well-known tetracycline resistance
systems It uses the proton-motive force to drive
out the antibiotic against its concentration
gradient by proton-coupled antiport.
FQ
FQ
H
FQ
H
H
Intrinsic FQ resistance in streptococci reported
to be due to a homologue the PmrA protein
30
Major Facilitator Family Permeases
31
ABC transporters
Implicated in fluoroquinolone resistance in
streptococci (PatA, PatB) Multi-drug resistance
in many Gram-positive organisms
32
Microarray analysis

• No antibiotic for most genes M4 and M22 similar
  levels of gene expression
• After CIP exposure similar levels of gene
  expression except for- those involved in DNA
  repair, transcription etc. as found with
  Haemophilus influenzae (Gmuender et al., 2001) -
  ATPases - those involved in solute
  transport/efflux

Marrer et al., 2006 AAC 50 269-278
33
patA and patB

• Two genes, patA and patB (pneumococcal ABC
  transporter) constitutively over-expressed by
  M22, but not M4.
• patA and patB DNA sequences (pfam and SOSUI)
  suggest ABC transporters
• PatA predicted 6TMs (no model)
• PatB predicted 6TMs (Swissprot model based on
  Sav1886 transporter in S. aureus)

Predicted PatB
34
Induction of patA and patB microarray data
After 10 mins exposure to ciprofloxacin
patA
patB
M4
M22
35
Effect of inactivating patB
Recent data with M22 patA and patB inactivated
with magellen2 revealed full reversal of MDR.