Evgenii S.Severin
Russian Research Center for
Molecular Diagnostics and Therapy
RCMDT
MOLECULAR
PHYSIOLOGY INSIGHT
IN OVERCOMING
MULTIDRUG
RESISTANCE
GENOMES OF THE PATHOGENIC BACTERIA
Bacterium
(strain)
Disease
Sise of
genome,
(million bp)
Number
of genes
Mycobacterium
tuberculosis
(H37Rv)
Tuberculosis
4411
4000
Streptococcus
pneumoniae
Pneumonia,
meningitis
2000
1880
Klebsiella
pneumoniae
Pneumonia
5920
5800
Staphylococcus
aureus
Skin infectious,
pneumonia,
osteomyelitis
2878
2600
Salmonella
enteritidis
Gastroenteritis
(salmonellosis)
4680
4400
Pseudomonas
aeruginosa
Pneumonia,
gastrointestinal
infection,
sepsis
6264
5600
Circular map of the chromosome of
M. tuberculosis H37Rv
[S. T. Cole et al. Deciphering the biology of
Mycobacterium tuberculosis from the complete
genome sequence. Nature 393, 537-544 (1998)]
THE BASIC GENETIC MECHANISMS OF DRUG RESISTANCE
1.
Chromosomal mutations
2.
Plasmid or transposon mediated transport of resistanсe gene:
Bacterium received resistance gene
plasmid
Plasmid
donor
а
Bacterium
infected by
virus
b
gene transported
into plasmid or
chromosome
Virus
c
Dead bacteria
a – plasmid transport
b – transport by virus
c – transport of free DNA
MAJOR BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE
Bacterial
cell
plasmid
antibiotic
antibiotic
Pumping out
antibiotic
a
b
Enzyme,
degrading
antibiotic
c
Enzyme,
modifying
antibiotic
antibiotic
Genes of resistance:
a - code efflux pump (TetA - efflux proteins for tetracyclines)
b – code enzymes, which degrade antibiotics (β-lactamases
cleave β-lactam antibiotics)
c - code enzymes, which modify antibiotics (ADP-ribosyl
transferase provides ADP ribosylation of rifamycin)
QUINOLONES
Generation
Drug Names
Spectrum
1st
nalidixic acid
Gram- but not Pseudomonas species
2nd
ciprofloxacin
lomefloxacin
Gram- (including Pseudomonas
species), some Gram+ (S. aureus)
levofloxacin
Gram-, extended Gram+ and atypical
coverage
moxifloxacin
Same as 3rd generation with broad
anaerobic coverage
3rd
4rd
Essential structure of all
quinolone antibiotics
Mechanism of Action - inhibition of bacterial DNA Gyrase (Topoisomerase II)
Quinolone
3’
3’
5’
DNA Gyrase
1. Formation
of intermediate
Quinolone-GyraseDNA complex
5’
2. Promoting of cleavage
of bacterial DNA, inhibition
of DNA replication and
induction of bacterial death
ANTIBACTERIAL ACTIVITY AND PHARMACOKINETICS
OF LOMEFLOXACIN-LOADED PLGA NANOPARTICLES
Antibacterial activity of lomefloxacin and
lomefloxacin-nano
Zone of bacteria growth
inhibition, о mm
Lomefloxacin
- Lomefloxacin, 10% solution
- Lomefloxacin-nano,
10% solution
32
Biodistribution of lomefloxacin and
lomefloxacin-nano in organs of rats after
oral administration
30
28
AUC, (μg·h/g)
26
1400
24
1200
- Lomefloxacin
- Lomefloxacin-nano
1000
22
Escherihia Klebsiella Staphylo- Salmonella Pseudomonas
coli 1257 pneumonia
coccus
enteritidis aeruginosa
spp
aureus, 906 ATCC 9640
spp
800
600
400
Antibacterial activity of lomefloxacin-nano was greater
as compared to free form of lomefloxacin
200
0
liver
kidney
lung
spleen heart
blood
FIRST-LINE ANTI-TUBERCULOUS DRUGS
Isoniazid
Rifampicin
Ethambutol
Pyrazinamide
SECOND-LINE ANTI-TUBERCULOUS DRUGS
Levofloxacin
Cycloserine
Kanamycin
Capreomycin
Ethionamide
Rifabutin
MECHANISM OF ACTION AND RESISTANCE
TO ANTITUBERCULOSIS DRUGS
M. tuberculosis
INH
Mechanism of
action of antituberculosis drug
Mutation
Mechanism of
drug resistance
Isoniazid (INH)
katG
(catalaseperoxidase)
Loss of catalase
activity to
produce active
metabolites
of INH
rpoB (βsubunit of
RNA
polymerase)
Loss of RNA
polymerase
activity to bind
with RIF
embB
(arabinosyl
transferase)
Loss of arabinosyl
transferase
activity to interact
with EMB
pncA
(pyrazinamidase/
nicotinamidase)
Loss of
pyrazinamidase
activity to
produce active
form of PYZ pyrazinoic acid
(inhibits synthesis of
mycolic acid of cell
wall)
Rifampicin (RIF)
EMB
(binds to the β-subunit
of RNA polymerase
and inhibits
transcription)
Ethambutol (EMB)
(inhibits an arabinosyl
transferase and
biosynthesis of
arabinogalactan of cell
wall)
PYZ
RIF
Pyrazinamide
(PYZ)
(targets an enzyme
involved in fatty-acid
synthesis)
ACTUAL PROBLEMS OF MODERN ANTITUBERCULOSIS THERAPY
The main problems of current therapy:
Solution:
- Degradation of the drugs before reaching their
target;
- Large doses can cause toxic side effects;
- Emergence of multidrug-resistant tuberculosis
(MDR-TB) and extensively drug-resistant
tuberculosis (XDR-TB)
- New antibiotic development: rational drug
design based on genomics/proteomics;
- Use of drug delivery system based on
polymeric nanoparticles loaded with
antituberculosis drugs for sustained release
MAP OF GLOBAL DISTRIBUTION OF MULTI-DRUG RESISTANT TUBERCULOSIS
The distribution of
multidrug-resistant
tuberculosis in
Russia in 2009
~ 24%
ADVANTAGES OF NANO DRUG DELIVERY
FOR TREATMENT OF TUBERCULOSIS
Nano drug
Reduce the dosage of
antituberculosis drugs
Usual drug
Toxic level
Conc.
Plasma
Safe zone
Min. effective conc.
0
1
2
7 Time (days)
Drug-loaded nanoparticles
Macrophage
Capillary flow
M. tuberculosis
Nanoparticles
biodegradation
and drug release
Drug
-
Reduce dosage frequency
Minimise the toxicity of drugs
Reduce the cost of TB
treatment
Improve patient compliance
Targeting antituberculosis
drugs in infected Macrophages
Poly(lactide-co-glycolide) - Ideal Biodegradable Polymer
• No inflammatory or toxic response
• Is metabolized after fulfilling its purpose
• Is easily sterilized
• Acceptable shelf life
Applications
• Matrices for Drug Delivery Systems
- Nanoparticles, Microspheres
- Implants
• Medical Devices
- Sutures
- Stents
• Tissue Engineering Matrices
Electron micrograph
of PLGA microspheres
PLGA-based Formulations in the Marketplace
Product Name
Active ingredient
Distributor
Decapeptyl SR
Triptorelin
Ipsen-Beaufour
Lupron Depot®
Luprorelin
TAP
Suprecur®MP
Buserelin
Sanofi-Aventis
Somatuline®LA
Lanreotide
Ipsen-Beaufour
DESIGN OF TARGETED DRUG DELIVERY SYSTEMS
ON THE BASE OF PLGA-NANOPARTICLES
PLGA (рoly(D,L-lactide-co-glycolide)
Chemical structure of PLGA
*
CH3 O
O
OCH C
OCH2 C
*
y
n
x
Lactic
acid
Glycolic
acid
Scheme of polymeric
nanoparticles preparation
by emulsification method
Mixing of drug + PLGA
+ organic solvent
Addition of surfaceactive substance
Oil-in-water system
Metabolism of PLGA and PLA
Poly(lactide-coglycolide)
Poly(lactide)
Polyglycolide
Homogenization
Nano-dispersed oil-inwater system
Removal of organic solvent
Lactic Acid
Glycolic
Acid
Nanoparticles emulsion
Filtration, liophilization
Tricarboxylic Acid Cycle
Carbon dioxide and water
Nanoparticle
powder
ANTITUBERCULOSIS ANTIBIOTICS FOR PREPARATION
OF DRUG-LOADED NANOPARTICLES
Levofloxacin
Cycloserine
Rifampicin
Cycloserine – 12.5 %
PLGA-COOH (50/50) – 50 %
Practicle Size – 309±67 nm
Rifampicin – 8.5 %
PLA – 59 %
Practicle Size – 300±71 nm
Levofloxacin – 8.4 %
PLGA (50/50)– 59 %
Practicle Size – 339±40 nm
Protionamide
Capreomycin
Protionamide – 8.4 %
PLGA (50/50)– 59 %
Practicle Size – 367±70 nm
Capreomycin – 8.5 %
PLGA (50/50)– 59 %
Practicle Size – 358±55 nm
BIODISTRIBUTION OF DRUG-LOADED PLGA NANOPARTICLES
IN ORGANS OF MICE
AUC Rifampicin-nano/
Rifampicin, %
- Rifampicin
- Rifampicin-nano
200
150
100
50
blood
The accumulation of
fluorescent nanoparticles
in alveolar macrophages
(data of light and
fluorescent microscopy)
liver
spleen
lung
Free form of
fluorescent
agent in
macrophage
Nano-form of
fluorescent
agent in
macrophage
COMPARATIVE TOXICITY OF ANTIBIOTICS
ENCAPSULATED INTO PLGA NANOPARTICLES
(IN BALB/C MICE)
Antibiotic
Route of
administration
LD50 of drug,
(mg/kg)
LD50 of nano-drug,
(mg/kg)
Change of toxicity
Rifampicin
i/v
260
390
reduction
Rifabutin
i/v
320
451
reduction
Capreomycin
i/v
150
145
retention
Cycloserine
i/v
5
5
retention
Cycloserine
i/g
6900
>7000
reduction
Levofloxacin
i/v
1800
1650
increase
Lomefloxacin
i/g
4000
>4000
reduction
General toxicity of nanoform of antibiotics was decreased as compared
to free form of antibiotics
ANTITUBERCULOSIS ACTIVITY OF D-CYCLOSERIN and RIFAMPICIN
ENCAPSULATED INTO PLGA NANOPARTICLES
IN A MOUSE INFECTION MODEL WITH
MULTIDRUG-RESISTANT STRAINS OF M.TUBERCULOSIS
Number of mycobacteria,
colony-forming unit (CFU) per mouse
109
- Control (Contr)
- Cycloserine (C)
- Cycloserine-nano (C)
- Rifampicin (R)
- Rifampicin-nano (R)
21 days
108
107
200
times
106
1. Infection with
M. tuberculosis
3. Collection of organs
samples on day 21 after
infection
105
104
103
102
101
Contr C Cnano R
Rnano
2. Administration
of drug
On day 21 after infection antibacterial activity of Cycloserine-nano and Rifampicin-nano
was about 200 times greater than that of free form of drugs