molecular physiology insight in overcoming multidrug resistance evgenii s.severin russian research...
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MOLECULAR PHYSIOLOGY INSIGHT IN OVERCOMING MULTIDRUG RESISTANCE
Evgenii S.SeverinRussian Research Center for Molecular Diagnostics and Therapy
RCMDT
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)]
18802000Pneumonia, meningitis
Streptococcus pneumoniae
Pneumonia, gastrointestinal infection, sepsis
Gastroenteritis (salmonellosis)
Skin infectious, pneumonia, osteomyelitis
Pneumonia
Tuberculosis
Disease
56006264Pseudomonas aeruginosa
44004680Salmonella enteritidis
26002878Staphylococcus aureus
58005920 Klebsiella pneumoniae
40004411Mycobacterium tuberculosis (H37Rv)
Number of genes
Sise of genome,(million bp)
Bacterium (strain)
GENOMES OF THE PATHOGENIC BACTERIA
1. Chromosomal mutations
2. Plasmid or transposon mediated transport of resistanсe gene:
THE BASIC GENETIC MECHANISMS OF DRUG RESISTANCE
а
gene transported into plasmid or chromosome
Bacterium received resistance gene
plasmid
Plasmid donor
b
Virus
Bacterium infected by virus
c
Dead bacteria
c – transport of free DNA
a – plasmid transport
b – transport by virus
Bacterialcell
plasmid
antibiotic
Pumping out antibiotic
antibiotic
Enzyme,modifyingantibiotic
Enzyme,degradingantibiotic
antibiotic
c - code enzymes, which modify antibiotics (ADP-ribosyl transferase provides ADP ribosylation of rifamycin)
MAJOR BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE
a - code efflux pump (TetA - efflux proteins for tetracyclines)
b – code enzymes, which degrade antibiotics (β-lactamases cleave β-lactam antibiotics)
a b c
Genes of resistance:
QUINOLONES
Gram-, extended Gram+ and atypical coverage
levofloxacin 3rd
Same as 3rd generation with broad anaerobic coverage
moxifloxacin4rd
Gram- (including Pseudomonas species), some Gram+ (S. aureus)
ciprofloxacin lomefloxacin
2nd
Gram- but not Pseudomonas speciesnalidixic acid 1st
SpectrumDrug NamesGeneration
Mechanism of Action - inhibition of bacterial DNA Gyrase (Topoisomerase II)
Essential structure of all quinolone antibiotics
3’
5’
3’
5’
1. Formation of intermediate Quinolone-Gyrase-DNA complex
2. Promoting of cleavage of bacterial DNA, inhibition of DNA replication and induction of bacterial death
Quinolone
DNA Gyrase
AUC, (μg·h/g)
liver kidney lung spleen heart blood0
200
400
600
800
1000
1200
1400
- Lomefloxacin - Lomefloxacin-nano
Biodistribution of lomefloxacin and lomefloxacin-nano in organs of rats after
oral administration
ANTIBACTERIAL ACTIVITY AND PHARMACOKINETICS OF LOMEFLOXACIN-LOADED PLGA NANOPARTICLES
Zone of bacteria growth inhibition, о mm
30
28
26
24
22
32
Escherihia coli 1257
Klebsiella pneumonia
spp
Staphylo-coccus
aureus, 906
Salmonella enteritidis ATCC 9640
Pseudomonas aeruginosa
spp
- Lomefloxacin, 10% solution- Lomefloxacin-nano, 10% solution
Antibacterial activity of lomefloxacin and lomefloxacin-nano
Antibacterial activity of lomefloxacin-nano was greater as compared to free form of lomefloxacin
Lomefloxacin
Isoniazid Rifampicin Ethambutol Pyrazinamide
FIRST-LINE ANTI-TUBERCULOUS DRUGS
SECOND-LINE ANTI-TUBERCULOUS DRUGS
Levofloxacin
Cycloserine Rifabutin
Kanamycin Capreomycin Ethionamide
Loss of RNA polymerase activity to bind with RIF
rpoB (β-subunit of RNA polymerase)
Rifampicin (RIF)(binds to the β-subunit of RNA polymerase and inhibits transcription)
Loss of catalase activity to produce active metabolites of INH
katG (catalase-peroxidase)
Isoniazid (INH) (inhibits synthesis of mycolic acid of cell wall)
Loss of arabinosyl transferase activity to interact with EMB
embB (arabinosyl transferase)
Ethambutol (EMB)(inhibits an arabinosyl transferase and biosynthesis of arabinogalactan of cell wall)
Loss of pyrazinamidase activity to produce active form of PYZ - pyrazinoic acid
pncA (pyrazina-midase/ nicotina-midase)
Pyrazinamide (PYZ)(targets an enzyme involved in fatty-acid synthesis)
Mechanism of drug resistance
MutationMechanism of action of antituber-culosis drug
INH
RIF
PYZ
EMB
MECHANISM OF ACTION AND RESISTANCE TO ANTITUBERCULOSIS DRUGS
M. tuberculosis
ACTUAL PROBLEMS OF MODERN ANTITUBERCULOSIS THERAPY
MAP OF GLOBAL DISTRIBUTION OF MULTI-DRUG RESISTANT TUBERCULOSIS
The distribution of multidrug-resistant tuberculosis in Russia in 2009
~ 24%
Solution:
- 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
The main problems of current therapy:
- 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)
Targeting antituberculosis drugs in infected Macrophages
Capillary flow
Macrophage
Nanoparticles biodegradation and drug release
Drug-loaded nanoparticles
M. tuberculosis
Drug
ADVANTAGES OF NANO DRUG DELIVERY FOR TREATMENT OF TUBERCULOSIS
Reduce the dosage of antituberculosis drugs
- Reduce dosage frequency - Minimise the toxicity of drugs- Reduce the cost of TB
treatment- Improve patient compliance
0 1 2 7 Time (days)
Toxic level
Safe zone
Min. effective conc.
Conc.Plasma
Nano drug Usual drug
• No inflammatory or toxic response• Is metabolized after fulfilling its purpose• Is easily sterilized• Acceptable shelf life
Poly(lactide-co-glycolide) - Ideal Biodegradable Polymer
Applications• Matrices for Drug Delivery Systems - Nanoparticles, Microspheres - Implants• Medical Devices - Sutures - Stents• Tissue Engineering Matrices
PLGA-based Formulations in the MarketplaceDistributorActive ingredientProduct Name
Ipsen-BeaufourLanreotideSomatuline®LA
Sanofi-Aventis BuserelinSuprecur®MP
TAPLuprorelinLupron Depot®
Ipsen-BeaufourTriptorelinDecapeptyl SR
Electron micrograph of PLGA microspheres
DESIGN OF TARGETED DRUG DELIVERY SYSTEMS ON THE BASE OF PLGA-NANOPARTICLES
Scheme of polymeric nanoparticles preparation by emulsification method
Addition of surface-active substance
Oil-in-water system
Nano-dispersed oil-in-water system
Nanoparticles emulsion
Nanoparticlepowder
Homogenization
Removal of organic solvent
Filtration, liophilization
Mixing of drug + PLGA + organic solvent
Chemical structure of PLGA
*x
OCH C OCH2
OCH3
C
O
y*
n
Lacticacid
Glycolic acid
PLGA (рoly(D,L-lactide-co-glycolide)
Lactic Acid
Poly(lactide)Poly(lactide-co-glycolide)
Polyglycolide
Tricarboxylic Acid Cycle
Carbon dioxide and water
Glycolic Acid
Metabolism of PLGA and PLA
ANTITUBERCULOSIS ANTIBIOTICS FOR PREPARATION OF DRUG-LOADED NANOPARTICLES
Rifampicin Cycloserine
ProtionamideCapreomycin
Rifampicin – 8.5 %PLA – 59 %Practicle Size – 300±71 nm
Cycloserine – 12.5 %PLGA-COOH (50/50) – 50 %Practicle Size – 309±67 nm
LevofloxacinLevofloxacin – 8.4 %PLGA (50/50)– 59 %Practicle Size – 339±40 nm
Protionamide – 8.4 %PLGA (50/50)– 59 %Practicle Size – 367±70 nm
Capreomycin – 8.5 %PLGA (50/50)– 59 %Practicle Size – 358±55 nm
Free form of fluorescentagent in macrophage
Nano-form of fluorescent agent in macrophage
The accumulation of fluorescent nanoparticles in alveolar macrophages
(data of light and fluorescent microscopy)
- Rifampicin- Rifampicin-nano
blood liver spleen lung
200
150
100
50
AUC Rifampicin-nano/ Rifampicin, %
BIODISTRIBUTION OF DRUG-LOADED PLGA NANOPARTICLES IN ORGANS OF MICE
COMPARATIVE TOXICITY OF ANTIBIOTICS ENCAPSULATED INTO PLGA NANOPARTICLES
(IN BALB/C MICE)
reduction >40004000i/gLomefloxacin
increase 16501800i/vLevofloxacin
reduction >70006900i/gCycloserine
retention 55i/vCycloserine
retention 145150i/vCapreomycin
reduction 451320i/vRifabutin
reduction 390260i/vRifampicin
Change of toxicityLD50 of nano-drug, (mg/kg)
LD50 of drug, (mg/kg)
Route ofadministra-
tionAntibiotic
General toxicity of nanoform of antibiotics was decreased as compared to free form of antibiotics
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
Number of mycobacteria, colony-forming unit (CFU) per mouse
- Control (Contr)- Cycloserine (C)- Cycloserine-nano (C)- Rifampicin (R)- Rifampicin-nano (R)
Contr C Cnano R Rnano
109
108
107
106
105
104
103
102
101
200 times
1. Infection with M. tuberculosis
2. Administration of drug
3. Collection of organs samples on day 21 after infection
ANTITUBERCULOSIS ACTIVITY OF D-CYCLOSERIN and RIFAMPICIN ENCAPSULATED INTO PLGA NANOPARTICLES
IN A MOUSE INFECTION MODEL WITH MULTIDRUG-RESISTANT STRAINS OF M.TUBERCULOSIS
21 days