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