CHEMOTHERAPY FOR

TUBERCULOSIS
Dr John Tiong
Tuberculosis: World statistics
• Tuberculosis (TB) is a disease that has plagued mankind for centuries
and dates as far back as ancient Egyptian times  caused by a slow
growing type of bacterium called Mycobacterium tuberculosis.

• The disease is so contagious that approximately a third of the world

population was infected by TB in the 20th century with an estimate of 8
million new cases and 2 million deaths each year.

• Approximately 40% of total global cases were found in China and

India alone followed by 26% in Africa.

• The global TB death rate was 23% but in some African nations, TB
fatality was more than 50% due to higher incidence of AIDS.
Challenges to global TB eradication
• Resurgence is on the rise in highly industrialized countries in Europe and
United States owing to factors like international migration, strong
epidemiological coexistence between TB with HIV and drug
resistance.

• There are now reports of emergence of multi drug resistant (MDR)

strain of M. tuberculosis  decreased susceptibility to first-line drugs
such as isoniazid and rifampicin making disease eradication more
challenging.

• Even though MDR–TB has long been recognized as a serious threat in

TB treatment, it is not prioritized by the developing nations in
particular due to national financial constraint as well as lack of expertise
in this area.

• This is further complicated by the emergence of extensively drug

resistant (XDR)-TB defined as resistance to rifampicin, isoniazid, any
fluoroquinolone and one or more of the intravenous second-line
drugs (capreomycin, kanamycin and amikacin).
Mycobacteria
• Mycobacteria are slender, rod-shaped bacteria with lipid-rich cell
walls that stain poorly with the Gram stain but once stained, the walls
cannot be easily decolorized by treatment with acidified organic
solvents  acid-fast.

• The most widely encountered mycobacterial infection is tuberculosis

the leading cause worldwide of death from infection  Members of
the genus Mycobacterium also cause leprosy and other human
infections.

• Mycobacterial infections are intracellular and, generally, result in the

formation of slow-growing granulomatous lesions that are responsible
for major tissue destruction.
Tuberculosis: The disease
• TB was much more prevalent in the past than it is today and has
caused the deaths of around one billion people over the last two
hundred years due to the following factors:

The improvement of sanitation and living conditions

The introduction of chemotherapy in the 1950s

BCG vaccination which confers immunity up to 80% of immunized

individuals.
Tuberculosis: The prognosis
There are 3 possible outcomes following initial infection:
• The first and the most optimistic outcome would be where sufficient
immunity is mounted to rid the host of the bacteria and the host fully
recovers from the disease.

• The other possibility will be where complete eradication of the

bacteria cannot be achieved by host‘s immune system but a defense
barrier is formed against the disease. The patient maybe
symptomless while the bacteria enter into dormancy but if the host‘s
immune system weakens at some point of his/her life, active TB will
develop. This is known as latent TB.

• The full blown stage of the disease may result in death if it goes
untreated.
Tuberculosis: Pathogenesis
• The disease is spread by coughing, talking, spitting or sneezing which
spreads the mycobacteria through the air in tiny aerosolized droplets
of water which are inhaled in to the lungs.

• The bacteria are then engulfed by macrophages in the alveoli where

approximately 95% being killed. The remnants which survive will
continue to replicate in the established niche within the macrophages
and can spread throughout the body.

• Multiplication of M. tuberculosis persists with eventual lysis of the

infected cells by the bacterial phospholipases, releasing the
pathogens which are then phagocytosed by further macrophages.

• Additional immune cells, especially T-cells, may be mobilised against

the infected macrophages when they move into tissues to form the
granuloma leading to the development of necrosis.

• In the full blown stage of the disease, the bacteria will spread around
the lungs causing destruction to the lung tissues and possibly death.
Complicated treatment
• Limitation with current TB treatment is its complexity which entails a
cocktail of anti-TB drugs  precise combination over a stipulated
duration that might compromise patient compliance

• There are other problems associated with killing the bacterium using
the available drugs which mostly kill the bacteria only when they are
growing.

• Inaddition the drugs have to penetrate a very complicated

mycobacterial cell wall which is sometimes described as “waxy”
because of its complex fatty acid barrier which makes getting a drug
molecule into the cytoplasm extremely difficult.
Complicated treatment
• Additionally, after treatment, the bacteria that are not killed outright
can lie in a dormant state waiting to be reactivated. These bacteria
are called “persistors” and the reason for reactivation is not
understood which makes destroying them very difficult.

• It is therefore vitally important that all of the growing bacteria are killed
in the first instance to destroy as many bacteria as possible which will
also help prevent drug-resistance from occurring which is proving to
be a major problem in developing countries.
Cell wall of Mycobacterium tuberculosis
Current treatment regimen
• TB is treated in two phases. There is an initial phase and a
continuation phase and depending upon the patients ability to
comply with the drug regime, these phases may be carried out either
unsupervised or supervised.

• The latter is known as DOT (directly observed treatment) where the

drugs are given to the patient three times per week in order to
maintain the strict regime that must be observed.

• The ultimate aims of the treatment regimen are to contain the

infection by clearing the pathogens as quickly as possible from the
body and to prevent the emergence of drug resistance.
Initial phase
• Here there is the concurrent use of at least three drugs which is
designed to reduce the bacterial population as rapidly as possible in
order to prevent resistance.

• These drugs are generally given as a combination preparation or

“triple therapy” unless one of the component drugs cannot be given
because of intolerance or potential drug resistance.

• The three drugs normally used in combination are isoniazid (INH),

rifampicin (RIF) and pyrazinamide (PZA).

• INH is effective against actively growing M. tuberculosis, killing

approximately 95% of the pathogen within the first 2 days.
Initial phase
• RIF is responsible for the killing of sporadically active bacterial
population

• PZA is needed to eliminate pathogen with low metabolic activity.

• Ethambutol (EMB) may also be used during the initial phase but is
normally omitted from the regime if risk of resistance to INH is low, for
example if a patient has not been treated for TB before or those who
have not been in contact with drug-resistant strains of the disease.

• Streptomycin (SM) may be used in cases where resistance to INH

has been established before treatment begins but it is generally
associated with higher incidence of toxicity.
Initial phase
• The initial phase drugs are normally used for two months but this
may be extended if necessary until full susceptibility towards the
drugs is confirmed after sensitivity testing which takes 6-8 weeks.

• In the case of DOT the drugs will be administered individually

because of dosage changes which must be taken into account when
taking the drugs only three times per week rather than daily as in the
non-supervised regime.
Continuation phase
• After the initial phase, a further four months of chemotherapy is
carried out using preferably a combination of RIF and INH.

• RIF is important to prevent possible relapse since it is effective

against persistor whereas INH is used to preclude the possibility of
rifampicin resistance.

• Treatment may be extended but only in cases of meningitis or for

resistant strains which may also require modification of the regime.

• Prevention of TB, or chemoprophylaxis, may also be achieved using

INH for six months for those who have evidence of previously
untreated TB and are receiving treatment with immunosuppressants
or with cytotoxics.
Continuation phase
• In the cases of patients who may be susceptible, and have been in
contact with the index case or those who have been tested tuberculin-
positive, INH alone or in combination with pyrazinamide can be
used.

• It must be noted that in cases where patients may be intolerant to the

desired regime of drugs or there are cases of drug resistance, other
drugs need to be used to treat the infection.
Types of anti-TB drugs
• The main drugs used to treat TB can be divided into two categories:
 Bacteriostatic e.g. EMB and para-aminosalicylic (PAS)
 Bactericidal e.g. INH, RIF, SM and fluoroquinolones (FQ)

• The distinction, however, between cidal and static activity is relative

because the activity of some of these drugs changes depending upon
the conditions to which they are subjected.

• For e.g. pyrazinamide is largely bacteriostatic although it is

bactericidal against growing bacteria.

• It is also noted that cidal drugs exhibit higher activity in reducing the
number of bacilli in lesions than the static drugs.
Types of anti-TB drugs
• The first-line drugs INH, RIF, PZA and EMB exhibit superior
efficacy with acceptable toxicity, where as the second-line drugs
SM, PAS, FQ, ETH, prothionamide (PTH) and cycloserine (CS)
have either less efficacy, increased side effects or both.

• The drugs kanamycin, amikacin and thioacetazone may also be

used as second-line agents.
First line drugs
Isoniazid (INH) – 1952
N

H2N
N O
H

• INH was discovered via a purely synthetic strategy in the quest to find
a new agent to combat the disease.

• It is bacteriostatic at concentrations of 0.025-0.05 mg/ml in vitro but at

higher concentrations is bactericidal. Isoniazid is bacteriostatic for
dormant bacilli.

• This is a prodrug and requires activation before destroying the

bacteria which it does by inhibiting the enoylreductase enzyme,
InhA which is responsible for the biosynthesis of mycolic acid.
Isoniazid (INH) – 1952
• INH is first activated by the catalase-peroxidase enzyme katG
which oxidizes the isonicotinic acyl group of the molecule to either an
isonicotinic acyl anion or radical.

• These forms then react with either an NADH cation or radical whilst
in the active site of InhA to form an isonicotinic acyl-NADH
complex.

• This new complex is tightly bound to the active sites of the InhA
resulting in its inhibition hence stopping the production of mycolic
acids (and so the cell wall of the bacteria).

• It is effective against intracellular bacteria.

• When it is used alone, resistant organisms rapidly emerge.

The activation of INH to the isonicotinic acyl-NADH
complex that binds to the active site of InhA

Isonicotinic Isonicotinic
acyl anion isoniazid acyl radical
Mn2+ N Mn2+
N N
KatG KatG

H2N
O N O O
H

NADH

NH2 H NH2 H H NH2 H

O O NAD
NAD+ O radical
N N N
R R R

NH2
NH2 H N N
O N
Isonicotinic O O O N
acyl-NADH R= O OP OP O O
N O- O-
R OH OH OH OH
Isoniazid (INH) – 1952
• Resistance is associated with several different chromosomal
mutations, each of which results in one of the following: mutation or
deletion of KatG (producing mutants incapable of prodrug
activation), or over-expression of InhA.

• Cross-resistance does not occur between isoniazid and other

antitubercular drugs.

• INH is well absorbed orally and distributes well throughout the body
and its metabolism occurs initially by liver N-acetyltransferase.
This means that patients who are poor acetylators can experience
toxicity as acetylation of INH is first required before the hydrolysis can
occur and the drug cleared by the kidneys.
Isoniazid (INH) – 1952
Adverse effects are related to the dosage and duration of
administration which include:
 Peripheral neuritis - Peripheral neuritis (manifesting as paraesthesia
of the hands and feet), which is the most common adverse effect,
appears to be due to a relative pyridoxine deficiency. Most of the toxic
reactions are corrected by supplementation of 25 to 50 mg per day of
pyridoxine (vitamin B6)
 Hepatitis and idiosyncratic hepatotoxicity: Potentially fatal
hepatitis is the most severe side effect associated with isoniazid.
Ethambutol (EMB) – 1961
HO
H
N
N
H
OH

• This drug exhibits bacteriostatic activity against M. tuberculosis in

vitro or in macrophages at 1 mg/ml.

• Stepwise resistance of the drug occurs when the drug is administered

alone but cross-resistance with other antimicrobials is rare so its
principle role is as a companion drug to prevent resistance.

• Ethambutol inhibits arabinosyl transferase - an enzyme that is

important for the synthesis of the mycobacterial arabinogalactan cell
wall.
Cell wall of Mycobacterium tuberculosis
Ethambutol (EMB) – 1961
• Approximately 80% of EMB is absorbed orally and is well distributed
throughout the body including the central nervous system before
being metabolized to inactive metabolites which are readily excreted
in urine.

• Penetration into the central nervous system (CNS) is therapeutically

adequate in tuberculous meningitis.

• The most important adverse effect is optic neuritis, which results in

diminished visual acuity and loss of ability to discriminate between red
and green  Visual acuity should be periodically examined.
Discontinuation of the drug results in reversal of the optic symptoms.
Pyrazinamide (PZA) – 1952
O O O
N N
NH2 NH2 OH

N N N
pyrazinamide (PZA) nicotinamide pyrazinoic acid (POA)

• This drug is a synthetic analogue of nicotinamide

• It has high sterilizing activity in vivo and is responsible for shortening

the therapy to six months.

• It is most active at acid pH in vitro, but shows no activity at

physiological pH.
Pyrazinamide (PZA) – 1952
• The exact mechanism of action of this unclear, however, it is known
that PZA is a prodrug which requires activation to pyrazinoic acid
(POA) by the PZase/ nicotinamidase enzyme.

• Pyrazinamide is active against tubercle bacilli in the acidic

environment of lysosomes, as well as, in macrophages.

• It is also known that resistance to PZA rapidly evolves if the drug is

used alone and is associated with mutation in the pncA gene which
encodes the PZase/ nicotinamidase enzyme.

• PZA is well absorbed orally and distributes well throughout the body
reaching concentration levels above that needed to kill the tubercle
bacilli.
Pyrazinamide (PZA) – 1952
• It is metabolized by the liver with metabolites, mainly POA, being
excreted by the kidneys. Dosages of the drug may need changing if
liver and renal function are compromised

• The most common side effects with PZA are liver toxicity and urate
retention which may predipose to gout.
Rifampicin (RIF) – 1966
O HO

O OH O
OH OH
MeO NH

N
O
O OH N
O

• This drug is a broad spectrum semi-synthetic derivative of the

complex macrocyclic antibiotic rifamycin B, produced by
Streptomyces mediterranei.

• An important feature about RIF is that it is active against both actively

growing and slowly metabolizing non-growing bacilli, the latter feature
is thought to be important in reducing the treatment from 12-18
months to 9 months.
Rifampicin (RIF) – 1966
• RIF is bactericidal against M.tuberculosis at 0.005-0.2 mg/ml

• It inhibits RNA synthesis by binding to the bacterial DNA-dependent

RNA polymerase b-subunit encoded by the rpoB gene.

• Resistance occurs rapidly when used alone and arises because of a

mutation in a defined 81-bp region of the rpoB gene and can cause
cross resistance to other rifamycins such as rifabutin and rifapentine
which have very similar structures.

• RIF is well absorbed orally giving peak plasma concentrations of 7-8

mg/ml after a dose of 600mg and distributes well throughout the body.

• Its lipophilic nature allows it to distribute throughout the central

nervous system, penetrate phagosomes and makes it a good
candidate to treat the meningitis form of the disease.
Rifampicin (RIF) – 1966
• It is deacylated to an active form which undergoes biliary excretion
and enterohepatic recirculation.

• Due to autoinduction of RIF metabolism, biliary excretion increases

with continued therapy whereby subsequent excretion of the drug
takes place primarily to the gastrointestinal tract with lesser amounts
in urine.

• It may cause the red-orange coloration of body fluid (such as tears

and urine)  Permanent staining of contact lenses will occur.
Second line drugs
Streptomycin (SM) – 1944
NH
NH
H HN NH2
H2N NH H
OH H

H OH
H
O
H O
CHO H
CH3 H
OH
O
OH O
CH2OH
NHCH3
H H
OH H

• SM is an aminoglycoside antibiotic isolated from the bacteria

Streptomyces griseus and was introduced in the 1940s as the first
drug to reduce TB mortality.

• Due to its hydrophilic nature owing to the presence of several

hydroxyl groups and two guanidine groups, this drug is poorly
bioavailable orally and has to be administered by an intramuscular
injection and is virtually excluded from the central nervous system.
Streptomycin (SM) – 1944
• It is bactericidal against M. tuberculosis in vitro and resistance
develops rapidly when given alone.

• The drug works by interfering with protein synthesis as it

inhibits the initiation of mRNA translation as well as
damaging the cell membrane.

• The main site of action is in the small 30S subunit of the

ribosome. Mutation to this site will give rise to SM resistance as
well as for other aminoglycoside antibiotics such as kanamycin,
amikacin, viomycin and capreomycin cross-resistance.

• The toxicity of SM is like that of the other aminoglycosides but

with less renal and acoustic toxicity (affects the hearing).
Ethionamide (ETH) and Prothionamide
(PTH) - 1956
S NH2 S NH2

N N
ethionamide prothionamide
(ETH) (PTH)

• These drugs are very similar in structure and action to INH and they are
also prodrugs that undergo catalase-peroxidase activation to inhibit the
enzyme InhA thus preventing the synthesis of mycolic acids and cell wall
biosynthesis.

• Resistance also occurs by mutation of the oxygenase enzymes and InhA

and it is tuberculostatic at 0.6-2.5 mg/ml making it less potent that INH.

• They are well absorbed orally and well distributed throughout the body
and are metabolised by the liver with metabolites excreted via the kidney.
Ethionamide (ETH) and Prothionamide
(PTH) - 1956
• Adverse effects that limit its use include hepatotoxicity, peripheral
neuropathies and optic neuritis.

• Supplementation with vitamin B6 (pyridoxine) may lessen the severity

of the neurologic side effects.
Cycloserine (CS) – 1952
H
N HO O
O O

NH2 NH2
H H
cycloserine
(CS) D-alanine

• This drug inhibits M. tuberculosis between 5–20 mg/ml again by

preventing cell wall biosynthesis.

• It works by mimicking alanine which is the natural substrate for the

enzyme D-alanine racemase (Alr) and D-alanine:D-alanine ligase
(Ddl) thus preventing the synthesis of peptidoglycan. Alr serves to
convert L- aniline to D-alanine and mutation in the active site of this
enzyme have been proposed to be responsible for the resistance of
CS.

• It is one of several alternatives for re-treatment regimes or for

treatment of primary drug resistant TB although has poor activity
against some MDR-TB strains.
Para-Aminosalicylic Acid (PAS) – 1946
O OH

OH

NH2

• This drug is bacteriostatic against M. tuberculosis and is not

completely absorbed orally due to its hydrophilic nature as dictated by
its functional groups attached to the benzene ring.

• PAS works by inhibiting a salicylate kinase enzyme which is

responsible for synthesizing iron regulating molecules called
siderophores which are necessary for the survival of bacteria
although it has been suggested that they also interfere with folic
acid metabolism.
Fluoroquinolones – 1963
HN N O N O
N N
N N N N N N
OH
OH OH OH
F F F
O O
O O O O O O
nalidixic acid cipfloxacin ofloxacin levofloxacin

• The fluoroquinolones, specifically, ciprofloxacin, moxifloxacin and

levofloxacin have an important place in the treatment of multidrug-
resistant tuberculosis.

• Some atypical strains of mycobacteria are also susceptible.

• Ciprofloxacin, ofloxacin and levofloxacin are the best studied of the

quinolones and work by inhibiting DNA synthesis targeting the DNA
gyrase at concentrations well within the achievable plasma levels.

• Resistance to the FQs has been found to occur when administered on

their own, however, ofloxacin in combination with other second line
drugs, may be used as a treatment for resistant TB.
New drugs
Bedaquiline
• This drug candidate was disclosed to the world in January 2005 by
the Johnson & Johnson Co and represented the first specifically
designed drug to treat TB since rifampicin.

• It contains two chiral centres essential for the activity of the

compound giving it the correct three dimensional geometry needed
for binding in the active site of the mycobacterial ATP-synthase
which it targets.

• This enzyme is responsible for the production of adenosine

triphosphate (ATP) and its inhibition disrupts trans-membrane energy
transfer through the cell hence killing it.
Bedaquiline
• It exhibits activity against susceptible M. tuberculosis with an MIC of
0.03 mg/ml and is active against MDR-TB isolates between 0.01 and
0.09 mg/ml suggesting that its resistance pathway is different to that
of other drugs and that it may be of great use in the fight against
resistant forms of the disease.

• The has been granted FDA approval in 2012 to Johnson and Johnson
for its use in the treatment of pulmonary multi-drug resistant in
combination with other susceptible drugs (in adults).

• The most common adverse drug reactions reported are nausea,

arthralgia and headache.
 Nitroimidazopyran (PA-824)
• This compound is a new nitroimidazole derivative developed by
PathoGenesisChiron Co after observations by Indian scientists that 5-
nitroimidazole had good in vitro and in vivo activity against M.
tuberculosis.

• PA-824 has been shown to inhibit the bacteria with an MIC of 0.015-
0.25 mg/ml and possibly more importantly has shown activity against
non-replicating bacteria indicating its use for tackling mycobacterial
persistence.
•
• PA-824 is a prodrug that is activated by F420-dependent glucose-6-
phosphate dehydrogenase and nitroreductase enzymes within the
bacilli.

• It is postulated that the active metabolites interfere with the enzymes

which oxidize hydroxyl fatty acids to generate M. tuberculosis
ketomycolic acids.
Nitroimidazopyran (PA-824)
• There is no sign of cross resistance with the drug suggesting that a
novel resistance mechanism is involved as indeed it is active against
MDR-TB.

• This compound is jointly being developed by the Global Alliance for

TB drug development and there have been no signs of undesired
toxic effects that are often associated with drugs possessing nitro
groups.
 THANK YOU!!!

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