Pharmacology –

Tuberculosis; available treatment and latest developments of therapy

Contents

Title Page numbe

r

Introduction...................................................................................................................................5

Pathophysiology of tuberculosis.................................................................................................5

Available treatment......................................................................................................................7

Combination therapy..................................................................................................................10

Novel anti-tuberculosis treatment.............................................................................................10

Summary.....................................................................................................................................13

Reference list..............................................................................................................................14

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 Figure content page

Title Page number

Figure 1: Pathogenesis of Tuberculosis in the Infected Host................................................6

Figure 2: Pathophysiology of tuberculosis disease...............................................................6

Figure 3: Molecular structures of rifampicin (a), pyrazinamide (b), ethambutol (c) and
isoniazid (d)...........................................................................................................................7

Figure 4: Model of the pyrazinamide mechanism of action..................................................8

Figure 5: Mechanism of action of Isoniazid..........................................................................9

Figure 6: Development of anti-tuberculosis drugs since 1940 versus the reported

worldwide MDR-TB cases..................................................................................................12

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 Table content page

Title Page number

Table 1: Examples of types of reactive species produced by isoniazid................................9

Table 2: Novel compounds introduced for tuberculosis treatment.....................................11

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Question 2

Tuberculosis; available treatment and latest developments of therapy

Introduction

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis

(MTB). Robert Koch in 1882 demonstrated that the MTB bacillus was the cause of
tuberculosis. MTB is an aerobic, rod shaped bacteria which has a unique cell wall
structure. The cell wall contains mycolic acid, fatty acid, peptidoglycan and
arabinogalactan; a polysaccharide. Some common symptoms of the disease are coughing,
chest pain, weight loss and fatigue. MTB is transmitted through aerosol transmission by
airborne droplets which are generated by coughing, sneezing or talking of an active
tuberculosis individual. Entering of the bacterium to the lung would cause inflammation in
the respiratory system which has a potential of spreading to other places such as the bones,
lymphatics and meninges (Knechel, 2009). Tuberculosis has become a major health
concern around the world with an estimated of 9.6 million cases of tuberculosis and with
around 1.5 million deaths in 2014 in the world. Currently, the highest prevalence of
tuberculosis is observed in South East Asian countries (Okorie et al., 2016).

Pathophysiology of tuberculosis

Once the MTB is inhaled, the infectious droplets settle mainly through the upper airways
which contain the mucus secreting goblet cells. Initially as a physical innate defense the
mucocilliary system fights the foreign substance entrapped by mucus by beating them
with the surface cilia. The mycobacteria which breach the above will be surrounded by
macrophages in the alveoli and is up taken by the macrophages in different mechanisms;
one such way is the MTB lipoarabinomannan acting as a ligand for the macrophage
receptor. Additionally the complement protein C3 opsonizes the MTB by binding to the
cell wall and enhancing the recognition of the MTB by macrophages. Phagocytosis
performed by these macrophages results in a reaction cascade which either gives rise to
successful control of the infection; latent tuberculosis or progression of the disease; active
tuberculosis (Figure 1). The MTB multiply slowly once every 25-32 hours by cell division
within the macrophage after ingestion. Macrophages initially produce cytokines which
attract T-lymphocytes and proteolytic enzymes to the site. Then macrophages present the

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MTB antigen on their surface to the T cells in order to initiate the T cell mediated
immunity. This immune process continues for 2-12 weeks. Afterwards, the formation of
granuloma around MTB occurs which creates a micro-environment that limits the
replication and spread of MTB. Then necrosis is initiated. However, some bacilli can
adapt to survive necrosis. By 2-3 weeks the lesion is known as caseous necrosis which
appears as a soft cheese. The lesion has low pH, nutrient and oxygen levels which limit
the growth of MTB causing latency of the disease. The events which occur after inhalation
of MTB is summarized in Figure 2 (Knechel, 2009).

Figure 1: Pathogenesis of Tuberculosis in the Infected Host (Long and Schwartzman,

2014).

Figure 2: Pathophysiology of tuberculosis disease (Crevel, Ottenhoff and Meer, 2002).

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Available treatment

Tuberculosis treatment could be categorized into two phases as the initial intensive phase
(IP) and the continuous phase (CP). IP is carried out for six months and then the CP is
executed if the fourth month culture report of the patient is negative, unless additionally IP
is continued for another month. The duration of CP is 18 months (Chhabra et al., 2012).
Tuberculosis treatment mainly includes four different first line defence drugs, namely
rifampicin, isoniazid, pyrazinamide and ethambutol (Figure 3). Additionally, there are
other second line drugs such as Ethionamide and Cycloserine which is used to treat
tuberculosis when the first line drugs fail to cure the disease. The drugs used for treatment
should be able to penetrate the MTB in order to reach their intracellular targets
(Sacksteder et al., 2012). Rifampicin was introduced in 1972 and is a rifamycin derivate.
The chemical structure is shown in Figure 3 (a). Rifampicin is one the most effective anti-
tuberculosis antibiotic which acts against growing and non-growing MTB (Palomino and
Martin, 2014). Rifampicin targets the beta-subunit of the DNA dependent RNA
polymerase, thus preventing transcription occurring. Therefore inhibition of bacterial
RNA synthesis takes place. The action of rifampicin could be bactericidal or
bacteriostatic. The Minimum Inhibitory Concentration (MIC) of rifampicin is 0.05–0.1
mg/L (Chemother, 2011).

Figure 3: Chemical structures of rifampicin (a), pyrazinamide (b), ethambutol (c) and
isoniazid (d) (Shewiyo et al., 2012).

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Pyrazinamide is a prodrug which is similar to nicotinamide. It was discovered in 1952 and
was introduced to tuberculosis chemotherapy in the early 1950s. The use of pyrazinamide
allowed to lessen the treatment period from nine to six months and is highly specific for
MTB. The drug passively enters MTB by diffusion and is converted to the active form
pyrazinoic acid by the enzyme pyrazinamidase (Pzase) which is coded by pncA gene in
MTB. Then pyrazinamide is excreted via a weak efflux pump. Outside the cell a
proportion of the acid gets uncharged and protonated (HPOA). In acidic conditions, the
HPOA is readily reabsorbed and the influx of HPOA can overcome the efflux of
pyrazinoic acid which would cause accumulation of pyrazinoic acid in the cell. Due to the
influx of protons, the cytoplasm becomes acidic which causes inhibition of normal cell
enzymatic functions. Pyrazinoic acid is capable of disrupting the MTB membrane
energetics and interferes with the energy required for the survival of MTB ultimately
causing inhibition of membrane transport as shown in Figure 4. The MIC of the drug is 16
to 50 mg/L (Chemother, 2011).

Figure 4: Model of the pyrazinamide mechanism of action (Zhang et al., 2013).

Isoniazid is also a prodrug which upon activation by the primary mycobacterial catalase-
peroxidase enzyme (katG) and MnCl2 produces highly reactive species such as reactive
oxygen, organic species and electrophilic species (Table 1). MTB possess long chain
branched fatty acids known as mycolic acids which makes the mycobacterial outer
membrane unique. The active drug targets the enzyme NADH dependent fatty-acid enoyl-
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acyl carrier protein reductase (InhA) involved in the mycolic acid synthesis by covalently
binding to the NADH present in the active site of the protein. InhA catalyses a NADH
dependent reduction reaction of 2-trans-enoyl-ACP, the reaction is similar to the final
elongation reaction of mycolic fatty acids. Thus inhibition of the alpha-mycolic acid
synthesis takes place as illustrated in Figure 5 (Jena et al., 2014). Other intracellular
targets of isoniazid are a complex of an acyl carrier protein (AcpM) and the β-ketoacyl-
ACP synthase (KasA). The MIC of isoniazid is 0.02–0.2 mg/L (Somoskovi, Parsons, and
Salfinger, 2001).

Figure 5: Mechanism of action of Isoniazid (Adapted from Wright, 2012).

Table 1: Examples of types of reactive species produced by isoniazid (Adapted from Jena
et al., 2014).
Reactive species Example

Reactive oxygen species (ROS) Superoxide, peroxide, hydroxyl radical, nitric oxide

Reactive organic species Isonicotinic acyl radical or anion

Electrophilic species -

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Kanamycin is an aminoglycoside which is used as a second line anti-tuberculosis drug. It
binds to the 30s subunit of the ribosome and causes a disturbance in the initiation of
protein synthesis by misreading the mRNA thereby causing inhibition of protein synthesis.
Kanamycin has a bactericidal action. Kanamycin is generally administered intravenously;
as poorly absorbed due to the first-pass metabolism when given through oral route
(Chhabra et al., 2012). The MIC of kanamycin is 2 to 4 mg/L (Chemother, 2011).

Combination therapy

Tuberculosis is associated with combination therapy which involves the use of two or
more drugs for treatment. It is not one specific treatment as a vast number of treatment
regimens are involved. This is considered as common cause for patient compliance in
tuberculosis (Kumar, Negi and Rawat, 2015). The first successful combination therapy
regimen included three drugs; isoniazid, streptomycin and para-aminosalicylic acid (PAS)
which showed a low relapse rate as such as 4% (Kerantzasa et al., 2017). Currently, fixed
dose combinations are recommended for active tuberculosis. One such example is the
complete first line four drugs regimen administration for two months and administration
of rifampicin and isoniazid for another 4-6 months. The application of synergic drug
combinations possesses various advantages with concern to the treatment. The efficacy of
the treatment could be increased without expanding the toxicity of individual drugs.
Moreover combination therapy reduces the toxic side effects of drugs. For an example
rifampicin is a toxic drug, but in combination therapy it is not used in the most effective
dose, even that it produces a therapeutic effect due to the synergic action (Ramón-García
et al., 2011). Combination therapy also prevents monotherapy and reduces the treatment
failure, relapse and the drug resistance by the patient which increases the treatment
effectiveness. The International Union against Tuberculosis and Lung Disease (IUATLD)
too recommends the use of fixed drug combinations as treatment (Albanna et al., 2013).

Novel anti-tuberculosis treatment

Tuberculosis is considered as one of the leading causes of deaths in the world. A major
issue concerning this is the inadequate treatment and the resistance to certain drugs such as
rifampicin and isoniazid. Most of the drugs currently being used have been discovered
about 50 years back. Within the past decade novel drugs which was clinically tested and
approved were introduced (Table 2). The statistical approach taken to compare the

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Multiple Drug Resistance-Tuberculosis (MDR-TB) and the development of anti-
tuberculosis drugs within the past decades is illustrated in Figure 5 (Olaru et al., 2015).

Table 2: Novel compounds introduced for tuberculosis treatment (Adapted from Olaru et
al., 2015).
Mode of
Mechanism of administration Study
Drug Drug class Function
action and current phase
dosage
Oral, 400 mg
for 2 weeks,
Bedaquiline Inhibits ATP Sterilising
Diarylquinoline 200 mg thrice a III
(TMC207) synthase Bactericidal
week for 22
weeks
1,2- Inhibits cell Oral, 300 mg
SQ109 Bactericidal II-a
ethylenediamine wall synthesis per day
Blocks 50s
Oral or
subunit of the
intravenous,
Linezolid Oxazolidinone ribosome and Bactericidal II
300–600 mg
inhibits protein
per day
synthesis
Oral, 100 mg
Inhibits per day for 2
Nitro-dihydro-
Delamanid mycolic acid months, then Bactericidal III
imidazooxazole
synthesis 200 mg per day
for 4 months
Inhibits DNA Oral or
Gatifloxacin Fluoroquinolone Bactericidal II
gyrase intravenous

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 Figure 6: Development of anti-tuberculosis drugs since 1940 versus the reported
worldwide MDR-TB cases (Olaru et al., 2015).

Bedaquiline is a novel drug which was approved by the European Medicine Agency
(EMA) in 2014. It is a diarylquinoline which inhibits mycobacterial Adenosine tri-
phosphate (ATP) synthase; a cause for the depletion of intracellular ATP. Bedaquiline has
the ability to act on active and dormant MTB both. The recommended dose of bedaquiline
for adults is 400mg a day for two weeks and followed by 200mg thrice a week for 24
weeks. Phase II evidence of clinical trials illustrates that bedaquiline treatment
significantly reduces the time of sputum culture conversion and also it non-significantly
reduces the resistance of other combined drugs thereby favouring treatment process. The
trial cure rate of bedaquiline according to the World Health Organization (WHO) is 58%
(Olaru et al., 2015). The MIC of the drug is 0.004 to 0.13 μg/mL (Field, 2015).

Delamanid is derivative of metronidazole and a nitroimidazopyran. As a pro-drug, it

requires activation by a F420 dependent mycobacterial co-enzyme and upon activation,
the methoxy-mycolic acid and keto-mycolic acid synthesis of the cell wall is inhibited.
The MIC of the drug is 0.006 to 0.024 g/mL. Delamanid has a bactericidal property. The
phase IIa and IIb clinical trial data presents that the short term (2 months) use of the drug
shows a favourable result of 48.5% and the long term use shows a favourable outcome of
57.3% positive results in MDR- tuberculosis patients. Dilamanid is also considered as less
toxic and lacks interactions with other antiviral drugs which make it more liable for
treatment (Lewis and Sloan, 2015).

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SQ109 is another novel anti-tuberculosis drug which is active on both drug susceptible
and resistant tuberculosis. SQ109 is considered as an ethambutol analogue. It targets the
MmpL3; a membrane protein of mycobacteria which transports trehalose monomycolate.
Thus by targeting the MmpL3, SQ109 inhibits the protein synthesis. This will also reduce
the mycolic acid assembly into the MTB cell wall. The drug has been approved by phase I
and early phase II clinical trials and is presently undergoing phase II-b trials. The MIC of
SQ109 ranges within 0.2 to 0.78 μg/mL and has a bactericidal action. Studies show a
synergistic effect of SQ109 with some drugs such as bedaquiline enhances the treatment
process when used as a combination (Sacksteder et al., 2012).

Summary

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis that

commonly affects the lungs. In healthy people, tuberculosis often has no symptoms as the
immune system has the ability to fight back and paves a pathway to latency. Tuberculosis
has been a major threat to life on earth. The main reason for the above situation is the lack
of treatment for tuberculosis. The recent approval of anti-tuberculosis drugs such as
bedaquiline, delamanid and SQ109 has paved a pathway for the cure of tuberculosis and to
manage the drug resistance. Presently there is only one vaccine for tuberculosis, namely
Bacille Calmette-Guerin (BCG) vaccine. To achieve the elimination of tuberculosis new
effective vaccines are also required. The vaccine development could be approached in
three different pathways such as the further improvement of the BCG vaccine, production
of a therapeutic vaccine to reduce the treatment period or production of a boost strategy
vaccine in order to boost the treatment process (Ahsan, 2015). In spite the progress
achieved so far in the anti-tuberculosis therapy, other preventive methods of transmitting
the active disease and early diagnosing are required to ensure future success in reducing
the global burden of tuberculosis.

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