Rev. sci. tech. Off. int. Epiz., 2001, 20 (1), 55-70
Treatment of mycobacterial infections
W . W . Barrow
Senior Microbiologist, Mycobacteriology Research Unit, Southern Research Institute, 2000 Ninth Avenue South,
Birmingham, Alabama 35205, United States of America
New address on 30 March 2001: Professor and Sitlington Chair in Infectious Diseases, Department of
Veterinary Pathobiology, Oklahoma State University, College of Veterinary Medicine, 250 McElroy Hall,
Stillwater, Oklahoma 74078-2007, United States of America
Summary.
Treatment of m y c o b a c t e r i a l infections differs f r o m t h a t of other bacterial diseases
due t o several properties possessed by the m y c o b a c t e r i a and the host. A hallmark
of m y c o b a c t e r i a is the complex lipid-rich cell envelope t h a t protects t h e organism
f r o m both t h e host response a n d a n t i m y c o b a c t e r i a l therapy. In addition,
m y c o b a c t e r i a are f a c u l t a t i v e intracellular parasites w h i c h generally cause a
more chronic t y p e of disease. These properties a d d greater constraints t o
efficient therapy. To be effective, drugs must be able t o penetrate t h e host
m a c r o p h a g e and preferably have r e d u c e d t o x i c i t y and be effective at l o w doses
to a l l o w prolonged therapy. T h e author presents t h e general properties of t h e
p a t h o g e n / h o s t relationship in m y c o b a c t e r i a l infections, in addition t o t h e
t h e r a p e u t i c c h o i c e s available a n d t h e m e c h a n i s m s of action involved in
t r e a t m e n t . The evolution of t e c h n o l o g y f o r a n t i m y c o b a c t e r i a l t h e r a p y is illustrated
by a discussion of n e w strategies being developed f o r t h e t r e a t m e n t of
m y c o b a c t e r i a l infections.
Keywords
Antimycobacterials - Drugs - Mycobacteria - Pathogenesis - Treatment.
Introduction
latency. Although information about the latent state is limited,
it is rationalised that the organism persists in an altered
metabolic state, probably relying upon enzymatic pathways
not used (or used only to a limited degree) during a normal
life-cycle in the host. This would suggest possible new drug
targets that could be included in future drug discovery efforts.
Mycobacterial infections represent some of the most difficult
diseases to treat in humans and animals. Because of the high
lipid content and complexity of the cell envelope,
mycobacteria are refractory to most antimicrobial agents (6,
88). In addition, mycobacteria are facultative intracellular
parasites, capable of surviving and persisting within the host
macrophage (6). This is the very cell that is largely responsible
for eliminating pathogenic microbes. Given this intracellular
infestation, agents that are to be effective in controlling and/or
killing the pathogen must also be able to penetrate the host
cell that harbours the mycobacteria. Intracellular effectiveness
is therefore an additional attribute necessary for adequate
antimycobacterial therapy. Initial screening of potential
antimycobacterial agents should, therefore, also include
appropriate experimentation to determine intracellular
efficacy.
The chronic nature of mycobacterial infections generally
necessitates prolonged therapy over several months. An ideal
agent should therefore have low toxicity and be effective at
low dose levels. The importance of these considerations is
further amplified because mycobacterial infections are treated
with multiple drug regimens ( 1 1 6 , 125). Multiple drug
regimens are necessary to prevent the development of drug
resistant strains, an event that occurs at different frequencies
depending upon which drug is considered. However, the use
of multiple drugs increases the likelihood of toxic side-effects
that may influence therapeutic success.
In addition to intracellular survival, some mycobacteria
(e.g. Mycobacterium tuberculosis) are capable of persisting in
the host tissue in a state of dormant existence referred to as
The purpose of this paper is to review the various
antimicrobics that are used to treat mycobacterial infections.
In general, the same agents are used to treat mycobacterial
56
infections in both animals and humans. Information in the
published literature is limited with regard to dosing and
therapeutic regimens for various animal species. In some
cases, animals are euthanised rather than being treated.
However, chemotherapy has been reported to be successful in
treating cases of tuberculosis in monkey colonies. I n one case,
rhesus monkeys (Macaca
mulatta)
received isoniazid,
rifampin and ethambutol once daily for twelve months, with
the conclusion that chemotherapy could provide an
alternative to destruction of the animals (125). In another
case, an epidemic of tuberculosis occurred in a non-human
primate breeding colony containing cynomolgus monkeys
(Macaca fascicularis) and rhesus monkeys (116). The animals
were treated with a combination of streptomycin and
isoniazid, with similar conclusions to the former study,
namely that chemotherapy was a viable alternative to
euthanasia (116).
Dosing and treatment schedules vary and these will not be
discussed in this paper. Instead, each of the agents will be
described in terms of mode of action and in vitro and in vivo
efficacy.
In
addition,
the
development
of
new
antimycobacterials will be discussed with special emphasis on
drug discovery and design, in addition to delivery systems
being developed for improved treatment of mycobacterial
infections. The aim is to present the relevant information to
enable the reader to analyse existing data effectively and
derive a personal evaluation of potentially useful
antimycobacterials for use in a specific area of therapy.
Pathogenesis
Intracellular parasitism
As stated earlier, mycobacteria are facultative intracellular
parasites (6). Unlike obligate intracellular parasites
(e.g. viruses, chlamydia, rickettsia, etc.) which are completely
dependent upon an intracellular environment, mycobacteria
are capable of existing both intracellularly and extracellularly
within the infected host. This is extremely important from a
therapeutic standpoint because effective therapy requires
another level of competency which is not necessary for strictly
extracellular parasites. To be effective against mycobacteria,
drugs must also be able to penetrate host macrophages, in
addition to the cell envelope of the pathogenic organism.
Testing for antimycobacterial agents must therefore include
efficacy studies involving macrophages. Generally, this type of
testing can be grouped into one of two categories. Some
investigators utilise macrophages derived from animals, for
example, peritoneal, bone marrow or alveolar macrophages.
The other category of testing involves cell lines. One of the
most commonly-used animal cell lines is the murine J774, an
adherent macrophage cell line that has been used extensively
for antimycobacterial drug screening.
Rev. sci. teck Off. int. Epiz, 20 (1)
Evaluation of intracellular efficacy requires the initial infection
of macrophages with the mycobacterium in question (126).
The mycobacterium should be able to replicate intracellularly
within the time frame of the experimental protocol (generally
from five to seven days) and produce a significant increase in
colony forming units (CFUs) which can be used for statistical
analysis to compare the treated and control groups (126).
Toxicity evaluations should be included to verify that
inhibition of growth is due to the drug under evaluation and
not the result of lysing macrophages that may falsely indicate
positive drug action. For example, some mycobacteria may
not replicate in the tissue culture medium after being released
from lysed macrophages. This would result in an apparent
decrease in CFUs when compared to the non-treated control,
and hence an incorrect evaluation of the drug being tested.
While not all species of mycobacteria have been tested using
these systems, it is very likely that any mycobacterial species
can be utilised, providing that the investigator establishes the
necessary parameters for the species in question. Thus,
antimycobacterial drug testing can potentially be performed
with any mycobacterium. It is also very likely that other
animal macrophage systems could be developed to evaluate
potential antimycobacterial action. Ideally, intracellular
efficacy evaluations would be performed with macrophages
derived from the species to be treated with the drug. As this
may not always be possible, commonly-used systems, such as
the J774 murine cell line, should be adequate if development
of appropriate animal macrophage systems is not feasible (6,
35, 82, 89, 126, 127). Hence, the development of other
macrophage assays could potentially be very useful, but the
use of established procedures should not be precluded.
Another important aspect of intracellular efficacy evaluations
for antimycobacterial drags involves the variation of
environmental parameters found within the host and host
macrophages. Although exact parameters may not have been
established for this intracellular environment, it has generally
been observed that growth of mycobacteria in a host is quite
different from that observed in vitro (123). This difference is
due in part to the choice of substrates available to the infecting
mycobacteria. This is important because a drug target (e.g. an
enzyme in a key pathway) that may be upregulated in vitro
may not necessarily be present at the same level when the
mycobacteria grow intracellularly. It is possible that the drug
target may not be expressed at all in the intracellular growth
pattern. If that is the case, the potential drug would not be
expected to produce the optimum effect when used to treat
the animal in question.
Thus, intracellular efficacy evaluations are an important part
of the development and screening of any antimycobacterial
drug. Although this type of testing may be tedious and require
a different level of expertise, the process is essential to
properly
evaluate
the
therapeutic
potential
of
antimycobacterial drugs.
57
Rev. sci. tech. Off. int. Epiz., 20 (1)
Dormancy (latency)
As mentioned in the Introduction, the ability of mycobacteria
to persist in a dormant state is also an important consideration
for optimum mycobacterial therapy. In this discussion,
M. tuberculosis will be used as the representative organism.
Studies involving specific aspects of dormancy are limited ( 2 0 ,
120). However, evidence from an in vitro system suggests that
various stages occur in the 'shiftdown' and 'shiftup' of
M. tuberculosis as the organism enters and exits dormancy
(117, 119). During the 'shiftdown', the organism apparently
goes through two or more stages. In the first stage, the
organism shifts from rapid to slow replication (117). The
second stage involves a complete shutdown o f replication
without death of the organism. Three markers have been
described in this process, as follows:
a) changes in tolerance to anaerobiosis
b) production of a unique antigen
c) an increase in glycine dehydrogenase production ( 1 1 7 ,
118,121).
During the 'shiftup' process, initial production of ribonucleic
acid (SNA)
occurs, followed by the initiation of
deoxyribonucleic acid (DNA) synthesis (119), indicating that
during dormancy, the metabolic events that occur serve a
completely different purpose. This would suggest that targets
for optimal therapy would be different from those found in an
active disease state and would therefore require different
antimicrobics and therapeutic regimens ( 1 2 2 ) . Investigations
of dormancy are currently in progress and hopefully a more
specific evaluation will eventually result. However, an
awareness of this variation in metabolism is important
because it most certainly indicates a different assessment for
therapy.
Antimycobacterial agents
Antimycobacterial agents will be discussed as two major
groups. The first group consists of antimycobacterials that
require some type of activation by the mycobacteria. The term
used to describe this type of compound is 'prodrug'. A
prodrug is a compound that is converted to an active
derivative by biotransformation or non-enzymatic processes
such as hydrolysis, oxidation or reduction (7). Generally,
these compounds are more selective for mycobacteria because
the targets are associated with mycobacteria and not other
microorganisms. These targets are generally involved in the
synthesis of unique structures found in the cell envelope of
mycobacteria, including mycolic acid, arabinogalactan and
lipoarabinomannan.
The second group consists of
antimycobacterials that do not require activation by
mycobacteria and have a broader spectrum of activity. This is
due to the fact that the targets of these drugs are found in
other bacteria as well as mycobacteria. These compounds
affect a specific target, such as an enzyme in a key pathway or
a subunit on the bacterial ribosome.
Antimycobacterials requiring activation by
mycobacteria
Isoniazid
Isoniazid (isonicotinic acid hydrazide [INH]) is an analogue of
nicotinamide and, amongst various mycobacteria, is most
effective against M. tuberculosis. This is a very potent and
specific drug for M. tuberculosis, with relatively low toxicity
(56). Early studies described the ability of INH to inhibit
mycolic acid synthesis and the hypothesis which developed
suggested this mechanism as the primary cause of
mycobacterial cell death ( 5 6 , 124). Later work by Takayama
and associates ( 9 9 ) elucidated mycolic acid metabolism and
demonstrated that INH inhibits the desaturation of C and
C acids, thus leading to an accumulation of saturated fatty
acids. More recent studies have shown that resistance to INH
is associated with mutations of the inhA gene ( 3 , 1 8 , 7 6 , 1 3 0 ) .
Isonicotinic acid hydrazide is one of several antimycobacterial
agents that requires activation for effectiveness. Catalaseperoxidase activity, encoded by the katG gene (32, 7 1 , 7 2 , 77,
78), is responsible for oxidation of INH which eventually
results in toxicity to M. tuberculosis, after several intermediarysteps. Despite the wealth of information that has been
obtained, the precise target for the action of INH remains
unknown (3). Although INH is most effective against
M. tuberculosis, some activity is apparent against a few
non-tuberculous mycobacteria, in particular M. kansasii, but
not against M. avium (24).
2 4
2 6
Ethambutol
Ethambutol (EMB) is a synthetic drug that was developed
from N,N'-diisopropylethyl-enediamine and has been
demonstrated to have activity against M. tuberculosis (104).
The drug is active against M. tuberculosis, Al. kansasii and
some other non-tuberculous mycobacteria, including
Al. avium (24). Ethambutol prevents the polymerisation of
cell wall arabinan by inhibiting the introduction of
D-arabinose into arabinogalactan and lipoarabinomannan (60,
6 1 , 75, 1 0 0 , 1 0 1 ) .
Pyrazinamide
Pyrazinamide (PZA) is the amide of pyrazinoic acid that is
metabolised to 5-hydroxypyrazinamide after absorption from
the gastrointestinal tract ( 5 6 ) . Pyrazinamide is active against
M. tuberculosis, but not other members of the M. tuberculosis
complex (e.g. M. bovis), nor non-tuberculous mycobacteria
such as M. avium ( 2 4 , 5 3 , 56). Although the exact mechanism
of action of PZA. is not known, studies have shown that PZA. is
converted to pyrazinoic acid by nicotinamidase (63). One
hypothesis suggests that PZA functions as a prodrug of
pyrazinoic acid (95). Additional studies have suggested that
susceptibility of M. tuberculosis to PZA is due to a deficient
pyrazinoic acid efflux mechanism, whereas resistance is due
to a highly active efflux pump (131).
58
Rev. sci. tech. Off. int. Epiz., 20 (1)
Antimycobacterials not requiring activation by
mycobacteria
Rifamycins
Rifamycins are a group of bactericidal antimicrobials that were
originally isolated from a species of Streptomyces
(Nocardia
mediterranei by the Lepetit Laboratories in Italy (24). One of
the most important of these was later synthesised in 1965 and
is known as rifampin (RIF), 3-4-(4-methyl-piperazinylimino-methylidene)-rifamycin SV (24). Rifampin has a wide
range of activity, including M. tuberculosis, M. kansasii,
M. marinum and, to a lesser degree, M. avium ( 2 4 ) . In
addition, RIF has activity against other microorganisms,
including a variety of Gram-positive and Gram-negative
bacteria. Rifabutin (RBT) is a semi-synthetic derivative of
rifamycin S (spiro-piperidyl-rifamycin) (15), and has greater
activity than RIF for M. tuberculosis, M. kansasii, M. marinum,
M. xenopi, M. haemophilum,
and particularly M. avium
complex
(22, 3 0 , 9 1 , 109). Rifapentine
(RFP),
3-(4-cyclopentyl-piperazinyl-imino-methyl)-rifamycin-SV (24),
has better activity than RIF against M. tuberculosis and
M. avium complex (22, 2 9 , 3 0 , 3 1 , 54, 62).
The target for the rifamycins is the beta subunit of the
DNA-dependent RNA polymerase, coded for by the rpoB gene
(81). Rifampin has one of the lowest minimal inhibitory
concentrations (MIC) for susceptible strains of M. tuberculosis
(51) and has the ability to kill both the actively growing
extracellular mycobacteria and the slower growing
semi-dormant extracellular mycobacteria found in caseous
material (42). Rifampin is also effective for M. tuberculosis
growing intracellularly (42) and is the least toxic of the major
antituberculosis drugs. Rifampin has the fastest onset of action
(15 min-20 min), as compared to the other antituberculosis
drugs, and is the most capable of killing the tubercle bacilli
during growth spurts (42). In addition, in unselected
populations of tubercle bacilli, mutants that are resistant to
rifampin are the least common (1 in 1 0 are resistant to
rifampin, as compared to 1 in 1 0 for isoniazid and
streptomycin and 1 in 1 0 for ethambutol [27]).
8
polypeptide chains. The aminoglycosides are broad spectrum
antibiotics, having activity against both Gram-positive and
Gram-negative bacteria, in addition to mycobacteria.
Macrolides
Macrolides are a group of broad-spectrum bacteriostatic
antibiotics, some of which have activity against certain
mycobacteria. The mechanism of action is inhibition of
protein synthesis, which is accomplished by binding of the
drug to the 50S ribosomal subunit ( 3 3 ) . The macrolides
include erythromycin, azithromycin, clarithromycin and
roxithromycin.
The macrolides appear to have better activity against
non-tuberculous infections, such as those caused by the
M. avium complex ( 9 , 1 0 , 1 6 , 5 2 , 6 7 , 1 0 2 , 1 2 9 ) , than against
M.
tuberculosis
( 1 1 0 , 128). Reports indicate
that
clarithromycin also has activity against M. ulcerans (86) and
M. marinum (14).
Fluoroquinolones
The fluoroquinolones are heterocyclic carbonic acid
derivatives which are structurally related to nalidixic acid.
Included in this group are ciprofloxacin, clinafloxacin,
gatifloxacin,
grepafloxacin,
levofloxacin,
ofloxacin,
moxifloxacin, sitafloxacin and sparfloxacin. The primary
target for the quinolones is the bacterial DNA gyrase
(topoisomerase II) (56). Binding of the drug to the enzyme
prevents subsequent processes that depend upon the proper
maintenance of DNA superhelical twists, such as replication
and transcription (17).
Generally, the fluoroquinolones are active against several
mycobacteria, including
M. tuberculosis,
M.
bovis,
M. fortuitum, M. kansasii, M. peregrinum and M. leprae ( 4 9 ,
5 7 , 5 8 , 1 0 7 , 1 0 8 , 111). These drugs are not very active against
M. avium complex, M. intracellulare,
M.
marinum,
M. chelonae and M. abscessus ( 4 9 , 57, 111).
6
5
Aminoglycosides
Aminoglycosides represent one of the oldest groups of
antimycobacterials.
These
bactericidal
agents
are
characterised
by
combinations
of
six-membered
aminocyclitol rings with varying attached side chains. The
first to be discovered was streptomycin. The antimicrobic
activity of streptomycin was reported in 1 9 4 4 by Albert
Schatz, a doctoral graduate student of Dr Selman Waksman at
the time (93). As a result of the discovery, Dr Selman
Waksman was awarded the Nobel Prize in 1 9 5 2 . Other
aminoglycosides include kanamycin and amikacin. The mode
of action for the aminoglycosides is inhibition of protein
synthesis. Inhibition is accomplished by irreversible binding
of the drug to the bacterial 30S ribosomal subunit (binding
sites vary for each aminoglycoside), thus freezing' the
initiation complex and preventing further elongation of
Oxazolidinones
Oxazolidinones are a relatively new group of totally synthetic
antimicrobial compounds that were developed in the late
1970s by DuPont ( 1 , 4 7 ) . These compounds inhibit protein
synthesis, presumably at a site that precedes chain elongation,
or more specifically, before the interaction of fMet-transfer
RNA (fMet) and 3 0 S ribosomal subunits with the initiator
codon (39, 4 0 ) . Some of these agents have shown activity
against M. tuberculosis in vitro (2, 4) and in a murine model
(25). Further testing is needed to evaluate the full potential in
treating mycobacterial infections and also toxicity.
Antimetabolites
An antimetabolite is a compound which is sufficiently similar
in structure to a normal metabolite that it can occupy the
enzyme binding site in place of the metabolite, thus inhibiting
a biosynthetic pathway (94). Antifolates are a group of
antimetabolites which have been utilised for antimicrobial
drug therapy in the past. A target for this class of drugs is
59
Rev. sci. tech. Off. int. Epiz., 20 (1 )
dihydrofolate reductase (DHFR), a key enzyme in the folate
metabolic pathway which is necessary for the biosynthesis of
RNA, DNA and protein. The enzyme is an important target for
medicinal chemistry (13), and inhibitors of DHFR have been
used in chemotherapy for cancer (methotrexate [11]),
bacterial infection (trimethoprim [41]), and malaria
(pyrimethamine [92]). All cells contain DHFR, and the
activity of this enzyme is necessary for the maintenance of
intracellular folate pools in a biochemically active reduced
state (70). Inhibition of DHFR is effective because binding
affinities for substrate analogues are so great that these
analogues are not readily displaced by the natural substrates
(70). Inhibition results in the depletion of intracellular
reduced folates necessary for one-carbon transfer reactions
which are important for biosynthesis of RNA, DNA and
protein. Although some bacteria have an uptake system for
folates, most have to synthesise folates de novo. Reduction of
dihydrofolate to tetrahydrofolate is a universal requirement
(50).
Investigations of the antimycobacterial properties of a group
of
antifolate
compounds
that
are
derivatives of
2,4-diamino-5-methyl-5-deazapteridine (DMDP) have been
performed in the Mycobacteriology Research Unit at the
Southern Research Institute, Alabama, United States of
America (USA). These lipophilic compounds have structures
similar to the piritrexim/trimetrexate class of antifolates and
have demonstrated activity against M. tuberculosis (96) and to
an even greater extent, against M. avium ( 9 6 , 9 7 ) . The
M. avium folA gene, which codes for DHFR, has been
identified, cloned and expressed for use in enzyme assays
designed for drug screening and identification ( 1 3 2 , 133).
Subsequently, the recombinant M. avium DHFR was used to
screen more than seventy DMDP derivatives and a
structure/activity relationship was identified that resulted in
the synthesis of a DMDP derivative with reduced toxicity for
human DHFR, specificity for M. avium DHFR, and
intracellular efficacy in a M. avium-infected macrophage
model (98). Other workers have shown an enthusiasm in the
development of improved derivatives of this class of inhibitor
with regard to mycobacteria (26, 5 9 , 6 9 , 7 4 , 103). Further
evaluation and testing will be necessary to develop more
effective DMDP derivatives for clinical trials; these studies are
in progress.
of appropriate radiolabelled precursors into respective
mycobacterial macromolecular components. Thus, for
protein synthesis, incorporation of [ C]-phenylalanine will
be assayed, whereas for RNA and DNA synthesis,
incorporation of [ C]-uracil will be assayed.
14
14
The use of radiolabelled thymine or thymidine should be
avoided in monitoring DNA synthesis in M.
tuberculosis,
because of the presence of thymidine phosphorylase which
hydrolyses the substrate (12, 118). Use of thymidine will
result in misleading incorporation data. It is therefore
necessary to use radiolabelled uracil, which is incorporated
into both RNA and DNA, and to distinguish between
radiolabelled RNA and DNA by hydrolysing the RNA prior to
DNA precipitation from the cells using potassium hydroxide
(118). In the case of DNA, uracil is methylated prior to
incorporation into the macromolecule ( 4 8 ) , therefore uracil
that is radiolabelled at the 5-position with [ H] should not be
used because the label will be lost during the methylation
process.
3
14
In the following examples, [2- C]-uracil was used for
incorporation into RNA and DNA. Additional parameters to
be considered in these assays are a negative control (e.g. a
polymyxin such as polymyxin B which inhibits membrane
function), a solvent control (e.g. equivalent amount of solvent
necessary for dissolving the drug being tested), and a positive
control for the biosynthetic process to be tested
(e.g. streptomycin for protein synthesis, rifampin for RNA
synthesis and ofloxacin for DNA synthesis). The assay, which
is depicted in Figures 1, 2 and 3, involves a short time period
(1 h-5 h). Appropriate drugs are added to an actively growing
culture of M. tuberculosis H37Ra (avirulent strain), following
addition of the appropriate radiolabelled component. Samples
of mycobacteria are taken at various time intervals and
processed for radiolabel incorporation by precipitation with
trichloroacetic acid, followed by measurement of radioactivity
using a scintillation counter.
Protein synthesis
Macromolecular aspects of inhibition by
antimycobacterial drugs
For
the protein synthesis assay, streptomycin (an
aminoglycoside) is used as the inhibitor of protein synthesis
(i.e. positive control). As discussed above, aminoglycosides
inhibit protein synthesis by binding to the bacterial
30S
ribosomal
subunit, thus preventing elongation
of
polypeptides.
The radiolabel in this case is
[ C]-phenylalanine.
In this section, only those antimycobacterials that have a
specific mechanism of action (i.e. those not requiring
activation by the mycobacteria) will be discussed. The
following examples are included to give a representation of
the
effects
of certain
antimycobacterials
at
the
macromolecular level. These examples will include inhibition
of protein synthesis (e.g. aminoglycosides), RNA synthesis
(e.g. rifamycins) and DNA synthesis (e.g. fluoroquinolones).
The experimental procedures are designed to determine the
inhibitory activity of each drug by observing the incorporation
As illustrated in Figures l a and l b , inhibition is not
immediate, occurring in these examples at approximately 2 h
post treatment. Since translation of the already attached
messenger RNA (mRNA) will continue following attachment
of the drug to the appropriate receptor on the 3 0 S ribosomal
subunit, the round of protein elongation in progress will
proceed until completion. Once that round of translation has
finished, protein synthesis by the affected ribosome will cease
because binding is irreversible. By adjusting
the
14
60
Rev. sci. tech. Off. int. Epiz., 20 (1)
a) Inhibition of protein synthesis by streptomycin sulphate (SSO4) at 1 µg/ml,
5 µg/ml and 10 µg/ml
b) Inhibition of protein synthesis by streptomycin sulphate (SS0 ), rifampin (RIF)
and ofloxacin (OFL)
4
Fig.1
Effects of inhibitors on protein synthesis in Mycobacterium tuberculosis H37Ra
Mycobacterium tuberculosis H37Ra was cultured in Middlebrook 7H9 broth (10% oleic acid-albumin-dextrose-catalase [OADC], 2 % glycerol) until exponential
14
phase was obtained. [ C]-phenylalanine was added, followed by the respective inhibitors (time 0). Samples containing viable mycobacteria were removed and
proteins precipitated with trichloracetic acid (TCA, 10%). Radioactivity, as determined using a scintillation counter, is expressed as counts per minute (CPM)
concentrations of the drug, a dose response can be observed
(Fig. la). The MIC of streptomycin for M. tuberculosis H37Ra
is 0.5 µg/ml-1.0 µg/ml. In Figure l b , the effect of
streptomycin is compared to that of rifampin and ofloxacin.
Rifampin has an immediate effect on protein synthesis,
whereas ofloxacin acts more slowly (Fig. l b ) . This is
explained by the fact that rifampin inhibits RNA synthesis
which is immediately necessary for protein synthesis.
Ofloxacin inhibits DNA supercoiling which will eventually
affect protein synthesis, but not in this time frame.
a) Inhibition of RNA synthesis by rifampin (RIF) at 0.004 µg/ml, 0.02µg/mland
0.04 µg/ml
Ribonucleic acid synthesis
For the RNA synthesis assay, RIF (a rifamycin) is used as the
inhibitor of RNA synthesis (i.e. positive control). As discussed
above, rifamycins inhibit RNA synthesis by binding to the
beta-subunit of the DNA-dependent RNA polymerase. The
radiolabel used in this case is [ 2 - C ] -uracil.
14
Inhibition of RNA synthesis by rifampin is apparent early in
the process (Figs 2a and 2b), as expected by the rapid onset of
action of the drug. Adjustment of drug concentration around
b) Inhibition of RNA synthesis by rifampin (RIF), streptomycin sulphate (SS0 ) and
ofloxacin (OFL)
4
Fig. 2
Effects of inhibitors on ribonucleic acid (RNA) synthesis in Mycobacterium tuberculosis H37Ra
Mycobacterium tuberculosis H37Ra was cultured in Middlebrook 7H9 broth ( 1 0 % oleic acid-albumin-dextrose-catalase [OADC], 2 % glycerol) until exponential
14
phase was obtained. [2- C]-uracil was added, followed by the respective inhibitors (time 0). Samples containing viable mycobacteria were removed and nucleic
acids precipitated with trichloracetic acid (TCA, 10%). Radioactivity, as determined using a scintillation counter, is expressed as counts per minute (CPM)
61
Rev. sci. tech. Off. int. Epiz., 20 (1)
a) Inhibition of DNA synthesis by ofloxacin (OFL) at 0.5 µg/ml, 2.5 µg/ml
and 5.0 µg/ml
b) Inhibition of DNA synthesis by ofloxacin (OFL), streptomycin sulphate (SS0 ) and
rifampin (RIF)
4
Fig. 3
Effects of inhibitors on deoxyribonucleic acid (DNA) synthesis in Mycobacterium tuberculosis H37Ra
Mycobacterium tuberculosis H37Ra was cultured in Middlebrook 7H9 broth ( 1 0 % oleic acid-albumin-dextrose-catalase [OADC], 2 % glycerol) until exponential
14
phase was obtained. [2- C]-uracil w a s added, followed by the respective inhibitors (time 0). Samples containing viable mycobacteria were removed and RNA
was hydrolysed with potassium hydroxide (K0H), prior to D N A precipitation with trichloracetic acid (TCA, 10%), following removal of RNA by K0H. Radioactivity,
as determined using a scintillation counter, is expressed as counts per minute (CPM)
the MIC, which in this case is 0.004 µg/ml, results in a dose
response (Fig. 2a). This inhibitory action also affects synthesis
of proteins (Fig. l b ) and DNA (Fig. 3b). The effect of
streptomycin and ofloxacin on RNA synthesis, in this time
frame, are only minimal (Fig. 2b).
primarily because the formulations are designed to facilitate
phagocytic uptake by host macrophages. As mycobacteria are
intracellular parasites, these techniques have proven useful in
the therapy of certain mycobacterial diseases. Two
formulations which have been used for delivery of
antimycobacterial drugs are liposomes and microspheres.
Deoxyribonucleic acid synthesis
In the DNA synthesis assay, ofloxacin (a fluoroquinolone) was
used as the inhibitor of DNA synthesis. As discussed above,
fluoroquinolones bind to the bacterial DNA gyrase, thus
preventing proper maintenance of DNA superhelical twists.
Ultimately, this results in inhibition of replication and
transcription. The radiolabel for these experiments was
[2- C]-uracil.
14
Action of ofloxacin on DNA synthesis is immediate, with loss
of radiolabel incorporation being seen after one hour (Figs 3a
and 3b). The MIC of ofloxacin for M. tuberculosis H37Ra is
0.5 µg/ml and the dose response for concentrations around
this MIC is given in Figure 3a. Although this inhibition will
ultimately affect other parameters such as RNA and protein
synthesis, these effects are not observed within the time frame
of these experiments (Figs l b and 2b). Deoxyribonucleic acid
synthesis is affected by both rifampin and streptomycin
(Fig. 3b).
Delivery systems
In recent years, techniques for improved delivery of
antimicrobics have been developed. These techniques have
been beneficial in the therapy of mycobacterial infections,
Liposomes
Liposomes are small phospholipid vesicles which are
composed of concentric lipid bilayers with alternating
aqueous compartments. These artificial vesicles have
permeability properties which are similar to biological
membranes. Delivery of various antimicrobics has been
reported for therapy of mice infected with M. avium (8, 2 3 ,
3 4 , 3 5 , 3 6 , 4 3 , 6 8 , 7 9 , 8 0 , 8 5 ) and M. tuberculosis (28, 8 3 ,
115). Use of liposomes allows for delivery of drugs to
macrophages, but allows no flexibility with regard to timing of
drug release.
Microspheres
Microspheres are discrete particles, the biological agent can
either be encapsulated within the microsphere or attached to
the surface (87). Microspheres are generally formulated with
polymers, such as poly(lactide-co-glycolide) (PLG), that allow
significant flexibility with regard to programmed release ( 5 ,
87). Degradation of PLG microspheres results from hydrolysis
and not enzymatic processes (105). These properties enable
the formulation of microspheres that release a drug for a
period of days or months (small microspheres: 1 µm-10 µm
in diameter) or for a year or more (large microspheres:
10 µ m - 1 5 0 µm), which would not be possible with liposomes
62
(87). An additional benefit of microspheres is the ability to
administer a greater quantity of drug than liposomes, on a
weight basis comparison (83, 8 7 , 1 1 5 ) .
The chemical formulation of microspheres (PLG) is based
upon the formulation for synthetic sutures (45). The PLG and
also its metabolic products, lactic acid and glycolic acid, are
known to be biocompatible ( 1 1 2 , 113, 114). Microsphere
technology has been used for sustained delivery of a variety of
substances, including antigens, steroids, peptides, proteins
and antibiotics (21, 37, 3 8 , 5 5 , 9 0 , 1 0 6 ) .
The
intracellular efficacy
of small
rifampin-loaded
microsphere formulations has been demonstrated in the
Mycobacteriology Research Unit at the Southern Research
Institute. These formulations have been shown to be effective
against M. tuberculosis-infected macrophages by significantly
reducing levels of intracellularly replicating mycobacteria (5).
The small microspheres are better at delivering effective doses
of rifampin intracellularly than equivalent doses of free drug
(5). Subsequently, the effective use of both small and large
rifampin-loaded microspheres was demonstrated in treating
M. tuberculosis-infected mice ( 8 7 ) . The microsphere
formulations, administered in one or two doses during a
one-month period, were able to achieve a reduction in
mycobacteria similar to that obtained with a daily drug
regimen of rifampin equivalent to 10-20 mg/kg/day.
Programmed release of rifampin was obtained throughout the
twenty-six-day experimental period (87). The results suggest
that these types of drug delivery formulations can be used for
therapy of mycobacterial infections and will allow reduced
dosing and targeted delivery to host macrophages (5, 8 7 ) .
Use of microsphere technology for treatment of animal
diseases is very feasible, particularly with regard to reducing
dosing intervals and toxicity. Recent studies at the Southern
Research Institute with non-human primates (cynomolgus
monkeys of approximately 4 kg) have revealed that as much
as 4 g of a small rifampin-loaded microsphere formulation
(1 µm-10 µm in diameter, 5.8% [wt/wt] loading of rifampin)
can be injected intravenously (2 g given twice at one
week intervals) without adverse effects on the animal
(D.C. Quenelle and W.W. Barrow, unpublished data). The
small microsphere formulation demonstrated sustained
programmed release of drug over thirty days. In some
experiments, animals were treated with intravenous injections
(4 g total) along with 2 g of a larger microsphere formulation
injected subcutaneously (27 wt% loading of rifampin). In
these experiments, release of rifampin was observed for up to
forty-eight days without any adverse reactions observed in the
animals (i.e. body weights, histopathology, etc.) and without
any biologically significant changes in liver enzyme activity
(i.e. >1.5-fold change for alanine aminotransferase, aspartate
aminotransferase and alkaline phosphatase). Bioassay results
demonstrated blood levels, urine levels and more importantly,
concentration of rifampin in various tissues (e.g. liver, spleen
Rev. sci. tech. Off. int. Epiz., 20 (1)
and lungs) (E.L.W. Barrow and W . W . Barrow, unpublished
data). These preliminary studies suggest that a microsphere
drug delivery protocol offers an economically feasible and
effective approach for treating companion and domestic
agriculture species. Animals would be handled much less
frequently and the outlay for drugs would be decreased since
required dosages would be considerably reduced. Hence, this
approach could reduce significantly both the cost and stress of
treatment. For food animals, however, the impact on the
withholding period prior to using the animal products would
have to be evaluated.
New strategies for
antimycobacterial drug
development
Traditionally, drug discovery has been primarily dependent
upon the screening of large collections of previously
synthesised chemical compounds or products occurring
naturally (46). However, since the early 1990s, new and
improved techniques have evolved that will increase the
opportunities for new drug discovery and improve the ability
to synthesise more selective and active compounds for
treating mycobacterial infections. Some of the new
technological platforms that are being developed are
discussed below.
Genomics
The sequencing of a number of bacterial genomes has been
achieved and work on others is progressing. Given the
numerous genomic projects currently underway, the
development of new and better drugs should be possible for a
number
of
pathogenic
microorganisms,
including
M. tuberculosis
(19). This is extremely important for
M. tuberculosis because of the increasing numbers of drug
resistant strains that are developing (64, 8 4 ) . Although
approximately 1 6 % of the estimated 4 , 0 0 0 encoded proteins
of M. tuberculosis have still not been identified, 4 0 % have
been associated with known biochemical functions ( 7 , 1 9 ) . As
suggested by Barry et al. (7), mycobacterial research has now
shifted 'from gene hunting to interpretation of the biology of
the whole organism'. Given this expanding genetic knowledge
base, it should be possible to utilise sophisticated technologies
to identify new drug targets and design new and improved
therapeutic regimens for mycobacterial infections.
One such technology that will be helpful in observing
differential gene expression is the DNA-based microarray
technique. This technology should allow the analysis of the
differential metabolic patterns of mycobacteria in vivo and in
vitro (7). This technology can also be used to predict
mechanisms of action of drugs (7).
63
Rev. sci. tech. Off. int. Epiz., 20 (1)
Proteomics
The next level of technological platforms being developed for
drug discovery and development involves techniques that can
analyse protein arrays. This technology is referred to as
proteomics, a term that was coined in 1 9 4 4 by a postdoctoral
researcher from Australia by the name of Marc Wilkins ( 4 4 ) .
The term proteomics refers to the proteins that are expressed
by a genome (44). Unlike DNA and RNA arrays, proteomics
addresses the final gene product, i.e. the protein. The benefit
of this technology is that the major shortcoming of DNA chip
technology (i.e. failure to consider pre-translational events
and post-translational modifications of proteins) is overcome
(44). In the case of antimycobacterial drug development, this
has been shown to be critical. An example of this was recently
presented by Barry et al. (7), in a discussion of the mechanism
of INH action. A covalent complex of INH with two different
proteins, AcpM and KasA had been described (73). Although
both related proteins were transcriptionally up-regulated, the
actual interaction between the proteins and INH would not
have been revealed without a proteomic experiment (7).
Initially, the major tool used for proteomics was
two-dimensional gel electrophoresis, a tedious and laborious
technique which is time-consuming and is only useful for
proteins that exist in fairly large proportions in the cell ( 4 4 ) .
However, in the last few years, instrumentation companies
have been able to exploit the use of mass spectrometry (MS)
for improved analysis of the protein arrays. In 1989, matrixassisted laser desorption/ionisation (MALDI) was introduced,
in addition to electrospray ionisation (ESI) (44). These two
developments greatly expanded the range of proteins that
could be analysed by MS ( 4 4 ) . Subsequently, a more rapid
method of identifying proteins from MS was introduced,
namely: protein mass fingerprinting ( 4 4 ) . Currently,
companies
are
developing
more
sophisticated
instrumentation to render proteomics more feasible and
useful for drug discovery ( 4 4 ) . Although this type of
technology is fairly new, it holds great promise for future drug
discovery and development.
Combinatorial chemistry and structure-activity
relationship
Combinatorial chemistry and structure-activity relationship
(SAR) represent two additional technological platforms that
are useful for the discovery and design of new drugs. The role
of combinatorial chemistry is to synthesise large 'libraries' of
related compounds that can be assayed by means of a
high-throughput screening technique, a process which has
been of great benefit in the task of random searching for drug
candidates (65). Generally, these libraries are based upon a
lead compound which has shown in vitro activity against the
organism in question (7). Subsequently, a more focused
library is generated around the most active substructures, to
improve binding efficiency (7). This approach significantly
accelerates the process of synthesising new drug candidates
(65).
Structure-activity
relationship
involves the
use of
crystallography and nuclear magnetic resonance (NMR) to
assess the three-dimensional structure of the target protein to
enable the production of inhibitors that are complementary to
the binding site (66). With the information derived from these
sources, improved binding affinities can be obtained, thus
optimising activity for the target protein (e.g. an enzyme in a
mycobacterial biosynthetic pathway), while decreasing
selectivity for the similar host protein, if applicable. Ideally, a
drug should have a perfect geometric fit of the ligand to the
binding site. As binding affinity information is obtained, new
drugs can be synthesised and tested in appropriate in vitro and
in vivo assays. One of the failings of SAR is the inability of the
procedure to predict bioavailability and metabolic stability
(66), characteristics which have to be accomplished by
appropriate animal studies.
Conclusion
A number of effective and reliable antimycobacterial drugs are
currently available for treatment of several mycobacterial
infections. The primary reason for the loss of efficacy of some
of these drugs is the emergence of drug resistant strains.
Development of resistance is an important variable to be
considered in the eradication of mycobacterial disease and
should be treated very seriously. However, the recent
advances in genomics and the development of new and
improved biotechnological platforms suggest a promising
future with regard to development of effective drugs for
control and/or eradication of mycobacterial diseases.
Acknowledgements
Some of the research discussed in this review was funded by
grants AI38185 (Treatment of TB with Microencapsulated
Drugs'; Principal Investigator: W . W . Barrow), AI41348
('Antifolates against Mycobacterial Infections in AIDS';
Principal Investigator: W . W . Barrow) and A I 4 3 2 4 1 ('Purine
Analog Anti-mycobacterial Drug Development', Principal
Investigator: W . B . Parker; Co-Investigator: W.W. Barrow)
from the National Institute of Allergy and Infectious Diseases
(NIAID), National Institutes of Health (N1H), USA. The
author would like to thank Dr L.G. Wayne (Tuberculosis
Research Laboratory, Department of Veterans Affairs Medical
Center, Long Beach, California, USA), for his advice and
consultation
regarding
radiolabelling
of DNA
in
M. tuberculosis. Special thanks to E. Barrow for helpful
comments and suggestions in the writing of this manuscript.
64
Rev. sci. tech. Off. int. Epiz., 20 (1)
Traitement des infections mycobactériennes
W . W . Barrow
Résumé
Le traitement des infections mycobactériennes diffère de celui appliqué à
d'autres maladies bactériennes en raison de plusieurs propriétés spécifiques des
mycobactéries et de leurs hôtes. L'un des traits distinctifs des mycobactéries est
l'enveloppe cellulaire complexe et riche en lipides qui protège le
micro-organisme contre la réponse de l'hôte et contre les produits
thérapeutiques antimycobactériens. Par ailleurs, les mycobactéries sont des
parasites intracellulaires facultatifs qui provoquent généralement un type de
maladie plus chronique. Ces propriétés sont autant d'obstacles à une bonne
thérapie. Pour être efficaces, les médicaments doivent pouvoir pénétrer dans les
macrophages de l'hôte ; ¡I est également souhaitable qu'ils soient peu toxiques et
qu'ils soient actifs à de faibles doses afin de permettre une thérapie à long t e r m e .
L'auteur explique les propriétés générales de la relation agent pathogène/hôte
lors des infections mycobactériennes, et examine les différentes options
thérapeutiques ainsi que les mécanismes sur lesquels repose le traitement.
L'évolution récente des techniques thérapeutiques antimycobactériennes est
illustrée par une description des nouvelles stratégies mises en œuvre pour
soigner ces infections.
Mots-clés
Antimycobactériens - Médicaments - Mycobactérie - Pathogénie - Traitement.
Tratamiento de infecciones micobacterianas
W . W . Barrow
Resumen
Debido a algunas de las propiedades de las micobacterias y de sus huéspedes, el
tratamiento de las infecciones micobacterianas difiere del de otras
enfermedades bacterianas. Una de las principales características de las
micobacterias radica en la compleja envoltura celular, rica en lípidos, que
protege al microorganismo tanto de la respuesta del huésped como de las
terapias antimicobacterianas. Esas bacterias son además
parásitos
intracelulares facultativos, que suelen provocar un tipo de enfermedad de
carácter más crónico. Esas propiedades dificultan considerablemente la
aplicación de una terapia eficaz. Para que resulten efectivos, los f á r m a c o s han de
ser capaces de penetrar también en los macrófagos del huésped, y es preferible
además que tengan poca toxicidad y resulten eficaces a dosis bajas, haciendo
posible de esta manera la aplicación de terapias prolongadas. El autor describe
las propiedades generales de la relación huésped-patógeno que se establece en
las infecciones micobacterianas, así como las alternativas terapéuticas
existentes y los mecanismos de acción de los tratamientos. Con la descripción de
nuevos métodos de tratamiento de estas infecciones, el autor ilustra las
posibilidades terapéuticas que trae consigo el progreso de la tecnología.
Palabras clave
Antimicobacterianos - Fármacos - Micobacterias - Patogenia - Tratamiento.
•
Rev. sci. tech. Off. int. Epiz., 20 (1)
65
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