Chemotherapy Strategies for Treating Human Trypanosomiasis in Africa

This report provides a detailed analysis of chemotherapy strategies employed in the treatment of Human African Trypanosomiasis (HAT), also known as sleeping sickness, a disease prevalent in sub-Saharan Africa. It begins by highlighting the challenges in disease prevention and the reliance on chemotherapy due to antigenic variation of the parasite. The report then examines the pathogenesis of HAT, differentiating between the early and late stages of infection, and explaining how the parasite evades the host's immune response. A significant portion of the report is dedicated to discussing the chemotherapeutic drugs used, including eflornithine, pentamidine, suramin, melarsoprol, and the Nifurtimox-eflornithine combination (NECT), detailing their mechanisms of action at the molecular level. The report explores assays like DNA staining and flow cytometry, to understand how these drugs impact the trypanosomes. The report concludes by summarizing the current state of chemotherapy in HAT treatment, emphasizing the importance of understanding drug mechanisms for improving therapeutic outcomes, and suggesting future research directions.

Running Head: TREATMENT OF HUMAN TRYPANOSOMIASIS IN AFRICA BY
CHEMOTHERAPY STRATEGIES
Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Name of the Student:
Name of the University:
Author’s Note:

1Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Abstract
Chemotherapy continues to majorly impact the reduction of the disease burden caused by
trypanosomatids. But it should be noted that the mode of action of the anti-trypanosomal
drugs remains completely clear and is only partially characterised. There are five current
drugs that are used for the treatment of the Human African Trypanosomiasis (HAT), they are
eflornithine, pentamidine, suramin, melarsoprol and Nifurtimox-eflornithine combination
(NECT). The assays that were exploited to detect the mechanism of action of the current
drugs included DNA staining followed by visualising the results by microscopy as well as
quantitative image analysis, flow cytometry, TUNEL for monitoring the DNA replication.
Eflornithine is an ornithine decarboxylase inhibitor. Melarsoprol inhibits mitosis. Nifurtimox
caused reduction in the abundance of the mitochondrial protein. Pentamidine causes
progressive loss of the kinetoplast DNA as well as perturbs the membrane potential and
suramin inhibits the cytokinesis. Cytology based profiling aids in effective understanding of
the mechanism of action of the drugs.

2Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Table of Contents
Introduction...........................................................................................................................3
Background...........................................................................................................................3
Aims......................................................................................................................................4
Discussion.............................................................................................................................4
Pathogenesis of Human African Trypanosomiasis (HAT)................................................4
Chemotherapeutic drugs against Human African Trypanosomiasis (HAT).....................5
NECT: Nifurtimox–Eflornithine Combination Treatment................................................8
Mode of Action of the Chemotherapeutic drugs at the Molecular Level........................10
Perspective...........................................................................................................................16
Conclusion...........................................................................................................................16
Summary.............................................................................................................................17
References...........................................................................................................................18

3Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Introduction
Human Africa trypanosomiasis (HAT) also known as sleeping sickness is a centuries-old
disease that negatively impacted the economy as well as the physical suffering in the sub-
Saharan Africa. Statistical data states that approximately 50 million people are at risk of
acquiring the disease considering an area of 10 million square kilometres (Büscher et al.,
2017). The major concern is that there is no effective vaccination for the prevention of the
disease. The creation of vaccines targeting trypanosomes is also a challenge due to the
antigenic variation observed in the species (Keating et al., 2015). This is the reason how the
parasite can evade the immune responses. There is only availability of chemotherapies that
account for the anti-trypanosomiasis measures. This article enlightens on the various
chemotherapeutic drugs exploited for effective treatment of trypanosomiasis.
Human African trypanosomiasis (HAT) also called as sleeping sickness, causes infection
by the parasite Trypanosoma that is transmitted by the vector tsetse fly. The parasite has two
subspecies, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense; and it can
lead to fatal consequences if left untreated (Wangwe et al., 2019). Of late there is an observed
reduction in the number of cases that has been reported of patients affected with African
trypanosomiasis but the treatment is a challenge for the clinicians. The treatment of the CNS
stage disease is considered to be toxic hence, diagnostic staging for differentiating the early
stage from the late stage of the disease when the CNS is invaded is indeed crucial but is
challenging. Eflornithine is combined with nifurtimox drug and acts as the first-line treatment
for late-stage Trypanosoma brucei gambiense. New drugs are in pipeline for the treatment of
CNS human African trypanosomiasis, and is giving rise to the cautious optimism (Santos et
al., 2015).

4Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Background
Human Africa trypanosomiasis (HAT) is a disease that is caused by hemoflagellates
belonging of the subgroup of Trypanosoma brusei known as Trypanosoma brusei gambiense
and Trypanosoma brusei rhodesiense which are the Gambian and the Rhodesian form
respectively (Rassi et al., 2017). Trypanosoma brusei gambiense is the first subspecies
responsible for the disease and it accounts for almost of the 98% of the reported cases of
sleeping sickness and is also responsible for cause of chronic infection (Jones and Avery,
2015). Trypanosoma brusei rhodesiense is responsible for causing acute infection and
accounts for only 2% of the cases reported (Kennedy, 2019). The tsetse fly, Glossina sp. is
the vector and aids in transmission of these parasites between the domestic as well as the wild
animals and the human beings. The other modes of infection can be accidental infection in
the laboratory or mother to child infection (Stich, 2015).
Aims
The aim of this article is to understand the action of the chemotherapeutic drugs against
the human African trypanosomiasis at the molecular level and discover the most effective
chemotherapy for the treatment of the disease.
Discussion
Pathogenesis of Human African Trypanosomiasis (HAT)
There are two stages in the clinical course of Human African Trypanosomiasis (HAT),
they are the early stage or the first stage, at this stage the parasite is present in the peripheral
circulation of the infected individual and it has not invaded the central nervous system (CNS)
(World Health Organization, 2019). In the second stage also known as the late stage in which
the parasite invades the blood-brain barrier leading to infection of the central nervous system.
The early stage causes eliciting an immune response against the pathogen (Chappuis, 2018).

5Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
At this stage there are uninterrupted waves of the parasitaemia, which leads to survival of
some parasites that evading the immune response, subsequently. The trypanosomes exploit
antigenic variation to escape the immune response (Bottieau and Clerinx, 2019). In the
process of antigenic variation, the trypanosome switches the variable surface glycoprotein
(VSG) coat to a new VSG coat which is not recognized by the immune system of the host.
This is a continual action that exhausts the defence system of the host. The process of the
evasion also involves the endocytosis of VSG-antibody complexes that allows the complexes
to escape the process of detection by antibodies that are involved in the complement-
mediated killing (Fairlamb and Horn, 2018). The mechanism of antigenic variation ensures
sufficient time for the trypanosomes to spend in the host system promoting the proliferation
and the transmission to other host system through tsetse fly that serves as the vector for
transmission of these trypanosomes. This causes infection of host systems that been already
infected with the trypanosomes and recognises the VSGs (West, 2019). The symptoms of
early stage of the disease involves fever, headaches, joint pain and irritation of the skin at the
site of infection which is a rare case though. The most important symptom is the fever which
lasts for almost about a week and may recur in a span of days or a month. The waves of
parasitaemia and the counter responses of the immune system triggers the fever (Wangwe et
al., 2019) (Santos et al., 2015).
The later stage of the trypanosomal infection is also called as the meningo-encephalitic
stage, which involves parasite invading the blood–brain barrier, entering the CNS and settling
in cerebrospinal fluid (CSF) (Winkelmann and Raether, 2016). The second stage symptoms
of infection involve confusion and poor coordination, tremors, general motor weaknesses,
irritability as well as aggressive behaviour. The most significant symptom of second stage of
the infection is disruption of the natural circadian sleep or the wake rhythm of the body, this
results in irregular and fragmented patterns of sleeping (Docampo and Moreno, 2017). Hence

6Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
the name of the disease is ‘sleeping sicknesses. If the disease is left untreated or it is treated
inadequately, the HAT infections can result in death. The subspecies of Trypanosoma brusei
determines the acute or chronic nature of trypanosomiasis infection.
Chemotherapeutic drugs against Human African Trypanosomiasis (HAT)
The effective chemotherapeutic drugs exploited to treat trypanosomiasis in the early stage
includes Pentamidine which is a water soluble aromatic diamidine which has been used since
the early times of 1930s (Steverding and Rushworth, 2017). This drug is effective against
Trypanosoma brusei gambiense but it is comparatively less effective in Trypanosoma brusei
rhodesiense. The treatment in the early stage exploits the use of 7-10 intramuscular
injections. The African trypanosomes exploits the nucleoside transporter P2 that takes up the
drug pentamidine. There are two other transporters discovered of late that uptake pentamidine
to atleast 50% (Strauss et al., 2018). Pentamidine impacts the kinetoplast that is the
mitochondrial DNA but is ineffective on the nuclear DNA. It is also observed that
pentamidine is also considered as a reversible inhibitor of the S-adenosylmethionine
(AdoMet) decarboxylase, which is an enzyme in the polyamine biosynthetic pathway but it is
not the primary mechanism of action (Santos et al., 2015). But it should be noted that this
chemotherapeutic drug does not kill the trypanosomes and even post treatment with the drug
it causes them to persist in the bloodstream forms.
Berenil is a diminazene aceturate that was discovered by Hoechst is an aromatic diamidine
exploited for treatment of bovine trypanosomiasis. This chemotherapeutic drug is effective in
the early stages of Trypanosoma brusei gambiense and Trypanosoma brusei rhodesiense
(Wenzler et al., 2016). This drug can be used in combination with melarsoprol for the late-
stage disease. This drug also acts in the kinetoplast DNA and intervenes with RNA editing
and trans-splicing (Mazzeti et al., 2018). In comparison to pentamidine, berenil is more
effective and acts as a non-competitive inhibitor of S-adenosylmethionine decarboxylase. It

7Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
results in elevation of the putrescine and elevation of the spermidine content in the parasite. It
is taken up by the P2 nucleoside transporter like pentamidine. But the use of berenil in
comparison to the pentamidine is well tolerated in humans (Rathore et al., 2016).
Suramin is a polysulphonated naphthylamine based compound which is derived from
naphthylamine dyes like trypan red and trypan blue. It is observed that the effectivity of the
drug is comparatively more when administered in the patients intravenously via injection than
by oral administration (Lombardo and Batlle, 2018). It is not absorbed by the intestines when
administered orally. This chemotherapeutic drug has several side effects as well like renal
problems, anaemia, nausea and anaphylactic shock. It is effective against the bloodstream
forms of the parasite (Fairlamb and Patterson, 2018). This chemotherapeutic drug acts on the
glycosomal enzymes that is exploited in the glycolysis pathway. This drug has been proved to
be highly effective with no resistance on Trypanosoma brusei rhodesiense. There is no
resistance against suramin is majorly because it inhibits multiple enzymes as well as
numerous metabolic pathways (Singh et al., 2016).
Melarsoprol is an organic arsenical drug was extensively exploited until the usage of the
combination treatment of nifurtimox eflornithine that got introduced in the medical world in
2009 (Leroux and Krauth-Siegel, 2016). The major cause of the replacement of this drug was
due to the elevated levels of toxicity exhibited by this chemotherapeutic agent. It is
considered to be a potent trypanocide. This drug has been proved to be effective against
Trypanosoma brusei rhodesiense and it is evident based on the literature studies that 96% of
the diseases are cured after a year of treatment with this antibiotic (Njamnshi, Gettinby and
Kennedy, 2017). This chemotherapeutic drug intervenes with the process of redox
metabolism as well as the glycolysis in the trypanosomes and causes effective lysis of the
same.

8Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Alpha difluromethylornithine, DFMO also known as eflornithine is considered as the
newest of the anti-trypanosomiasis mono therapies. It is claimed to be effective in the first
and the second stage of the disease that has been caused by Trypanosoma brusei gambiense
but it is not effective for the treatment of the Trypanosoma brusei rhodesiense (Franco,
Scarone and Comini, 2018). It is also considered as the potential chemotherapeutic agent for
the treatment of cancer. It also inhibits the enzyme ornithine decarboxylase (ODC), that are
exploited for the synthesis of polyamines (Epting et al., 2017).
Table 1: Five most recent effective chemotherapeutic drugs against Human
African Trypanosomiasis (HAT)
Drugs Mechanism Advantages Disadvantages
Pentamidine
(Pentamidine
isethionate)
This drug
accumulates in the
trypanosomes and
disrupts the
mitochondrial
process.
This drug is effective
against the stage I
Trypanosoma brucei
gambiense
It is ineffective
against stage II
Trypanosoma brucei
gambiense and both
stages of
Trypanosoma brucei
rhodesiense
Suramin
(Bayer 205,
Germanin)
This enzyme binds to
the glycosomal
enzymes and
interferes with the
glycolysis.
This drug is effective in
the stage I infection by
Trypanosoma brucei
rhodesiense
It is ineffective
against both the
strains of
Trypanosoma brucei
in the stage II of the
disease.
Melarsoprol
(Mel B)
It intervenes and
impacts the
trypanosomal redox
metabolism and the
process of glycolysis.
This drug is effective
against both the
subspecies and in either
of the stages.
This drug is toxic and
causes post treatment
reactive
encephalopathy
(PTRE). The
resistance of
trypanosome is

9Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
considered to be high
of about 30%.
Eflornithine
(difluoromethy
lornithine)
It causes inhibition of
ODC, also disrupts
the proliferation and
vulnerability to the
oxidative attack.
This drug is effective
against stage II
Trypanosoma brucei
gambiense
The treatment with
this drug is time
consuming and is
ineffective against
both the stages of
Trypanosoma brucei
rhodesiense
NECT
(Nifurtimox-
eflornithine
combination)
Eflornithine inhibits
the ODC and
Nifurtimox induces
it.
This has a high cure rate
for both the stages of
Trypanosoma brucei
This combination
drug has a potential to
be resistant to the
treatment executed in
the field.
NECT: Nifurtimox–Eflornithine Combination Treatment
NECT, Nifurtimox–Eflornithine Combination Treatment is considered as the most recent
chemotherapy against Trypanosoma brusei gambiense infection. Literature studies have
already shown that Nifurtimox has a synergism level with melarsoprol and this combination
therapy has also shown to be more effective in the treatment of trypanosomiasis, with
minimal or almost negligible relapses in comparison to the nifurtimox and the melarsoprol
monotherapies (Baker and Welburn, 2018). A major concern about utilization of nifurtimox–
melarsoprol combination therapy is that it is safe. The safety of this combination drug therapy
might be a question since there are lack of more published research data. There had been a
comparative analysis of the combination therapy of nifurtimox-melarsoprol and nifurtimox-
eflornithine was executed in a randomized clinical trial. The results from the trial exhibited
that the combination therapy of nifurtimox–eflornithine combination therapy was far better in
comparison to the combination therapy of nifurtimox–melarsoprol with respect to both

10Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
effectiveness as well as safety (Wilkowsky, 2018). The report of this clinical trial paved way
for the future tests that involved a case series that happened in Uganda, which is a phase III
randomized clinical trial in Republic of Congo and most recently, a phase III non-inferiority
trial, that compared the NECT to the standard regimen of eflornithine monotherapy
(Steverding, 2015). All the three experimental trials that were executed were a part of a wider
neglected tropical diseases control programme that was initiated and funded by the Médecins
sans Frontieres. The experiments subsequently were followed by report that stated the
addition of the NECT to the list of important medicines of the World Health Organization for
the treatment of neglected tropical diseases (Franco et al., 2017).
It is to be noted that shortly post the inclusion of NECT, it was distributed amongst the
different NSSCPs in the countries endemic for Trypanosoma brusei gambiense. The
treatment regimen of the NECT involved daily three oral doses of the drug nifurtimox for ten
days totally and fourteen infusions of the drug eflornithine for total seven days. This was
comparatively as well as significantly lesser than the fifty-six doses in a span of fourteen days
that is required for the eflornithine monotherapy treatment (Franco et al., 2017). It was also
noted that the medical care giver could efficaciously and effectively utilize the combination
therapy who previously was trained in utilization of the eflornithine drugs. Lastly, the
reduced dosages of each of the drug meant that more drug quantity could be transported at
comparatively lesser cost, when compared to only eflornithine (Aksoy et al., 2017). Hence,
all these advantages cumulatively made NECT the frontline drug for treatment of the stage II
T. b. gambiense and even accounted for almost 59% of all the considered cases in the year
2010. But the main disadvantage of the treatment with NECT is that it is comparatively
labour-intensive and implementation logistically is complicated (Mesu et al., 2018). The
treatment regimen mentioned previously exploits at least four nurses to provide the
eflornithine infusions to patients. It should also be noted that doctor should firstly prescribe

11Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
the therapy and post administration observe the adverse reaction of the patient. The medical
personnel are required to have specific training in handling eflornithine is also a factor that
needs to be considered. The drug therapy has side effects like vomiting, nausea, headaches,
abdominal pain, joint pains, insomnia and seizures. But the side effects are comparatively
lesser comparison to the other drugs. Fortunately, the side effects are less severe than the
previous drugs (MacGregor et al., 2019).
Mode of Action of the Chemotherapeutic drugs at the Molecular Level
In the combination treatment of NECT, Nifurtimox is exploited for the treatment of the
sleeping sickness in the second stage and it causes the involvement of the central nervous
system. The combination therapy with melarsoprol was initially used but due to the toxicity
caused by the later in the system the combination is replaced by nifurtimox-eflornithine
combination therapy which is safer to use and comparatively easier than the usage of
eflornithine alone. Hence, NECT is considered as the first line treatment for the second stage
of African trypanosomiasis also known as sleeping sickness (Spaulding, Gallerstein and
Ferrins, 2019).
Nifurtimox and fexinidazole both are nitro pro-drugs that gets activated by the putative
ubiquinone nitro reductase (NTR) that is located in the mitochondria of the parasite. The
chemotherapeutic drug, Nifurtimox aids in formation of a metabolite that is a nitro-anion
radical in the body of the host that reacts with the nucleic acids of the pathogen and results in
breakdown of their DNA (Njamnshi, Gettinby and Kennedy, 2017). This drug is similar to
the nitrofuran agents which are antibacterial agents possessing a furan ring with a nitro group.
The nifurtimox undergoes a reduction reaction and aids in creating an oxygen radical like the
superoxide (Mesu et al., 2018). These radicals are toxic to the pathogen. It should be noted
that the mammalian cells are protected from the superoxide due to the presence of the
catalase, glutathione, superoxide dismutase and peroxidases. The accumulation of the

12Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
hydrogen peroxide to the cytotoxic levels may result in the death of the parasite. Further
studies are required to investigate whether these drugs kill the pathogens primarily by
disruption of the mitochondrial function or whether the toxic metabolites gains access to
targets that are outside the mitochondria (Spaulding, Gallerstein and Ferrins, 2019).
Nifurtimox is stated to be a pro-drug that gets activated by the mitochondrial nitroreductase
(NTR), but it is still unclear whether the toxic metabolites formed access targets outside the
mitochondrion, or whether the killing of the trypanosome is primarily due to the disruption of
the mitochondrial functions. Based on the literature study it can be stated that nifurtimox
drug, like pentamidine, also causes disruption of the MitoTracker signal. There were no
defects in the kinetoplast observed and the staining pattern of MitoTracker for nifurtimox was
distinct when compared with pentamidine. There was loss of the nuclear-encoded ATP-
synthase subunit on treatment with Nifurtimox but pentamidine-treated cells indicated that
there was still expression of the ATP-synthase, but it was not imported, when membrane
potential was disturbed (Mesu et al., 2018). Thus, it can be suggested that severe disruption
of the mitochondrial structure and function by the nifurtimox was consistent with the damage
to targets in the organelle where it was activated. The other nitro pro-drug fexinidazole,
currently is under clinical trials, is also activated by the mitochondrial nitroreductase (NTR).
These mode of action of this drug might also be through similar anti-trypanosomal
mechanisms (Njamnshi, Gettinby and Kennedy, 2017).
The mode of action of the anti-trypanosomal drugs are partially characterised. The
eflornithine drug is considered to be a specific inhibitor of the ornithine decarboxylase.
Melarsoprol inhibited mitosis and nifurtimox aided in reduction of the abundance of the
mitochondrial protein. Pentamidine drug triggered progressive loss of the kinetoplast DNA as
well as the disruption of the membrane potential of the mitochondria and suramin inhibits the
cytokinesis in the cell. Suramin enters the trypanosomes by the process of endocytosis that

13Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
involves a blood stream stage specific invariant surface glycoprotein ISG75, as well as the
variant surface glycoprotein (Mesu et al., 2018). The action of suramin is enhanced by the
import of ornithine via the amino acid transporter and the breakdown of ornithine by
ornithine decarboxylase enzyme in such a way that eflornithine is antagonistic. Under in vitro
condition the drug suramin causes inhibition of the enzyme pyruvate kinase and it binds to
the ADP/ATP binding sites in the other enzymes. Melarsoprol is an arsenic drug which enters
the trypanosomes through an adenosine transporter and an aquaglyceroporin, AQP2 and
forms a stable adduct called Mel T, with trypanothione antioxidant. Similarly, pentamidine
also enters the trypanosomes via the aquaglyceroporin, AQP2. The drug pentamidine
prevents the permeability of the glycerol of AQP2 and this step negatively impacts the
viability of the parasite. This DNA binding drug becomes immensely concentrated in the cell
and causes the trypanosome mitochondrial membrane to collapse. Pentamidine drug is
considered to remain as a low nanomolar anti-trypanosomal agent that acts against the
pathogens that lack the mitochondrial DNA. But this displays a resistance of 2.5-fold.
Melarsoprol drug forms a potentially toxic adduct with the trypanothione, which is an
unusual glutathione form that contains two glutathione molecules attached by a spermidine
linker. This trypanothione is observed in protozoan parasites like Leishmania and
Trypanosomes (Spaulding, Gallerstein and Ferrins, 2019). It is observed that melarsoprol
increases the proportion of the cells containing replicated but unsegregated nuclear genome
which indicates a defective mitosis phenomenon. The identification of the multiple putative
kinases in a loss-of-function screen for the resistance to melarsoprol indicates a role for
signalling cascade in the susceptibility of melarsoprol susceptibility. The putative kinases
proteins include mitogen-activated protein kinases MAPK, MAPKK and MAPKKKs and
based on the evidences it can be stated that there is less defect in the mitosis when the
putative MAPK and MAPKK are depleted. It can be considered that these kinase enzymes

14Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
might regulate the mitosis negatively as a part of the quality control post the DNA replication
(Mesu et al., 2018).
The chemotherapeutic drug eflornithine enters the trypanosomes via the amino acid
transporter AAT6 and causes inhibition of the ornithine decarboxylase. There is another drug
of recent known as acoziborole that targets the mRNA maturation factor called CPSF3. The
main aim of the anti-trypanosomal drugs is to selectively target the trypanosomes or reduce
the parasite viability in the cell culture or the animal model.
The profiling based on the cytology facilitates the efforts of the antibiotic discovery and a
cellular assays selection have also been applied previously to the anti-trypanosomal
compound; The cytology-based profiling for Trypanosoma brusei is reported to probe the
mode of action of all the five anti-trypanosomal drugs that are used in the patients. A panel of
assays are described that assess the cell cycle progression, the nuclear and mitochondrial
DNA content, the mitochondrial DNA replication, the nuclear DNA damage, mitochondrial
membrane potential, and the structure and function of the lysosome. By exploiting some
assays, it can be stated that each of the drug tested induces specific and distinct cellular
perturbations, yielding novel insight into the mode of action of the anti-trypanosomal drugs.
The cytostatic and cytocidal compounds that are identified exploiting phenotypic
approaches may target nucleic acids, proteins, membranes or other metabolites. These
compounds may also exhibit poly-pharmacology, which is defined as a process of killing
cells by multiple pathways. The compounds that are developed as target-based therapies may
destroy trypanosomes by the ‘off-target’ mechanisms (Spaulding, Gallerstein and Ferrins,
2019). The determination of the mechanism of action is still considered as a major challenge
for these compounds and drugs and also aid in an improved understanding of how these
compounds kill pathogens can guide new opportunities in terms of the development of more

15Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
potent compounds, delivering the compounds to their targets efficiently or by devising
combination therapies to minimise the occurrence of resistance. Eflornithine drug attacks the
trypanosomes by blocking enzyme ornithine decarboxylase but the mechanism of action of
the other anti-trypanosomal drugs are yet to be confirmed. Cytology-based profiling aids in
providing a rapid as well as effective means for yielding insight into the mode-of-action of
the drug (Kansiime et al., 2018).
Suramin causes production of cells with more than two nuclei which indicated defect in
the cytokinesis with continued mitosis. Based on the experimental evidences in the research
study it is stated that these multinucleated cells retained the membrane potential of the
organelle by getting stained with the MitoTracker dye. The terminal dUTP nick end labelling
(TUNEL) assay and the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI)-staining
executed in these multinucleated cells determine that the nuclear: nuclear replication and the
nuclear: kinetoplast replication still remained synchronised in the cells. But it should also be
noted that the TUNEL assay could not reveal any damage to the nuclear DNA that is induced
by the drug that was being exploited in the experiment. But TUNEL assay was considered to
be an excellent marker for the kinetoplast replication cycle especially the S-phase and
produced signals that were robust and consistent with presence of DNA-ends at the antipodal
sites on the kinetoplast during the minicircle DNA replication (Kansiime et al., 2018).
The chemotherapeutic agent pentamidine is considered a DNA-binding drug which causes
the trypanosomal mitochondrial membrane potential to collapse and also promotes the loss of
kinetoplast DNA. This drug accumulates in the mitochondria and prevents translation process
to occur. Additionally, metabolomic studies also indicate that the pentamidine is not likely to
act by inhibiting any of the specific metabolic pathways. It is to be noted that the loss of the
kinetoplast DNA through the pentamidine drug is progressive, that leads to a progressive loss
of the maxicircles and minicircles; the minicircles are present in multiple copies per

16Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
kinetoplast. This progressive loss of the kinetoplast DNA can be captured by the TUNEL
assay and the DAPI staining methods (Kansiime et al., 2018). The kinetoplast DNA loss is
expected to cause membrane-potential disruption because the A6-subunit of the ATP-
synthase, is required for maintaining the membrane potential, and it is encoded by the
kinetoplast DNA. Hence, it can be summed up that the primary target of pentamidine is
kinetoplast DNA which involves the inhibition of the action of mitochondrial type II
topoisomerase. But it is still unclear whether the loss of the mitochondrial membrane-
potential is solely due to the defect in the kinetoplast or whether it reflects an independent
response to the drug (Spaulding, Gallerstein and Ferrins, 2019).
The arsenicals that are themselves mutagens and are used for the treatment of leukaemia.
Evidence based studies show that arsenic causes damage in the DNA of the pathogens. The
mechanism of action of the pathogen involves the MAP kinase pathway activation and
similar incidence occurs in the mammalian cells (Patterson and Fairlamb, 2019). The mitotic
arrest occurs due to the induction of the mitotic spindle checkpoint. Though there has been
no evidence of the damage of nuclear DNA in the T. brucei following the treatment with
melarsoprol but there was an evident mitotic defect which was melarsoprol-dependant as well
as kinase-dependant (Bruhn et al., 2015). The mechanism of action of arsenic involves a
common kinase signalling cascade that leads to mitotic arrest in the trypanosomes as well as
the mammalian cells. The target protein similar to Myt1 kinase contains a guanylate cyclase
domain and a putative transmembrane domain. Thus, these research based journals throw
insight on how genetic as well as cytological approaches can converge in yielding knowledge
about the mechanism of action of the drug (Du et al., 2016).
The cytology based approaches are considered to be rapid and cost effective for the
quantitative profiling of the cellular responses to the drugs. The drugs may be also considered
as a chemical probe for exploring the parasite biology (Macedo et al., 2017). Based on the

17Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
systematic cytology-based approaches exploiting the anti-trypanosomal drugs of uncertain
mechanism of action, findings indicate the target organelles and structures for the drugs-
pentamidine, nifurtimox and melarsoprol; the mitochondrion, the kinetoplast and the nucleus,
respectively (Cullen and Mocerino, 2017). It is confirmed from primary assays that the
mitochondrial ATP-synthase is destructed by the nifurtimox and melarsoprol causes the
induction of the mitotic defect. The approach may be also applicable to other
trypanosomatids that are parasitic in nature like Trypanosoma cruzi and Leishmania. There
might be other approaches that should reveal those susceptible pathways that can be
prioritised and targeted by anti-trypanosomal therapies (Begolo and Clayton, 2016).
Perspective
The greatest opportunity in this field is that the mode of action of the chemotherapeutic
drugs are characterised partially. The anti-trypanosomal drugs has been administered since
ages but the mechanism of action of these drugs are still considered a challenge and there are
requirements to gain knowledge on how these drugs destroy the protozoan pathogen which
would aid in guiding the new opportunities for development of modern drugs to combat the
protozoan disease as well as to prevent the same. There are limited research on the usage of
the combination drugs which indeed have great potential to reduce the resistance of the
pathogen to the drugs. The future studies can investigate on the action of the drugs which
primarily kill the pathogen or the metabolites gain access to the targets that are located
outside the mitochondria.
Conclusion
Trypanosomiasis is a cause of epizootic problems and complex public health observed in
the developing countries of Africa. Though there has been considerable progress being made
the major challenges like paucity in resources and lack of agreement possess a threat in the

18Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
way to effective treatment of the disease. The five chemotherapeutic drugs that are discussed
in this article are the modern pharmaceutical compounds that combat the two stages of the
disease caused by the pathogen Trypanosoma brusei. NECT is a combination drug therapy
that can be exploited effectively for the treatment of the stage II of trypanosomiasis.
Unavailability of the vaccines is also a concern. Chemoprophylaxis is the only effective way
for treatment of the disease. There is further scope to research on the mode of actions of the
drugs.

19Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
Summary
African trypanosomes causes lethal and devastating diseases in the humans as well as
livestock. The pathogens are transmitted to the host body through vectors, the tsetse fly and
once inside the host body it circulates and grows in the tissue fluids as well as blood. There
are different drugs that are available for treatment of the disease but mechanism of action of
the drugs are not vastly known despite their use since old times (Thomas et al., 2018). The
exposure of the trypanosome to each of the drug discussed in this article revealed the cellular
structures, compartments and the growth phases that are affected. It was investigated that two
major DNA structures and the mitochondria were impacted post administration of the drugs
but the mode of action was completely different (Kansiime et al., 2018). There was another
chemotherapeutic drug that disrupted the growth of the cell at a distinct point of the growth
cycle. A drug which was arsenic-based, and related to the anti-leukaemia drugs, disturbed the
nuclear DNA division cycle, and his indicates that arsenicals may kill parasites as well as the
cancer cells by a similar mechanisms. Thus, ‘chemical-biology’ profiles of the drugs
illuminate distinct killing mechanisms against the trypanosome. Research studies can be
exploited for assessing the new drugs and insights may aid in the improvement of the anti-
parasite therapy (Spaulding, Gallerstein and Ferrins, 2019).

20Treatment of Human Trypanosomiasis in Africa by Chemotherapy Strategies
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