pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 1
REVIEW
published: 28 Sep embe 2016
doi: 10.3389/ pha .2016.00351
Edi ed by:
Ha y P. De Koning,
Uni e si y o Glasgow, UK
Re iewed by:
And ei Ad ian Tica,
Uni e si y o Medicine and Pha macy
o C aio a, Romania
Cons an in Ion Mi cioiu,
Ca ol Da ila Uni e si y o Medicine
and Pha macy, Romania
*Co espondence:
José A. Ga cia-Salcedo
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
D ug Me abolism and T anspo ,
a sec ion o he jou nal
F on ie s in Pha macology
Recei ed: 24 July 2016
Accep ed: 16 Sep embe 2016
Published: 28 Sep embe 2016
Ci a ion:
Ga cia-Salcedo JA,
Unci i-B oce a JD, Val e de-Pozo J
and So iano M (2016) New
App oaches o O e come T anspo
Rela ed D ug Resis ance
in T ypanosoma id Pa asi es.
F on . Pha macol. 7:351.
doi: 10.3389/ pha .2016.00351
New App oaches o O e come
T anspo Rela ed D ug Resis ance
in T ypanosoma id Pa asi es
Jose A. Ga cia-Salcedo1,2*, Juan D. Unci i-B oce a1, Ja ie Val e de-Pozo1,2 and
Miguel So iano2,3
1Unidad de En e medades In ecciosas y Mic obiología, Ins i u o de In es igación Biosani a ia, ibs.GRANADA, Hospi ales
Uni e si a ios de G anada – Uni e sidad de G anada, G anada, Spain, 2Cen o de Genómica e In es igación Oncológica –
P ize /Uni e sidad de G anada/Jun a de Andalucía, G anada, Spain, 3Depa amen o de Ag onomía, Uni e sidad de Alme ía,
Alme ía, Spain
Leishmania and T ypanosoma a e membe s o he T ypanosoma idae amily ha cause
se e e human in ec ions such as leishmaniasis, Chagas disease, and sleeping sickness
a ec ing millions o people wo ldwide. Despi e e o s o e adica e hem, mig a ions a e
expanding hese in ec ions o de eloping coun ies. The e a e no accines a ailable and
cu en ea men s depend only on chemo he apy. D ug esis ance is a majo obs acle
o he ea men o hese diseases gi en ha exis ing d ugs a e old and limi ed, wi h
some ha ing se e e side e ec s. Mos esis ance mechanisms de eloped by hese
pa asi es a e ela ed wi h a dec eased up ake o inc eased e lux o he d ug due o
mu a ions o al e ed exp ession o memb ane anspo e s. Di e en new app oaches
ha e been elabo a ed ha can o e come hese mechanisms o esis ance including
he use o inhibi o s o e lux pumps and d ug ca ie s o bo h ac i e and passi e
a ge ing. He e we e iew new o mula ions ha ha e been success ully applied o
ci cum en esis ance ela ed o d ug anspo e s, opening al e na i e ways o sol e
d ug esis ance in p o ozoan pa asi ic diseases.
Keywo ds: ypanosoma id pa asi es, d ug anspo , su ace anspo e , e lux pumps, d ug esis ance,
nanoca ie s
INTRODUCTION
Pa asi ic p o ozoa a e esponsible o some o he mos p e alen diseases in humans, h ea ening
he li es o nea ly 1.250 million people a ound he wo ld (Lozano e al., 2012). Among hem,
ypanosoma ids a e esponsible o diseases such as human A ican ypanosomiasis, Chagas
disease and leishmaniasis (Table 1). Toge he , hese neglec ed diseases cause mo e han 52.000
a ali ies annually and he loss o 5 million disabili y-adjus ed li e yea s in de eloping coun ies
wo ldwide (Lozano e al., 2012;Ho ez e al., 2014).
Despi e hei high p e alence, he e is no e ec i e accine agains any o hese pa hogens and
hei ea men is based only on chemo he apy. Howe e , he ac ual numbe o e ec i e d ugs is
e y low, mos o hem ha ing high oxici y, a si ua ion ha has wo sened by he de elopmen o
esis ance o almos all o hem. E en he newes d ugs a e being comp omised by he eme gence
o esis ance in pa asi ic ypanosoma ids (Cojean e al., 2012;Zingales e al., 2015).
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
The ecen call o elimina ion and e adica ion o neglec ed
opical diseases equi es esea ch om mul iple on s, including
de eloping s a egies o he e icien deli e y o cu en
medicines and o o e come d ug esis ance.
THE DISEASES AND THEIR
PATHOGENESIS
Human A ican T ypanosomiasis
A ican ypanosomiasis is a ec o -bo ne disease ha h ea ens
millions o people li ing in impo e ished u al pa s o sub-
Saha an A ica, whe e hey a e a isk o in ec ion. I is caused by
he lagella ed p o ozoa T ypanosoma b ucei and ansmi ed by
se se lies o he genus Glossina. The subspecies T. b. gambiense
and T. b. hodesiense cause human A ican ypanosomiasis,
also called sleeping sickness (Table 1). In addi ion, he pa asi e
in ec s domes ic animals, causing nagana, a de as a ing disease
o li es ock in A ica. P obably, no o he disease excep mala ia,
HIV and ube culosis, has hinde ed he de elopmen o a
con inen as has ypanosomiasis in A ica. The disease p esen s
wo s ages, he ea ly s age o hemolympha ic phase and he la e
s age o neu ological phase which is cha ac e ized by in asion
o he cen al ne ous sys em. The Eas A ican a ian is a
ch onic disease which akes yea s o p og ess, while Wes A ican
ypanosomiasis is an acu e in ec ion in which he la e s age
de elops in a ew weeks o mon hs a e in ec ion (Welbu n e al.,
2016). Human A ican ypanosomiasis is a al i he e is no a
chemo he apy in e en ion (Ma hews, 2015).
Leishmaniasis
Leishmaniasis is a ec o -bo ne disease caused by di e en
species o he genus Leishmania, ( ypanosoma idae amily) and
ansmi ed by phlebo omine sand lies. Clinical mani es a ions
a y om local skin lesions and des uc i e mucosal
in lamma ion o dissemina ed isce al in ec ion (kala-aza ),
depending on in ec ing species and hos ac o s (Mu ay e al.,
2005;T ipa hi e al., 2007). Epidemiology and immunopa hology
a e also di e se wi h mul iple endemic egions in a eas o he
opics, sub opics, and sou he n Eu ope. A o al o 350 million
people a e conside ed a isk wi h an es ima ed 1.5–2 million
new cases pe yea , up o 500.000 o which a e isce al and
abou 1.5 million a e mucocu aneous. Visce al leishmaniasis
is a al i un ea ed, causing 20.000 o 30.000 dea hs annually
(Table 1), whe eas cu aneous leishmaniasis has a endency o
spon aneously sel -cu e (den Boe e al., 2011). All o ms o he
disease sha e h ee pa hogene ic ea u es: issue mac ophages a e
a ge ed cells and suppo in acellula pa asi e eplica ion; he
hos immunoin lamma o y esponse egula es he ou come o
disease; and he e is a pe sis en issue in ec ion (Mu ay e al.,
2005). Due o i s highe impac on global heal h we will ocus in
his e iew on isce al leishmaniasis and he Leishmania species
esponsible o he disease (L. dono ani and L. in an um).
Chagas Disease
One o he mos se ious heal h p oblems in he Ame ican
con inen is Chagas disease, caused by he in acellula pa asi e
T. c uzi (Table 1). In ec ion is ansmi ed by edu iid insec
ec o s, bu can also esul om e ical ansmission om
mo he o e us (Ca lie e al., 2015), by o al inges ion
o con amina ed ood o d ink (Noya e al., 2015), blood
ans usions (Young e al., 2007) and o gan ansplan s (Kun
e al., 2009).
Chagas disease is endemic o La in Ame ica whe e mo e han
6 million people a e in ec ed, and as a esul o mig a ion is an
eme ging disease in adi ionally non-endemic coun ies (Cou a
and Vinas, 2010;Gascon e al., 2010). While in ec ion can emain
asymp oma ic o many yea s, an es ima ed 30% o indi iduals
in ec ed wi h T. c uzi de elop po en ially a al ca diomyopa hy,
and gas oin es inal ac lesions (Nunes e al., 2013).
CURRENT TREATMENTS
Human A ican ypanosomiasis he apy elies only on ou
d ugs: pen amidine, su amin, mela sop ol and e lo ni hine, also
known as DFMO and ea men is dependen on he subspecies
and disease s age (Table 1) (Delespaux and de Koning, 2007;
Babokho e al., 2013). Pen amidine and su amin a e used o ea
ea ly s age ypanosomiasis, when he pa asi e is es ic ed o he
blood/lympha ic sys em. Pen amidine is he i s -line ea men
TABLE 1 | T ypanosoma id diseases.
Disease human A ican ypanosomiasis Visce al leishmaniasis Chagas disease
Causa i e agen s T ypanosoma b ucei gambiense
T ypanosoma b ucei hodesiense
Leishmania dono ani
Leishmania in an um
T ypanosoma c uzi
A eas o endemici y Wes and cen al A ica (T. b. gambiense) Eas
and sou he n A ica (T. b. hodesiense)
India, Bangladesh, Nepal, Sudan, E hiopia,
and B azil
Cen al and Sou h Ame ica
Dea hs pe annum ∼10 000 ∼30 000 ∼12 000
Cu en on -line he apies Ea ly s age: pen amidine and su amin
La e s age: mela sop ol, e lo ni hine
mono he apy a and NECT
(ni u imox-e lo ni hine combina ion he apy)
Ampho e icin B and lipid o mula ions
Mil e osine
Pen a alen an imonials
Pa omomycin
Acu e: ni u imox and benznidazole
Inde e mina e and ch onic s ages: no
s anda d ea men s
T anspo e s associa ed o
esis ance
Aquaglyce opo in 2 (pen amidine and
mela sop ol)
TbAAT6 (e lo ni hine)
ABC anspo e s (ni u imox)
ABC anspo e s: PRP1 (pen amidine and
an imonials)
Aquaglyce opo in 1 (an imonials)
LdMT (mil e osine)
ABC e lux pumps
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
o T. b. gambiense in ec ions and su amin co e s he ea men
o T. b. hodesiense ypanosomiasis. Nei he o hese compounds
c osses he blood-b ain ba ie , being useless o ea cen al
ne ous sys em in ec ions.
Mela sop ol and e lo ni hine a e used in he la e s age o
he disease, once he pa asi e has in aded he cen al ne ous
sys em. Mela sop ol was in oduced in he mid-20 h cen u y and
is cu en ly he only e ec i e d ug agains he la e s age o human
A ican ypanosomiasis caused by bo h subspecies. E lo ni hine
was he las d ug o be in oduced o ea human A ican
ypanosomiasis 50 yea s ago. The d ug c osses he blood-b ain
ba ie bu lacks e ec i eness agains T. b. hodesiense.
The ecen ly launched e lo ni hine-ni u imox combined
he apy is he sa es ea men o la e-s age ypanosomiasis
(P io o e al., 2009;Babokho e al., 2013). The combina ion
o e lo ni hine and ni u imox allows signi ican educ ion
o e lo ni hine dose and ea men du a ion. Howe e , like
e lo ni hine alone, his combined he apy is only e ec i e
in he second s age o T. b. gambiense in ec ions. This
lea es mela sop ol, an a senical de i a e ha causes eac i e
encephalopa hy in abou 10% o ea ed pa ien s, as he only
e ec i e d ug agains bo h T. b. hodesiense and T. b. gambiense
la e-s age in ec ions. Because o his, he inc easing a e o
mela sop ol ea men ailu es is ala ming.
In he case o isce al leishmaniasis, he comme cially
a ailable i s -line d ugs o he ea men o he disease
a e: pen a alen an imonials, ampho e icin B, mil e osine,
pen amidine and pa omomycin (Table 1) (Monge-Maillo and
Lopez-Velez, 2013;Elmahallawy and Agil, 2015). Like human
A ican ypanosomiasis, isce al leishmaniasis ea men
egimens a e based on Leishmania species and geog aphic
egion.
The e a e only wo d ugs now a ailable o Chagas disease,
ni u imox and benznidazole (Table 1). Bo h a e e y e ec i e
i gi en soon a e in ec ion. Howe e , ea men ailu es a e no
in equen (Munoz e al., 2013;Pin o e al., 2013) and bo h d ugs
cause ad e se e ec s (Jackson e al., 2010). Benznidazole is he
i s choice o Chagas disease ea men due o lowe side e ec s
han ni u imox.
MODE OF ACTION, SURFACE
TRANSPORTERS AND RESISTANCES
To exe i s ac ion, a d ug mus be able o in e ac inside he
cell wi h i s a ge /s a a concen a ion su icien o inhibi i s
biological ac i i y. The eme gence o esis ance is associa ed
wi h his p ocess. Pa asi es ha e e ol ed nume ous ways o
o e come he oxici y o d ugs. Mos o hem a e based on
educing he d ug concen a ion ha can access he a ge
o , al e na i ely, modi ying he a ge molecule. Thus, d ug
esis ance migh in ol e mu a ions causing he loss o hei
up ake sys em (Figu e 1). Once inside, d ugs may be modi ied
and inac i a ed, exc e ed o eloca ed in o acuoles. P od ugs
equi e ac i a ion o become e ec i e and supp ession o loss
o he ac i a ing p ocess may lead o esis ance. Ano he
mechanism o d ug esis ance is he al e a ion o d ug/ a ge
in e ac ion by inc easing he numbe a ge molecules o
hei modi ica ion. T ypanosoma ids employ hese s a egies o
de elop esis ance.
Su amin, a polysulphona ed naph hylamine-based com-
pound, is a highly nega i ely cha ged molecule ha accumula es
inside he ypanosome p obably bound o human se um
p o eins such as lipop o eins, albumin, globulins and ib inogen
(Vans e kenbu g e al., 1993). The mode o ac ion o his d ug
is no well es ablished and is a ibu ed o inhibi ion caused by
elec os a ic in e ac ions wi h essen ial p o eins, enzymes e c. The
manne in which he d ug en e s he cell was pos ula ed wo
decades ago o be ecep o -media ed endocy osis, bu i was only
ecen ly ha e idence in his ega d has been p o ided. Following
an RNAi app oach, se e al genes whose down- egula ion con e s
esis ance o su amin ha e been iden i ied (Als o d e al., 2012).
Su p isingly, mos o hese genes code o p o eins ha a e pa
o he endocy ic machine y and he endosomal compa men .
This inding has led ISG75 being p oposed as he majo su ace
ecep o o su amin up ake (Figu e 2). Al hough su amin was
he i s d ug used o ea ypanosomiasis ea ly las cen u y,
esis ance cases in he ield ha e no ye been epo ed.
Pen amidine is a diamine ha apidly accumula es inside
ypanosomes agains a concen a ion g adien up o millimola
concen a ions (Dampe and Pa on, 1976a,b;Be ge e al.,
1995). I is known ha pen amidine binds DNA, accumula es
in he mi ochond ia and collapses he mi ochond ial memb ane
po en ial bu i s p ecise mode o ac ion has no been ye
sa is ac o y esol ed.
Mela sop ol, an a senical de i a e, is uns able once in
ci cula ion in he human body and is apidly p ocessed o he
mo e s able o m mela sen oxide (Keise e al., 2000). Once
inside he pa asi e, mela sen oxide eac s wi h ypano hione, a
me aboli e exclusi e o ypanosomes which is esponsible o
de oxi ica ion o pe oxides and o he edox balance o he cell
o ming a oxic adduc known as Mel T.
I is well es ablished ha pen amidine and mela sop ol en e
he cell using he same anspo e . The adenosine/adenine
anspo e P2 was he i s desc ibed anspo e associa ed
wi h mela sop ol (Figu e 2) (Ca e and Fai lamb, 1993;Mase
e al., 1999) and pen amidine up ake (Ca e e al., 1995;De
Koning, 2001). Howe e , dele ion o he gene gene a ed a cell
wi h ma ginal esis ance o bo h d ugs (Ma o u e al., 2003).
Recen ly aquaglyce opo in 2, a membe o a amily o su ace
channel p o eins in ol ed in he anspo o wa e and small
non-cha ged solu es, has been iden i ied as he anspo e
esponsible o esis ance o high concen a ions o pen amidine
and mela sop ol (Als o d e al., 2012;Bake e al., 2012).
Al hough esis ance o pen amidine has no been o icially
epo ed, mela sop ol esis ance is a common issue. A ecen
s udy on T. b. gambiense demons a ed ha ea angemen s o
he aquaglyce opo in 2/3 locus accompanied by aquaglyce opo in
2gene loss also occu s in he ield, and ha T. b. gambiense
ca ying such mu a ions co ela es wi h a esis ance o
pen amidine and mela sop ol (G a e al., 2013). Pen amidine
ailu es ha e commonly been dismissed as misdiagnosis o
la e-s age sleeping sickness. Thus, we specula e ha esis ance o
pen amidine is likely p esen in he ield.
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FIGURE 1 | Common mechanisms o d ug esis ance.
Pen amidine has also been used as an al e na i e he apy
o an imonial- e ac o y pa ien s in isce al leishmaniasis. The
an ileishmanial mechanisms o pen amidine include inhibi ion
o polyamine biosyn hesis by inhibi ing a ginine and polyamine
up ake (Basselin e al., 2000), DNA mino g oo e binding
(Basselin e al., 1998), and inhibi ion o espi a o y chain complex
II (Meh a and Shaha, 2004). Thus, he main a ge o he d ug
seems o be he pa asi ic mi ochond ia.
Resis ance o pen amidine in Leishmania has been epo ed
bu he mechanisms a e no clea ly de ined. Pen amidine
esis ance p o ein 1 (PRP1), a membe o he ATP-binding
casse e (ABC) anspo e supe amily plays a ole in esis ance
o pen amidine and c oss- esis ance o i alen an imonials in
Leishmania when o e exp essed (Figu e 3) (Coelho e al., 2003,
2006, 2007, 2008). PRP1 is an in acellula p o ein possibly
associa ed wi h he ubulo esicula elemen ha is linked o he
exo- and endo-cy osis pa hway (McCon ille e al., 2002;Coelho
e al., 2006).
E lo ni hine, is an inhibi o o o ni hine deca boxylase, an
essen ial enzyme o he i s s ep o polyamine syn hesis and he
o ma ion o ypano hione (Bacchi e al., 1980). The d ug c osses
he blood-b ain ba ie and i s en y in o ypanosomes is also
anspo e -media ed. Se e al g oups ha e ecen ly iden i ied he
amino acid anspo e TbAAT6 as esponsible o e lo ni hine
up ake showing ha i s los is associa ed o d ug esis ance
(Figu e 2) (Vincen e al., 2010;Bake e al., 2011;Schumann
Bu ka d e al., 2011). Howe e , his mechanism o esis ance o
e lo ni hine s ill emains o be con i med in esis an pa asi es
om clinical isola es.
Pen a alen an imonials a e conside ed as p o-d ugs
which a e u he con e ed o i alen an imonials o
an imoni es, he ac i e o ms o he d ug. The mechanism
o ac ion seems o be mul i ac o ial, including inhibi ion o
mac omolecula biosyn hesis (Be man e al., 1985), likely by
a ge ing glycolysis and a y acid oxida ion (Be man e al., 1987).
DNA agmen a ion and ex e naliza ion o phospha idylse ine
on he ou e su ace o he plasma memb ane ha e been also
epo ed (Se eno e al., 2001;Lee e al., 2002;Sudhandi an and
Shaha, 2003). Howe e , he speci ic a ge s in hese pa hways
ha e no been iden i ied. Me abolism o hiol is essen ial in he
mechanism o ac ion o an imonials. T ypano hione is he majo
hiol in Leishmania. An imoni es exhibi in i o inhibi ion o
ypano hione educ ase, an enzyme esponsible o p o ec ion
o pa asi es om hos eac i e oxygen and ni ogen species
(Wyllie e al., 2004).
The ou e o en ance o an imonial d ugs in o Leishmania
emains elusi e. The su ace anspo e aquaglyce opo in 1
has been shown o acili a e i alen an imonial en ance, and
i s o e exp ession ende s Leishmania cells hype sensi i e o
an imonials (Gou bal e al., 2004).
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
FIGURE 2 | D ug anspo e s in T. b ucei.
FIGURE 3 | D ug anspo e s in Leishmania. K, kine oplas ; N, nucleus.
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
Mechanisms esponsible o an imonial acqui ed esis ance
in clinical isola es ha e been s udied o se e al decades, bu
he esul s a e puzzling. Clinical isola es a e cha ac e ized by
p esen ing high a iabili y in he mechanism as well as in
he deg ee o esis ance (Mukhe jee e al., 2007). Resis an
Leishmania cells o an imonials ha e been selec ed in i o and
some esis ance mechanisms ha e been sugges ed, including
educed accumula ion (Dey e al., 1994) and loss o educ ion o
he pen a alen me al (Shaked-Mishan e al., 2001).
ABC anspo e s use he hyd olysis o ATP o ansloca e
a a ie y o compounds ac oss biological memb anes. Some
ABC anspo e s play a majo ole in esis ance o umo s
o an icance d ugs (Fle che e al., 2016), and an ibio ic
esis ance in pa hogenic mic oo ganisms (Klein e al., 2011;
Lep ohon e al., 2011). P e ious s udies ha e epo ed he
ole o ABC anspo e s in Leishmania-acqui ed an imonial
esis ance (Figu e 3). Gene ampli ica ion o ABC pumps has
been associa ed wi h esis ance o an imonials in labo a o y-
de i ed and clinical isola e- esis an pa asi es (Fe ei a-Pin o
e al., 1996;Haimeu and Ouelle e, 1998;Haimeu e al.,
2000;Mukhe jee e al., 2007;Mandal e al., 2009;Manzano
e al., 2013, 2014;Pe ea e al., 2016). O e exp ession o ABC
anspo e s egula es he elimina ion o an imoni es h ough
wo di e en ou es: hei seques a ion in o he in acellula
acuola compa men o di ec e lux ac oss he plasma
memb ane. A di e en mechanism includes he modula ion
o mac ophage d ug anspo e s and me abolizing enzymes
ela ed o esis ance o an imonials. In ec ion wi h an an imoni e-
esis an Leishmania s ain up egula ed mul id ug esis ance-
associa ed p o ein 1 (MRP1) and pe meabili y glycop o ein
(P-glycop o ein) in hos cells, leading o he inhibi ion o
in acellula d ug accumula ion by educing an imoni e in lux
(Figu e 3) (Mukhe jee e al., 2007;Mandal e al., 2009).
An imonial esis ance is also associa ed wi h aquaglyce opo in 1
anspo e . Down- egula ion o aquaglyce opo in 1 is co ela ed
wi h lowe an imoni e up ake, leading o a dec ease in d ug
concen a ion wi hin he pa asi e (Figu e 3) (Ma quis e al.,
2005).
Ampho e icin B is a polyene d ug wi h selec i e ac i i y
agains ungi, Leishmania and T. c uzi due o i s highe a ini y
o e gos e ol, he p edominan s e ol in hese pa hogens o e
choles e ol, he p edominan s e ol in mammalian hos cells.
Ampho e icin B induces he o ma ion o small memb anous
po es ha al e memb ane pe meabili y owa d ca ions, wa e ,
and glucose molecules (Ramos e al., 1996).
Mechanisms o d ug esis ance o ampho e icin B de eloped
by Leishmania a e a ious and include: (i) a change in memb ane
luidi y; (ii) a dec ease in hiol and eac i e oxygen species
le els likely due o a hype ac i i y ypa edoxin cascade; and
(iii ) an inc ease in d ug e lux om he cell (Pu kai e al.,
2012). An RT-PCR analysis o clinical isola es o L. dono ani
has shown he up egula ion o se e al genes implica ed
in ypano hione biosyn hesis and he ypa edoxin cascade.
Fu he mo e, he mRNA le el o he ABC anspo e MDR1 was
ound o be ou - old highe in R-s ains which suppo ed he
obse a ions o inc eased d ug e lux (Figu e 3) (Pu kai e al.,
2012).
Mil e osine (hexadecylphosphocholine) is a memb ane-
ac i e alkyl phospholipid ha was o iginally de eloped as an
an icance d ug. Cu en ly, i is used as he i s -line ea men
o isce al leishmaniasis and was conside ed a majo s ep
o wa d in an ileishmanial he apy (Jha e al., 1999;Sunda
e al., 2006). The p ecise mode o ac ion emains la gely
unknown. Mil e osine educes he lipid con en and augmen s
he phospha idyle hanolamine con en in he pa asi e memb ane,
sugges ing a pa ial inhibi ion o phospha idyle hanolamine-
N-me hyl ans e ase which causes a delay in cell p oli e a ion
(Loiseau and Bo ies, 2006). Mil e osine also igge s an apop osis-
like cell dea h in Leishmania (Pa is e al., 2004;Alza e e al., 2008;
Khadem a an e al., 2011;Ma inho Fde e al., 2011) bu i is
unknown how his happens.
Mil e osine ac i i y is due o in acellula accumula ion o
he d ug, which is egula ed by wo anspo e s, he mil e osine
anspo e LdMT, a no el P- ype ATPase membe o he
aminophospholipid anslocase amily and i s be a subuni
LdRos3 (Figu e 3) (Pe ez-Vic o ia e al., 2003;Pe ez-Vic o ia
F.J. e al., 2006). Repo ed esis ances a e associa ed wi h a
se e ely diminished in acellula d ug concen a ion, due o
he o e exp ession o e lux pumps and, mainly, o he ailu e
o mil e osine anspo e s (Pe ez-Vic o ia J.M. e al., 2006).
Fu he mo e, low exp ession o he LdRos3 subuni is he cause
o na u al esis ance o mil e osine obse ed in Leishmania
(Sanchez-Cane e e al., 2009). Clinical esis ances ha e been
ecen ly isola ed and mos o hem a e associa ed wi h punc ual
mu a ions in he LdMT anspo e (Cojean e al., 2012).
Pa omomycin (aminosidine) is an aminoglycoside p o-
duced by S ep omyces imosus wi h bo h an ibac e ial and
an ileishmanial ac i i y (No, 2016). The d ug was licensed in 2006
and ield esis ance has no ye been epo ed.
Ni u imox and benznidazole a e ni ohe e ocyclic d ugs
ca ying a ni o g oup linked o an a oma ic ing ha unc ion
as p od ugs and mus unde go a educ i e ac i a ion wi hin he
pa asi e o ha e cy o oxic e ec s. This eac ion is ca alyzed
by NADH-dependen bac e ial-like ni o educ ase and
d ug-induced esis an pa asi es a ise as a consequence o
mu a ions in his enzyme (Wilkinson e al., 2008;Campos e al.,
2014). In addi ion, P-glycop o ein e lux and P-glycop o ein
ATPase ac i i y ha e been desc ibed in T. c uzi esis an o
ni ohe e ocycles, implica ing ABC e lux pumps in d ug
esis ance (Campos e al., 2013). Resis ance o ni ohe e ocycles
seems o be due o quali a i e di e ences in P-glycop o ein
unc ion (Mu a e al., 2001) and P-glycop o ein gene
o e exp ession (F anco e al., 2015;Zingales e al., 2015).
STRATEGIES TO SPECIFICALLY
OVERCOME TRANSPORT RELATED
DRUG RESISTANCE
E lux Pumps Inhibi ion
Among he di e en d ug esis ance mechanisms p e iously
desc ibed, hose based on dec easing he d ug concen a ion
a he a ge si e by d ug mo emen h ough he memb anes
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
appea o play an impo an ole in Leishmania and T. c uzi.
Hence, inhibi ion o he ac i i y o ABC anspo e s ep esen s
an in e es ing s a egy o con olling d ug esis ance.
A numbe o compounds ha e been iden i ied o inhibi
P-glycop o eins which a e as di e se as calcium channel blocke s,
calmodulin an agonis s, p o ein kinase inhibi o s, an ibio ics,
hyd ophobic pep ides, ho mone de i a i es, and la onoids.
Thei e ec i eness in e e sing acqui ed d ug esis ance in
Leishmania a ies g ea ly be ween species and cell cycle s ages
due o ma ked di e ences in he exp ession o hei a ge , ABC
anspo e s.
Ve apamil, a calcium channel blocke , has been employed
o e e se d ug esis ance o an imony associa ed wi h he
o e exp ession o se e al P-glycop o eins and MRP in
Leishmania and T. c uzi s ains ob ained in he labo a o y
(Neal e al., 1989;BoseDasgup a e al., 2008) and om clinical
isola es o Leishmania (Valia han e al., 2006). Ve apamil is
also able o o e come esis ance o pen amidine in Leishmania
(Basselin e al., 2002;Coelho e al., 2008) bu i s e ec i eness is
species dependen (Mukhe jee e al., 2006).
Calmodulin an agonis s ha e been shown o e e se
d ugs esis ance in pa asi es. Pheno hiazine de i a es such
as chlo p omazine inc eased sensi i i y o an imonials in
mul id ug esis an L. dono ani and L. majo s ains in i o
(el-On e al., 1986). These compounds ha e an ip o ozoal ac i i y
pe se (Pea son e al., 1984;We bo e z e al., 1992). Howe e ,
he s udy o pheno hiazine de i a es has been abandoned due
o he high a e o oxici y ounded in i s leading molecule
chlo p omazine.
S a ins, also known as HMG-CoA educ ase inhibi o s, a e
d ugs cu en ly used o lowe he le el o low-densi y lipop o ein
(LDL) in he blood p e en ing a he oscle osis and ca dio ascula
disease. Besides his lipid-lowe ing ac i i y, s a ins also inhibi
ABC anspo e s. Lo as a in, a membe o he s a in amily,
in combined he apy wi h he an i ungal d ug miconazole,
educed Leishmania cell g ow h and mac ophage in ec ion,
al hough he p oposed mechanism o ac ion by lo as a in was
h ough inhibi ion o s e ol biosyn hesis (Haughan e al., 1992).
Lo as a in has been shown o inhibi MRP1 and P-glycop o ein
in L. dono ani allowing an imony accumula ion and pa asi e
killing wi hin mac ophages (Mooke jee Basu e al., 2008).
Mo eo e , p e ea men wi h lo as a in in hams e s in ec ed wi h
an an imonial- esis an L. dono ani cell line e e sed acqui ed
esis ance o an imonials.
Fla onoids cons i u e a class o na u al inhibi o s o
P-glycop o ein and ela ed ABC anspo e s in Leishmania
(Pe ez-Vic o ia e al., 1999;Wong e al., 2007;Kennedy
e al., 2011). Que ce in, a la onoid ha egula es human
ABC anspo by educing P-glycop o ein syn hesis educed
mil e osine accumula ion in Leishmania (Cas anys-Munoz e al.,
2007). Syn he ic la onoid dimme s ha e been used o e e se
d ug esis ance o an imony and pen amidine in L. dono ani by
inc easing in acellula d ug accumula ion (Wong e al., 2007).
Sesqui e penes a e na u al p oduc s ha o e come mul id ug
esis ance in Leishmania including esis ance o mil e osine.
Sesqui e pene C3 ully sensi ized a mul id ug esis an
Leishmania by inc easing in acellula d ug concen a ion
h ough he modula ion o new ABC anspo e ac i i y
(Pe ez-Vic o ia e al., 2001).
D ug T anspo e s
A di e en s a egy o de ea d ug esis ance associa ed wi h
su ace anspo e s is he use o d ug deli e y sys ems.
Nanopa icles o biodeg adable and biocompa ible polyme s
a e widely used o his pu pose. Speci ic cell-su ace a ge ing
has been shown o be an e ec i e s a egy o o e come
d ug esis ance associa ed wi h he loss o unc ion o su ace
anspo e s esponsible o d ug up ake in T. b ucei. PEGyla ed
PLGA nanopa icles we e coupled o a single-domain hea y-
chain an ibody agmen (nanobody) ha speci ically ecognizes
he su ace o he pa asi e (Figu e 4) (S ijlemans e al., 2004).
Nanopa icles we e loaded wi h pen amidine, he i s -line d ug
o T. b. gambiense acu e in ec ion. An in i o and in i o
e ec i eness assay demons a ed ha he o mula ion was able
o signi ican ly educe he e ec i e d ug concen a ion (A ias
e al., 2015). Mo eo e , an imp o ed e sion o his nanoca ie
educed he cu a i e dose 100- old and mos signi ican ly,
ci cum en ed d ug esis ance as a esul o mu a ions in
aquaglyce opo in 2 (Unci i-B oce a e al., 2015), he su ace
channel p o ein ha media es pen amidine up ake in T. b ucei
(Bake e al., 2012). This s udy ep esen s a p oo -o -concep as
a s a egy o o e coming d ug esis ance linked o he loss o
unc ion o su ace anspo e s.
Cell-pene a ing pep ides, also known as pep ide ansduc ion
domains, memb ane ansloca ing sequences and T ojan
pep ides, a e sho pep ides wi h he abili y o en e almos any
cell. In he la e 1980s i was obse ed ha he ans-ac i a o
o ansc ip ion (TAT) o he HIV i us as well as a chemically
syn hesized e sion o TAT was able o en e cells (F ankel and
Pabo, 1988;G een and Loewens ein, 1988). Cell-pene a ing
pep ides a e highly ca ionic and usually ich in a ginine and lysine
amino acids. Thei excep ional p ope y o deli e ing in o he cell
a wide a ie y o ca goes such as p o eins, oligonucleo ides and
e en 200 nm liposomes has made hem a ac i e candida es o
anspo d ugs o he in e io o cells ( an den Be g and Dowdy,
2011). The ca go is associa ed wi h he pep ides ei he h ough
chemical linkage ia co alen bonds o h ough non-co alen
in e ac ions, and hey ge inside cells p ima ily ia endocy osis
bu also by di ec memb ane pene a ion.
Conjuga ion o mil e osine o he cell pene a ing pep ide
TAT has been applied as an e icien s a egy o de ea d ug
esis ance associa ed wi h lack o unc ion o he LdMT
su ace anspo e in Leishmania (Luque-O ega e al., 2012).
Func ionalized mil e osine ca ying a hiol g oup was conjuga ed
o TAT48−60 h ough ei he a disulphide o a hioe he bond.
D ug conjuga es we e able o en e and kill bo h p omas igo e
and in acellula amas igo e o ms o a Leishmania- esis an
s ain. In acellula elease o he d ug was no a equi emen
since he mos me abolically s able hioe he conjuga e e ained
signi ican leishmanicidal ac i i y compa ed o he less s able
disulphide conjuga e.
The s a egy was also applied o kill T. b ucei, a pa asi e
na u ally insensi i e o mil e osine due o he lack o he su ace
anslocase sys em esponsible o d ug up ake (Luque-O ega
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
FIGURE 4 | S a egy o o e come d ug esis ance associa ed o mu a ions in su ace anspo e in T. b ucei based on nanobody-coa ed
pen amidine loaded nanopa icle.
e al., 2012). This epo p o ides p oo -o -mechanism o he use
o cell-pene a ing pep ide conjuga es o bypass d ug esis ance
due o diminished in acellula d ug concen a ion in pa asi es,
and open he possibili y o ex end chemo he apy in o o he
pa asi es in insically de oid o memb ane ansloca ion sys ems.
STRATEGIES WITH POTENTIAL TO
OVERCOME RESISTANCE
Cyclodex ins a e cyclic oligosaccha ides, wi h a hyd ophilic
ou e su ace and a hyd ophobic in e io ca i y. Cyclodex ins
a e able o o m wa e -soluble inclusion complexes wi h many
poo ly soluble lipophilic compounds, and ha e been widely
used o enhance d ug solubili y and educe d ug oxici y
(Zhang and Ma, 2013). Mela sop ol, a highly oxic d ug o
ea ing he la e s age o human A ican ypanosomiasis,
has been success ully occluded wi hin wo di e en ypes
o cyclodex in: β-cyclodex in and andomly me hyla ed-
β-cyclodex in (Rodge s e al., 2011). These compounds e ained
ypanocidal ac i i ies in i o and cu ed he la e s age o he
disease in a mu ine model.
The en ance ou e o mela sop ol-cyclodex in complexes
is p obably di e en han ha o ee mela sop ol. En y o
he d ug wi h cyclodex in is likely ia endocy osis whe eas
ha o ee mela sop ol is ia he aquaglyce opo in 2 channel.
As men ioned be o e, esis ance o mela sop ol in clinical
isola es co ela es wi h mu a ions in aquaglyce opo in 2 su ace
anspo e . The e o e, al hough i has no been es ed, i is qui e
possible ha he mela sop ol-cyclodex in o mula ion could
ci cum en mela sop ol esis ance.
Biopha maceu ical issues a e mo e complex in he case o
in acellula pa asi es such as Leishmania o T. c uzi. The
design o d ug deli e y sys ems mus ake in o accoun aspec s
like passage h ough physical ba ie s and cell memb anes,
he mechanism o cellula up ake, s abili y, ac i i y and
kine ics o he d ug in he a ge cell en i onmen . The
pha macodynamic esponse o an ipa asi ic d ugs is go e ned
in in acellula pa asi es by he d ug concen a ion eached
inside hos cells a he han ha in he ci cula o y sys em
(Da e e al., 2007). In gene al, d ug deli e y sys ems inc eases
e icacy and educe side e ec s ela i e o ee d ug. I
has also been epo ed ha hese sys ems can inc ease
suscep ibili y o d ug in esis an pa asi es by inc easing d ug
concen a ion inside a ge ed hos cells, bu he e a e e y ew
speci ic s udies abou ci cum en ing d ug esis ance (Yasinzai
e al., 2013). Howe e , I has been desc ibed he use o
nanode ices o encapsula e al e na i e d ugs o ea men o
esis ance o a d ug. (Yasinzai e al., 2013;Gu ie ez e al.,
2016).
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Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance
Leishmania is a good model o be a ge ed by hese
nano o mula ions because i only pa asi izes mac ophages which
a e esponsible o he clea ance o blood pa icula e ma e ials
in i o. Liposomes, niosomes, nanoemulsions, nanodiscs,
ans e somes, solid lipid nanopa icles, polyme ic and me alic
mic o/nanopa icles and o he deli e y sys ems ha e been
ex ensi ely in es iga ed o he ea men o leishmaniasis (Da e
e al., 2007;Gup a e al., 2010;Yasinzai e al., 2013;Gu ie ez e al.,
2016). Liposomes a e he mos equen ly used d ug deli e y
sys ems o leishmaniasis ea men . I is desc ibed ha liposomes
deli e hei con en s in o he mac ophage cy oplasm by using
hei memb ane o he plasma memb ane o he cells, inc easing
he d ug concen a ion inside i . Di e en ypes o liposomes
ha e been de eloped o bo h passi e and ac i e a ge ing
o mac ophages. These o mula ions included con en ional
liposomes and a sonoliposomes (con aining a senolipids wi h
co alen ly linked a senic) o passi e a ge ing and suga -bea ing
liposomes (mannose, o neoglycop o ein); posi i ely cha ged
liposomes (con aining phospha idyl choline o s ea yl amine);
pep ide-g a ed liposomes (wi h mac ophage-ac i a ing pep ides
like u sin o he chemo ac ic pep ide -Me -Leu-Phe) and
immunoliposomes (IgG-coupled liposomes) o ac i e a ge ing
o mac ophages. A comme cial p epa a ion o Ampho e icin B
in liposomes (AmBisome) was app o ed o ea men o isce al
leishmaniasis as he i s choice o he ea men o esis ance
associa ed wi h an imonials (Ng e al., 2003). I has also been
shown ha liposomal mil e osine imp o ed he suscep ibili y o
mil e osine- esis an Leishmania p omas igo es (Papagianna os
e al., 2005). In i o in es iga ions on a sonoliposomes
e ealed ha hey we e ac i e agains ampho e icin B and
mil e osine- esis an L. dono ani a ela i ely low concen a ions
o a senolipids (An imisia is e al., 2003).
Va ious polyme ic nanopa icula e o micella sys ems o
polyalkylcyanoac yla e, polylac ic acid, poly (lac ic co-glycolic
acid), poly(ε-cap olac one), chi osan, albumin, gela in, lipid
and ino ganic nanopa icles (gold, sil e , i anium oxide,
e c) we e also es ed o he ea men o leishmaniasis
(Da e e al., 2007;Gup a e al., 2010;Pham e al., 2013;
Yasinzai e al., 2013;Gu ie ez e al., 2016). Some pa icula e
nanoca ie s can c oss biological ba ie s by endoci osis-like
mechanisms and could be app op ia e o a ge in acellula
pa asi es like Leishmania. Ul as uc u al s udies indica ed
ha nanopa icles we e concen a ed in he phagolysosome,
qui e close o Leishmania amas igo es, eleasing he d ug
in o he mac ophage cy oplasm (Fusai e al., 1997). PLGA
nanopa icles loaded wi h he an ipa asi ic d ug and og apholide
and s abilized he excipien i amin E-TPGS (D-a- ocophe yl
polye hylene glycol 1000 succina e) which is an inhibi o o
he P-glycop o ein e lux pump showed high e ec i i y agains
wild and mul id ug esis an L. dono ani s ains in i o
(Mondal e al., 2013). In his con ex , pipe olac am A loaded
hyd oxyp opyl-β-cyclodex in nanopa icles ha e also p o ed
e ec i e agains wild and mul i esis an L. dono ani in i o
(Bha acha ya e al., 2016). P e ious s udies showed ha
some me al and me al oxide nanopa icles ha e an imic obial
ac i i y by gene a ion o eac i e oxygen species. These
nanopa icles ha e been assayed agains d ug- esis an bac e ia
and Leishmania pa asi es (Allah e diye e al., 2011;Das
e al., 2013;Jebali and Kazemi, 2013). A e exposu e o
nanopa icles, he highes an ileishmanial ac i i y was obse ed
o Ag-nanopa icles, ollowed by Au, TiO2, ZnO, and MgO
nanopa icles. Bo h ul a iole and in a ed ligh inc eased he
an ileishmanial p ope ies o all nanopa icles. Un o una ely,
despi e hei signi ican po en ial in an imic obial applica ions,
he oxici y o me al oxide nanopa icles has es ic ed hei
use. Ne e heless, ecen s udies using c ys alline nanopa icles
o ZnCuO showed be e biocompa ibili y indica ing ha me al
oxide nanopa icles in esiden non- oxic o m ha e conside able
po en ial o an ibac e ial and an ipa asi ic applica ions in he
u u e (Nadhman e al., 2015).
T ans e some is a nanoca ie designed o d ug deli e y
ac oss he skin ba ie . T ans e some o mula ions bea ing
ampho e icin B ha e been es ed agains sensi i e and esis an
clinical isola es o L. dono ani and compa ed wi h he
con en ional liposomal o mula ion and ee ampho e icin B.
T ans e some o mula ions we e signi ican ly mo e ac i e han
con en ional liposomes and ee ampho e icin B sugges ing
imp o ed pene a ion and be e pa i ioning in skin laye s.
T ans e somes we e also able o pa ially e e se esis ance
o ampho e icin B. Ne e heless, po en ial u ili ies o hese
no el o mula ions as an al e na i e chemo he apeu ic app oach
o he ea men o esis an leishmaniasis necessi a e u he
in es iga ions (Singodia e al., 2010).
In he case o Chagas disease, he e is ew esea ch wo ks
published in he con ex o d ug deli e y sys ems and only
in some o hem esis ance is men ioned. The e a e wo
main challenges in Chagas disease he apy using d ug
ca ie s, he i s one is o each dissemina ed in acellula
pa asi es (in ca diac, skele al and smoo h muscle, glial cells,
e c.) a he apeu ic doses and he second is he selec i i y
o a ge cells since cu en d ugs a e oxic. Di e en
nano- o mula ions ha e been assayed, including liposomes,
polyalkylcyanoac yla e nanosphe es, poly(e hyleneglycol)-co-
poly(lac ic acid) nanopa icles, dend ime s, and o he lipid
o mula ions. In gene al, hese o mula ions had limi ed e icacy
ela i e o ee d ug wi h he only ad an age o educing
oxici y (Rome o and Mo illa, 2010;Salomon, 2012;Mo illa
and Rome o, 2015). Low endocy ic ac i i y o hos o ms o he
pa asi e ( ypomas igo e and amas igo e), a ge selec i i y and
he selec ed nanoca ie could be esponsible o his lack o
e ec i eness. Ne e heless imp o emen s in nanoca ie s design
like pH-sensi i e o mula ions, passi e and ac i e a ge ing,
p od ug dend ime s, ni ic oxide- eleasing nanopa icles, e c.,
could imp o e d ug e icacy (Rome o and Mo illa, 2010;Mo illa
and Rome o, 2015;Seab a e al., 2015).
CONCLUDING REMARKS
Diseases caused by ypanosoma ids a e a se ious heal h and
socioeconomic p oblem, in pa icula in de eloping coun ies.
The lack o e ec i e accines, old and unsa e d ugs, along wi h
he eme gence and sp ead o esis ance, has ecen ly led o he
launch o campaigns suppo ing he ba le agains hese diseases.
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