New Approaches to Overcome Transport Related Drug Resistance in Trypanosomatid Parasites

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). F on ie s in Pha macology | www. on ie sin.o g 1Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 2 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 F on ie s in Pha macology | www. on ie sin.o g 2Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 3 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. F on ie s in Pha macology | www. on ie sin.o g 3Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 4 Ga cia-Salcedo e al. App oaches o O e come D ug Resis ance 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). F on ie s in Pha macology | www. on ie sin.o g 4Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 5 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. F on ie s in Pha macology | www. on ie sin.o g 5Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 6 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 F on ie s in Pha macology | www. on ie sin.o g 6Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 7 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 F on ie s in Pha macology | www. on ie sin.o g 7Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 8 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). F on ie s in Pha macology | www. on ie sin.o g 8Sep embe 2016 | Volume 7 | A icle 351 pha -07-00351 Sep embe 26, 2016 Time: 16:52 # 9 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. F on ie s in Pha macology | www. on ie sin.o g 9Sep embe 2016 | Volume 7 | A icle 351