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New Approaches to the Development of Anti-Protozoan Drug Candidates:
a Review of Patents
Elaine F. F. da Cunha,* Teodorico C. Ramalho,* Daiana T. Mancini, Emanuella M. B. Fonseca and Aline A. Oliveira
Departamento de Química, Universidade Federal de Lavras, 37 2 0 0 - 0 0 0 Lavras- M G , Brazil
A s doenças causadas p or p rotozoários af etam h oj e em dia um a g rande p arcela da p op ulação m undial, p rov ocando m uitas m ortes e ex ercendo g rande inluência na q ualidade de v ida e no desenv olv im ento de m uitos p aíses. E ssas doenças af etam p rincip alm ente p aíses p ob res e p or isso, a p esq uisa e o desenv olv im ento de nov os f árm acos são neg lig enciados. D e f ato, a m aioria das drog as usadas no tratam ento dessas doenças data de décadas p assadas e ap resentam m uitas lim itações, incluindo o ap arecim ento da resistência às drog as. E ste artig o tem com o f oco os m ais recentes desenv olv im entos p ub licados no cam p o de p atentes, entre 2001-2008, com esp ecial atenção a p rom issores com p ostos atuando contra trip anossom íase, leish m aniose, m alária, tox op lasm ose, am eb íase, g iardíase, b alantidíase e p neum ocistose.
Protozoan inf ections are p arasitic diseases th at aff ect h undreds of m illions of p eop le w orldw ide,
b ut h av e b een larg ely neg lected f or drug dev elop m ent b ecause th ey af f ect p oor p eop le in p oor reg ions of th e w orld. M ost of th e current drug s used to treat th ese diseases are decades old and
h av e m any lim itations, including th e em erg ence of drug resistance. T h is rev iew w ill f ocus on th e m ost recent dev elop m ents, f rom 2001 to 2008, p ub lish ed in th e ield of p atents and p ub lications, p ay ing p articular attention to p rom ising com p ounds acting ag ainst try p anosom iasis, leish m aniasis,
m alaria, tox op lasm osis, am eb iasis, g iardiasis, b alantidiasis and p neum ocy stosis, th eir ch em istry and b iolog ical ev aluation, and to new ch em ical and p h arm aceutical p rocesses.
Keywords: neg lected disease, p otential antip rotozoan com p ounds, p atents
1 . I ntroduction
Neg lected p arasitic diseases are a g roup of trop ical inf ections w h ich are esp ecially endem ic in low -incom e p op ulations in dev elop ing reg ions of m any countries. D esp ite af f ecting m illions of p eop le around th e w orld, causing m any death s and h av ing a g reat and lim iting inf luence on th e q uality of lif e, th e selection of new
m olecular targ ets and th e dev elop m ent of m ore ef icient drug s ag ainst neg lected diseases are scarce ( T ab le 1) .
Now aday s, am ong th em , zoonotic diseases attract m uch attention. T h ose are deined as diseases sh ared b y anim als and h um ans. I n f act, w ildlif e serv es as a reserv oir f or m any diseases com m on to dom estic anim als and h um ans. G enerally , disease is m ore easily p rev ented th an treated. T h is discussion rev iew s com m on zoonotic diseases,
including th ose ailm ents th at are of ten erroneously cited as b eing closely link ed to w ildlif e.
Sev eral sp ecies of p rotozoans inf ect h um ans and inh ab it th e b ody as com m ensals or p arasites. Protozoa h av e traditionally b een div ided b ased on th eir m eans of locom otion, alth oug h th is ch aracter is no long er b eliev ed to rep resent g enuine relationsh ip s:2,3 (i) F lag ellates
( Z oom astig op h ora) : F lag ellates are ch aracterized b y h av ing one or m ore lag ella. Parasitic sp ecies g enerally
h av e m ore f lag ella th an th ose th at are f ree liv ing . Path og ens: G iardia intestinalis, T richomonas vag inalis,
T ry panosoma cruzi, Leishmania donovani; (ii) A m oeb ae
( actinop ods, rh izop oda) : A m oeb ae m ay b e div ided into sev eral m orp h olog ical categ ories b ased on th e f orm and structure of th e p seudop ods. I t can liv e in a num b er of p laces
w ith in th e h um an b ody , b ut m ost are f ound in th e intestine. T h ose in w h ich th e p seudop ods are sup p orted b y reg ular array s of m icrotub ules are called actinop ods, and f orm s in w h ich th ey are not, are called rh izop ods, f urth er div ided into lob ose, ilose, and reticulose am oeb ae. Path og ens:
an “ap ical com p lex ” of m icrotub ules at one end of th e cell th at m any p ref er to th e old nam e of sp orozoans. A ll th e m em b ers of th e p h y lum are p arasites. T h ey do not h av e a set b ody p lan lik e th e oth er p arasitic p rotozoans, alth oug h th ey are ch aracterized b y h av ing com p lex lif e-cy cles w ith an alternation of sex ual and asex ual g enerations. Th e
m ost w ell k now n of all th e sp orozoans are th e org anism s w h ich cause th e m alaria disease -P lasmodium f alciparum,
P lasmodium vivax, P lasmodium malariae and P lasmodium
ovale- and p neum ocy stosis; (iv) C iliates ( ciliop h ora) :
T h ese are larg er p rotozoans, g row ing to ov er 100 µm. T h ey
h av e h undreds of tiny cilia w h ich b eat in unison to p rop el it th roug h th e w ater. O f ten cilia are f used tog eth er in row s or tuf ts ( called cirri) and are used f or sp ecial f unctions
such as f ood g ath ering . I n addition to locom otion, th e Param ecium and oth er ciliates lik e th e Stentor use cilia to sw eep f ood dow n into th eir central ch annel or g ullet. T h ere is only one sp ecies of p ath og enic ciliate k now n to p arasitise h um ans: Balantidium coli; (v) M icrosp oridia: T h ey are so
dif icult to diag nose, v ery little w ork h as b een done into th eir im p ortance in h um an disease, alth oug h th ey are k now n to b e a m aj or cause of p roductiv ity loss in aq uaculture f acilities such as p raw n f arm s. M icrosp oridia h av e also b een im p licated in causing disease in im m unocom p romised h osts.
I n th is p ap er, w e f ocused on som e diseases, such as try p anosom iasis, leish m aniasis, tox op lasm osis, g iardiasis,
am oeb iasis, b alantidiasis and m alaria. I n th is contex t,
w e b eliev e th at a rev iew of p atents on th e h ig h lig hted inv entions and innov ativ e ideas in th is ield could b e of im p ortance f or scientists, m anag ers and decision-m ak ers in th e p h arm aceutical industry .
2 . T argeting New Anti-Protozoan Drug
Candidates
2 .1 . T reatment of A f rican try panosomiasis
A f rican try panosomiasis is a p arasitic disease of p eop le and anim als, caused b y p rotozoa of th e sp ecies
T ry panosoma b rucei g amb iense or T . b rucei rhodesiense
and transm itted b y th e tsetse ly b ite. T h e ly is inf ected w h en it b ites an inf ected h ost and th e try p anosom e dev elop s
in th e v ector, culm inating in inf ection transm ission b y its saliv a.
T h e ch em oth erap y of H um an A f rican try panosomiasis
( H A T ) is restricted to th e clinically ap p rov ed drugs: suram in (1) , p entam idine (2) , m elarsop rol (3) and elornith ine (4) .
Suram in and p entam idine are restricted to th e treatm ent
of early -stag e H A T p rior to central nerv ous sy stem ( C NS) inf ection, w h ile m elarsop rol is used once th e p arasites
h av e p enetrated th e C NS. E f lornitine is not ef f ectiv e ag ainst rh ondesiense inf ections. T h ese drug reg im ens can cause tox icity and p oor ef icacy . T h us, th e dev elop m ent of nov el com p ounds to p rov ide nov el treatm ent ag aint H A T is necessary .
T h e m aj or ly sosom al cy steine p roteinase of H A T is a candidate targ et f or nov el ch em oth erap y f or sleeping
sick ness. L y sosom al cy steine p roteases, g enerally know n as
th e cath ep sins, w ere discov ered in th e irst h alf of th e 20th century . Quib ell and W atts,4 -10 at th e A m ura T h erap eutic
L tda, describ ed a series of tetrah y drof uro [ 3 ,2-b] p y
rrol-3 -one analog ues as cath ep sin K inh ib itor.C ath ep sin K
ex h ib its th e h ig h est cap ab ility to deg rade com p onents of th e ex tracellular m atrix .11 C om p ounds of g eneral f orm ula
5 m ay b e conv eniently considered as a com b ination of
T ab le 1 . T h e m ain neg lected p arasitic diseases1
D isease O rg anism Scop e T h erap y needs
M alaria P lasmodium sp p . 5 00 m illion inf ections annually C ircum v enting drug resistance L eish m aniasis Leishmania sp p . 2 m illion inf ections annually Saf e, orally b ioav ailab le drug s,
esp ecially f or th e v isceral f orm of th e disease
R y p anosom iasis ( sleep ing sick ness, C h ag as’ disease)
T . b rucei ( sleep ing sick ness) ;
T . cruzi ( C h ag as’ disease)
H A T : 3 00,000 cases annually C h ag as: 16 m illion ex isting
inf ections
Saf e, orally b ioav ailab le drug s, esp ecially f or th e ch ronic p h ases
of disease
Sch istosom iasis Schistosoma sp p > 200 m illion ex isting inf ections Back up drug sh ould resistance to p raziq uantel arise
G iardiasis G iardia lamb lia; M illions of cases of diarrh ea annually
W ell-tolerated drug s
A m eb iasis E ntamoeb a histoly tica M illions of cases of diarrh ea annually
W ell-tolerated drug s
Pneum ocy stosis P neumocy stis carinii or
P neumocy stis j iroveci
- T rim eth op rim -sulf am eth ox azole of ten in conj unction w ith
th ree b uilding b lock s ( P1, P2, and P3 ) th at resp ectiv ely occup y th e S1, S2 and S3 b inding sites of th e p rotease. T h e com p ounds 6,9 710 and 88 ex h ib ited m ore th an 80%
inh ib ition at a concentration of 1 µm ol L-1.
C y steine p rotease inh ib itors w ere also describ ed b y M erck F rosst C anada L td. f or th e treatm ent of try p anosom iasis.12-16 C om p ounds such as 9 and 1 0 w ere
claim ed in th e p atent, h ow ev er no b iolog ical data were p resented. I n addition, Sm ith K line Beech am C orp oration describ ed a nov el sub stituted 3 ,7-diox o-azep an-4 -y lam ide,17
diazep ino[ 1,2-b] p h th alazine 1 1,18 ox o-am ino-sulf ony
l-azap anes 1 2,19 5 -sub stituted-6-ox o-[ 1,2] diazep ane, 1 3,20
th eir p h arm aceutical com p ositions, p rocesses f or their p rep aration and m eth ods of th eir use f or inh ib iting a p rotease, p articularly a serine p rotease and a cy steine p rotease such as cath ep sin K and f alcip ain, and f or th e
treatm ent of p arasitic inf ections including try p anosom iasis. F or instance, th e com p ound 1 2 is ob tained according to th e sch em e 1.
Based on th e 2 structure, X av ier, U niv ersity of L ouisiana,
disclosed b isb enzam idines and b isb enzam idox im es analog s as com p ounds ef f ectiv e ag ainst inf ection b y p arasitic h em of lag ellates, in p articular T ry panosoma b rucei g amb iense and T ry panosoma b rucei rhodesiense.21 I t
h as b een p ostulated th at arom atic b isb enzam idines are selectiv ely tak en up b y transp orters into try p anosom es, w h ere th ey b ind strong ly to D NA in th e nucleus and m itoch ondria, inh ib iting th e top oisom erases I I , th us th e
b e inh ib ited.22 I n m ice inf ected w ith T . b rucei, treatm ent
w ith th e com p ound 1 5 at a dose of 5 m g k g-1 per day resulted
in a m ean surv iv al in day s, of 18 ( ex cluding cured anim als) and 5 of th e 6 treated anim als w ere cured.
2 .2 . T reatment of A merican try panosomiasis
A merican T ry panosomiasis is a trop ical p arasitic disease caused b y th e lag ellate p rotozoan T ry panosoma cruzi. T h e
disease m ay also b e sp read th roug h an insect v ector, b lood transf usion and org an transp lantation, p arasite contam inated
f ood ing estion, and f rom a m oth er to h er f etus. Nif urtim ox (1 6) or b enznidazole (1 7) is used in th e acute C h ag as disease p h ase and in reactiv ation in im m unosup ressed p atients. A noth er irref utab le sig n ref ers to th e treatm ent of lab oratory accident v ictim s, w h ich dem ands a v ery early start f or th e adm inistration. T h e action m ech anism of 1 7
m ay dep end on th e f orm ation of nitro anion radicals, b ut p recise details are not k now n. L ittle is also k now n reg arding m ode of action of b enznidazole. Because of th e adv erse ev ents associated w ith th ese m edications, p atients b eing treated req uire caref ul m onitoring . I n a p atent ap p lication, R eed and co-w ork ers,23 ,24 at th e I nf ectious D isease R esearch
I nstitute, claim ed a f usion p rotein usef ul f or th e diag nosis of T ry panosoma cruzi inf ection w ith in a b iolog ical sam p le. M ore sp eciically , th e inv ention is related to th e use of
T . cruzi antig enic p oly p ep tides and f usion p oly p ep tides ( T cF and I T C -6) in m eth ods f or screening indiv idual and b lood sup p lies f or T . cruzi inf ection. A lso included are
details of a k it w h ich uses th e inv ention to detect T . cruzi
inv olv ing th e f usion p rotein. I t is a p rotein created th roug h
th e j oining of tw o or m ore g enes w h ich orig inally coded f or sep arate p roteins. T h e reactiv ity of sera f rom T . cruzi
-inf ected indiv iduals and control sera f rom non--inf ected
indiv iduals ag ainst th e f usion p oly p ep tides T cF and I T C -6 w as determ ined b y E L I SA . T h e data sh ow th at th e invention is cap ab le of recog nizing sera th at are neg ativ e or low w ith T cF .23 ,24
Quib ell and W atts, at A m ura T h erap eutics L im ited, describ ed nov el tetrah y drof uro[ 3 ,2-b] p y rrol-3 -one
com p ounds (1 8) , th eir salts, h y drates, com p lex es or
p rodrug s. A p rocess f or th eir p rep aration, com p ositions
com p rising th em and th eir use as inh ib itors of C A C 1 cy steine p roteases, p articularly cath ep sin K , in the
treatm ent of C h ag as disease w ere also describ ed.4 -10
C om p ound 1 8 ex h ib ited g ood p rim ary D M PK ( dy strop h ia m y otonica-p rotein k inase) p rop erties along w ith p rom ising
activ ity in an in vitro cell-b ased h um an osteoclast assay
of b one resorp tion. C om p ound 1 9 ex h ib ited > 80% inh ib ition at a concentration of 1 µm ol L-1. I n addition,
h ex ah y drof uro[ 3 ,2-b] p y ridine-3 -one, h ex ah y dro-2-ox
a-1,4 -diazap entalene and h ex ah y drop y rrolo[ 3 ,2-c] p y razole
w ere also sy nth etized and p h am acolog ically tested.4 -10
I n vitro activ ity w as ob serv ed ag ainst a rang e of C A C 1
cy steiny l p roteinases, h ow ev er tetrah y drof uro[ 3 ,2-b] p y rrol
analog s sh ow ed a b etter in vitro activ ity v alue.25
I n a f urth er p atent ap p lication w ith cath ep sin K inh ib itors ag ainst C h ag as disease or A m erican
try p anosom iasis, Sm ith K line Beech am C orp oration rep orted new p rom ising triox o-[ 1,2] th iazep any lam ide deriv ativ es.26 T h ese com p ounds are sub stituted
asy m m etrical im idodicarb onim ide diam ides deriv ed f rom h y drox y lam ines. T h ey include th e structure 2 0 as one of
sev eral com p ounds sp eciically claim ed, b ut b iolog ical data are not p resented in th is p atent.
2 .3. T reatment of g iardiasis
G iardiasis is a disease caused b y G iardia lamb lia and
only th e cy st f orm is inf ectious b y th e oral route. Peop le catch G iardia b y eating f ood or drink ing w ater w h ich h as b een contam inated b y th e org anism -usually f rom f eces. W h en th ere is a lot of G iardia p resent, th is g enerates
G iardiasis h as b een recently included in th e W H O Neg lected D iseases I nitiativ e.27 G iardia lamb lia inf ection
in h um ans is f req uently m isdiag nosed. M ultip le stool ex am inations are recom m ended, since th e cy sts and trop h ozoites are not consistently sh ed. G iv en th e dif icult nature of testing to ind th e inf ection, including m any f alse neg ativ es, som e p atients sh ould b e treated on th e b asis of em p irical ev idence; treating b ased on sy m p tom s. H um an inf ection is conv entionally treated w ith m etronidazole (2 1) ,
tinidazole (2 2) or nitazox anide (2 3) .28 Nitroim idazoles are
th e m ost ef f ectiv e drug s av ailab le and no drug w as rep orted to b e unsaf e, causing only m ild to m oderate and transient side ef f ects. Suk et al.29 rep orted th e desig n, sy nth esis,
and activ ity of a p otent and non-tox ic second g eneration anti-g iardial ag ent th at w as desig ned as an inh ib itor of cy st w all sy nth ase. I n th e f orm ation p rocess of th e G iardia
cy st w all, th e U D P-G alNA c is p oly m erized b y cy st w all sy nth ase into a p oly sacch aride, w h ich , in conj unction
w ith p oly p ep tides, f orm s th e ilam entous outer cy st w all of G iardia.3 0 T h e com p ound 2 4, a p h osp h onox in, w as th e
m ore p otent inh ib itor of G iardia cy st f orm ation and sh ould h av e clinical p otential as a new anti-g iardia drug .
C urrently ( 2001-2008) , th ere are no p atents of nov el anti-g iardiasis com p onds. H ow ev er, olig onucleotide m olecules f or detection of G . lamb lia w ere disclosed
b y M acq uarie R esearch L im ited.3 1 Pref erab ly , th e
olig onucleotide m olecule h y b ridises sp eciically uniq ue to 18S rD NA / rR NA seq uences of G . lamb lia under
m edium to h ig h string ency conditions. T h e sam p le can b e env ironm ental, f rom w ater sources, f rom w aste m aterials, f rom m edical and b ody luids.
2 .4 . T reatment of b alantidiasis
Balantidiasis is a disease caused b y th e larg e ciliated p rotozoan, Balantidium coli and tetracy cline or iodoq uinol
is usually used in th e treatm ent. H um an inf ection occurs w h en h um an contact w ith p ig s is p articularly close. I ng estion of cy sts or trop h ozoites f rom th e inf ected anim al or h um an f eces is th e inf ectious f orm .
Balantidiasis disease is treated w ith m etronidazole (2 1)
or tetraciclin (2 5) f or 5 -10 day s. I t is usually ef f ectiv e, b ut w ith out antib iotic m ortality lev els. I n th e treatm ent of acute b alantidiasis, tetracy cline is th e drug of ch oice to elim inate
B. coli trop h ozoites in h um ans. T h e disease is esp ecially
dang erous in im m unocom p rom ised p atients as p ulm onary p arench y m a inv olv em ent is p ossib le. B. coli is rare in
W estern E urop e and North A m erica and alternativ e drug s f or th e treatm ent of b alantidiasis m ay b e 5 -nitroim idazole drug s such as tinidazole (2 2) and ornidazole (2 6) .3 2 A s w ell
as in th e case of G . lamb lia, currently ( 2001-2008) , th ere are no new p atents f or anti-b alantidiasis drug s.
2 .5 . T reatment of ameob iasis
A m oeb iasis is an inf ection of th e intestine ( g ut) caused b y an am oeb a called E ntamoeb a histoly tica, th at are f ound
in contam inated f ood or drink . Virulent strains such as
E ntamoeb a coli and E ntamoeb a hartmanni, can p roduce
m ild diarrh ea and dy sentery . O nly a f ew cy sts are needed to cause inf ection. A m oeb ic cy sts resist iodine and ch lorine
if concentration of th ese ch em icals is too low .
D if f erent drug s are av ailab le to treat am eb iasis. O ral antip arasitic m edication is th e standard treatm ent.
F or asy m p tom atic inf ections, iodoq uinol (2 7) or
p arom om y cin (2 8) are drug s of ch oice. F or m ild, m oderate, or sev ere intestinal disease, and f or ex traintestinal inf ections
(e.g ., h ep atic ab scess) th e drug s of ch oice are m etronidazole (2 1) or tinidazole (2 2) , im m ediately f ollow ed b y treatm ent w ith iodoq uinol (2 7) , p arom om y cin (2 8) , or dilox anide
f uroate (2 9) . C en and L v , J iang su H ansoh Ph arm aceutical C o L im ited, describ ed nov el op tically p ure α-sub stituted
2-m eth y l-5 -nitroim idazole-1-eth anol deriv ativ es and th eir use f or th e treatm ent of am oeb iasis.3 3 I n an assay testing
resistance activ ity in rats, com p ound 3 0 sh ow ed E D5 0 v alue 7 m g k g-1 ag ainst A moeb a dy senteriae, w h ereas
m etronidazole sh ow ed 25 m g k g-1, and ornidazole sh ow ed
10 m g k g-1.
T h iosem icarb azones and th eir m etal com p lex es h av e b een ex tensiv ely studied during recent y ears due to th eir
w ide v ariety of b iolog ical activ ities.3 4 C ertain drug s ev en
rep orted th e sy nth esis of 5 -nitrof uran 2-carb ox aldeh y de th iosem icarb azones (3 1) and th eir sub seq uent b identate C uI I com p lex es (3 2) . T h ese com p ounds w ere screened
f or th eir anti-am oeb ic activ ities ag ainst H K - 9 strain of
E ntamoeb a histoly tica in in vitro ex p erim ents and f ound th at th e ch elation induced sig niicant ch ang es in th e b iolog ical activ ity of th e lig ands and som e cop p er sh ow ed b etter I C5 0 v alue th an m etronidazole in vitro.
2 .6 . T reatment of P lasmodium f alciparum inf ection
M alaria is a m osq uito-b orne disease caused b y sm all, one-celled p arasites called P lasmodium th at inf ect and
destroy red b lood cells. F our dif f erent p lasm odia can cause disease in h um ans: P lasmodium. f alciparum, P lasmodium malariae, P lasmodium ovale, and P lasmodium vivax.
Peop le g et m alaria f rom th e b ite of an inf ected m osq uito. A m osq uito can b ecom e inf ected w h en it b ites a p erson w h o h as m alaria org anism s in th e b lood.
T h e ef f ectiv eness of antim alarial drug s dif f ers w ith dif f erent sp ecies of th e p arasite and w ith dif f erent stag es
of th e p arasite’s lif e cy cle. D rug s include ch loroquine,
m ef loq uine, p rim aq uine, q uinine, p y rim eth am ine-sulf adox ine ( F ansidar) , and dox y cy cline. T w o asp ects h av e stim ulated new ef f orts reg arding th e dev elop m ent of ch em oth erap y , v accines and sanitary studies on m alaria: th e resistance to currently av ailab le antim alarial drug s and th e inef icacy of antim alarial v accines.3 5 C lark et al,3 6 at th e
Sm ith K line Beech am C orp oration, describ ed a p rocess ab le to p rep are com m ercial q uantities of b enzof uran-2-carb ox y lic
acid { (S) -3 -m eth y l-1-[ ( 4S,7R) -7-m eth y l-3 -ox o-1-( p y
ridine-2-sulf ony l) -azep an-4 -y l-carb am oy l] -b uty l} -am ide, 3 3, and analog s. T h e m eth od starting f rom th e cy cloaddition of 3 -ch loro-1-b utene w ith p otassium p h th alim ide in th e p resence of an alk ali m etal carb onate to f orm com p ound 3 6
N-( alf a-m eth y lally l) p h th alim ide. T h e com p ound 3 6 reacts w ith alk y ldiam ine f ollow ed b y azeotrop ic distillation w ith eth anol and g aseous H C l treatm ent to g iv e (R) -3 -am
ino-1-b utene h y droch loride, 3 7. 2-C h lorosulf ony l p y ridine is coup led w ith 3 7 in th e p resence of a th ialk y lam ine b ase to f orm th e p y ridine sulf onam ide 3 8. M itsunob u reactions of ( 2S,3R) -1,2-ep ox y -4 -p ent3 -ol lead to th e nitrog
en-p rotected een-p ox ide f rag m ent 4 0. T h e reaction of sulf onam ide f rag m ent 3 9 and ep ox ide f rag m ent 4 0 p rov ides 3 3. T h ere are tw elv e ox idation reaction step s to y ield th e inal p roduct ( Sch em e 2) . T h e com p ound 3 3 is th e R elacatib used f or treatm ent of m alaria.
Sm ith K line Beech am C orp oration h as also p atented new p rom ising dih y drotriazine deriv ativ es.3 7 T h ese
com p ounds deriv ed f rom ox o-am ino-sulf ony l-azap anes 4 1
also are cath ep sin K inh ib itors and usef ul f or th e treatm ent of P . f alciparum inf ection. E ach R2 is H , C
1-8alk eny l,
C2-8alk y ny l, or sub stituted C1-4alk y l. R3 is indep endently H ,
C1-8alk y l, C2-8alk eny l, C2-8alk y ny l or sub stituted C1-6alk y l. R4 is H , op tionally sub stituted C
1-7alk y l, C2-7alk eny l,
C2-7alk y ny l, C3 -7cy cloalk y l, or C3 -7cy cloalk eny l. R5 is
op tionally sub stituted C3 -7cy cloalk y l, C3 -7cy cloalk eny l,
ary l, h eterocy cloalk y l. A dditionally , Sm ith K line Beech am C orp claim ed nov el diox o-azep an (4 2)3 7 and am ino-azep an
(4 3)3 8-4 3 deriv ativ es and th eir salts, a p rocess f or th eir
p rep aration, com p ositions containing th em and th eir use in
m eth ods f or inh ib iting p roteases, p articularly cath ep sin K and f alcip ain.3 7-4 4 F or instance, th e sch em e f or p rep aration of
th e com p ound 4 2 w ith sub stituent C H2-cy cloh ex y l is sh ow n in Sch em e 3 . T h ose com p ounds w ere tested f or treating diseases ch aracterized b y b one loss such as osteop orosis, p eriodontitis and g ing iv itis, and diseases ch aracterized b y p arasitic inf ections p articularly m alaria, try p anosom iasis and leish m aniasis. I n th e p atent,4 5 th e C orp oration related
a m eth od of treatm ent of p arasitic diseases, esp ecially m alaria. T h e com p ound 4 4 ex h ib ited a K i v alue of 1.9 nm ol L-1 ag ainst th e f alcip ain cy steine p rotease.
U S A rm y M edical R esearch I nstitute of I nf ectious D iseases p atented nov el tricy clic com p ounds th at reduced side ef f ects and restore th e clinical ef icacy of antim alarial drug s, including m eloq uine (4 5) and ch loroq uine (4 6) .4 6
T h eir salts and p rodrug s, and th eir use as ch em osensitizing ag ents and f or m odulating resistance to a drug , p articularly an antim alarial drug is also claim ed. I C5 0 v alues w ere determ ined f or each candidate com p ound alone and in
com b ination w ith ch loroq uine. T h e f ractional inh ib itory concentration indices ( F I C , actual I C5 0 of one com p ound
in th e p resence of a second com p ound b ut is ex p ressed as a f raction of its I C5 0 w h en used alone) f or 1: 1 com b inations w ere determ ined using a ch loroq uine-resistant P . f alciparum
W 2 clone ( T ab le 2) . T h e com b ination of th e com p ounds
4 7 b and 4 7 e and ch loroq uine sh ow ed th e b est M D R ( m ultidrug resistance) -rev ersing activ ity . C h loroq uine w ith com p ounds containing saturared (4 7 i and 4 7 m) and
unsaturated sev en-m em b ered central ring s (4 7 q and 4 7 r) p ossessed sim ilar activ ity . T h e com p ounds 4 7 s and 4 7 t w ere
S cheme 3 .
not as p otent as th eir tricy clic ring counterp arts. A F I C index of less th an 1.0 rep resents sy nerg y or p otentiation and a F I C index g reater th an 1.0 rep resents antag onism ( Tab le 2) .
M ep h a Ph arm a A G studied th e adm inistration of th e com b ination of artesunate (4 8) and m eloq uine (3 0)
on h um ans w ith acute, uncom p licated P . f alciparum
m alaria. A rtem isinin and its deriv ativ es are th e m ost rap idly acting antim alarial drug s, h ow ev er th e duration of antim alarial activ ity is sh ort.4 7 T h us, in th ose studies, p atients
w ere random ized to receiv e according ly in: G roup A - artesunate 200 m g per day and m eloq uine 25 0 m g per day
sim ultaneously once daily f or 3 day s; G roup B - artesunate 200 m g per day and m eloq uine 75 0 m g ( no dose on th e 1st
day , 25 0 m g on th e 2nd day and 5 00 m g on th e 3rd day ) once
daily f or 3 day s. T h e cure rate af ter 14 day s and 28 day s w as m onitored. T h e results sh ow th at g roup A h ad a 100% cure rate and g roup B h ad a cure rate of 9 8% af ter 14 day s. A lso in g roup A , th e low incidence rate of early v omiting and ov erall v om iting w as ob serv ed.
class p rotein ( ScarB1) f or th e th erap y and/ or p rop h y lax is of a m alaria inf ection.4 8 ScarB1 ap p ears to m ediate H D L
-transf er and up tak e of ch olesterol. I t w as ob serv ed th at inh ib itors of ScarB1 f unction inh ib it th e g row th of p rotozoa in liv er cells, th us, inh ib itors of ScarB1 can b e used to treat inf ectious diseases inv olv ing liv er cells and b ecause th e ScarB1 is ex p ressed in ery th rocy tes, in h em atop oietic cells. H um an h ep atom a cells w ere treated and th e inluence
of th e com p ounds on p rolif eration and inf ection w ith
P lasmodium sp orozoites w as calculated as p ercentag e of
th e p late m ean f or all sam p les, w ith th e m ean set to 100% . I n each com p ound a score w as assig ned b etw een 0 and 4 f or inh ib ition of inf ection. T h e com p ounds 4 9, 5 0 and 5 1
sh ow ed score 4 ( corresp onding to an I C5 0 of 1 µm ol L-1
or low er) , 1 ( I C5 0 b etw een 3 and 4 µm ol L-1) and 0 ( I C 5 0
b etw een 4 µm ol L-1 or larg e) , resp ectiv ely .
F ansidar is a com b ination of sulf adox in (5 2) and p y rim eth am ine (5 3) used f or th e treatm ent of ch loroq uine (4 7) resistent P . f alciparum.4 9 C ouncil of Scientif ic
and I ndustrial R esearch p atented a k it of α,ß-arteeth er ( an artem isinin deriv ativ e) , f or increasing th e sites of action on th e p arasites and th us w ill b e m ore ef f ectiv e in controlling th em . T h e result sh ow s th at intram uscular α,ß -arteeth er ( 7.5 m g k g-1) and th e com b ination of sulf adox in
and p y rim eth am ine ( 5 m g k g-1) p roduced 100% curativ e
ef icacy com p ared to th e indiv idual com p ounds alone.4 9
T h e H olding C om p any F or Biolog ical Products A nd Vaccines dev elop ed a scorp ion v enom (P andinus
imperator) drug th at h as th e ab ility to stop th e dev elop m ent
of asex ual lif e cy cle f rom ring to sch izont stag e.5 0 D oses
of th e v enom stim ulated th e im m une sy stem p roducing sp eciic im m une resp onse ag ainst m alaria p arasite. Th e T ab le 2 . F I C ( f ractional inh ib itory concentration - 1: 1 com b ination of drug and ch loroq uine) indices of new m odulators in P . f alciparum W 2 clone
com p ound X n Y 1 R R2 F I C
4 7 a S 4 - C H3 C H3 0.23
4 7 b S 4 - C2H5 C2H5 0.23
4 7 c S 4 Py rrolidiny l - - 0.21
4 7 d S 4 Pip eridiny l - - 0.4 9
4 7 e S 4 M orp h oliny l - - 0.3 9
4 7 f S 4 4 -M eth y lp ip eraziny l - - 0.4 0
4 7 g C2H4 4 - C H3 C H3 0.23
4 7 h C2H4 4 - C2H5 C2H5 0.3 9
4 7 i C2H4 4 Py rrolidiny l - - 0.3 2
4 7 j C2H4 4 Pip eridiny l - - 0.4 5
4 7 k C2H4 4 M orp h oliny l - - 0.3 1
4 7 l C2H4 4 4 -M eth y lp ip eraziny l - - 0.5 2
4 7 m C2H4 4 - C2H5 C2H5 0.5 3
4 7 n C2H4 4 Py rrolidiny l - - 0.3 3
4 7 o 4 - C2H5 C2H5 0.4 8
4 7 p 4 Py rrolidiny l - - 0.4 5
4 7 q C2H4 5 - C2H5 C2H5 0.4 4
4 7 r C2H4 5 Py rrolidiny l - - 0.4 8
4 7 s C2H4 6 - C2H5 C2H5 0.4 8
v enom is f ree of side ef f ects and does not cause h istam ine release. C uriously , th e scorp ion v enom , is used as a carrier to deliv er radioactiv e iodine into tum our cells lef t b eh ind af ter surg ery , h as rem ov ed th e b ulk of th e tum our. T h e P . f alciparum ery th rocy tic stag e causes sev eral
m illion death s y early , p rim arily in A f rica. T h us, National I nstitutes of H ealth dev elop ed a nov el v accine f orm ulation com p rising a P . f alciparum ery th rocy te b inding p rotein, BA E BL .5 1 BA E BL p oly nucleotide is used to induce an
im m une resp onse to a P lasmodium p arasite, b ecause it h as h om olog y to E BA -175 , a P . f alciparum recep tor
th at b inds sp eciically to g ly cop h orin A on ery th rocy tes. L ik e E BA -175 , th e ery th rocy te recep tor f or BA E BL is destroy ed b y neuram inidase and try p sin, h ow ev er BA E BL can b ind to ery th ocy tes th at lack g ly cop h orin A and G erb ich ery th rocy tes ( p redent g ly cop h orin C and ab sent g ly cop h orin D ) b ind BA E BL m uch m ore w eak ly th an norm al ery th rocy tes. T h e com p ound of th is inv ention can
b e em p loy ed in adm ix ture w ith conv entional ex cip ients and can b e com b ined w ith oth er activ e ag ents, e.g ., v itam ins.
I n addition, J ensen et al.5 2 isolated VA R 4 , VA R 5 or VA R 6
p oly p ep tide ( f rom var g enes) and its nucleic acid f or use
as v accines f or th e diag nosis and treatm ent of m alaria.T h e p roteins are resp onsib le f or inducing th e im m unog lob ulin G ( I g G ) antib ody w ith sp eciicity to P . f alciparum ery th ocy te
m em b rase p rotein 1 ( Pf E M P1) on inf ected red b lood cells. T h us, sev ere m alaria is caused b y p arasites ex p ressing a sub set of Pf E M P1 m olecules and antib odies directed ag ainst th ese w ill b e resp onsib le f or th e p rotection ag ainst sev ere disease acq uired early in lif e.
Y et in v accine p rep aration, C laudianos et al.,5 3 at th e
I m p erial C olleg e I nnov ations L im ited, claim ed nov el secreted scav eng er recep tor ( Pf SR ) p roteins f rom th e
P . f alciparum g enom e datab ase, and th eir use in th e p rep aration of v accines ag ainst m alaria and in diag nosis. Pf SR p rotein w as sh ow n to h av e 64 % h om olog y to p roteins f ound in th e m ouse m alarial p arasites P . b erg hei ( Pb SR ) and
P . y oelii ( Py SR ) , w h ilst nucleic acid com p arisons sh ow ed 74 % h om olog y . I t contains L im ulus clotting f actor C , lip id b inding , cy steine rich scav eng er recep tor and p entrax in
dom ains. T h e p rotein causes disrup tion of th e norm al ex p ression of th e p roteins, so th e m alaria p arasite dies at an early stag e in its lif e cy cle. Parasite disrup tion via drug s
or v accines is th eref ore m ore lik ely to b e ef f ectiv e w h en p arasite num b ers are low , i.e., at th e early inf ectiv e stag es:
sp orozoites in h um an and ook inete in m osq uito.
W alter and E liza H all I nstitute of M edical R esearch dev elop ed a v accine f or inducing an im m une resp onse to
P . f alciparum using a g ly cosy lp h osp h atidy linositol ( G PI ) inositolg ly can dom ain or its deriv ativ e or eq uiv alent.5 4 G PI
h as b een identiied as a candidate tox in of p arasite orig in.
E x p erim ents using G PI in m ice w ith cereb ral m alaria sh ow ed relativ ely low p arasitem ia lev els b etw een day s 5 to 12 p ost-inf ection.
2 .7 . T reatment of Leishmaniasis
C utaneous leish m aniasis is caused b y Leishmania mex icana and L. b rasiliensis in th e A m ericas, and L. tropica
in th e O ld W orld; and b y v isceral leish m aniasis L. donovani, L. inf antum and L. chag asi. Sandf ly v ectors transm it cutaneous leish m aniasis. T reatm ent rem ains inadeq uate b ecause of drug tox icity ( sodium antim ony g luconate) , long courses req uired, and f req uent need f or h osp italization.
F or th e treatm ent of L eish m aniasis th e currently used drug s are lim ited to f our. T h e irst line com p ounds are th e tw o p entav alent antim onials, sodium stib og luconate and m eg lum ine antim oniate. T h ey w ere used f or th e irst tim e in 19 4 7 and 19 5 0, resp ectiv ely . F ailures and relap ses occur
in all f orm s of leish m aniasis and constitute ap p rox im ately 10-25 % of cases. I f th ese drug s are not ef f ectiv e, th e second
line com p ounds of p entam idine (2) and am p h otericin B
(5 4) are used, w h ich w ere introduced in 19 4 0 and 19 5 9 , resp ectiv ely .
F uertes and J im enez5 5 at th e M olog en H olding A G
D ana-F arb er C ancer I nstitute, claim ed to h av e discov ered a
new D NA -ex p ression construct f or treatm ent of inf ections w ith leish m aniasis and a corresp onding v accine. T h e D NA ex p ression constructs m ay code f or one or m ore Leishmania
antig ens, including th e p 3 6 L A C K antig en. p 3 6 A ntibodies w ere determ ined in m ice f ollow ing im m unization.
D uran et al.5 6 ( U niv ersidad C om p lutense de M adrid)
describ ed a nov el m eth od of ob taining w ater-disp ersib le alb um in m icrosp h eres containing am p h otericin B. T h e inv ention is b ased on th e atom isation of th e alb um in w ith th e drug th at is m icroencap sulated f or treatm ent of leish m aniasis and not req uiring th e p re-f orm ation of an em ulsion, av oiding th e use of oils and org anic solv ents. T h is com p osition is less tox ic, h as f ew er side ef f ects th an current am p h otericin B f orm ulations, such as F ung izone®,
artem isinin (5 6) or a deriv ativ e f or treating leish m aniasis is related b y T rop m ed G m b H .5 7-5 9 T h e inclusion of
p rocaine or lidocaine in th e f orm ulation reduces the p ain of inj ection. A rep resentativ e tab let com p osition
com p rises th e f ollow ing com p ounds: 3 5 0 m g of dim inazene diaceturate (5 5) , 25 0 m g of ch loroq uine (4 7)
and 4 00 m g of antip y rine (5 7) .
T h e I nstitut de R ech erch e p our le D ev elop p em ent rep orted th e use of niacin ( v itam in B3, 5 8 a, R = O H ) or
nicotinam ide (5 8 b, R = NH2) f or treating p arasitic diseases. Nicotinam ide is currently in trials as th erap y to p rev ent cancer recurrence and insulin-dep endent ( ty p e I ) diab etes. I n p articular, th ey can b e used to treat p rotozoan p arasitic diseases such as leish m aniasis in im m unodep ressed p atients.60 I t h as b een recently dem onstrated th at
nicotinam ide is a sub strate of SI R 2-lik e enzy m es in vitro.61
T h e data p resented th at nicotinam ide strong ly inh ibited th e
p rolif eration of b oth p rom astig otes and am astig otes w ith p rom astig ote f orm s sh ow ing less sensitiv ity to nicotinam ide
th an am stig otes. A lso th e p atent rep orted th e com p osition and adm inistration in com b ination w ith anoth er activ e ag ent selected f rom , e.g ., am p h otericin B, p arom om y cin, m elarsop ol and antim onials, using oral, intrav enous, top ical
or intralesional routes. I n addition, th e I nstitut describ ed in a p atent, th e nucleic acid constructs, w h ich com p rise nucleic acid encoding an im m unog enic p rotein f rom a p rom astig ote or am astig ote of Leishmania.62,63
A m ine-b orane com p ounds ex h ib it h ig h sim ilarity to org anic com p ounds m ainly due to th e atom ic radii and th e ch aracteristics of th e B–N b ond, w h ich resem b le th ose of carb on-carb on b onds. T h us, nov el f am ilies of am ine-b orane com p ounds, including luorinated am inoine-b oranes, nov el b is-am inob oranes and am inob oranes h av ing saturated and unsaturated alk y l ch ains are p rov ided b y
Soreq Nuclear R esearch C enter I srael A tom ic E nerg y
C om m ission and Y issum R esearch D ev elop m ent C o.64
Processes f or p rep aring , p h arm aceutical com p ositions and m eth ods utilizing th ese nov el com p ounds are also p rov ided. R adiolab eled am inob oranes and uses th ereof in radioim ag ing (e.g ., PE T ) and radioth erap y are f urth er p rov ided. T h ey are used f or th e treatm ent of leish m aniasis and dem onstrated ex cellent antim icrob ial activ ity with
reduced adv erse side ef f ects and im p rov ed p h arm acokinetic p roile. T h e in vitro anti-leish m anial ef f ect of com p ound 5 9
( dim eth y l-undecy l-am ine cy anob orane) w as determ ined on p rom astig otes and am astig otes. A ccording to T ab le 3 , th e m easured radioactiv ity , w h ich is indicativ e of th e p arasite, dim inish es rap idly w ith th e increased dosag e of am ine-b orane, indicating a strong anti-leish m anial ef f ect.
I n a f urth er p atent, Soreq Nuclear R esearch C enter
I srael A tom ic E nerg y C om m ission and Y issum R esearch D ev elop m ent C o. disclose m odiied p oly m er conj ug ates. T h e conj ug ate of a p oly m er (e.g ., p oly sacch aride) and a drug
(e.g . am p h otericin B, 5 4) reduced th e tox icity relativ e to th e unm odiied p arent com p ound, increased th e solub ility and retained sub stantially th e sam e deg ree of th erap eutic activ ity of th e unm odiied p arent com p ound.65 I t is an antip arasitic
com p osition ag ainst L. donovani and f orm ulated in th e f orm
of a nanop article, m icellar disp ersion and a lip osom e. T h e test w as p erf orm ed in dex tran-A m B im ine (6 0) conj ug ate w ith or w ith out eth anolam ine ( T ab le 4 ) . T h e result sh ow s th at th e conj ug ate h ad an I C5 0 v alue of 0.25 µg m L-1 com p ared to
th e D ex tran-A m B im ine alone ( 1.2 µg m L-1) .
Valtion T ek nillinen T utk im usk esk us rep orted th e use of b etulin (6 2) deriv ativ es or th eir salts ag ainst leish m aniasis T ab le 3 . C oncentration of th e am ine-b orane is g iv en in µg m L-1 versus th e counts per m inute ( cp m ) recorded f or th e p arasite sam p le
m g m L-1 cp m
0 869 9
3 .6 8003
11 789 8
3 3 5 3 7
T ab le 4 . I n vitro activ ity ag ainst Leishmania donovani, cy totox icily and h em oly sis of conj ug ates
C om p ound A ntip arasitic activ itya I C 5 0/ ( µg A m B m L-1)
T ox icityb I C 5 0/ ( µg A m B m L-1)
H em oly sisc/ ( µg A m B m L-1)
F ree A m B (5 4) 0.05 9 16
D ex tran-A m B am ine (6 0) 1.2 14 00 > 5 00
D ex tran-A m B im ine (6 1) 0.3 200 25 0
D ex tran-A m B– eth anolam ine im ine 0.25 4 00 > 5 00
aI C
5 0 v alues w ere deriv ed f rom th e activ ity test of A m B and dif f erent dex tran-A m B conj ug ates ag ainst L. donovani. bI C
5 0 v alues w ere deriv ed f rom cy tox icity test A m B and dif f erent dex tran-A m B canj ug ates ag ainst th e m urine R A WcH em oly sis w as ev aluated v isually af ter 1 h incub ation at 3 7 ºC 264 .7 cell line. w ith sh eep ery th rocy tes.65
in p h arm aceutical industry ap p lications.66 Betulin is th e
m ost ab undant triterp enoid of th e lup ane series and is f ound
in th e b ark of som e tree sp ecies, p articularly in b irch b ark (Betula) . I t is a com p onent of v arious f ood additiv es and cosm etic p roducts. T h e activ ity of th e b etulin deriv ativ es w as tested f or b oth L. donovani and L. tropica sp ecies
( T ab le 5 ) .
K em in Ph arm a E urop e p atented th e use of b icy clic carb oh y drates f or th e treatm ent of leish m aniasis.67
C om p ound 6 3 and 6 4, b icy clic carb oh y drates w ith
T ab le 5 . A ctiv ity in vivo of th e com p ounds tested f or L. donovani. I nitially , th e concentration of th e com p ounds w as 5 0 µm ol L-1. A m p h otericin B ( 1 µm ol L-1) w as included as p ositiv e control. Broth containing D M SO w as used as neg ativ e control
C om p ound Nam e I nh ib ition of L. donovani / ( % )
6 2 Betulin 3 5 .0
6 2 a 28-A cetate of b etulonic alcoh ol 4 0.6
6 2 b 28-M eth y lester of b etulonic acid 4 0.1
6 2 c Betulinic aldeh y de 65 .0
6 2 d Betulin 3 ,28-diox im e 72.4
6 2 e Betulin 28-ox im e 66.8
6 2 f Betulonic alcoh ol 4 4 .0
6 2 g Betulin 3 -acetox y ox im e-28-nitrile 66.4
6 2 h Betulin 28-acetic acid m eth y lester 9 5 .3
6 2 i 20,29 -H y drob etulonic acid 73 .4
6 2 j 2,3 -D ideh y dro-3 -deox y b etulin 13 .2
6 2 k Betulonic acid 9 7.6
6 2 l Betulinic acid 3 9 .8
6 2 m 28-A sp artateam ide dim eth y lester of b etulonic acid 69 .3
6 2 n Betulonic aldeh y de 4 6.2
6 2 o Betulin 28-N-acety lanth ranilic acid ester 5 9 .2
6 2 p b etulin 28-ch ry santh em ate 13 .4
6 2 q Betulin 28-carb ox y m eth ox y m enth olester 16.6
p ositiv e control: am p h otericin B ( 1 µm ol L-1) 5 5 .4
neg ativ e control: b roth + D M SO 0.0
h alog en containing ary l g roup s, p ossessed sig nif icant activ ity ag ainst L. donovani, w ith I C5 0 v alues of 1.01 and < 0.9 8 µm ol L-1, resp ectiv ely , com p ared w ith
0.4 7 µm ol L-1 f or m iltef osine 6 5. T h e use of 6 5 in
leish m aniasis th erap y sh ould b e caref ully considered.68
ory zalin 6 669 p ref erab ly less cy totox ic to norm al cells th an
ory zalin.70 T ub ulin, a critical euk ary otic p rotein resp onsib le
f or f orm ation of th e m itotic sp indle h as b een im p licated as th e targ et of dinitroaniline analog s. C om p ounds 6 7 and 6 8
are sig niicantly m ore p otent th an com p ound 6 6 in b lock ing
leish m anial tub ulin assem b ly ( T ab le 6) . T h e com p ound
6 8 h ad Kd ( dissociation constant) v alues of 5 7 µm ol L-1
leish m anial tub ulin, and th e corresp onding v alue f or ory zalin w ere 170 µm ol L-1. T h ese data are consistent w ith
th e h y p oth esis th at tub ulin is th e targ et of th e dinitroanilines in Leishmania.
I n 2006, D e Souza and C h eq uer Bou-H ab ib71 related
a p rop h y lax is v accine f or b lock ing transm ission of
Leishmania inf ections. I t is b ased on L. donovani sap onin
and p rom astig otes or am astig otes f ractions called Fucose
M annose lig and ( F M L , an isolated g ly cop rotein com p lex ) .
L eish m une® v accine ( F M L v accine) dev elop ed 9 2-9 5 % p rotection ag ainst canine v isceral leish m aniasis and sh ow ed
im m unoth erap y p otential. I n 2008, due to h ig h p roduction cost of L eish m une®, G azzinelli and co-w ork ers72 dev elop ed
a v accine f orm ulation com p rising am astig ote A 2 antig en.
T h e antib odies p roduced b y th e dog s in th e v accination p rocess w ith th is antig en are non-reactiv e to serolog ic diag nostic inf ection tests. T h e f orm ulation is com posed of
5 0 to 200.00 m g m L-1 of recom b inant A 2-H I S ( rA 2) p rotein
of Leishmania, p roduced in E . coli, 0.125 to 0.5 00 m g m L-1
of Sap onin, 1.00 m L of q .s.p b uf f ered saline solution and 0.01 m L of th im erosal. T h eref ore, th e m ain innov ation of th is v accine f orm ulation is th e p roduction of antib odies sp eciic ag ainst th e v accine antig en. T h e m ain innov ation of th is f orm ulation is th at th e anim als v accinated p resent serolog ic reaction ag ainst th e antig en, b ut rem ain seroneg ativ e in th e reaction w ith th e total p arasite ex tract, e.g ., th e antib odies
do not react w ith th e ex tract of th e p rom astig ote form s of
Leishmania in th e E L I SA tests, f or ex am p le.
R osalind F rank lin U niv ersity of M edicine & Science disclosed th e use of a liv e m utant Leishmania as a suicidal
v accine.73 T h e m utant can b e selected f rom natural
Leishmania sp ecies or constructed b y g enetic eng ineering .
T h e inv ention also ex p loits th e v irtual ab sence of h em e b iosy nth esis p ath w ay in try p anosom es to identif y or construct th e m utant p orp h y ric Leishmania as suicidal
v accines. T h e v accinated h am sters sh ow ed ca.10 f old
reduction of p arasite loads th an th e control g roup .
2 .8 . T reatment of pneumocy stosis
Pneum ocy stosis is a p neum onia th at is ch aracterized b y th e accum ulation of v ery larg e num b ers of a eucary otic sing le-celled org anism called P neumocy stis carinii, w h ich
h as not b een cultured. Pneum ocy stosis also occurs in m any
oth er m am m alian sp ecies. I t is not y et estab lish ed w h eth er
P neumocy stis carinii is a f ung us or a p rotozoan.
P neuocy stis carinii causes P . carinii pneumonia
( PC P) in p eop le w ith dep ressed im m une sy stem s such as A I D S p atients, p atients underg oing ch em oth erap y , or transp lant p atients b eing treated w ith im m unosup p ressants.
T h e inf ection is treated w ith p entam idine (2) and th e
com b ination of trim eth op rim (7 7) and sulf am eth ox azole, h ow ev er th ere are side ef f ects and th e m ortality rate rem ains h ig h . A n alternativ e treatm ent is th e p rep aration containing m eth ioninase in com b ination w ith an antib iotic,
eith er p entam idine (2) or com b ination of trim eth op rim (7 7) and sulf am eth ox azole related b y A ntiC ancer I nc.74
I nf ection b y P . carinii can b e treated b y adm inistering m eth ioninase op tionally in com b ination w ith additional th erap eutic ag ents, such as antib iotics, eith er p entam idine or com b ination of trim eth op rim and sulf am eth ox azole. T h e use of m eth ioninase dep letes p lasm a lev els of m eth ionine and dep riv es th e p arasites of S-adenosy lm eth ionine.
I n a f urth er p atent ap p lication and dev elop m ent, th e p rep aration of ten carb am ate and carb onate p rodrug s of T ab le 6 . A ctiv ity in vivo of O ry zalin and new dinitroniline com p ounds
ag ainst L. donovani and leishimanial tub ulin in vitro
C om p ound I C5 0 vs.
L. donovani
p rom astig otes / ( µm ol L-1)
I C5 0 vs.
L. donovani
am astig otes / ( µm ol L-1)
I nh ib ition % of
leishimanial tub ulin assem b ly
at 20 µm ol L-1 com p ound
4 4 .1 72.5 5 4
17.8 20.1 89
b isam idinop h eny l f urans w as p atented b y T h e U niv ersity of North C arolina at C h ap el H ill.75 C om p ounds w ere tested
ag ainst P . carinii inf ected im m unosup p ressed rats. T h e m ore p otent com p ound, 6 9, inh ib ited cy st f orm ation in th e lung b y 100 and 9 7.9 % inh ib ition at doses of 22 and 3 3 µm ol k g-1 per day , resp ectiv ely .
A series of p rim aq uine-deriv ed im idazolidin-4 -ones w ere screened f or th eir in vitro activ ity ag ainst P . carinii b y
Vale et al.76 O ne of th e tested im idazolidin-4 -ones, 7 0, w as
slig h tly m ore activ e ( I C5 0 = 1.9 µm ol L-1) th an th e p arent
p rim aq uine ( I C5 0 = 2.5 µm ol L-1) .
D ih y drof olate reductase ( D H F R ) is a k ey enzy m e in th e treatm ent of Pneum ocy stosis. I ts role in th y m idine
b iosy nth esis ( F ig ure 1) is th e reduction of 7,8-dihy drof olate
( D H F ) to 5 ,6,7,8-tetrah y drof olate ( T H F ) using th e cof actor nicotinam ide adenine p h osp h ate ( NA D PH ) . A f ter reduction, serine h y drox y m eth y ltransf erase ( SH M T ) cataly ses the reg eneration of 5 ,10-m eth y lenetetrah y drof olate; and deox y uridine m onop h osp h ate ( dU M P) is m eth y lated to g iv e deox y th y m idy late ( dT M P) in a reaction cataly sed b y th y m idy late sy nth ase ( T S) . T h is reaction conv erts m eth y lene-tetrah y drof olate b ack to dih y drof olate, com p leting th e cy cle. T h eref ore, th e inh ib ition of
D H F R p rev ents th e b iosy nth esis of th y m idine and, as a conseq uence, D NA b iosy nth esis.77 Nov el sub stituted
2,4 -diam ino-5 -b enzy lp y rim idine tested as inh ib itors of
P . carinii D H F R , w as claim ed b y F orsch et al.78 I n sum m ary
( T ab le 7) , th e results indicated th at, 5 ´-( 5 -carb oxy - 1-p enty ny l) sub stitution is m ore f av orab le w h en th e b enzy l ring is 3′,4′,5′-trisub stituted th an w h en it is
2′,5′-disub stituted. C om p ound 7 3 h as th e h ig h est sp ecies selectiv ity f or D H F R P . carinii. F or th is reason th is nov el analog ue m ay b e v iew ed as a nov el lead f or f urth er structure-activ ity op tim ization of D H F R b inding . R osow sk y et al.79 rep orted a concise new route allow ing
easy access to iv e 2,4 -diam inop y rido[ 2,3 -d] p y rim idine
F igure 1 . R ole of D H F R in th e sy nth esis of th y m ine.
T ab le 7 . I nh ib ition of P . carinii D H F R b y 2,4 -diam ino-5 -( 3 ′,4′-dim eth ox y -5′-sub stituted b enzy l] p y rim idines w ith a carb ox y alk y ny l or carb ox y p h eny lalk y ny l g roup in th e side ch ain
C om p ound 5 0 / ( nm ol LI C -1)
28
13 00
1.0
1200
analog s containing a sub stituted p h eny l or oth er arom atic ring attach ed to C 6 via b y a sh ort b ridg e. T h e deriv ativ es
w ere tested ag ainst P . carinii D H F R , h ow ev er none of th e q uinazolines or p y ridop y rim idines tested ( I C5 0
0.087-72 µm ol L-1) w ere m ore p otent ag ainst th e P . carinii
enzy m e th an th e structurally related ref erence com p ound p iritrex im (7 7, I C5 0 = 0.013 µm ol L-1) . I n th e p atent
ap p lication, F orsch and R osow sk y ,80 at th e D ana-F arb er
C ancer I nstitute I nc., sy nth esized sev eral lip op h ilic D H F R inh ib itors h av ing an arom atic g roup and a h eteroarom atic g roup link ed b y a m eth y lene g roup in th e treatm ent of
P . carinii. T rim eth op rim (7 7) and p iritrex im (7 6) are
lip id-solub le antif olates th at h av e b een used clinically f or th e p rop h y lax is and treatm ent of P . carinii inf ections in
p atients w ith A I D S. I n addition, R osow sk y and F orsch ,81
sy nth esized nov el 7 7 deriv ativ es ( T ab le 8) , com p ositions
com p rising th em and th eir use as D H F R inh ib itors. T h e m ost p otent of th e O-alk y l deriv ativ es ag ainst P . carinni
D H F R w as com p ound 7 8 c ( n = 6) . M oreov er th ere w as an increase in p otency as th e leng th of th e 5′-o-alk y l g roup increased f rom n = 4 to n = 6, f ollow ed b y a decrease in p otency as th is leng th increased f rom n = 6 to n = 8. T h e m ost p otent of th e 5 ´-o-(ω-carb ox y alk y l) analog s w as 60g ( n = 4 ) , 24 5 tim es m ore p otent th an 7 7.
T h e activ ity of f our triazoly l analog ues (7 9 a-d) w as ev aluated as inh ib itors of P . carinii D H F R b y C h an et al.82
T h e tw o m ost p otent com p ounds are 8 0 a and 8 0 b, ag ainst
th e P . carinii D H F R . F our com p ounds w ith a nitrog en b ridg e ( b enzanilides and b enzy lam ines) w ere also tested f or activ ity . C om p ounds in th e b enzanilide series (8 0 a,b) are less p otent th an th e b enzy lam ines (8 1 a,b; T ab le 9 ) . W ith an I C5 0 v alue of 0.12 m m ol L-1, com p ound (8 1 a) is
th e m ost p otent m em b er of th e g roup as an inh ib itor of
P . carinii D H F R . I nteresting ly , m eth y lation of th e am ino b ridg e (8 1 b) sh ow ed a 10-f old reduction of activ ity tow ards
th e P . carinii enzy m e and nearly a 7-f old g ain of p otency tow ards th e rat liv er D H F R .
H allb erg et al.83 rep orted a nov el series of D H F R
inh ib itors, w h ere th e m eth y lenam ino-b ridg e of non-classical inh ib itors w as rep laced w ith an ester f unction.
T h e m ost p otent of th e new ester-b ased D H F R inh ib itors, th e 1-nap h th y l deriv ativ e (8 2) , ex h ib its an I C5 0-v alue of
110 nm ol L-1, and is less activ e th an trim etrex ate (8 3)
( I C5 0 = 4 2 nm ol L-1) .
C ush ion and co-w ork ers84 sy nth esized a series of
p entam idine (2) cong eners and screened f or th eir in vitro
activ ity ag ainst P . carinii. T h e A T P assay w as used to ev aluate th e ef f ects of th is p entam idine on th e v iab ility of T ab le 9 . I nh ib ition of P . carinii D H F R b y b enzanilides and b enzy lam ines deriv ativ es
C om p ound 1 R R2 I C
5 0 / (µm ol L -1)
7 9 a H C H2Ph 5 .18
7 9 b H Ph 3 .5 3
7 9 c H SiM e3 10.5
7 9 d H H 24 .8
C om p ound R 5 0 / (I Cµm ol L
-1)
8 0 a Ph 10.8
8 0 b C6H2( O M e)3 1.3
C om p ound R 1 R I C
5 0 / (µm ol L -1)
8 1 a H C H2Ph 0.12
8 1 b M e C H2Ph 1.07
T ab le 8 . I nh ib ition of P . carinii D H F R b y trim eth op rim deriv ativ es
C om p ounds R 5 0 / ( µm ol LI C -1)
7 8 a M e, n = 4 19
7 8 b M e, n = 5 14
7 8 c M e, n = 6 5 .6
7 8 d M e, n = 7 4 4
7 8 e M e, n = 8 5 1
7 8 f C O2H , n = 3 0.25
7 8 g C O2H , n = 4 0.04 9
7 8 h C O2H , n = 5 0.80
7 8 i C O2H , n = 6 2.6
7 8 j C O2H , n = 7 7.1
7 8 k C O2H , n = 8 4 .8
7 7- trim eth op rim 12
A 5 4 9 ep ith elial lung cell m onolay ers deriv ed f rom a h um an carcinom a. A ll diam ide deriv ativ es (8 4 a-e, T ab le 10) , including th e m ost p otent ag ents 8 4 c and 8 4 e, sh ow ed no cy totox icity at 100 tim es th e I C5 0 concentration ag ainst
P . carinii. T h is study identiied th e sim p le sm all m olecules
8 4 c and 8 4 e as tw o v ery p rom ising th erap eutic leads, w h ich deserv e f urth er p re-clinical and clinical testing .
I n a p atent ap p lication, W alzer et al.,85 U niv ersity of
C incinnati, disclosed a m eth od of com b ating inf ectious ag ents, such as P neumocy stis p neum onia, and a m eth od of treating a sub j ect in need of such treatm ent. T he m eth od com p rises adm inistering a b is-b enzam idox im e to th e sub j ect. T h e m ost activ e com p ound of th e series, com p ound 8 5, h ad an I C5 0 v alue of 0.00087 µg m L-1 ag ainst
P . carinii. I n com p arison, p entam idine (2) h ad an I C5 0 v alue of 0.3 00 µg m L-1.
2 .9 . T reatment of tox oplasmosis
T ox op lasm osis is a disease caused b y p rotozoan
T ox oplasma g ondii. T ransm ission to m an occurs via
ing estion of m eat containing cy sts or tach y zoites, ing estion of oocy sts b ecom e inf ectiv e af ter sp orulation or transp lacentally .
T ox oplasma g ondii (T . g ondii) is an ub iq uitous p arasitic p rotozoan th at inf ects up to one-th ird of th e U S p op ulation and up to 9 0% of certain E urop ean p op ulations.86 R isk
f actors f or inf ection include ex p osure to inf ected cats,
consum p tion of rare m eats and contact w ith contam inated soil. M edications th at are p rescrib ed f or tox op lasmosis are p y rim eth am ine (5 3) ( an antim alarial m edication) and sulf adiazine (8 6) ( an antib iotic) . T h us, th e need f or new ch em oth erap eutics activ e ag ainst T . g ondii is th eref ore acute. Sanoi-A v entis dev elop ed h erb im y cin (8 7) deriv ativ es, a b enzoq uinone ansam y cin antib iotic, as h eat sh ock p rotein 9 0 ( H sp 9 0) inh ib itors, usef ul f or th e treatm ent of tox op lasm osis.87 H sp 9 0 is a m olecular ch ap erone and
is one of th e m ost ab undant p roteins ex p ressed in cells. I t is h ig h ly conserv ed and ex p ressed in a v ariety of dif f erent org anism s f rom b acteria to m am m als. I n an in vitro assay ,
8 8 h ad an I C5 0 of 0.9 28 µm ol L-1 f or th e inh ib ition of th e
A T Pase activ ity of H sp 82.
O ne f eature th at disting uish es T . g ondii f rom its h um an h ost is its inab ility to sy nth esize p urine “salv ag e”.88
D if f erently f rom its h um an h ost, th e T . g ondii recov ers
p urine p recursors f rom th e adenosine k inase, enzy m e w h ich conv erts adenosine in adenosine m onop h osp h ate ( A M P) ( F ig ure 2) . T h roug h it, all oth er p urine nucleotides can b e sy nth esized. T h en, th e inh ib ition of th e adenosine k inase activ ity interrup ts th e p urine recov ery route ( “salv ag e”) , of f ering a num b er of p otential targ ets f or th e antip arasite ch em oth erap y . I t is k now n in th e literature th at b enzy lth ioinosine analog ues are sub strates f or th e p arasite adenosine k inase, b ut not f or h um an T ab le 1 0 . I nh ib ition of P . carinii D H F R b y diam ide deriv ativ es
C om p ound L ink er 5 0 / ( µm ol LI C
-1) ; ( µg m L-1)
8 4 a 5 .3 ; 2.3
8 4 b 1.2; 0.6
8 4 c 0.003 ; 0.0013
8 4 d 22.8; 0.0013
adenosine k inase.89 R ais et al.9 0,9 1 describ ed a series
of 6-b enzy ladenosine analog ues th at act as p otent and selectiv e sub strates f or T . g ondii adenosine k inase. 6-Benzy lth ioinosine analog s ( T ab le 11) w ere identiied as ex cellent sub v ersiv e sub strates of T . g ondii adenosine k inase.
C urrently , th ere are no tests w h ich can dif f erentiate b etw een oocy st ing estion v ersus tissue cy st ing estion as th e inf ection route.9 3 R ecom b inant p roteins h av e b een
dev elop ed b y U S D ep artm ent of A g riculture f or th e detection of T . g ondii oocy st p roteins f or ex am p le in b iolog ical luids. I solates and recom b inant T . g ondii-sp eciic p roteins
( rD G P5 p ) can b e adsorb ed to th e surf ace of m icrotiter p lates or to im m unob lotting m em b ranes and used in an E L I SA f orm at f or detection of antib odies. R ecom b inant antig ens
also can b e used to p rep are m onoclonal antib odies wh ich selectiv ely identif y T . g ondii oocy st p roteins. I n addition, p rim ers directed to T . g ondii-sp eciic reg ions of th e D NA seq uences can b e p roduced f or sensitiv e detection of th e
p arasite b y p oly m erase ch ain reaction. U sing E L I SA and M A T , ( m icroscop ic ag g lutination test) assay s w ere p erf orm ed on 127 h um an sera k now n to h av e resulted f rom oocy st ex p osure, iv e sera f rom inf ections resulting f rom cong enital inf ection, and 76 sera f rom M A T negativ e indiv iduals. F rom th e 76 uninf ected sera, all w ere neg ativ e
using th e recom b inant E L I SA and f rom th e 127 oocy st induced inf ections, th e recom b inant antig en E L I SA detected
119 , and did not detect 8. Sera f rom 4 0 oocy st inf ected p ig s and 4 5 tissue cy st inf ected p ig s also w ere tested using E L I SA and M A T . F rom th e 4 0 p ig s inf ected w ith oocy sts, 3 9 w ere detected using E L I SA assay .
I n th e dev elop m ent of v accine p atents, G lax oSm ith K line B i o l o g i c a l s p a t e n t e d a v a c c i n e c o m p o s i t i o n
com p rising th e tox op lasm a p rotein, SA G 3 , th e m aj or g ly cosy lp h osp h atidy linositol ( G PI ) -anch ored surf ace p rotein of T . g ondii.9 4 SA G 1 m ediates th e h ost-cell
inv asion and m ono/ p oly clonal antib odies directed to SA G 1 inh ib ited inv asion of cells b y tach y zoites, p rob ab ly b y interf ering at th e p arasite attach m ent lev el.9 5 T h e b inding
of SA G 1 and SA G 3 to C H O K 1 cells ( C h inese h am ster ov ary cells) w as ex am ined. J ust SA G 3 disp lay s af inity f or sulf ated p roteog ly cans. T h e p resence of alternativ e
cellular recep tors used b y T . g ondii w as already sug g ested,
as inh ib ition of p arasite attach m ent b y solub le G A G s or b y h ep arinase treatm ents of h ost cells w as not com p lete. T h e studies sh ow th at solub le h ep arin ( g ly cosam inog ly can) inh ib ited SA G 3 b inding b y up to 9 0% . D erm atan sulf ate, a g ly cosam inog ly can th at is a structural com p onent of
certain b ody tissues also inh ib ited th e b inding of SA G 3 , b ut ch ondroitin sulf ate A , a ch ondrin deriv ativ e, did not. I n a f urth er p atent ap p lication on v accines, K y ush u T L O C o L td [ K y ush u U niv ersity ] dev elop ed a nov el f used D NA v accine p roduced b y constructing a f used g ene com p rising a g ene f rom an intracellular p rotozoal p arasite, such as tox op lasm a, w ith a ub iq uitin g ene.9 6 T h is v accine is said to
f orm an ub iq uitinated p ath og en antig en w h ich is p rocessed w ith a p roteasom e w h ich strong ly induces th e p roduction of C D 8+ T cells.
3 . Current and F uture Developments
Neg lected trop ical diseases are w idely related to pov erty
and disadv antag e. T h e p oorest p op ulations of ten liv ing in T ab le 1 1 . I nh ib ition of T . g ondii A K b y 6-b enzy ladenosine deriv ativ es
C om p ound Structure 5 0 / ( µm ol LI C -1)
8 9 a o-M eth y l-Benzy lth ioinosine 7.7
8 9 b o-C h loro-Benzy lth ioinosine 6.7
8 9 c m-M eth y l-Benzy lth ioinosine 8.2
8 9 d m-T riluorom eth y
l-Benzy lth ioinosine
8.7
8 9 e m-Nitro- Benzy lth ioinosine 6.2
8 9 f p-M eth y l-Benzy lth ioinosine 7.8
8 9 g p-Brom o-Benzy lth ioinosine 14 .3
8 9 h p-M eth ox y -Benzy lth ioinosine 3 .5
8 9 i p-tert-Buty l-Benzy lth ioinosine 23 .3
8 9 j p-A cetox y -Benzy lth ioinosine 15 .0
8 9 k p-C h loro-Benzy lth ioinosine 8.11
8 9 l p-Nitro-Benzy lth ioinosine 12.0
8 9 m p-C y ano-Benzy lth ioinosine 4 .3
8 9 n p-F luoro-Benzy lth ioinosine 10.3
8 9 o 2,4 -D ich loro-Benzy lth ioinosine 7.3
8 9 p 2-C h loro-6-luoro-Benzy lth ioinosine
8.7
8 9 q m,p-D ich loro-Benzy lth ioinosine 10.4
8 9 r o,m,p-T rim eth y l-Benzy lth ioinosine 3 1.1
