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R.J. MARTIN
Depa,/men/ o/ l',e,li,i,al Vete,'i,m,y .Sciences. R. CDOS.V.S., Su,n,,,e,hall. University' of Edinbu,gh, Edinburgh Etq9 I QH, UK
SUMMARY
Modes of action of anthelmintic drugs are described. Some anthelmintic drugs act rapidly and selectively
on neuronmscular transmission of nematodes. Levamisole, pyrantel and morantel are agonists at nicotinic
acetylcholine receptors of nematode muscle and cause spastic paralysis. Dichlorvos and haloxon are
organophosphorus cholinesterase antagonists. Piperazine is a GABA (T-amino-bu~ric acid) agonist at
receptors on nematode muscles and causes flaccid paralysis. The avermectins increase the opening of
glutanmte-gated chh)ride (GltlC1) channels and produce paralysis of phmTngeal pumping. Praziquantel
has a selective efl'ect on the tegument of trematodes and increases permeability of calcium. Other
anthehnintics have a biochemical mode of action. The benzimidazole drugs bind selectively to ~-tubulin
of nematodes, cestodes and fluke, and inhibit nficrotubule formation. The salicylanilides: rafoxanide,
oxych)zanide, brotianide and closantel and the substituted phenol, nitroxynil, are proton ionophores.
Clorsuhm is a selective antagonist of fluke phosphoglycerate kinase and mutase. Diethylcarbamazine
blocks host, and possibly parasite, enzymes involved in arachidonic acid metabolism, and enhances the
innate, nonspecific immune system.
Kx-~WOnl)S: Mode of action; anthehnintics; levamisole; dichlorvos; piperazine; avermectins; praziquantel;
benzimidazoles; salicylanilides; clorsulon; diethylcarbamazine.
Parasitic nematodes affect animals and man caus- Levamisole, lmtamisole, pyrantel, morantel,
ing considerable suffering and poor growth. oxantel, bephenium and thenium
Fig. 1 shows the chemical structures of nicotinic
Effective anthehnintic drugs, used to treat and
control these infestations, must have selective anthelmintics. There are the imidazothiazoles
toxic effects on these parasites. Unfortunately (levamisole and butamisole); the tetrahydropyrim-
idines (pyrantel, morantel and oxantel); the quat-
with the increased use of these compounds,
anthelmintic resistance has appeared and ernat T ammonium salts (bephenium and
theniuna) and tile pyrimidines (methyridine).
increased in fl-equency (Prichard, 1994). If resist-
These compounds act selectively as agonists at syn-
ance to a particular anthehnintic has occurred, it
aptic and extrasynaptic nicotinic acetTlcholine
is likely that another anthelmintic with the same
receptors on nematode muscle cells (Fig. 2) and
mode of action will also be ineffective although
produce contraction and spastic paralysis. The
other anthehnintics with another mode of action,
electrophysiological effects of levamisole,
may still be effective. Clearly then, it is important
pyrantel, morantel and oxantel have been studied
to have an understanding of the mode of action of
in greatest detail.
anthehnintics in order to inform the selection of
effective therapeutic agents. This rexqew describes
modes of action of anthelmintic drugs (Table I).
Table I.
Summary table o f the m o d e s o f action o f anthelmintic drugs
(;e,eric drueff name Iqxam/de,~ ,1 ,w,me (". K. & l '.&A. Trade .\'mm,.s
Nicotinic agonists Icvanlisole Nilverm
Illllanlisolt' Slyqllill
pVlatltel Stl(ingid
ii1( ii+~lll tcl P;u'alct'l
I)cphcnhlili
lht'llillni ( :anlll)ar
nlcthvridinc
Butamisole Levamisole
CH H 0
a / H N/H
N
CHa
S
CHa
%)
Thenium CH:I
Methyridine
0 - - CH 2 - - CH,2 - - N+ -- CH._,
Bephenium
I
CHa
CH a
~ /CH 2 - CH 2 - O - - CH 2
C> O -- CH 2 -- CH 2 --
'
N +
1
CHa
-- CH2
C?
Fig. 1. C h m n i c a l structures of nicotinic a n t h c h n i n t i c s .
~ Syncylium
eao
~'ry Arm Syncytium
s cord
I J
200 I.tm
1 mm
Ventral nerve cord
(b)
Posterior Anterior
Dorsal
DE3
T ~
.% -YY Y V Y Y Y nerve
cor
Left ./
commissure
J. ~ k v .t ,k 2~ ,k
v ., o w v v v Ventral
YY YY YY YY YY Y Y Y Y Y
nerve
7
T cord
Y g Y
g 7
7 V
T
Fig. 2. Diagrams o f tile n e u r o n m s c u l a r o r g a n i z a t i o n of n e m a t o d e somatic muscle. (a) Diagram o f a s(mmlic muscle cell
showing: the c o n t r a c t i l e spindle region; tile b a l l o o n - s h a p e d bag that c o n t a i n s tile n u c l e u s a n d glycogen granules; tile a r m
which is a process o f tile muscle a n d r e a c h e s to o n e o f tile n e r v e c o r d s w h e r e i, divides into t]ngers Ihal I()lnl all
electrically-coupled c o m p l e x with the fingers o f a d j a c e n t muscle cells a n d collectiveh' is k n o w n as the svncvtitml. T h e
muscle cell possesses synaptic nicotinic acetylcholine receptors, a n d synaptic GABA r e c e p t o r s at tile svncvtial region a n d
extrasynaptic acetylcholine a n d G,MIA r e c e p t o r s over tile surface o f the muscle. (b) Diagram o f the dorsal a n d ventral
n e r v e cord showing the cell r e p r e s e n t e d in a s e g m e n t . All tile cell b o d i e s o f tile m o t o r n e t t r o n e s are c o n t a i n e d in tile
ventral n e r v e cord. Each s e g m e n t has t h r e e r i g h t - h a n d c o m m i s s u r e s a n d o n e left-hand c o m m i s s u r e . Each c o m m i s s u r e is
m a d e up o f o n e or two a x o n s or d e n d r i t e s o f tile m o t o r n e u r o n e s that pass r o u n d the body to c o n n e c t tile ventral a n d
dorsal n e r v e cord. W i t h i n each s e g m e n t t h e r e are 11 m o t o r nero-ones that are divided into seven a n a t o m i c a l types (DEI,
DE2, DE3: dorsal excitato~3.' m o t o r n e u r o n e s . DI: dorsal i n h i b i t o r y m o t o r n e u r o n e s . VI: ventral i n h i b i t o r y m o t o r n e u r o n e .
VI & V2: ventral excitatocv n t o t o r n e u r o n e s . T h e r e m a i n i n g a x o n s in the ventral nelwe cord are i n t e r s e g m e n t a l n e u r o n e s .
Tile excitatory m o t o r n e u r o n e s are c h o l i n e r g i c a n d tile i n h i b i t o r y m o t o r n e u r o n e s are GABAergic. (c) Diagrmn of a cross
section o f the body just caudal to the p h a u , ngeal muscle. It shows the relative locations of tile n e r v e cords, lateral lines, gut
a n d muscle cells.
tration of the anthelmintic. This property of pro- preparation (Robertson & Martin, 1993) using the
ducing open channel block has been established patch-clamp technique. The ion-chalmel currents
using single-channel recording techniques activated by low levamisole concentrations were
(Robertson & Martin, 1993). shown to cart3, cations and to have similar kinetics
to acetylcholine-activated channels. The levami-
Single-channel currents activated by nicotinic sole activated channels have a mean open time of
anthelmintics 1.34 ms and a conductance in the range 20-45 pS.
Initially, levamisole-activated channel currents At higher concentrations of levamisole, a flicker-
[Fig. 3(a)] were recorded fi'om the muscle vesicle ing open channel block that increases on hyper-
MODES OF ACTION OF ANTHELMINTI(" DRUGS 15
Table II
Voltage sensitivity o f dissociation constants o f o p e n channel block by the nicotinic anthelmintics
Membrane I33 l.evamisole 2KI, l5,rantel Kl~ Morantel KR Oxantel
-50 mV 123 I.tXJ 37 IJ.Xl 12 p.xt 18.5 I.tM
-75 mV 46 IJM 20 I.t~l 1 ~,xl 7.5 law
Voltage-sensitivlty of h]~ e-fold every 20 mV e-fold eve D' 40 mV e-fbld every 22 mV e-fold ever), 29 mV
/~ is the dissociation constant: the lower the concentration of this constant the more potent it is. At the concentration
of the KB the channel is blocked for 50% of its open time so tile response would be reduced by a half.
16 THE VETERINARYJOURNAL. 154, 1
(a }Levamisole
15
10
0
%
1 pA L
0 5 10 15 20 ms 0
(b~
1PA3~ms ............ i
~~
............................................ i ...... C
(c) Pyrantel
C
15-
O
10-
%
C
O
0-
0 5 10 15 20 ms
Fig. 3. Patch-clamp records of silagle-channel currents activated by levamisole. The patch-clamp technique allows the
activit3' of single ion-channels in the membrane of the cell to be recorded as the channel opens and closes. Small ( l x l 0 -v-'
A) current pulses are recorded with this technique. The currents are rectangular in shape; C is the closed state and O is
the open state. (a) Histogram of open-times and records of levamisole-activated single-channel currents. Cell-attached
patches. Openings downward. Patch potential -75 mV; 10 laM levamisole in the patch pipette. Mean open time: 1.34 ms.
(b) A flickering burst demonstrating channel block produced by 30 last levamisole at -75 mV, cell-attached patch. The
comb effect of the channel current is characteristic of a drug moving into and blocking the ion-channel. (c) Histogram of
open-times and records of pyrantel-activated single-channel currents. Cell-attached patch; openings downward; patch-
potential -75 mV; 0.1 I.tM micromolar pyrantel in the patch-pipette. Mean open-time: 1.09 ms. The pyrantel channel open
times are on average slightly shorter than those of levamisole.
MODES OF ACTION OF ANTHELMINTIC DRUGS 17
Dichlo,wos
mutants with modified nicotinic acetylcholine
receptors on muscle (Lewis el al., 1980; Lewis et o H
al., 1992). (2) Accomnaodation and recovery of CH30\ li I C]
/p--o--c~-c /
parasites after long periods of exposure to the
CH30 C1
anthelmintics (Lewis et al., 1980; Lewis et al.,
1992), and which may be explained by nicotinic
receptor desensitization.
Haloxon
These resistant or recovered nematodes no
longer respond to the nicotinic anthehnintics, but 0
interestingly, may still respond to acetylcholine
(Coles et al., 1975). One explanation for these C1CH2CH2 0 /
resuhs is that in addition to tile nicotinic receptor
on nematode muscle, there are other non-nic-
otinic cholinergic receptors present on muscle
not stimulated by the nicotinic anthelmintics.
Such receptors may facilitate contraction by being CH3
coupled via G-proteins to vohage-activated
channels. Fig. 4. (:heroical s t r u c t u r e o f d i c h l o r v o s a n d h a l o x o n .
ORGANOPHOSPORUS CHOLINESTERASE
Genetics of resislance to nicotinic anlhelmintics INHIBITORS
Tile genetics of resistance to the anthelmintic
levamisole has been studied in the laboratory Dichlmvos, haloxon
using the small fi'ee-living soil nematode, Caenor- Compounds like dichlorvos and haloxon are
habdili.~ eh,gans. Several hundred levamisole-resist- selective organophosphorus anti-cholinesterases
ant alleles have been identified that were isolated (Fig. 4), and have an anthehnintic action, as well
ill several screens (Lewis el al., 1980; Lewis e/ al., as all insecticidal, action. Dichlorvos and haloxon
1992). The genes responsible tbr this resistance can control insect parasites, as well as hehninth
inclucled: h,v-1, unc-29 and unc-38 that encode tile parasites. The mode of action of these compounds
proteins subunits that make up the nicotinic ion is to block tile action of the parasite enzyme, ace-
channels of the nematode. In addition, other tylcholinesterase, leading to the excessive build up
genes including: un, c-50, unc-63 and unc-74 that of tile neurotransmitter, acetylcholine. This mode
are believed to be involved in receptor biosynth- of action also predisposes towards toxicity in the
esis have been identified. host animal where acetylcholinesterase
Only two strains of lev-1 (21 and x61) were enzymes are also present. Because more selective
dominant when crossed over with wild-types (non- combined anthehnintic+insecticidal agents
resistant) (Fleming el al., 1994). The lev-1 (x21) (avermectins and milbemycin) are available, the
strain only inw~lves mutation at a single amino- organophosphorus compounds are now used less
acid, replacing glutanaic acid in the ion pore of frequently.
the receptor ion channel (ill alpha-7 at position The existence of cholinesterase, the enzyme
237) with a positively-charged lysine. This change that breaks down acetylcholine, was first described
is believed to be sufficient to change the ion chan- in Ascmis by Bueding (1952). The distribution of
nel from cationic to anionic, i.e., to convert tile cholinesterase was described by Lee (1962) who
receptor from an excitatory channel to all inhibi- used histochemical techniques. In the head
tory one. The ~,-1 (x61) swain contains tile region of Ascmis, most of the enzyme activity is
amino-acid leucine ill the pore of tile ion-channel associated with the contractile spindle region of
(in alpha-7 at position 247): this point mutation is the muscle and is in the extracellular matrix. Lee
expected to produce increased desensitization (1962) also described cholinesterase activity on
and reduced affinity for levamisole, rendering lev- the muscle arms near their endings on the nerve
anfisole less potent as an agonist. cords but not on the bag region of the muscle.
The genetics of levamisole resistance in para- In C. elegans, three classes of acetylcholinester-
sitic nematodes remains to be studied. ase are recognized: class A, class B, and class C,
GABA ,
,A, Although acetylcholinesterase is responsible for
/ \
COOH \ NH;? the breakdown of acehllcholine and involvecl in
motor action in nematodes, it is also secretecl into
the external environment in large quantities bv 4.
.s717077 and other parasitic nematodes. The fun&on
of llle secretecl aceh~lcholinesterase ma), be to
reduce the effects of host acetylcholine in the
intestine, perhaps clecreasing niucosal glanclulai
secretion by the host. The original hypothesis of a
biochemical hold-fast effect (recluced acetylcho-
line in the host intestine blocking peristalsis of the
gut) has now been rqjectecl. The identification of
the function of secreted cholinesterase hy para-
H
sitic nematodes, ancl the abilitv to antagonize this
enz\me, mav lead to the increased use of anti-
cholinesterases in the future to facilitate renio\xl
Piperazine of gut nematodes.
2 pA
J
100ms
GABA AGONIST
Ivermectin
T h e difference in p o t e n c y may be explained by
the fact that h i g h e r c o n c e n t r a t i o n s o f piperazine
H O ~ a ~OCH'~
are r e q u i r e d to p r o d u c e the same o p e n i n g rate o f
H3C O 0...1~ ~ CHa A .-CHa the c h a n n e l as that p r o d u c e d by GABA, and that
the average duration o f the c h a n n e l openings pro-
d u c e d by piperazine is m u c h shorter. T h e same
probability o f c h a n n e l o p e n i n g can be achieved
IlL o_._A with lower c o n c e n t r a t i o n s o f GABA than pipera-
h-o j. zine. Therapeutically, however, GABA would be
Abamectin ~" " ~
OCH.~ 0 ~ CH ineffective because it is not selective like pipera-
HO_ 2-. hl :' zine and is highly ionized and does not cross the
"~ OCHa OH
cuticle.
HaC/k" 0''~ 0 ~ [ ' ~ " CH, CHaR
FI LI/,q.~.fiIIBL. Z~t)le, C
20 THE VETERINARY JOURNAL, 154, 1
--- :::::::::::::::::::::::
i::ii!iiiiiiii::i
i i! i ~ p m o ::.::::.: p m 3 :: pm4 : pm3 :.::::.: ~ 2
E ~
Fig. 7. Diagram of the phalyngeal muscle of (J. eh'gan,~ showing tile location ~f the GIuC1 receptors ~m the pro4 muscle
ceils that are innervated by the m3 motor neurones. Also illustrated is the division o1 the p h a l ) n g e a l muscle into Ihe
regions: lerminal bulb: isthmus: carpus (sul)divided into metacarpus and procarpus). T h e o l h e l l l l ( l l ( } l " I l e l l l ' ( } l l e s (Ill l, m2,
m4 ~" mS) that innervate the muscle cells (pro3, proS, pro6, pro7) are shown. The i n t e r n e u r o n e s 15 thai inhibits m3, as well as
m4, is shown. Adapted t i o m Avery (I ~).t)~:~).
llllllIHltli!llTiii,Nl!lI,E!ll!
I,[!iIL!!I l ! ilt 5mVl__ the original GluCl-cd subunit r e m a i n s to be deter-
mined. However, the p r e s e n c e o f m o r e than o n e
GluCl-ot subunit indicates the p r e s e n c e o f m o r e
tttlillhlll,Ll,li&L,ll[tl[,tlktdlt[
tilhliltttt 30 s than o n e site of action for the avermectins. It
implies that the a p p e a r a n c e of resistance to aver-
mectins requires m u t a t i o n o f m o r e than o n e
300 ~IMGABA gene.
In addition to effects on the p h a r y n g e a l muscle,
ivermectin also has an effect on somatic muscle
a n d o p e n s non-GABA activated channels, a n d in
addition, inhibits GABA-activated channels
Fig. 9. (a) Effect of 890rim milbemycin D on the
memb,ane potential and input conductance. Milbemycin ( H o l d e n - D y e & Walker, 1990). T h u s again it
slowly p,oduccd an increase in input conductance that a p p e a r s that the a v e r m e c t i n s may have m o r e than
was not reversed on washing (not shown). (b) Effect of o n e site o f action in parasitic n e m a t o d e s .
application of 100 ItM t.-glutamate on membrane potential
and input conductance. Different preparation fi'om (a).
Glutamate (applied during horizontal bar) produced a
INCREASED CALCIUM PERMEABILITY
transient small hyperpolarization of 1 mV associated with
an input conductance change from 157 ItS to a peak of
429 ItS (AG: 272 ItS) desensitizing to 231 gS ( AG: 74 ItS) Praziquantel
after 4 rain. (c) After washing the preparation the input Fig. 11 shows the c h e m i c a l su-ucture o f pra-
conductance of the pharynx returned towards control ziquantel. Praziquantel has a selective toxic effect
levels (142 ItS) and the effect of 300 gM GABA was tested
on schistosome parasites, w h e r e its m o d e o f action
without effect (applied during the horizontal bar). The
lack of effect of GABA was not due to desensitization has b e e n studied m o r e extensively (Andrews et al.,
because subsequent application of 100~xt L-glutamate 1983; H a r n e t t , 1988) t h a n in cestodes. Many
increased the input conductance again (not shown). actions o f p r a z i q u a n t e l may be e x p l a i n e d by an
increased Ca `'+ p e r m e a b i l i t y o f parasite muscle
inhibit p h a r y n g e a l p u m p i n g that is p a r t of the a n d / o r t e g u m e n t a l m e m b r a n e s . Fig. 12 is a dia-
n e m a t o d e parasite f e e d i n g process. Zero-C1- g r a m o f the schistosome t e g u m e n t a n d u n d e r l y i n g
solutions were shown to abolish reversibly the glut- muscle that illustrates the k n o w n sites w h e r e Ca `'+
a m a t e - i n d u c e d c o n d u c t a n c e responses (Martin, crosses into a n d b e t w e e n intracellular c o m p a r t -
1996) a n d were used to d e m o n s t r a t e that the glut- ments, a n d t h e r e f o r e , the possible sites for pra-
a m a t e effect was m e d i a t e d by a CI- c h a n n e l s in ziquantel action.
Ascam.
'22 ]'HE VETERINARVJOURNAI~, 154. 1
12f (a)
/
100 ~-
60-
4O
/
2O
I I I l ~ I
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Milbemycin (prq)
5 mV
Fig. 10. (;luta,mlte potentiation effects ()f milbcmvcin D. (a) ( | ) Pl()l of peak A(; produced by. 30 p_~lghmm)aw againsl
milbemvcin co,lcentnuioq: (O) plot ()f resting input conductance ()f the phalynx agains! mill)cmvcin c().ccn,alion.
Resuhs fiom the experiment illustrated in (b), (c) & (d).(b) ('ontrol 30 ~.txlglutamate vcsp()nsc, peak A(;, is small. 16 ~.tS.
(c) In the pvescace of 30 .xt milbemvcin (D) the" peak A(; produced by 30 ~Xl gltmtmatc i.cvcased t() 27 l.tS. (d) 300 nxl
milbemvcin D increased the peak A(; to 86 laS. I . this pavticttlar prel)aration the gltmlmatt" resp().se was a small
depolarizing potential, indicating that the (:l-reversal p()te.tial was slightly depolarized relative, to the' m c m b r a . c
potential.
Fig. 12. Diagram of 1he s t r u c t u r e of the body wall of Schi.~losoma ma,soni showing the possible sites of action of
p r a z i q u a n t e l a n d meclaanisms of Ca'-" t r a n s p o r t into a n d o u t o t the t e g u m e n t . 1: Vohage-activated Ca +-'+ c h a n n e l . 2:
II'Jll'~lct'lhll~tl" i]lessell~el activated Ca +-'" chanllel. 3: E x t r a c e l h d a r r e c e p t o r o p e r a t e d Ca ~" c h a n n e l . 4: Non-selective cation
c h a n n e l also allowing t+ntz-v of Ca'-". 5 : N a ' / C a e+ e x c h a n g e r . 6: Ca `'+ ATPase pt, m p i n g out ( ' J * . 7: I n t r a t e g t m l e n t a l Ca e+
buffers. 8: IP:~ releasable store from the sarcol)lasmic rcticuhtm. 9: Ca*-'" i n d u c e d Ca'-" release c h a n n e l (CICR c h a n n e l ) . I0:
(;~(-'" ATPase pttmp: savcoplasmic e n d o p l a s m i c r e t i c t d u m Ca'-'- (SERCA). 11: Electrical j t m c t i o n b e t w e e n mttscle cell a n d
tegumetlt. 12: Electrical j u n c t i o n s b e t w e e n tnuscle cells. If praziqttantel acts like caffeine it would act o n site 9. 13: A
p r o t o n I)tttnp may set tq) a pH g r a d i e n t actress the t e g u m e n t a n d be r e q u i r e d for q o r m a l titnction. A p r o t o n i o n o p h o r e
wc+uld ttl)Set tiffs g r a d i e n t a n d lead to the d e m i s e c~t+the organism.
blocked by Mg '-'+, or La :~+ but not I)y Ni '-'+ Co e+ 01- will eliminate the effect of praziquantel on the
the calcium-channel blocker D-600 (Fetterer et al., muscle preparation in Ca'-'+-free solutions. These
1980; Wolde-Mussie et al., 1982). The application experiments suggest that caffeine and praziquan-
of a high K+ solution to depolarize the schisto- tel share the same site of action.
some preparation results in a rapid depolariz- In vertebrate smooth muscle preparations, caf-
ation, but the application of praziquantel resultsin feine acts to stimulate the o p e n i n g of a large cat-
a slower onset depolarization. From these observa- ion channel known as the CICR channel (calcium-
tions, it may be concluded that praziquantel some- induced calcium release channel) ( H e r r m a n n -
how results in an increase in Ca `-'+ permeability Frank et al., 1991) in the sarcoplasmic reticulum.
across parasite m e m b r a n e s via channels that are This channel is activated by cytosolic rises in Ca '-'+,
not activated by depolarization. These channels adenosine triphosphate (ATP) and caffeine but is
must be pharmacologically different fi'om those of inhibited by Mg '-'+. T h e CICR channel p r o d u c e s
the host animal or the praziquantel would affect regenerative Ca `-'+ release from the sarcoplasmic
the host animal. reticulum following entry of Ca `-'+t h r o u g h voltage-
Experiments on a snail smooth muscle prep- activated channels (Gregoire et al., 1989). Large
aration (Gardner & Brezden, 1984) have illus- cation channels, which might be similar to the
trated that caffeine mimics the effect of pra- CICR channel, have been observed in the tegu-
ziquantel in producing contraction, and that prior mental m e m b r a n e of schistosomes (Day et al.,
treatment of the muscle preparation with caffeine 1992).
24 THE VETERINARY .]OURNAL. 154, I
BENZIMIDAZOLES PRODRUG
NHCO2CH3
l "I N
N~C
NHCO2CH3
N
iL
S
NHCOCH2OCH3
H
Cambendazole: R = (CHa)2CHOCONH--
i NHCO2CH a
' ' I "~ SCH 3
N
CH 3 " N
H
H
Oxibendazole: R = CH~CHzCH20--
Albendazole: R = CHaCH2CH2S--
@
O
Oxfendazole: R = S---
@
O
El
Mebendazole: R = C m
Flubendazole: R = @,
F
0
S m
Fig. 13. Chemical structure of benzinfid~oles, benzimidazole carbamates, prodrug febantel and the antifluke drug
triclabendazole.
[3-Tubulin
100
IH TI300im
200
i i
400
Hinge
Q) a-tubulin ~ ( ~ 13 membered
+ ~-tubulin~ ~ D ~ : spiral made of
alternating ~-
and [3-tubulin
~ Vesicle
transport along
Fig. 14. Diagram of the 50 kDa ~-tubulin protein. It consists of three domains that are separated by protease action. In
the middle in a 'hinge' allowing the molecule to fold. The location of the amino acids 100, 200, 300 and 400 are shown.
Below this is a diagram representing the formation of microtubules by 0t- and ~-tubulin that polymerizes by forming a 13
membered hollow spiral. Each turn of the spiral allows the 0t- and ~-tubulin to pack ahernately along the length of the
microtubule. Polymerization takes place at the positive pole where temperature and GTP Mg '' and other factors favour
microtubule formation. The formation of microtubules is inhibited by binding of benzimidazoles to ~-tubulin to produce
'Capping' and inhibition of further microtubule formation. Breakdown occurs at the negative pole. The physiological
function of the microtubules include intracellular transport via special proteins, kinesins or dynesins (here represented by
the match-stick man).
A reduction in the nunfl)er o f isotype alleles fi)r series o f protein c o m p l e x e s on the inner mito-
]3-mbulin is associated with the a p p e a r a n c e of chondrial m m n b r a n e . This process leads to pro-
benzimidazole resistance (Roos e/ al., 1995). tons being p u m p e d out o f the m i t o c h o n d r i a
Resistance is associated with a progressive loss o f matrix p r o d u c i n g a p r o t o n motive force that is
alleles o f isotype 1 and a total loss o f isot?'pe 2 due to the pH gradient and t r a n s m e m b r a n e elec-
fi'om the n e m a t o d e population. A resistant popu- tric potential. ATP is synthesized when the pro-
lation o f Haemonchus co~tlorlus has b e e n charac- tons flow back into the m i t o c h o n d r i a l matrix
terized by only the presence o f a single [3-tubulin t h r o u g h an enzyme complex. This process of oxi-
allele r e f e r r e d to as allele 200. T h e a p p e a r a n c e o f dative phosphoD, lation takes place in the host ani-
resistance can be e x p l a i n e d by a loss o f susceptible mal (StD, er, 1995), as well as in the parasitic hel-
p h e n o t y p e s and the st, rvival of a resistant p h e n o - minths (Van den Bossche, 1972; McKellar &
type and its increased representation in the Kinabo, 1991).
r e m a i n i n g population. Lipid soluble substances that can car D' protons
In fungi, benzimidazole resistance is associated may shuttle across the inner m e m b r a n e o f the
with the a p p e a r a n c e o f a different form of [3-tubu- m i t o c h o n d r i a , and remove the p r o t o n gradient
lin (Fujinmra el al., 1992; J u n g el al., 1992) which and u n c o u p l e oxidative phosphorylation so that
is characterized by the a p p e a r a n c e of tvrosine the oxidation o f NADH and FADH is no longer
instead of phenylalanine in position 200. Because linked to the p r o d u c t i o n of ATP and may carry, on
m a m m a l i a n [3-tubulins have also tvrosine present without its production. Fig. 15 shows the position
in the 200 a m i n o acid position (Lewis el al., 1985) o f the p r o t o n that is able to dissociate froni a var-
it is unlikely that benzimidazole resistance may be iety of c o m p o u n d s . 2,4-Dilfitrophenol, carbonyl-p-
o v e r c o m e by changes in d r u g chemistvv. It would t r i f l u o r o - m e t h o x y p h e n y l h y d r a z o n e , (Stryer, 1995)
not be possible to design a selectively-toxic agent and laexachloroplaene, nitroxynil, oxvclozanide
against the ['ungi because the fungi and host ~-tub- (Corbett & Goose, 1971; Edwards el al., 1981b) are
ulins would both bind the benzimidazole to the all lipophilic c o m p o u n d s that are capable o f
same d e g r e e and st) be loxic to both species. A carr}.'ing p r o t o n s across m e m b r a n e s , and there-
similar i ) h e l m m e n o n may o c c u r with n e m a t o d e fore, may act to u n c o u p l e oxidative pbosphoD,1-
pm-asites. ation preventing the p r o d u c t i o n of the p r o t o n
gradient across the inner mitochondrial
membrane.
P R O T O N I O N O P H O R E S : SALICYLANILIDES T h e nleclaallism of action of the p r o t o n ionoph-
AND SUBSTITUTED PHENOLS. ores has been assnmed to be to selectively
u n c o u p l e oxidative pbosphoD'lation in parasite
Salic~,lanilides: closantel, raJbxanide, o.~yclozanide, m i t o c h o n d r i a . Pax and B e n n e t t (1989) have
bro/i'anide a~d substituted phenols: nitroxynil, e x p e r i m e n t a l evidence that the i n n e r m e m b r a n e
niclopholan, hexachorophene dibromsalan & o f the parasite may not be the site o f action of the
niclosa mide p r o t o n iollophores. T h e v observed that appli-
T h e plaarmacolog,3, o f a range o f anti-fluke cation of closantel to Schistosoma mansoni and Fasci-
drugs have been reviewed (McKellar & I<Anabo, ola hepatica p r o d u c e s a fall in t e g u m e n t a l pH
1991). In tiffs section, the m o d e of action of the (6.8-6.5) when m e a s u r e d with an intracellular
p r o t o n i o n o p h o r e s (often called oxidative phos- electrode, and that this c h a n g e in pH o c c u r r e d
pholwlase u n c o u p l e r s ) are considered. T h e chemi- after 10 rain and before an), c h a n g e in the pro-
cal structure salicylanilides and nitroxynil illus- duction o f ATP bv the parasite. T h e intra-tegu-
t,-ates that each anthelmintic molecule possesses a mental [',all in p H was associated with a reduction
d e t a c h a b l e p r o t o n (Fig. 15). These molecules are in motility o f the parasite and could explain the
vmT lipophilic and may shuttle protons across anti-parasitic action o f closantel. It was c o n c l u d e d
m e m b r a n e s , particularly the i n n e r mitochondrial by Pax and B e n n e t t (1989) that closantel was a
m e m b r a n e . Fig. 16 represents the process by m e m b r a n e active molecule and that it may affect a
which ATP p r o d u c t i o n occurs in the m i t o c h o n d - n u m b e r o f biochemical and physiological pro-
ria, that is c o u p l e d to the p r o t o n gradient across cesses. T h e s e processes are presumably sensitive to
the i n n e r m i t o c h o n d r i a l m e m b r a n e . Oxidative changes in intra-tegumental pH. T h u s the site o f
p h o s p b o u l a t i o n may be summarized as electrons action o f the p r o t o n i o n o p h o r e s may include the
fi'om NADH or FADH being conveyed t h r o u g h a t e g u m e n t r a t h e r than just the m i t o c h o n d r i a .
28 THE VETERINARYJOURNAL. 154, 1.
Oxyclozanide
C1 OH OH C1
CI CI CI
Rafoxanide
I OH C1
Closantel
This is explained by their strong plasma protein
I OH C1 binding which is more than 99% for the salicylani-
lides (Mohammed-Ali & Bogan, 1987) and 98%
CON__// \/x_._?__</ \~---Cl for nitroxynil (Alvinerie et al., 1995). The selective
anthehnintic action of these highly protein-bound
anthelmintics may be explained, in part, by their
I OH CI effect against blood-sucking parasites, concentrat-
ing the anthelmintic in the parasite without the
high tissue levels being produced in the host. The
Brotianide high level of protein binding may explain the
selective effect of these agents, and the fact that
C1 OH CI
CS o< r
well bled out carcasses have low tissue residue
levels (McKellar & Kinabo, 1991). Thus the mode
of action of this group of anthelmintic involves
the selective delivery of the proton ionophores to
the parasite because of the high level of plasma-
Br OCOCH
3 CI protein binding.
Nitroxynil DIAMPHENETHIDE
Diamphenethide
. / - - -
,,' / \
Clorsulon
C1
CI2 = C NH2
~\ /
H2NO2S SONH 2
Diethylcarbamazine
C9H5
/ \
CHaN NCON
\ / ....,
C2Hs
was likely to be a prima D, effect because it plmsphoglyce,'ate kinase (Schuhnan et al., 1982)
occurred early on and before the deterioration of and phosphoglyceromutase (Schulman &
the whole parasite. Interestingly, in the second Valentino, 1982) of Fasciola and inhibits the
paper of Edwards et al. (1981h) the effect of dopa- Emden-Meyerhoff pathways in fluke. As a result,
mine, a putative neurotransmitter in Fasciola had a there is a selective inhibition of glucose utiliz-
protective effect against diamphenethide. ation, and acetate and propionate formation. The
The action of diamphenethide remains to be inhibition of the Fasciola phosphoglycerate kinase
defined in greater detail but the study of Edwards is competitive (Fig. 18) with a ~ of 0.29 mM: clor-
el aL (1981a) showed that its biochenaical effects sulon competitively inhibiting the binding of ATP
contrasted with the proton ionophore, oxyclozan- and 3-phosphoglycerate to the phosphoglycerate
ide and appears to involve effects on malate kinase. Schulman et al. (1982) suggested that the
metabolism in Fasciola. large group on the six position of clorsulon pre-
vented the conformational change in the kinase
enzyme required for activity. There was also a
I N H I B I T I O N OF P H O S P H O G L Y C E R A T E good correlation between the I~ values.for antag-
KINASE A N D MUTASE onism of Fasciola phosphoglycerate kinase for a
range of compounds related to clorsulon and the
Clorsulon potency of these compounds as antifluke agents.
Clorsulon is 4-amino-6 trichloro ethenyl 1,3- This evidence supports the suggested mode of
benzenedisulphonamide (Fig. 17). Structurally, it action.
is similar to 1,3-diphosphoglycerate (Schulman et The inhibition of Fasciola phosphoglyceromut-
al., 1982) and consequently, inhibits the enzymes ase by clorsulon was studied by Schulman and
31) THE VETERINARY JOURNAl., 15-1, I
E-ATP
/ \
E1 -- I + E 3PG-E-ATP -- ADP-E-1,3DiPG "~-----~E-3DiPG + ADP ~ - ~ - E + 1,3DiPG
\ ,/
/
/ "
3PG-E
Fig. 18. Diagram o f the competitive a n t a g o n i s m o f 19hlsl)hoglyctwate kiqase (El o f l,'asciola hepatica by clorsulon (1).
DiphosphoiT1 glycerale: DiPG. 3-Plmsphol3l glyceratc: 3PC;.
Valentino (1982) who pointed out that this does not appear to be involved because nucle,
enzyme requires 2,3-diplaosphoglycerate for acti- athvmic mice, infected with BruKia pahanffi,
vation and the ch)se similarity of clorsuh)n to showed a marked reduction in microfilariae fol-
diphosphoglycerates could explain its inhibition lowing diethvlcarbamazinc treatment (Vickel T el
of the mutase enzwne. These authors puritied the al., 1986). Thus T cells and T-dependent
enzyme and compared some of its properties with ,'esponses (Ig(; and lgE) do not appear to be
mamnaalian l~hosphoglyceromutase that were not invoh'ed. The involvenlent of complement also
inhibited by clorsuhm. There appears to be no seems unlikeh' because clearing of microfilaria
recent studies on the mode of action of this series ii-om the nuc(e mice by diethvlcarbanmzine was
of compounds ahhough clinical trials and efficacy not inhibited by prio," treatment with cobra
studies have been made. venom lactor treatment (Vicke D' el al.. 1986).
MEMBRANE
PHOSPHOLIPIDS
SE 1 PLA 2 . . . . . 1~-GLUCOCORTICOIDS
.................. 1
r ........................
i i
i DIETHYLCARBAMAZINE i i. . . . . . . . . . . . . . . . . . . . . . . . . . .
"--r ...................... ARACHIDONIC , DIETHYLCARBAMAZINE
-1
ACID .-"-" 1_ _ andNSAIDS
LTD4
eosinophil;
mast cell
I
LTE 4
eosinophil;
mast cell
Fig. 19. Diagram of the pr(~duction of the leukotrienes and prostaglandins via lipoxygenase or cyclo-oxygenase fi-om
ar,tchidonic acid thal is produced from membrane phospholipids as a result ol phospholipase A=, acidity. The inhibitory
silc.s of action of gluccwo,ticoids, non-steroidal anti-inllammatola' drugs (NSAIDs) and diethvlcarbamazine are shown.
l)iethvlcmbamazine is shown inhil)iting the action of LTA, svnthase thal converts 5-hydr,>peroxyeicosatetraenoic acids (5-
HPE'I:E) and 5-1avdroxveicosatet,aenoic acids (5-HETE) to the let,kotrienes I.TAa and I+TA~ to LTC; in mast cells and
eosinophils. Dietl~vlcarbamazine is also shown inhibiting the cych>-oxygenase enzyme that converts a,achidonic acid to the
prostaglm]dins P(~G._, and P(;H._,. The subsequent production of P(;D_,. PGE=,, PGI=, & TY~-k,_,is also inhibited in the blood
vessels and ,nicrol]lari;~.
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