+

The I'eteHnaU.flmrna11997, 154, 11-34

Review
Modes of Action of Anthelmintic Drugs
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.

INTRODUCTION

Parasitic nematodes affect animals and man causing considerable suffering and poor growth.
Effective anthehnintic drugs, used to treat and
control these infestations, must have selective
toxic effects on these parasites. Unfortunately
with the increased use of these compounds,
anthelmintic resistance has appeared and
increased in fl-equency (Prichard, 1994). If resistance to a particular anthehnintic has occurred, it
is likely that another anthelmintic with the same
mode of action will also be ineffective although
other anthehnintics with another mode of action,
may still be effective. Clearly then, it is important
to have an understanding of the mode of action of
anthehnintics in order to inform the selection of
effective therapeutic agents. This rexqew describes
modes of action of anthelmintic drugs (Table I).
1(190-0233/t)7/04(1011-24/S 12.00/0

NICOTINIC AGONISTS

Levamisole, lmtamisole, pyrantel, morantel,

oxantel, bephenium and thenium
Fig. 1 shows the chemical structures of nicotinic
anthelmintics. There are the imidazothiazoles
(levamisole and butamisole); the tetrahydropyrimidines (pyrantel, morantel and oxantel); the quaternat T ammonium salts (bephenium
and
theniuna) and tile pyrimidines (methyridine).
These compounds act selectively as agonists at synaptic and extrasynaptic nicotinic acetTlcholine
receptors on nematode muscle cells (Fig. 2) and
produce contraction and spastic paralysis. The
electrophysiological
effects
of
levamisole,
pyrantel, morantel and oxantel have been studied
in greatest detail.

1997 Bailli6re Tindall

12

THE VEH.:RIN..\RY .IOL'RNAI.. 154, I

Table I.
Summary table o f the m o d e s o f action o f anthelmintic drugs

(;e,eric drueff name

Nicotinic agonists

Iqxam/de,~

Icvanlisole
Illllanlisolt'
pVlatltel
ii1( ii+~lll tcl
I)cphcnhlili
lht'llillni

,1 ,w,me

(".

K. & l '.&A. Trade .\'mm,.s

Nilverm
Slyqllill

Stl(ingid
P;u'alct'l
( :anlll)ar

nlcthvridinc

Acetylcholinesterase inhibitors

11;llOXOll

Mulitwttrma

dichhwvos

GABA agonist

pipura/hic

End.rid.

GiuCI potentiators

ivt'l+lllt'Clill
al)alllcclill
d~WalllCClill

[V(IIIR'C
l)t~('tOlll;tX
( :v(It'ctill

inoxidcciin
niill)cinvcin l)

Calcium permeability increase

[~-tubulin binding

praziqu,in tel

l)l~mcit

thial)cnda]<dc

Thibcnz.lc
Equibcn, N~vil)cn
koditac
Valb,lzcn
Rvc<d)cn

Callli)t,lld~iztilt +

~lxil)cndaz<dc
all)cndazolc
albcndazoic sulphoxidc
I{'nl)cndazoic
tlxl{.lldazoh.
lllt'l)('llda/ilit'

Ihd)cndazole
I~'bantcl
nclol)ilnin
lhioj)hanaic
triclabcnda/olc

Proton ionophores

chls<tntcl
i-alilyanidt+
oxvch izi.illidc
I)r(>tianidc
nill~,xvnil
niclopholan
]lt'xachlorophcnt.

Pilll[I('lll"

Svstamcx
Tchniu
Fhil)vn~l
~alVVtTIII

I Icpadcx
\V< wlnal:tc, Ncmal,tx
Fasincx

Flukivcr
Flukanidc. Ranidu
Zanil
In Fhlkombin and Vermadcx
Trodax, l)ov<.'n ix
BilevolL Dist<don
(:oopaphcnc, l)ismdin

dil)rllmosalan

nich;samidc

Inhibition of malate metabolism

dialnphent'lhidc

( '.ol-il)an

Inhibition of phosphoglycerate kinase

and mutase

C](II'SII](III

In h'omec Super

Inhibitor of arachidonic acid

metabolism and stimulation of innate
immunity

dicthvlcarl)amazine

(:aricide,Filaricide

Electrophysiological effects of nicotinic

anthelmintics in nematodes
Intracelhllar recordings made with

micropip-

e t t e s fl'mn Ascaris s u u m b o d y m u s c l e s h a v e s h o w n
t h a t b a t h a p p l i c a t i o n o f t e t r a m i s o l e , t h e D/l.
racemic mixture of levamisole, produces ntem-

M()DES OF ACTION ()F ANTHELMINTIC DRU(;S

Levamisole

Butamisole
CH

13

N/H

CHa
S

Pyrantel

Morantel

Oxantel

CHal

CIita

CI~:,

%)

CHa

Thenium

CH:I
Methyridine
0 - - CH 2 - - CH,2 - -

N+

--

CH._,

I
CHa
Bephenium

C>
Fig. 1.

CH a
O --

CH 2 --

CH 2 --

'

N +

--

CH2

CHa

/CH

2 - CH 2 - O - - CH 2

C?

C h m n i c a l structures of nicotinic a n t h c h n i n t i c s .

brahe del)olarization, an increase in spike fl'eqnency and contraction (Aceves el al., 1970).
Pvrantel and its analogues also produce depolarization, increased spike actMtv and contl-action
when applied to Ascaris muscle (Aubr)' el al., 1970)
suggesting that these compounds have a common
mode of action.
The two-microelectrode currelat-clamp and vohage-clamp teclmiques have been used to exalnine
m t t s c l e n l e m b r a n e conductance changes in Ascaris
produced by acetylcholine, levamisole, pyrantel
and morantel (Martin, 1982a; Harrow & Gration,
1985). These anthehnintics have I)een shown to
increase the meml)rane conductance and depolarize the membrane by opening non-selective cation
ion-channels that are permeable to both Na + and
K+. Simultaneous application of acetylcholine and
pyrantel showed that both agonists acted on the
same nicotinic receptors, and the relative potency
of the anthehnintics in bath application exper-

inlents was: morantel=pyrantel>levamisole>acetylcholine (Harrow & Gration, 1985).

In addition to effects of tile anthehnintics on
the membrane conductance of the muscle,
Harrow and Gration (1985) described the conductance dose-response curves for pyrantel and morantel as being 'bell shaped'. The effect of this concentration-effect relationship
is that
the
conductance-response increases then decreases as
the concentration of the anthelmintics rises. One
explanation tor this phenomena is that of open
channel block (Colquhoun & Sakmann, 1985) by
the anthelmintic. Levamisole, pyrantel, morantel
and oxantel are large organic cations, and could
enter the nicotinic ion-channel from the outside
and tl-y to pass through the channel like Na + or K+
ions but produce the block at the narrow region
of the channel, the selectivity filter. This block
would be voltage-sensitive and increase with hyperpolarization of the membrane and c o n c e n -

14

THE VETERINARY .JOURNAL 154, 1

(c)~

(a)

t ~ ~

Dorsalnelve cord

Syncylium

eao
~'ry

Arm

Syncytium
s cord

J
200 I.tm

1 mm

Ventral nerve cord

(b)
Posterior

Anterior

DE3

.%

-YY Y V Y

Dorsal
nerve

Y Y

cor

Left ./
commissure

J. ~ k v
v

YY

YY

.t ,k 2~ ,k

.,

YY

YY

YY

o w

Y Y Y Y Y

7
Y

g
g

Ventral
nerve
cord

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 producing open channel block has been established
using
single-channel
recording
techniques
(Robertson & Martin, 1993).

Single-channel currents activated by nicotinic

anthelmintics
Initially, levamisole-activated channel currents
[Fig. 3(a)] were recorded fi'om the muscle vesicle

preparation (Robertson & Martin, 1993) using the

patch-clamp technique. The ion-chalmel currents
activated by low levamisole concentrations were
shown to cart3, cations and to have similar kinetics
to acetylcholine-activated channels. The levamisole activated channels have a mean open time of
1.34 ms and a conductance in the range 20-45 pS.
At higher concentrations of levamisole, a flickering open channel block that increases on hyper-

MODES OF ACTION OF ANTHELMINTI(" DRUGS

polarization is obsel-ved [Fig. 3(b)]. At - 5 0 mV
the dissociation constant for the channel block
was 123 ~,xl. In addition to the flickering block,
clusters of openings were also observed at high
levamisole c o n c e n t r a t i o n s where openings were
separated by long (seconds) closed times: this
chlstering behaviour is similar to that asstuned to
be desensitization in vertebrates but was m o r e
voltage-sensitive and m o r e p r o m i n e n t at hyperpolarized potentials in Ascaris.
Pyrantel-activated channels [Fig. 3(c)] have also
been r e c o r d e d using the same preparation
(Robertson el al., 1992). Pvrantel-activated channel showed at least two distinguishable conductance levels: the main c o n d n c t a n c e level was near
40 pS and the smaller c o n d u c t a n c e level was near
'22 pS, suggesting the p r e s e n c e o f m o r e than o n e
type o f nicotinic r e c e p t o r in the m e m b r a n e .
Again, the channels o p e n e d by pyrantel were
shown to be p e r n m a b l e to m o n o v a l e n t cations and
channel-block o c c u r r e d at hyperpolarized potentials. T h e channel block o c c u r r e d m o r e readily
with pyrantel than with levamisole as shown by the
tact that the dissociation constant, /x]~, for levamisole was laigher than that o f pyrantel (levamisole
~ at - 5 0 ntV 123 ~txl; pyrantel K]~ at - 5 0 naV
37 btxl).
Tal)le II summarizes the c h a n n e l I)locking
properties o f levamisole and pyrantel and, in
addition, m o r a n t e l and oxantel. Some general
points may be m a d e by e x a m i n i n g Table II. It can
be seen that all the nicotinic anthehnintics prod u c e o p e n channel-block, a form o f self antagonisnt, and that levamisole is the least p o t e n t at
blocking its own channel. T h e signiticance of the
o p e n c h a n n e l block ntay relate to the ability o f
some n e m a t o d e s to resist the effects of nicotinic
anthehninitics: c h a n n e l block may o c c u r at such
low anthelmintic c o n c e n t r a t i o n s that normal concentrations o f anthelmintic are ineffective.
In Ascmis, the channel-blocking ability o f
pyrantel is greater than levamisole, but the
greatest channel-block is p r o d u c e d by morantel

15

with oxantel, occupying an i n t e r m e d i a t e position

between morantel and oxantel. T h e least p o t e n t
agonist in Ascaris is oxantel which p r o d u c e s the
lowest probabilitT o f channel o p e n i n g even at
high c o n c e n t r a t i o n s (Dale & Martin, 1995). haterestingly, oxantel is not effective therapeutically
against ascariasis but is used instead to treat
7)'ichuris infections. T h e efficacy o f oxantel against
7)ichuds but not against AscaHs may be due to differences in the nicotinic receptors o f the two
species o f n e m a t o d e : oxantel may be effective and
p r o d u c e o p e n i n g o f the TtJchuris nicotinic acetylcholine r e c e p t o r but not so effective on the Ascaris
receptor. T h e c o n c e n t r a t i o n s o f oxantel and
pyrantel along the intestine may also be a factor
affecting efficacies o f the drugs.
Non-nicotinic or 'muscarinic' choliner~c receptor~
in nematodes a n d resistance to anthelmintics
In a n u m b e r o f vertebrate preparations, acetylcholine is able to alter the probability of" o p e n i n g
o f v o l t a g e - d e p e n d e n t channels by acting via G-protein-coupled muscarinic receptors. An action in
n e m a t o d e s at acetylcholine receptors analogous
to vertebrate muscarinic receptors, m o d u l a t i n g
voltage-sensitive ion channels has yet to be fully
reported.
T h e r e is biochemical exidence for the presence
of 'muscarinic' receptors in Ascaris muscle:
(1) D o n a h u e et aL (1982) have obsevved that
effects of cholinergic stimulation inchtdes
increases in levels o f cyclic-AMP;
(2) Arevalo and Saz (1992) have observed that
acetylcholine increases levels o f phosphoD, lcholine, 1,2-diacylglycerides and phosphatidic
acid, and d e m o n s t r a t e d the p r e s e n c e o f phospholipase C activitT. D o n a h u e et al. (1982)
and Arevalo and Saz (1992) did not distinguish between 'muscarinic' or 'nicotinic'
cholinergic receptors.
Previous studies on n e m a t o d e s have shown that
resistance to nicotinic anthelmintics may take two
forms: (1) T h e selection o f genetically resistant

Table II
Voltage sensitivity o f dissociation constants o f o p e n channel block by the nicotinic anthelmintics

Membrane
-50 mV
-75 mV

Voltage-sensitivlty of h]~

I33 l.evamisole

2KI, l5,rantel

Kl~ Morantel

KR Oxantel

123 I.tXJ
46 IJM

37 IJ.Xl
20 I.t~l
e-fold eve D' 40 mV

12 p.xt
1 ~,xl

18.5 I.tM
7.5 law

e-fbld every 22 mV

e-fold ever), 29 mV

e-fold every 20 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

%
5

1 pA L
0

10

15

20 ms

(b~

1PA3~ms

............

~~

............................................ i

......

C
0

(c) Pyrantel
C
15O
10%
C

00

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; patchpotential -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

mutants with modified nicotinic acetylcholine

receptors on muscle (Lewis el al., 1980; Lewis et
al., 1992). (2) Accomnaodation and recovery of
parasites after long periods of exposure to the
anthelmintics (Lewis et al., 1980; Lewis et al.,
1992), and which may be explained by nicotinic
receptor desensitization.
These resistant or recovered nematodes no
longer respond to the nicotinic anthehnintics, but
interestingly, may still respond to acetylcholine
(Coles et al., 1975). One explanation for these
resuhs is that in addition to tile nicotinic receptor
on nematode muscle, there are other non-nicotinic cholinergic receptors present on muscle
not stimulated by the nicotinic anthelmintics.
Such receptors may facilitate contraction by being
coupled via G-proteins to vohage-activated
channels.

Genetics of resislance to nicotinic anlhelmintics

Tile genetics of resistance to the anthelmintic
levamisole has been studied in the laboratory
using the small fi'ee-living soil nematode, Caenorhabdili.~ eh,gans. Several hundred levamisole-resistant alleles have been identified that were isolated
ill several screens (Lewis el al., 1980; Lewis e/ al.,
1992). The genes responsible tbr this resistance
inclucled: h,v-1, unc-29 and unc-38 that encode tile
proteins subunits that make up the nicotinic ion
channels of the nematode. In addition, other
genes including: un, c-50, unc-63 and unc-74 that
are believed to be involved in receptor biosynthesis have been identified.
Only two strains of lev-1 (21 and x61) were
dominant when crossed over with wild-types (nonresistant) (Fleming el al., 1994). The lev-1 (x21)
strain only inw~lves mutation at a single aminoacid, replacing glutanaic acid in the ion pore of
the receptor ion channel (ill alpha-7 at position
237) with a positively-charged lysine. This change
is believed to be sufficient to change the ion channel from cationic to anionic, i.e., to convert tile
receptor from an excitatory channel to all inhibitory one. The ~,-1 (x61) swain contains tile
amino-acid leucine ill the pore of tile ion-channel
(in alpha-7 at position 247): this point mutation is
expected to produce increased desensitization
and reduced affinity for levamisole, rendering levanfisole less potent as an agonist.
The genetics of levamisole resistance in parasitic nematodes remains to be studied.

17
Dichlo,wos

o
H
CH30\ li
I
C]
/p--o--c~-c /
CH30

C1

Haloxon

0
C1CH2CH2 0 /

CH3
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
INHIBITORS

CHOLINESTERASE

Dichlmvos, haloxon
Compounds like dichlorvos and haloxon are
selective organophosphorus anti-cholinesterases
(Fig. 4), and have an anthehnintic action, as well
as all insecticidal, action. Dichlorvos and haloxon
can control insect parasites, as well as hehninth
parasites. The mode of action of these compounds
is to block tile action of the parasite enzyme, acetylcholinesterase, leading to the excessive build up
of tile neurotransmitter, acetylcholine. This mode
of action also predisposes towards toxicity in the
host
animal
where
acetylcholinesterase
enzymes are also present. Because more selective
combined
anthehnintic+insecticidal
agents
(avermectins and milbemycin) are available, the
organophosphorus compounds are now used less
frequently.
The existence of cholinesterase, the enzyme
that breaks down acetylcholine, was first described
in Ascmis by Bueding (1952). The distribution of
cholinesterase was described by Lee (1962) who
used histochemical techniques. In the head
region of Ascmis, most of the enzyme activity is
associated with the contractile spindle region of
the muscle and is in the extracellular matrix. Lee
(1962) also described cholinesterase activity on
the muscle arms near their endings on the nerve
cords but not on the bag region of the muscle.
In C. elegans, three classes of acetylcholinesterase are recognized: class A, class B, and class C,

,
/ \
COOH

GABA

,A,
\ NH;?

Piperazine

2 pA

100ms

GABA AGONIST

and are known

to be products of three separate

cind are-3 (Opperman
& Chang,
1992). The three classes of acetylcholinesterase
are sepal-able by their solubility in Triton, their
temperature
stability and sensitivity to cholinesterase antagonists. In C. elegcr??.r,the major form is
class B which is distributed in the head and body;
classes A and C have a distribution
biased towards
the head. Extraction of the different
classes for
biochemical
characterization
has produced
evidence that the enzyme may exist as a monomer,
dimer and tetramer. Defects in the ace-1, m-e-2 mcl
ace-j genes suggest that the functions of the different classes are supplementary
but that if there
are defects in all of the genes, then elevated levels
of acetylcholine
occur in C. eleguns and there is
motor
incoordination
(Opperman
& Chang,
1992).

genes:

owl,

Although acetylcholinesterase
is responsible for
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 acetylcholine in the host intestine blocking peristalsis of the
gut) has now been rqjectecl. The identification
of
the function of secreted cholinesterase
hy parasitic nematodes, ancl the abilitv to antagonize this
enz\me, mav lead to the increased use of anticholinesterases in the future to facilitate renio\xl
of gut nematodes.

uw2

Piperazine has a heteroq~lic ring structure (Fig.

.5), ancl unlike y-amino-butyric
acid (GABA), lacks
a carboxvl group. Howe\*er. these t~vo con~pouncls
act on the same receptor that is a ligancl-galecl Clchannel found on the synaptic ancl extrasynaptic
membrane
of nematocle muscle (Martin,
1980;
h4artin. 1982). Bath application
of GABA or piperazine increases the opening of the muscle membrme <:I- channels, hvperpolarizes the membrane
conductance
potential,
increases tile membrane
and produces spastic paralysis. Piperazine appears
to ha\pe a selective effect on large nematocles of
the gastrointestinal
tract, perhaps because the
high CO, environment
allows CO, to mimic the
missing carboxy] group of piperzine.
Piperazine
appears less active against nematodes that occup!
a high 0, environment.
Piperazine- and GABA-activated
channel currents (Fig. 5) ha\re been recorded
using cellattachecl and isolated outside-out patches (Martin,
1985). Although
the channels may have more
than one conductance
level, the most frequently
observed is 22 pS for both agonists. However, the
mean open time of the channels for piperazine
was shorter, 14 ms, than that produced by GABA,
which produced
channels that had mean open
times of 32 ms. GABA is more than lo-100 times
more potent than piperazine when conductance
dose-response relationships are examined cluring
bath application
of the agonists (Martin,
1985).

MODES OF ACTION OF ANTHELMINTI(" DRUGS

Ivermectin
H O ~

a ~OCH'~

H3C

0...1~ ~

CHa

.-CHa

IlL o_._A

h-o j.

Abamectin
OCH.~
HO_

2-.
"~

~" " ~
0 ~

hl

OCHa

HaC/k" 0''~ 0 ~ [ ' ~

OH

"

CH
:'

CH,

Doramectin

l! o._.'o

GLUTAMATE-GATED CHLORIDE (GLUCL)

RECEPTOR POTENTIATORS

Ivermectin, abamectin, doramectin, milbemycin D,

moxidectin

]__I ~
OH

H I - . . ~ CH:,
OH

Milbemyein D

H3C"

"]1

OH

Moxidectin

c_Y
0

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
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
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 prod 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
with lower c o n c e n t r a t i o n s o f GABA than piperazine. Therapeutically, however, GABA would be
ineffective because it is not selective like piperazine and is highly ionized and does not cross the
cuticle.

CHaR

HO.~ ~ . , .CHa

CHa

~CHa

Fig. 6.

19

~CHa

CHa

T h e avermectins (Fig. 6) are a g r o u p o f broadspectruna, macrocyclic, lactone antibiotic anthelmintics used to control n e m a t o d e parasites in
man and animals (Campbell & Benz, 1984), and
assumed to have the same m o d e o f action in both
(Shoop et al., 1995). T h e y are used to control
onchocerciasis (river blindness) in h u m a n s and
gastrointestinal, cardiac and respirato~-y n e m a t o d e
parasites o f domestic animals. T h e m o d e o f action
o f the avermectins is to selectively paralyse the
parasite by increasing muscle CI- permeability, but
the identity o f the channel targeted by the avermectins has been controversial, see Arena (1994)
for a r e c e n t review. Cloning o f a GluCl-otl and a
GIu('I-~3 subunit fi'om the model soil n e m a t o d e C
elegans, and co-expression o f the subunits in Xenopus oocytes, has led to the identification o f an
avermectin-sensitive GIuC1 ion-channel (Cully et
al., 1994). T h e effects o f avermectins, at low concentrations, are to potentiate the effect o f glutamate, and at h i g h e r concentrations, the avermectins o p e n the glutanmte-gated c h a n n e l directly.
T h e selective therapeutic effects o f the avennectins could be explained by an action o n a Glu CI-

Fig. 6. Chmnical structure of avermectin and

milbenlycin anthelmintics. Ivermectin is at least 80%
22,23 dihydroavermectin Bla (R is sec butyl) and not
more than 20% 22,23 dihydroavermectin Blb (R is
isopropyl). Abamectin is at least 80% avermectin Bla (R is
sec butTI) and not more than 20% avermectin Blb (R is
isopropyl).

FI LI/,q.~.fiIIBL. Z~t)le, C

20

THE VETERINARY JOURNAL, 154, 1

Terminal i
bulb
:

Isthmus

Carpus
Metacarpus

---

Procarpus

:::::::::::::::::::::::

i::ii!iiiiiiii::i
i
i! i

~ p m o

::.::::.: p m 3

::

pm4

pm3

:.::::.: ~

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)~:~).

Head

Pharynx

Nerve cord

Intestine

Fig. 8. Diagram of the preparation used to record the

electrophysiological effect of glutamate and milbemycin
D on the phaiTngeal m e m b r a n e potential and input
conductance. Two micropipettes were inserted into the
pharyngeal muscle: one was used to record m e m b r a n e
potential (V); the o t h e r was used to inject current (I).

ion-channel that is present in parasitic nematodes

but not present in the host animal.
Molecular experiments using the lacZ marker
suggest that the GluC1-]3 subunit of the glutamate

channel is exl)ressed ill tile I)halo'nx of C. e/egans

pM4 muscle (Fig. 7) (Laughton el al., 1995). The
location of the Glu(]l-0tl suhui-lit is n o t known.
The phai)'ngeal muscle is required ti)r feeding
and is known to receive an inhihitorv motor netttone, M3 (Fig. 7), that is not likely to be GABAergic (Laughton et al., 1995) but glutamatergic
(Avel)', 1993). The location of an avermectin-sensitive (;luC1 channel, has heen identilied using a
two microelectrode current clamp technique
(Martin, 1996) in the parasitic nematode, A. Mtttm
(Fig. 8). Experiments show that the pharyngeal
muscle of this parasite possesses glutamate receptors that gate chloride channels and that are sensitive to the avermectin analogue milhemycin D.

Effect of glutamate
Fig. 9 shows effects of the application of milbemycin D and glutamate oll Ascaris phal3,ngeal
muscle input conductance and membrane potential: glutamate [Fig. 9(b)] produces a transient
small hyperpolarization of 1 mV associated with
an increase in membrane conductance. The effect
of glutamate but not GABA [Fig. 9(c)] in pharyngeal AscaTis preparations is to produce a reversible
increase in input conductance associated with a
small change in membrane potential and to

MODES OF ACTION OF ANTHELMINTIC DRUGS

(a)

r" -7iillI[llililliitlllll

Effect of milbemycin D

. . . .

---

. . . . .

llll "

- -

5 mV _ _

30s

890 nM Milbemycin
(b)
.....

:,,,ii',iiii,,.,?d.Tifflntitl'tl[lillllllllillilil

100 laM Glut.

(c)

llllllIHltli!
llTiii,Nl!lI,E!ll!
l ! ilt 5mVl__
I,[!iIL!!I

tttlillhlll,Ll,li&L,ll[tl[,tlktdlt[
tilhliltttt
30 s

300 ~IMGABA

Fig. 9. (a) Effect of 890rim milbemycin D on the

memb,ane potential and input conductance. Milbemycin
slowly p,oduccd an increase in input conductance that
was not reversed on washing (not shown). (b) Effect of
application of 100 ItM t.-glutamate on membrane potential
and input conductance. Different preparation fi'om (a).
Glutamate (applied during horizontal bar) produced a
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)
after 4 rain. (c) After washing the preparation the input
conductance of the pharynx returned towards control
levels (142 ItS) and the effect of 300 gM GABA was tested
without effect (applied during the horizontal bar). The
lack of effect of GABA was not due to desensitization
because subsequent application of 100~xt L-glutamate
increased the input conductance again (not shown).
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
n e m a t o d e parasite f e e d i n g process. Zero-C1solutions were shown to abolish reversibly the gluta m a t e - i n d u c e d c o n d u c t a n c e responses (Martin,
1996) a n d were used to d e m o n s t r a t e that the gluta 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

Ascam.

21

T h e effect o f ivermectin a n d m i l b e m y c i n D, on
the C. elegans avermectin-sensitive g l u t a m a t e
r e c e p t o r e x p r e s s e d in Xenopus oocytes is to prod u c e a p o t e n t i a t i o n o f g l u t a m a t e effects a n d to
p r o d u c e a slow irreversible increase in c o n d u c tance o f the m e m b r a n e (Cully et al., 1994). T h e
effects o f m i l b e m y c i n D on the i n p u t c o n d u c t a n c e
o f the Ascaris p h a r y n g e a l p r e p a r a t i o n is to prod u c e a small c h a n g e in m e m b r a n e c o n d u c t a n c e
a n d to p o t e n t i a t e the effect o f low c o n c e n t r a t i o n s
o f g l u t a m a t e [Figs. (9a), 10(a), (b), (c) a n d (d)].
T h e location o f the GIuC1 r e c e p t o r or the avermectin b i n d i n g 0t-subunit in the n e m a t o d e
r e m a i n s to be e x p l o r e d fully. R e c e n t m o l e c u l a r
biological e x p e r i m e n t s with C. elegans on an ivermectin-resistant strain (avr-15) suggest that it is a
GluCl-0t2 subunit that is p r e s e n t in the p h a r y n g e a l
muscle of C. elegans n o t the GluCl-otl (review:
Cully el al., 1996). T h e location a n d function o f
the original GluCl-cd subunit r e m a i n s to be determined. 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
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 avermectins requires m u t a t i o n o f m o r e than o n e
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
( H o l d e n - D y e & Walker, 1990). T h u s again it
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
o n e site o f action in parasitic n e m a t o d e s .

INCREASED

CALCIUM

PERMEABILITY

Praziquantel
Fig. 11 shows the c h e m i c a l su-ucture o f praziquantel. Praziquantel has a selective toxic effect
on schistosome parasites, w h e r e its m o d e o f action
has b e e n studied m o r e extensively (Andrews et al.,
1983; H a r n e t t , 1988) t h a n in cestodes. Many
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
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 diag 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
muscle that illustrates the k n o w n sites w h e r e Ca `'+
crosses into a n d b e t w e e n intracellular c o m p a r t ments, a n d t h e r e f o r e , the possible sites for praziquantel action.

'22

]'HE VETERINARVJOURNAI~, 154. 1

12f

(a)
/

100 ~-

60-

4O

2O

0.1

0.2

0.3

(b)

0.4
0.5
Milbemycin (prq)

(c)

0.6

I
0.7

0.8

0.9

(d)

5 mV
30 JaMGlut.

30 p.xl Glut.
in 30 nM Milb.

30 p.~ Glut.
in 300 nM Milb.

1 rain

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.

Effect o'n Ca "-'+permeability

Praziquantel

.N

Fig. 11.

Chemical structure of praziqtmn tel.

P r a z i q u a n t e l is k n o w n to i n c r e a s e ' ' C a '-'+ influx

across the s c h i s t o s o m e t e g u m e n t a n d to cause
rapid
muscle
contraction
o f the
parasite
( M e h l h o r n el al., 1981). It is k n o w n that b a t h i n g
the parasites in Ca'-'+-fi'ee m e d i u n a blocks the praz i q u a n t e l i n d u c e d c o n t r a c t i o n (Fettever el al.,
1980; W o l d e - M u s s i e et al., 1982; T h o m p s o n el al.,
1984) b u t the effect o f b a t h i n g the p r e p a r a t i o n in
Ca"+-fi'ee b a t h s o l u t i o n is n o t i m m e d i a t e , r e q u i r i n g
m o r e that 10 rain to be effective. T h e effect o f praz i q u a n t e l o n Ca `-'+ influx suggests that the sites o f
a c t i o n are Ca '-'+ p e r m e a b l e ion c h a n n e l s in the
m e m b r a n e o f the t e g u m e n t a n d m u s c l e cell (Blair
et aL, 1994). T h e c o n t r a c t i o n s in s h i s t o s o m e s are
r e v e r s e d if p r a z i q u a n t e l is r e m o v e d a n d m a y be

MODES OF ACTION OF ANTHELMINTIC DRUGS

Double

Host (Blood)

membrane

Ca 2+ 5

I
'

ua
1 ,-, I

'V.,
"Na

23

"

.
_ " .

2+
,

C a 2+
-

"

"
.

"~

z,

Ca2+

"
"

- 2+
Ida

. .

- Ca2+
4

V
'

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+ 01the calcium-channel blocker D-600 (Fetterer et al.,
1980; Wolde-Mussie et al., 1982). The application
of a high K+ solution to depolarize the schistosome preparation results in a rapid depolarization, but the application of praziquantel resultsin
a slower onset depolarization. From these observations, it may be concluded that praziquantel somehow results in an increase in Ca `-'+ permeability
across parasite m e m b r a n e s via channels that are
not activated by depolarization. These channels
must be pharmacologically different fi'om those of
the host animal or the praziquantel would affect
the host animal.
Experiments on a snail smooth muscle preparation (Gardner & Brezden, 1984) have illustrated that caffeine mimics the effect of praziquantel in producing contraction, and that prior
treatment of the muscle preparation with caffeine

will eliminate the effect of praziquantel on the

muscle preparation in Ca'-'+-free solutions. These
experiments suggest that caffeine and praziquantel share the same site of action.
In vertebrate smooth muscle preparations, caffeine acts to stimulate the o p e n i n g of a large cation channel known as the CICR channel (calciuminduced calcium release channel) ( H e r r m a n n Frank et al., 1991) in the sarcoplasmic reticulum.
This channel is activated by cytosolic rises in Ca '-'+,
adenosine triphosphate (ATP) and caffeine but is
inhibited by Mg '-'+. T h e CICR channel p r o d u c e s
regenerative Ca `-'+ release from the sarcoplasmic
reticulum following entry of Ca `-'+t h r o u g h voltageactivated channels (Gregoire et al., 1989). Large
cation channels, which might be similar to the
CICR channel, have been observed in the tegumental m e m b r a n e of schistosomes (Day et al.,
1992).

24

THE VETERINARY .]OURNAL. 154, I

Parasite antigen exposure

It has also been found that curative doses of
praziquantel in infected animals resuhs in
antigens of schistosomes being exposed and
binding and penetration of host antigen cells to
the parasite after 17h treatment (Harnett &
Knsel, 1986; Brindley & Sher, 1987). The immune
system of the host, therefore, plays an important
role in the praziquantel-induced death of the
parasite. One explanation for the exposure of the
parasite antigen may be that increased cvtosolic
Ca `-'+ causes phospholipase C activation and then
activation of protein kinase C (Berridge & h-vine,
1984; Sigiya & Furuyanaa, 1990). The phosphoDqation of proteins by protein kinase C could then
destabilize the tegument causing the observed
vacuolation and antigen exposure (Wiest el al.,
1992). Once again the mode of action of praziquantel relates to an effect on Ca `-'+ permeability
of the tegumental nmmhranes. Praziquantel is
used fi'equently to treat tapeworm infestations but
less is known of the mode of action in this group
of parasites. It is presumed that there is a common
mode of action against trematodes and cestodes.
INHIBITION OF MICROTUBULE
FORMATION, ~-TUBULIN BINDING

Benzimidazoles: thiabendazole, cambendazole,

mebendazole, fenbendazole, oxibe~Mazole,
oxfendazole, albendazole, albendazo& sulphoxide,
parbendazole, flubendazole, t~4clabendazole & prodrugs: netobimin, febantel, thiophanate
Fig. 13 illustrates the chemical structures of the
benzimidazole anthehnintics and prodrugs. Prodrugs are converted to active benzimidazoles by
metabolic processes in the host animal so that it is
the active metabolites that are responsible for the
anthelmintic action. Triclahendazole, the flukicidal compound, is assumed to act hy the same
mechanism as the other benzimidazoles but a
clear explanation for its selective effect against
fluke is not known (McKellar & Kinabo, 1991) but
may be related to its high level of plasma protein
binding.

fl-tulmlin binding
Mebendazole (Borgers et al., 1975; Van den
Bossche & De Nollin, 1973) and flubendazole
(Van den Bossche & De Nollin, 1973) induce the
loss of cytoplasmic microtubules of the tegumental and intestinal cells of cestodes and nematodes, and this is followed by loss of transport of

secreta D, vesicles, a decreased glucose uptake and

an increased utilization of stored glycogen. In
Ascaris, mebendazole is taken up I)y phaiTngeal
and intestinal cells where it is tound in the cytoplasnaic fi'action bound to proteins with molecular
weights of about 50 and 100 kDa that represent
mollonlel-s and dimers of tubulin. O t l r current
understanding of the mode of action of the benzimidazoles thus started when it was recognized that
the effect of mebendazole on Ascads was to disrupt the microtubules of the intestinal cells producing an inability to take up ghtcose (Van den
Bossche, 1972; Van den Bossche el al., 1982). It
was found that benzimidazole anthelmintic competed with the binding site for [H :~] colchicine on
13-tuhulin and that potency of the I)enzimidazole
antlmhnintics correlated with the dissociation constant for hinding to nematode I]-mbulin (Lacey,
1990; Sangster et al., 1985).
Micrombules are intracellular organelles that
serve a variety of fimctions including movement of
chromosomes during cell division; providing the
structural skeleton to the cell; movement of intracellular particles including e n e r ~ naetaholites:
and exocvtosis. They are lound in both animal,
plant, fungi and some bacterial cells (SnTer,
1995).
Microtul3ules are composed of t w o 450 aminoacid proteins known as c~-tubulin and I]-tt!bulin.
Fig. 14 is a diagrammatic representation of [3-tuhttlin. It is composed of three domains: domain 1 is
34 kDa in size; domain 2 is 19 kDa in size and the
tail is 2 kDa in size. There are GTP binding sites
present which bind to sites I, II, III, and IV on the
I]-tubulila. The 13-tul)ulin is folded so that the GTPhinding sites form a pocket.

Formation of microtubules
The formation of mio'otubnles is a dynamic
process (Fig. 14.) Formation involves the polynmrization of tubulin at one end (the positive pole)
and the depolymerization at tile other end (the
negative pole). The microtubules that tbrm are
made up of 13 tubulin molecule rings (6 0f
tubulin+7 13-tubulin alternating with 7 0t-tubulin+6
[3-tubulin rings). Seventy-five to 85% of the mass
of microtubules is composed of the tubtdin proteins but in addition, microtubule-associated proteins (MAPs) are present stabilizing the structure.
A number of factors favour polymerization including GTP, Mg'-', and an increase in temperature (to
37C). A decrease in temperature (to 4C), the
presence of Ca `-'+or cahnodulin will favour depoly-

MODES OF ACTION OF ANTHELMINTIC DRUGS

25

BENZIMIDAZOLES

"I

PRODRUG

NHCO2CH3

N~C
NHCO2CH3

iL

NHCOCH2OCH3

Febantel: converts to fenbendazole

Thiabendazole:

R = H--

Cambendazole:

R = (CHa)2CHOCONH--

BENZIMIDAZOLE CARBAMATES
R

TRICLABENDAZOLE
CI

CI

'

'

.N.
[

NHCO2CH a

i
N

I
CH 3

"~
"

SCH 3

H
H

Oxibendazole: R = CH~CHzCH20-Albendazole: R = CHaCH2CH2S-Fenbendazole:

R =

(/,, - -

~'~

S--

@
@
@,
O

Oxfendazole: R =

S---

El

Mebendazole: R =

Flubendazole: R =

C m

0
S m

Fig. 13. Chemical structure of benzinfid~oles, benzimidazole carbamates, prodrug febantel and the antifluke drug
triclabendazole.
m e r i z a t i o n . Interestingly, c~- a n d [3-tubulin will
pol),merize into a n u m b e r o f s h a p e s (rings a n d
sheets) in a d d i t i o n to m i c r o t u b u l e s w h e n in vitro
p r e p a r a t i o n s are m a d e . T h e t b r m a t i o n o f microt u b u l e s m a y be i n h i b i t e d by substances that b i n d
to the l e a d i n g e d g e (the positive pole) o f p o l y m e r ization. This p r o c e s s o f i n h i b i t i o n is k n o w n as
' c a p p i n g ' , a n d c o l c h i c i n e , vinblastine, vincristine,

the mitotic i n h i b i t o r s a n d b e n z i m i d a z o l e s can d o

tiffs by b i n d i n g to ~-tubulin m o l e c u l e s .
T h u s the m o d e o f a c t i o n o f the b e n z i m i d a z o l e
a n t h e l m i n t i c s is the selective b i n d i n g to n e m a t o d e
~-tubulin, a n d c o n s e q u e n t i n h i b i t i o n o f m i c r o t u b u l e f o r m a t i o n . T h e effects are, t h e r e f o r e ,
slower in o n s e t t h a n the a n t h e l m i n t i c s t h a t act as
n e u r o t r a n s m i t t e r agonists, a n d i n c l u d e slow o n s e t

26

THE VETERINARYJOURNAL. 154, 1.

[3-Tubulin
Domain I: 34 KDa
100

Domain: II 19 KDa Tail: 2 KDa

IH TI300im

200

i i

400

Hinge

Microtubule formation from cx- and ~-tubulin

Q) a-tubulin
+ ~-tubulin~

~ ( ~
~ D ~ :

13 membered
spiral made of
alternating ~and [3-tubulin

Vesicle
transport along

Negative pole.
Microtubule
breakdown
increased by fall in
temperature and Ca ++.

Positive pole.
Microtubule
formation
increased
by GTP, Mg+,
temperature
inhibited by
benzimidazoles.

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).

starvation of the nematode (intestinal cell

disruption) and an inhibition of egg production.

Genesfor fl-tubulin and resistance

In nematodes, two isotypes of [3-tubulin have
been identified: isotype I and isotype II which

have s e p a r a t e g e n e s ( G u e n e t t e et aL, 1991;

G u e n e t t e et aL, 1992; L u b e g a et al., 1994). E a c h o f
these isotypes have alleles: u p to six f o r isotype I;
a n d u p to 12 f o r isotype II. It is n o t k n o w n if the
specific isotypes have d i f f e r e n t f u n c t i o n s in the
nematodes.

MODES OF ACTION OF ANTHEI.MINTIC DRUGS

A reduction in the nunfl)er o f isotype alleles fi)r

]3-mbulin is associated with the a p p e a r a n c e of
benzimidazole resistance (Roos e/ al., 1995).
Resistance is associated with a progressive loss o f
alleles o f isotype 1 and a total loss o f isot?'pe 2
fi'om the n e m a t o d e population. A resistant population o f Haemonchus co~tlorlus has b e e n characterized by only the presence o f a single [3-tubulin
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
resistance can be e x p l a i n e d by a loss o f susceptible
p h e n o t y p e s and the st, rvival of a resistant p h e n o type and its increased representation in the
r e m a i n i n g population.
In fungi, benzimidazole resistance is associated
with the a p p e a r a n c e o f a different form of [3-tubulin (Fujinmra el al., 1992; J u n g el al., 1992) which
is characterized by the a p p e a r a n c e of tvrosine
instead of phenylalanine in position 200. Because
m a m m a l i a n [3-tubulins have also tvrosine present
in the 200 a m i n o acid position (Lewis el al., 1985)
it is unlikely that benzimidazole resistance may be
o v e r c o m e by changes in d r u g chemistvv. It would
not be possible to design a selectively-toxic agent
against the ['ungi because the fungi and host ~-tubulins would both bind the benzimidazole to the
same d e g r e e and st) be loxic to both species. A
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
pm-asites.

P R O T O N I O N O P H O R E S : SALICYLANILIDES
AND SUBSTITUTED PHENOLS.

Salic~,lanilides: closantel, raJbxanide, o.~yclozanide,

bro/i'anide a~d substituted phenols: nitroxynil,
niclopholan, hexachorophene dibromsalan &
niclosa mide
T h e plaarmacolog,3, o f a range o f anti-fluke
drugs have been reviewed (McKellar & I<Anabo,
1991). In tiffs section, the m o d e of action of the
p r o t o n i o n o p h o r e s (often called oxidative phospholwlase u n c o u p l e r s ) are considered. T h e chemical structure salicylanilides and nitroxynil illust,-ates that each anthelmintic molecule possesses a
d e t a c h a b l e p r o t o n (Fig. 15). These molecules are
vmT lipophilic and may shuttle protons across
m e m b r a n e s , particularly the i n n e r mitochondrial
m e m b r a n e . Fig. 16 represents the process by
which ATP p r o d u c t i o n occurs in the m i t o c h o n d ria, that is c o u p l e d to the p r o t o n gradient across
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
p h o s p b o u l a t i o n may be summarized as electrons
fi'om NADH or FADH being conveyed t h r o u g h a

27

series o f protein c o m p l e x e s on the inner mitochondrial m m n b r a n e . This process leads to protons being p u m p e d out o f the m i t o c h o n d r i a
matrix p r o d u c i n g a p r o t o n motive force that is
due to the pH gradient and t r a n s m e m b r a n e electric potential. ATP is synthesized when the protons flow back into the m i t o c h o n d r i a l matrix
t h r o u g h an enzyme complex. This process of oxidative phosphoD, lation takes place in the host animal (StD, er, 1995), as well as in the parasitic helminths (Van den Bossche, 1972; McKellar &
Kinabo, 1991).
Lipid soluble substances that can car D' protons
may shuttle across the inner m e m b r a n e o f the
m i t o c h o n d r i a , and remove the p r o t o n gradient
and u n c o u p l e oxidative phosphorylation so that
the oxidation o f NADH and FADH is no longer
linked to the p r o d u c t i o n of ATP and may carry, on
without its production. Fig. 15 shows the position
o f the p r o t o n that is able to dissociate froni a variety of c o m p o u n d s . 2,4-Dilfitrophenol, carbonyl-pt 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)
and laexachloroplaene, nitroxynil, oxvclozanide
(Corbett & Goose, 1971; Edwards el al., 1981b) are
all lipophilic c o m p o u n d s that are capable o f
carr}.'ing p r o t o n s across m e m b r a n e s , and therefore, may act to u n c o u p l e oxidative pbosphoD,1ation 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.
T h e nleclaallism of action of the p r o t o n ionophores has been assnmed to be to selectively
u n c o u p l e oxidative pbosphoD'lation in parasite
m i t o c h o n d r i a . Pax and B e n n e t t (1989) have
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
o f the parasite may not be the site o f action of the
p r o t o n iollophores. T h e v observed that application of closantel to Schistosoma mansoni and Fasciola hepatica p r o d u c e s a fall in t e g u m e n t a l pH
(6.8-6.5) when m e a s u r e d with an intracellular
electrode, and that this c h a n g e in pH o c c u r r e d
after 10 rain and before an), c h a n g e in the production o f ATP bv the parasite. T h e intra-tegumental [',all in p H was associated with a reduction
in motility o f the parasite and could explain the
anti-parasitic action o f closantel. It was c o n c l u d e d
by Pax and B e n n e t t (1989) that closantel was a
m e m b r a n e active molecule and that it may affect a
n u m b e r o f biochemical and physiological processes. T h e s e processes are presumably sensitive to
changes in intra-tegumental pH. T h u s the site o f
action o f the p r o t o n i o n o p h o r e s may include the
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

CI

CI

OH

C1

CI

Rafoxanide
I

OH

C1

~ C O ! ~
I

OH

Fig. 16. Diagram of the mitochondria illustrating the

inhibitory effects of an oxidative phosphorylase uncoupler
on normal ATP production that is driven by the proton
gradient inside the mitochondria and produced by
tricarboxylic acid metabolism.

C1

Closantel
I

OH

C1
CON__//

OH

\/x_._?__</

\~---Cl

CI

Brotianide
C1

OH

Br

OCOCH
3

CI

CS o< r
CI

This is explained by their strong plasma protein

binding which is more than 99% for the salicylanilides (Mohammed-Ali & Bogan, 1987) and 98%
for nitroxynil (Alvinerie et al., 1995). The selective
anthehnintic action of these highly protein-bound
anthelmintics may be explained, in part, by their
effect against blood-sucking parasites, concentrating the anthelmintic in the parasite without the
high tissue levels being produced in the host. The
high level of protein binding may explain the
selective effect of these agents, and the fact that
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 plasmaprotein binding.

Nitroxynil

DIAMPHENETHIDE

Diamphenethide (Fig. 17) is more active against

immature Fasciola hepatica in the liver than the
adult Fasciola in the bile ducts (Kendall & Parfitt,
1973). Diamphenethide is a prodrug that is deacetylated in the host's liver to an active form which is
the monoamine and the diamine (Coles, 1976).
The effects of the diamphenethide amine (active
form) on Fasciola in vitro have been studied by
Edwards et al. (1981a). These authors described
how diamphenethide amine produces an elevation of malate concentrations, an intermediary
breakdown product of glucose metabolism. They
were not able to identify an action of diamphenethide on a particular enzyme in the glycolytic
pathway but suggested that the effect on malate

O2N

Fig. 15. Chemical structure of the salicylanilides

oxyclozanide, rafoxanide & closantel and the DNP
derivative nitroxynil. The location of the dissociating
proton (H) is shown in the shaded box.

The anthelmintics that have the longest half-life

in the body are the salicylanides and nitroxynil.

MODES OF ACTION OF ANTHELMINTI(" DRU(_;S

29

Diamphenethide
. / - - ,,'

CH3CONH --

/ . . . .

'i:,

/, 4 /

OCHzCHzOCH2CHeO

'

NHCOCH 3
/

.. /

Clorsulon
C1
CI2 = C
~\

NH2

SONH 2

H2NO2S

Diethylcarbamazine

CHaN

C9H5
NCON

....,
C2Hs

Fig. 17.

Chemical structure of diamphenethide,

clorsuhm and diethylcarbamazine.

was likely to be a prima D, effect because it

occurred early on and before the deterioration of
the whole parasite. Interestingly, in the second
paper of Edwards et al. (1981h) the effect of dopamine, a putative neurotransmitter in Fasciola had a
protective effect against diamphenethide.
The action of diamphenethide remains to be
defined in greater detail but the study of Edwards
el aL (1981a) showed that its biochenaical effects
contrasted with the proton ionophore, oxyclozanide and appears to involve effects on malate
metabolism in Fasciola.

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
KINASE A N D MUTASE
Clorsulon

Clorsulon is 4-amino-6 trichloro ethenyl 1,3benzenedisulphonamide (Fig. 17). Structurally, it

is similar to 1,3-diphosphoglycerate (Schulman et
al., 1982) and consequently, inhibits the enzymes

plmsphoglyce,'ate kinase (Schuhnan et al., 1982)

and
phosphoglyceromutase
(Schulman
&
Valentino, 1982) of Fasciola and inhibits the
Emden-Meyerhoff pathways in fluke. As a result,
there is a selective inhibition of glucose utilization, and acetate and propionate formation. The
inhibition of the Fasciola phosphoglycerate kinase
is competitive (Fig. 18) with a ~ of 0.29 mM: clorsulon competitively inhibiting the binding of ATP
and 3-phosphoglycerate to the phosphoglycerate
kinase. Schulman et al. (1982) suggested that the
large group on the six position of clorsulon prevented the conformational change in the kinase
enzyme required for activity. There was also a
good correlation between the I~ values.for antagonism of Fasciola phosphoglycerate kinase for a
range of compounds related to clorsulon and the
potency of these compounds as antifluke agents.
This evidence supports the suggested mode of
action.
The inhibition of Fasciola phosphoglyceromutase by clorsulon was studied by Schulman and

31)

THE VETERINARY JOURNAl., 15-1, I

E1

-- I + E

/
\

E-ATP

\
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

enzyme requires 2,3-diplaosphoglycerate for activation and the ch)se similarity of clorsuh)n to
diphosphoglycerates could explain its inhibition
of the mutase enzwne. These authors puritied the
enzyme and compared some of its properties with
mamnaalian l~hosphoglyceromutase that were not
inhibited by clorsuhm. There appears to be no
recent studies on the mode of action of this series
of compounds ahhough clinical trials and efficacy
studies have been made.

DIETHYLCARBAMAZINE
The cllelnical structure of diethylcarbamazine a
piperazine derivative is shown in Fig. 17. Elect,-ophysiological experiments following bath application to A. suum of high concentrations of
diethvlcarbamazine have shown that it does not
mimic the action of piperazine (Martin, 1985).

Effects of diethylcarbamazine
A major indication for diethylcarbamazine at
low doses is as an anti-filarial drug; it is a good
microfilaricide (including microfilariae of the dog
hearnvorm, Dirofilana immilis) but has limited
macrofilaricidal effects. The limited action of
diethylcarbamazine against some aduh hehninths
(e.g. cattle lung3vorm, Dicl~,ocauhts) requires
nearly 10 times the dose of the prophylactic antifilarial dose, and may therebre, invoh,e another
mode of action. This review covers the mode of
action of low closes of diethylcarbamazine; little is
known of the mode of action of high doses of
diethylcarbamazine.
Most studies suggest that diethylcarbamazine
has no direct action on filarial parasites (Johnson
el al., 1991). It appears to have no action at
reasonable concentrations in vilro. In marked contrast to in vitro experiments, it is known that in
vivo diethylcarbamazine is effective within 4 rain
following intravenous injection (Hawking &
Laurie, 1949). The specific immune response

does not appear to be involved because nucle,

athvmic mice, infected with BruKia pahanffi,
showed a marked reduction in microfilariae following diethvlcarbamazinc treatment (Vickel T el
al., 1986). Thus T cells and T-dependent
,'esponses (Ig(; and lgE) do not appear to be
invoh'ed. The involvenlent of complement also
seems unlikeh' because clearing of microfilaria
ii-om the nuc(e mice by diethvlcarbanmzine was
not inhibited by prio," treatment with cobra
venom lactor treatment (Vicke D' el al.. 1986).

Fffects on arachidonic a d d metabolism may be the

mode of attire1 of diellg, lcarbamazine
Diethylcarbanlazine has an antagonistic action
of the metabolic enzymes that ,netabolize arachidonic acid, tile products released as a resuh of
phospholipase A.., action on cell membranes. Fig.
19 illustrates the sites of action of diethvlcarbamazine proposed by Maizels and Denham (1992).
Ahhough 5-1ipoxygenase is often being cited as
being inhibited by dietlaylcarbamazine, tiffs may
not be the case. In a mast cell line, conversion of
5-HPETE to leukotriene (LT),&~ by I.TA~ svnthetase
was
blocked
by diethylcarbamazine
(Mathews & Murphy, 1982). Razin el aL (1984)
found that in mouse, 5-1ipoxygenase conversion of
arachidonic acid to 5-HPETE was not inhibited
but that LT(h and LTA_~production was inhibited.
Diethylcarbanlazine also appears (KanesaThasan el al., 1991) to inhibit endothelial production by cyclo-oxygenase of prostaglandin
(PG)I._, (prostacyclin) and PGE., production but
does not affect platelet production of thromboxane (TX)A._, by cyclo-oxygenase and the 12-1ipoxygenase products 12- ancl 15-HPETE. Interestingly,
microfilariae also produce their own PGI._, and
PGE,, and like endothelial cell of blood vessels,
diethylcarbamazine also inhibits the microfilarial
production (Kanesa-Thasan el al., 1991). The coadministration of glucocorticoids, that are known
to block phospholipase A,_, activity and the production of arachidonic acid (Fig. 19), together

M()DES

OF

:M71"ION

OF

ANTHEI.MINTIC

DRL'(;S

31

MEMBRANE
PHOSPHOLIPIDS

SE
r ........................

i DIETHYLCARBAMAZINE i
"--r

1 PLA 2 . . . . . 1~-GLUCOCORTICOIDS
..................
1

i. . . . . . . . . . . . . . . . . . . . . . . . . . .

ARACHIDONIC
ACID

......................

5-LIPOXYGENASE / J

~_,

.-"-"

-1

, DIETHYLCARBAMAZINE
1_ _
andNSAIDS

CYCLO-OXYGENASE

5-HPTE
5-HETE
/

LTA I SYNTHASE /

""""'~

PGGo and
PG-H+ '~.~"".

12- and 15LIPOXYGENASE

\ ~

~ PGE2
.\ macrophages; microfilaria

\\

LTA4

12 and 15
HPTE/HETE
platelets; neutrophils;
eosinopils.

LTC 4 SYNTHASE

LTC.I
eosinophil;
mast cell

PGD~
mast-cells

'\

PGI.,: PROSTACYCLIN
\ vascular endothelium and
\.. microfilaria

LTB4
neutrol~hils;
macropnages

TXA2: THROMBOXAN
platelets

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 (5HPE'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;~.

with d i e t h y l c a r b a m a z i n e , r e d u c e s the effect o f

d i e t l a y l c a r h a m a z i n e (Stingl el al., 1988). T h u s ,
d i e t h y l c a r b m n a z i n e s e e m s to inhibit p r o d u c t i o n
o f PGI=, a n d PGE=, hy microfilaria a n d e n d o t h e l i a l
cells. T h e n o r m a l level o f p r o d u c t i o n o f PG1._, a n d
PGE,_, that p r o d u c e s a tonic dilation o f b l o o d vessels a n d inhibits a g g r e g a t i o n o f neutroplails a n d
e o s i n o p h i l s is r e d u c e d by a d m i n i s t r a t i o n o f
diethylcarhamazine.
A plausible e x p l a n a t i o n o f the m o d e o f action
o f d i e t h y l c a r b a m a z i n e is that this c o m p o u n d alters
the m e t a b o l i s m o f a r a c h i d o n i c acids in host e n d o thelial cell a n d also in microfilariae that are susc e p t i b l e to t h e a c t i o n o f d i e t h y l c a r b a m a z i n e .
T h e r e is t h e n a v a s o c o n s t r i c t i o n , a m p l i f i e d e n d o thelial a d h e s i o n a n d i m m o b i l i z a t i o n o f the micro-

filariae a n d cytotoxic activig, by the h o s t platelets

a n d g r a n u l o c y t e s . It a p p e a r s that d i e t h y l c a r b a m a z ine activates an i n n a t e i m m u n e r e s p o n s e r a t h e r
t h a n an a d a p t i v e i m m u n e r e s p o n s e (Maizels &
D e n h a m , 1992). This m o d e o f a c t i o n can e x p l a i n
why d i e t h y l c a r b a m a z i n e has n o a c t i o n in vitro
against microfilariae a n d is effective in n o n i m m u n e animals.

ACKNOWLEDGEMENTS

I a m p l e a s e d to a c k n o w l e d g e the financial s u p p o r t
o f the W e l l c o m e T r u s t w h o have s u p p o r t e d m y
w o r k o n the e l e c t r o p h y s i o l o g y o f t h e p i p e r a z i n e ,

32

THE VETERINARY.JOURNAL, 154, 1

nicotinic a n t h e l m i n t i c s a n d the GIuCi r e c e p t o r s o f

Asca,is.

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