Parasitology Today, vol. 6. no.

4, I990

SW
N=C-NHCO,Me
H,NCH,CH,SO,H
NO, b netobimin
45
accomplished by treatment of the 5-methylisothiourea deriva-
tive (45) with taurine. It should be noted that netobimin as a salt
is freely soluble in water and, as such, has major advantages
over the benzimidazoles per se. This is an area of research that
has great promise.
We have attempted to show a few examples of benzimida-
zole syntheses that have shown great value to humankind.
Does the synthesis of new benzimidazoles still have merit, and
is research in this area still going on? The answer is yes.
Resistance to the now commercially available benzimidazoles
has developed and poor oral absorption remain problems
which have yet to be solved.
Acknowledgement
This study was supported by the filariasiscomponent of the World
BankiUNDPIWHO Special Program for Research and Training in
Tropical Diseases(1.D. 870387).
References
I Townsend, L.B. and Revankar, G.R. ( I970) Chem. Rev. 70,389438
2 Preston, P.N. (I 98 I ) Benzimrdazoles and Congeneric Tricyclrc Compounds
Port I andPart John Wileyand Sons
3 Grlmmett M.R. (I 984) In Comprehensive HeterocycCc Chemistry(Katritzky,
A.R. and Rees, C.W.. eds), (Vol. 5) pp 345498, Pergamon Press
4 Brown, H.D. et al. ( I96 I )/. Am. Chem. Sot. 83, I764 I765
5 Grenda, V.J.. Jones, R.E., Gal, G. and Sletzlnger, (1965)j. Org. Chem. 30,
259-26 I
6 Hoff, D.R. et al. ( 1970) Experrentia 26,550
7 Ellsworth. R.L., Hinkley, D.F. and Schoenewaldt, E.F. ( I97 I ) Chem. Abstr.
74,76424q (French Patent 20 I4 308)
8 Ellsworth. R.L., Hinckley. D.F. and Schoenewaldt, E.F. (I 97 I) Chem. Abstr.
74,76423p(French Patent 2 0 I4 422)
9 Actor, P. et al. ( I 967) Nature 2 I 5,32 I-322
IO Raeymaekers, A.H.M., Van Gelder, J.L.H., Roevens. L.F.C. and Janssen,
P.A.J. (I 978)Arzneim..Fonch. DrugRes. 28,587-594
I I Theodorides, V.I.. Gyurik, R.J., Kingsbury, W.D. and Parish, R.C. (1976)
Experientia 32,702
I 2 Theodorides, V.J. et al. ( I973)Br. Vet.J. I 29, vcvii
I3 Baeder, C. et al. (I 974)Experientia 30,753
I4 Averkin, E.A. et al. ( I975)J. Med. Chem. 18, I I64 I I66
15 Barker, A.C. and Foster, R.G. (1973) Chem. Abstr. 79,534lc; 25669k
(German Patent 2 246 605)
I6 Loewe, H., Kirsch, R., Urbanletz, J. and Duewel, D. (I 975) Chem. Abstr. 82,
I563 I I f(German Patent 2 332 398)
I7 Klonnlng, H.K. ( I975)Chem. Abstr. 84,4948x (US Patent 3 896 230)
I8 Olin, J.F.and Dains, F.B.( I930)]. Am. Chem. Sot. 52,3322-3326
I9 E.I. du Pont de Nemours and Co. (I 97 I ) US Patent 3 562 290
20 Actor, P. and Pagano, J.F.( I975)Chem. Abstr. 84,495Os (US Patent 28 403)
21 Widdig A. and Kuehle, E. (1971) Chem. Abstr. 74, 7864256e (German
Patent I 932 297)
22 Ram,& Wise,D.S.andTownsend, L.B.( l985)OPPIBriefs 17,21>218
23 Bochis, R.J.et al. ( I 98 I )/. Med. Chem. 24, I 5 I 8-I 52 I
24 Wollweber, H. et al. (I 978)Arznermittelforsch 28,2 193-2 I95
25 Elchler. D.A. ( I973)Br. Vet.]. l29,533-543
26 Vashi, D.B., Clark, J.N. and Lindo, N.A. (I 983) US Patent4 406 893
27 Delatour. P.. Cure, M.C., Benoit, E. and Garnier. F. (I 986)J. Vet. Pharmacol.
Ther. 9,23G234
Leroy Townsend and Dean W/se ore at the Department Medicinal of
Chemistry, College ofPharmacy and Deportment ofChemistry, University
ofMichigan, Ann Arbor, MI 48 109, USA.

Mode of Action of Benzimidazoles

E. Lacey

Benzimidazoles represent the only class of
truly broad-spectrum anthelmintics, how-
ever, they also show activity against fungi
and mammalian cells. This raises the ques-
tion as to why benzimidazoles can selec-
tively kill helminths and yet exhibit little or
no mammalian toxicity. In this paper,
Ernest Lacey examines this example of
selectivity of drug action to the ubiquitous
target of these drugs, the structural pro-
tein, tubulin.
With the discovery of thiabendazole in
I96 I, the general pattern of benzimida-
zoles (BZs) as a class of low-dose broad-
spectrum anthelmintics with a high
therapeutic index was established ’ The
subsequent cascade of patents during
the next 25 years led to the experimen-
tal or commercial development of a fur-
ther I5 BZs (see pp IO7- I I 2, this issue)
and BZ prodrugs. Central to the success
of BZs is their selective toxicity for hel-
minths. Since the mid- 1960s the mode of
action of BZs has been extensively inves-
tigated and our understanding of how
BZs act has undergone radical reappral-
sa12d. In a recent review, Lacey4 con-
eluded that despite the diverse effects of
BZs at the biochemical and cellular levels,
the primary mode of action of these
drugs involves their interaction with the
eukaryotic cytoskeletal protein, tubulin.
This paper examines the paradox of the
interaction of BZs with a ubiquitous pro-
tein and the evidence for their selective
toxicity for helminths.
Tubulin and Microtubules
The microtubule subunit, tubulin, is a
dimeric protein comprised of c1- and
P-subunits of approximately 50 kDa
each5. Structurally, both a- and P-tubu-
lins are heterogeneous proteins, prod-
ucts of multi-gene families as well as
post-translational modifications. The se-
quences of tubulins from a wide diversity
of species have been reported, and
show a high degree of homology5.
Microtubules exist in dynamic equilib-
rium with tubulin, the ratio of dimeric
tubulin to polymeric microtubules being
controlled by a range of endogenous re-
gulatory proteins and co-factors (Fig. I p.
@1990
This equilibrium can be altered, both in
vivo and in vitro, by exogenous sub-
stances known as microtubule InhIbItor%
Most, but not all, such inhibitors exert
their action by binding to tubulin to pre-
vent the self-association of subunits onto
the growing microtubules. This results in
a ‘capping’ of the microtubule at the
associating end while the microtubule
continues to dissociate from the oppos-
ing end, with a net loss of microtubule
length. One implication of this phenom-
enon is that it is not necessary for inhibi-
tors to bind all tubulin dimers to inhibit
polymerization, it is sufficient for them
simply to’cap’the microtubule.
Microtubule inhibitors are a group of
structurally diverse compounds pro-
duced by fungi, plants, marine organisms,
possibly higher eukatyotic animals and,
more recently, synthetically. They show
a wide spectrum of selective and non-
selective toxicity against eukaryotic
genera. Some of the well-characterized
microtubule inhibitors, such as vinblas-
tine and vincristine, have found a use in
cancer chemotherapy, but most are too
toxic for therapeutic use.
Elsevter Swnce Publishers Ltd. (UK) 0169470719~02.00
Parasitology Today, vol. 6, nc. 4, I990
Benzimidazoles and Tubulin
Borgers and De Nollin6 first reported
the disintegration of the normal micro-
tubule matrix in intestinal cells of Ascaris
suum treated with mebendazole. Sub-
sequent studies7t* confirmed this obser-
vation in other BZ-sensitive species of
helminths. At about the same time, other
researchers at janssen Pharmaceutics
reported that nocodazole (an analogue
of mebendazole in which the S-benzene
group is replaced with a thiophene
moiety) was a potent inhibitor of the
polymerization of mammalian tubulin’.
Further evidence for a tubulin-de-
pendent mechanism for BZ action
was obtained with selected commer-
cially available BZs in polymerization
studies, using both bovine and ovine brain
tubulins”,’ ‘. Competitive ligand-binding
studies with [3H]colchicine confirmed
that BZs interacted within the colchicine-
binding domain”. Structure-activity
relationships for BZs as inhibitors of
L I110mouse leukaemia cell growth have
been correlated with inhibition of the
polymerization of ovine brain tubu-
jinl2.13
The kinetics of the binding of meben-
dazole and nocodazole to brain tubulin
have been described by several groups
using gel filtration and dialysis tech-
niques4.
Origins of Benzimidazole
Selectivity
The potent activity of ‘BZs as inhibitors
of mammalian tubulin polymerization in
vitro is paradoxical, given our under-
standing of their pharmacology in vivo.
The paradox can be condensed into two
questions: ( I ) why is mammalian toxicity
so low, given the potent inhibitory activity
of BZs on microtubules? (2) How can BZs
interact at a common site within a ubiqui-
tous eukaryotic protein to kill helminths
selectively? Indeed, in Table I the com-
parison between the ,activity of BZs
to cause 50% inhibition of the growth of
L’z’o cells in vitro and 50% inhibition of
nematode egg-hatch (a primary screen
for BZ activity) suggests that only the
parasite should survive treatment! How-
ever, recently developed in vitro tech-
niques for assessing la.rvicidal activity
showed that there ‘was selectivity
between mammalian cells and nema-
todes in a manner consistent with the
dose differential observed in vivo,
between efficacy and host toxicity4.
A hypothesis to account for the selec-
tivity of BZs was first advanced by Fried-
man and Platzer14, who repot-ted that
Fig. I. Typical microtubules reach a dynamic equilibrium with tubulin. controlled by endogenous
concentrations of co-factors such as GTP, M$+, microtubule-associated proteins (MAPS),Ca2+
and calmodulin. Regulation in vitro, by increasing temperatures, enhances polymerization,
whereas reducing the temperature induces depolymerization. Addition of inhibitors causes
‘copping’ and halts polymerization, a//owing only depolymerization with the loss of microtubule
structure.
fenbendazole was 250-fold and meben- tortus was stable to charcoal extraction.
dazole was 400-fold more potent as Furthermore, lower levels of charcoal-
inhibitors of [3H]colchicine-binding to stable binding were found in isolates of
the tubulin of embryonic A. suum than to H. contortus known to be resistant to
mammalian tubulin; BZs thus exhibit an BZs compared to BZ-sensitive isolates.
apparently higher affinity for helminth Decreased [3H]BZ binding also corre-
tubulin. However, using direct binding lated to the status of resistance of
studies with [3H]mebendazole and A. Trichostrongylus colubriformis and Oster-
suum intestinal tubulin, Kohler and Bach- tagia circumcincta2’.
mann” failed to find any change in BZ This raises the question of whether
affinity. They concluded that selectivity species selectivity may also be correlated
was related to the differing pharmaco- to charcoal binding stability. Comparison
kinetics of BZs within the host and the of charcoal-stable binding to meben-
parasite. Using an alternative approach, dazole in various species is shown in
Dawson et al.16 examined the compara- Table 2. Although high levels of charcoal-
tive activity of BZs as inhibitors of the stable [3H]mebendazole binding were
polymerization of purified tubulin from shown for H. contortus, no such binding
sheep and Ascardio gal/i. Selectivity was was detected for ovine brain tubulin. Yet
observed for the weak mammalian in- the binding of [3H]mebendazole (and
hibitors, oxfendazole and thiabendazole, other [3H]benzimidazole carbamates)
where their low affinity for mammalian can be readily detected by less drastic
tubulin resulted in insufficient ‘capping’to techniques such as gel filtration or dialy-
inhibit polymerization. sis4. The parasite tubuliwbenzimidazole
Davidse and Flach17 reported that the carbamate complex, like the mammalian
binding of the unsubstituted benzimi- brain tubuliwcolchicine complex, is
dazole carbamate, carbenazim, to the pseudo-irreversible in that, although
fungus Aseergillus nidulans, was stable to binding is non-covalent, once bound, the
extraction with charcoal. Their assay was ligand cannot be easily removed without
developed from the studies of Sherline denaturingthe protein.
et al. ‘13with [3H]colchicine. Although col- Charcoal-stable binding is character-
chicine is not considered among the istic of all S-substituted benzimidazole
most potent of microtubule inhibitors, it carbamates. For the unsubstituted ana-
forms a tight, pseudo-irreversible com- logues thiabendazole and carbenazim,
plex with tubulin that enables the little or no charcoal-stable binding to
colchicine-tubulin complex to survive helminth tubulin is observed (E. Lacey,
extraction with charcoal. Lacey and Pri- unpublished). This finding suggests that
chard” subsequently reported that the charcoal-stable binding per se is not the
binding of mebendazole, fenbendazole, sole determinant of BZ selectivity, since
oxfendazole, albendazole, oxibendazole both compounds are moderately potent
and parbendazole to Haemonchus con- inhibitors of [3H]mebendazole binding
II4 ParasitologyToday,vol. 6, no. 4, I990

Table I. Compilation of approximate in vivo dose and in vitro data for the inhibition of dazole are associated with high levels of
mammalian tubulin polymerization, L ,zra cell growth, [3H]mebendazole (MBZ) binding to binding, and vice versa.
H. contortus L3 larval tubulin, egg-hatch and larval development activity for a selection of However, the correlation between
commercially developed benzimidazolesa charcoal-stable binding and in vivo effi-
Drugb 150 LD50 IC50 EDSO LD50
cacy is not complete. For example, both
Wym;-i;tW $~zd C3H$fBZ (7e;g) (larvae) the filarial nematodes Dirofilaria immitis
Carbenazim 0.094 and Onchocerco gibsoni bind high levels
Parbendazole 3:l - 0:30 0:33 0.0 I4 of [3H]mebendazole, but there is little or
Oxibendazole 2.2 0.15 0.30 - no efficacy against the adult parasite. To
Albendazole 6.9 - 0.21 0.38 0.034
understand these apparent contradic-
Ricobendazole NI 100 - 2.3 NI 100 I .6
tions it is necessary to consider two
Mebendazole 6.1 0.31 0.19 4. I 0.048
aspects of the BZ-tubulin interaction.
Flubendazole 2.5 0.17 -
Fenbendazole 5.4 0.47 0.18 NI IO 0.019
First, although nominally nematocidal,
Oxfendazole NI 100 Il.3 I.3 NI 100 0.57 BZ toxicity in vitro has been documented
Thiabendazole 549.0 5.5 0.33 <0.04 only against developing stages of the
Cambendazole 64.0 - 0.54 - parasite. Despite extensive evidence of
impairment in non-developing stages,
a Data taken from Ref. 4 and Lacey et al. (unpublished). All dataquoted in bM unless otherwise stated.
b Concentration of drug (PM) required to inhibit by 50%.
overt toxicity over comparable times
I = inhibition of tubulin polymerization, LD = lethal dose to growing L, aI cells or nematode lavae, has not been reported. Second, in the
IC = inhibitory concentration to prevent [3H] mebendazole binding, ED = effective dose that inhibits absence of overt toxicity, the pseudo-
egg-hatch, NI = no inhibition at stated concentration. irreversible binding is rendered rever-
to helminth tubulin, yet essential1 inac- predictive for in vitro larvicidal activity. It sible by ‘sink’ conditions in the host and
Y is interesting that of the 60 non- the rate of tubulin turnover within the
tive against mammalian tubulin ([ H]col-
chicine binding or polymerization) at I 00 carbamate BZs examined, thiaben- parasite. While these processes are
PM (Table I ). dazole was the most potent inhibitor of probably slower than the dissociation of
[3H]mebendazole binding, underlining its the complex, efficacy is dependent on the
initial selection in vivo as an anthelmintic. rate of elimination of the parasite from its
Tubulin as the Site of BZ Action
Several known mammalian microtu- preferred site. lfthis is longerthan the rate
bule inhibitors also inhibit the binding of of dissociation, recovery will occur. Con-
Demonstration of the selectivity of [3H]mebendazole to helminth tubulin versely, if the rate of elimination is rapid,
binding of BZs between helminth and and have highly correlatable ovicidal and as in the gastrointestinal tract, higher than
mammalian tubulins provides a funda- larvicidal activity, consistent with hel- expected efficacy could occur with low
mental argument for tubulin as the site of minth tubulin involvement4. Preliminary levels of charcoal-stable binding.
BZ action. However, it is also necessary investigations of other unrelated anthel-
to substantiate the role of tubulin in the mintics (eg. the avermectins, levamisole, Resistance
BZ-parasite interaction. This can be vali- salicylanilides) have shown that none
dated by comparative structure-activity inhibit [3H]mebendazole binding to
relationships of BZ-analogues, by binding tubulin in amounts that account suf- Drug resistance in any organism is
studies of these and unrelated com- ficiently for their anthelmintic activity defined by a change in the drug’s phar-
pounds, and by comparison of the data (E. Lacey, unpublished). macodynamics (absorption, distribution,
on species selectivity and BZ-selected, metabolism, excretion and site of
resistant isolates, obtained in viva and in action). Resistance therefore need not
Species Selectivity
vitro, with [3H]benzimidazole carbamate necessarily occur at the site of action, but
binding. showing that changes occur at a putative
To establish a correlation between a Since the extent of charcoal-stable BZ site of action is a powerful tool for re-
biochemical event (binding) and a phar- binding offers an explanation of the ori- solving the mode of action. The obser-
macological end-point (in this case, death gin of BZ selectivity between parasite vation that resistance to BZs detected by
or failure to thrive), it is essential to and host, it is of interest to consider both in vitro and in vivo techniques
remove the host from the assay system, selectivity within different species of hel- was correlated with the extent of
since the host will affect the availability of minths. From Table 2, it can be readily [3H]mebendazole binding is also a strong
the drug via absorption, excretion and observed that low doses of meben- confirmation of tubulin as the site of
metabolism. Potency in an egg-hatch
assay can be correlated to drug- Table 2. Comparison of dose rates efficacious in vivo,and [3H]mebendazole charcoal-stable
resistance and [3H]BZ binding, but is binding datafor aselection of benzimidazole-sensitive and -insensitive speciesa
limited to a narrow range of drug hydro-
Species Dose(mg/kg) [3H]MBZ
phobicities where partitioning through
(pmoles/mg)
the egg shell is possible. Methods that
Trichostrongyluscolubriformis cl2.5 71
assess larvicidal action by inhibiting the < 12.5 63
Haemonchus contortus
development of the egg to the L3 elimin- Ostertagio circumcincta < 12.5 65
ate this artefact. With the exception of Nematodirus spathiger I5 25
thiabendazole, correlation between Fasciofdhepotica 100 IO
LD50 values in developing larvae, and the Taenia pisifirmis 200 IO
inhibition of binding of [3H]mebendazole (multiple doses)
to tubulin (I&, values: Table I ), is appar- Taenia hydatigena 160-320 5
ent. Affinity for tubulin as assessed by a (multiple doses)
Sheep brain tubulin >I280 <I
compound’s ability to inhibit the binding
of [3H]mebendazole is therefore highly a Data from Ref.4.
ParasitologyToday, vol. 6, no. 4, I990
action of BZs (see pp 125-l 27, this
issue). Indeed, reduced c:harcoal-stable
[3H]mebendazole binding has been
observed in all the BZ-resistant isolates
tested to date4**‘.
However, it is important to note that
for other postulated modes of action, for
example inhibition of fumarate re-
ductase and glucose uptake, limited
studies with resistant isolates have also
shown reduced activity4. ‘Thus, a differ-
ence in biochemical activity between iso-
lates is not in itself conclusive proof of a
mode of action. Drug selection (or any
lethal selection procedure) can lead to
coincident selection of unrelated physio-
logical processes. This is complicated by
our lack of understanding of how a pri-
mary action elicits secondary biochemi-
cal and physiological effects. The events
leading to failure to thrive and toxicity
are among the most poorly character-
ized mechanisms linking drug binding to
efficacy. To assign a site of action un-
equivocally based solely on resistance
studies, it is essential to show a change in
an isolated protein for a range of eco-
logically distinct sensitive and resistant
isolates. In this respect it is unfortunate
that the scope of our present knowledge
of isolate variability within biochemical
The Metabolism of Benzimidazole
Anthelmintics
D.W. Gottschall, V.J. Theodorides
The benzimidazole carbumotes are irnport-
ant broad-spectrum drugs for the control
of helminth parasites in momma/s. David
Gottschall, Vassilios Theodorides and
Richard Wang explain that the metabolism
of these compounds depends heavily on
the substituent present on carbon-5 of the
benzimidazole nucleus and involves a wide
variety of reactions. Worl: in vitro has
shown that two major enzyrne systems, the
cytochrome f-450 fami/y and the micro-
somal flavin monooxygenases are primarily
responsible for these biotfonsformations.
The parent compound is general/y short-
lived and its metabolites predominate in
the tissues and excreta of treated animals.
The metabolic pathways can be exploited
therapeutically to overcome the problems
of poor water solubility and absorption of
benzimidazoles by the development and
use of more soluble prodrugs.
Throughout their lifetime mammals
become exposed to a large number of
compounds foreign to normal interme-
diary metabolism. As a result, an intricate
0 ,990. Elsewr Scence PubIsherr Ltd. (UK) 0 I694707/9’3502
and physiological pathways is confined
mostly to drug-selected isolates.
Concluding Remarks
The mode of action of BZs can be
directly linked to an interaction with
tubulin in four important aspects of the
drug-parasite interaction, ( I ) structure-
activity relationships, (2) unrelated com-
pounds, (3) species selectivity and (4)
drug resistance. Selectivity within the BZ
class of anthelmintics is achieved by a
shift in the BZ-tubulin equilibrium lead-
ing to an increased stabilization of the
bound complex. Our understanding of
the link between efficacy and the mode
of action has advanced, and it will be
interesting to observe whether this leads
to the development of new chemical
entities that exploit this mode of action.
References
I Brown, H.O. et al. (I 96 I )I. Am. Chem. Sot. 83,
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acol. Chemother. I 9,67-I 28
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therapy ofPo&tic Diseases Plenum Press
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and R. Wang
system of detoxication mechanisms has
evolved that combines enzyme-
catalyzed metabolic conversions, non-
specific chemical reactions, and fine-
tuned excretion pathways. This process
is generally based on the transformation
of lipophilic xenobiotic compounds to
more polar hydrophilic products that
can be easily eliminated ’
The metabolism of foreign com-
pounds is commonly divided into two
phases. Phase I reactions include aliphatic
and aromatic hydroxylations, N-, 5, and
0-dealkylations, N- and S-oxidations
and a number of reductive type pro-
cesses. Phase II reactions, on the other
hand, are characterized by conjugation
with natural constituents such as amino
acids, sulfate, carbohydrates, bile salts,
and glutathione. Conjugation reactions
frequently occur at the site of the newly
introduced functional groups from Phase
I metabolism, although this is not a pre-
requisite (for a general discussion and
specific examples, see Ref. 2).
Although these metabolic reactions
00
II5
6 Borgers, M. and De Nollin, S. ( I975)J. Pa&to/.
60, I IO-122
7 Borgers. M. et of. (1975) in Microtubules and
Microtubule Inhibiton (Borgers. M. and De Bra-
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Elsevier
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of Porosites and Host-Parasite Relationshrps
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Ernest Locey is at CSIRO.Division of Animal
Health, M&laster Laboratory, Private Bag
No. I, Glebe,NSW 2037, Australia.
are generally associated with the detoxi-
cation of chemicals, the same enzyme
systems can in some cases increase tox-
icity through the creation of highly re-
atiive intermediates. These activated
species, once generated, can react fur-
ther with natural constituents (proteins,
lipids, etc.) forming covalently bou!d
adducts. The presence of these adducts
can disrupt normal cell function with a
resultant increase in specific organ tox-
icity, teratogenicity, or even death3.
Aside from considerations of toxicity,
metabolites may be produced that have
either increased, or decreased pharma-
cological activity relative to the parent
molecule. This can h&e a direct impact
on decisions regarding the administered
dose and, in the larger sense, can directly
affect overall product efficacy.
Thus the elucidation of the metabolic
profile of a specific compound becomes
increasingly important for the under-
standing and evaluation of overall prod-
uct safety. The following discussion
focuses on (I) the observed metabolic