Comparison of inhibition of polymerisation of mammalian tubulin and helminth ovicidal activity by benzimidazole carbamates

Veterinary Pat-astrology, 23 (1987) 106-119 Elsevier Science Publishers B.V., Amsterdam

106 - Printed

in The Netherlands

COMPARISON OF INHIBITION OF POLYMERISATION MAMMALIAN TUBULIN AND HELMINTH OVICIDAL BY BENZIMIDAZOLE CARBAMATES

E. LACEY’ , R.L. BRADY’,

R.K. PRICHARD’

OF ACTIVITY

and T.R. WATSON’

‘MeMaster Laboratory, CSIRO Division ofAnimal Health, Priwte Bag No. 1, Glebe, N.S. W. 2037 (Australia) aInstitute of Parasitology, MacDonald College, McGill University, Sre.-Anne-de-Belleuue, P.Q. H9X I CO (Canada) 3Pharmacy Department, Uniwrsiry of Sydney, Sydney, N.S.W. 2006 (Ausrralia) (Accepted

for publication

28 January

1986)

ABSTRACT Lacey, E., Brady, R.L., Prichard, R.K. and Watson, T.R., 1987. Comparison of inhibition of polymerisation of mammalian tubulin and helminth ovicidal activity by benzimidazole carbamates. Vet. Parasirol., 23: 105-119. The correlation between the inhibition of hatching of Haemonchus contortus eggs and inhibition of mammalian tubulin polymerisation by benzimidazole carbamates bas been investigated. The hatching process was observed to be independent of the biomass (eggs plus debris) over a 6-fold range and the early (E, -E,) stages of egg development, but was dependent on the concentration of co-solvent (DMSO) and time of incubation. Benzimidazole carbamates with strong inhibitory activity against mammalian tubulin were potent inhibitors of egg hatch, while non-inhibitors failed to prevent hatching. It is postulated that the primary mode of action of these drugs on nematode eggs is the inhibition of microtubule-dependent processes within the developing egg. The implications and limitations of this correlation are discussed.

INTRODUCTION

The mode of action of substituted benzimidazoles (BZs, Fig. 1) as antifungal and anthelmintic agents has been investigated since their introduction in the early 1960s (Davidse and Flach, 1977; Prichard, 1978). Although a number of mechanisms of action, such as inhibition of protein and nucleotide synthesis (Clemons and Sisler, 1971; Hamerschlag and Sisler, 1973), inhibition of fumarate reductase (Prichard, 1970) and inhibition of glucose uptake (Duwel, 1977) have been postulated for individual members of this class, the observation that mebendazole induced disintegration of the microtubular network in parasites has generated sustained interest in the biochemical pharmacology of BZs (Borgers and De Nollin, 1975; Borgers et al., 1975). Subsequent studies (Hoebeke et al., 1976; Friedman and Platzer, 1978;

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0 1987 Elsevier

Science

Publishers

B.V.

106

Ireland et al., 1979; Laclette et al., 1980; Lacey and Watson, 1985a,b) have led to the hypothesis that BZs, and in particular benzimidazole carbamates (BZCs, Fig. lb), act primarly by inhibiting polymerisation of tubulin to form microtubules. The anthelmintic, antifungal or antitumour activity of individual members of this class is thought to be due to an inherent specificity of the drug for tubulin of the target species (Friedman and Platzer, 1980; Kohler and Bachmann, 1980; Dawson et al., 1984). N

)5NH-i

R

N

b.

B

NHC-Oc$ R

Fig. 1. Structures of substituted

benzimidazoles

(a) and substituted

benzimidazole

carbamates (b).

Attempts to correlate the in vitro activity of BZs against mammalian tubulin to in vivo anthelmintic potency have met with only limited success (Friedman and Platzer, 1978). While the failure to obtain a significant correlation could be due to species specificity, one inherent problem in correlating in vitro assay results to in vivo activity is the role of host metabolism. This problem is significant with BZs which are extensively metabolised in vivo (Duwel, 1977; Gyurik et al., 1981). To investigate this, we have compared the inhibition of polymerisation of mammalian tubulin and inhibition of helminth egg hatch for a series of commercially available BZCs and their major metabolites. The egg hatch or ovicidal assay (Le Jambre, 1976) is an established technique for assessment of BZ resistance in nematode populations (Coles and Simpkin, 1977 ; Simpkin and Coles, 1978; Kirsch, 1978; Hall et al., 1978a; Boersema, 1984). The in vitro nature of this technique eliminates the influence of host metabolism, thereby allowing a direct comparison of tubulin polymerisation inhibitory activity and nematode toxicity. While the exact relationship between ovicidal activity and anthelmintic potency is not well understood, the relative ovicidal activity of commercial BZ anthelrnintics against eggs of nematodes of varying in vitro susceptibility is reflected by anthelmintic efficacy in vivo (Hall et al., 1978a,b). MATERIALS

AND METHODS

Chemicals Pure samples of the commercial BZs (Table I) were obtained as gifts from The major metabolites of mebendazole their respective manufacturers.

107

(MBZ),methyl[ 5( 6)a hydroxybenzylbenzimidazoL2yl] carbamate (OH-MBZ) MBZ) and 2-amino-5(6)-benzoylbenzimidazole (NH2-MBZ), were prepared according to the literature (Allan et al., 1980). Methyl[ 5(6)-phenylsulphonylbenzimidazoL2yll carbamate ( FBZ-S02) was prepared by the oxidation of either oxfendazole (OFZ) or fenbendazole (FBZ) with excess hydrogen peroxide in glacial acetic acid. Methyl[ 5(6)(4’-hydroxy) phenylthiobenzimidazol-2-yl] carbamate (OH-FBZ) was prepared according to the method of Averkin et al. (1975), replacing thiophenol with 4-mercaptophenol. Methyl[5(6)-(4’-hydroxy)phenylsulphinylbenzimidazol-2yl] carbamate (OH-OFZ) and methyl[ 5( 6)( 4’-hydroxy)phenylsulphonylbenzimidazol-2yl] carbamate (OH-FBZ-S02) were prepared by procedures analogous to FBZ-SO2 synthesis. The amino derivatives, 2-amino-5(6)-phenylthiobenzimidazole (NH2FBZ), 2-amino-5(6)-phenylsulphinylbenzimidazole (NH?-OFZ) and 2-amino5(6) phenylsulphonylbenzimidazole (NH?-OFZ-SO*), were prepared by alkaline hydrolysis of FBZ, OFZ and FBZ-S02, respectively. Albendazole metabolites were prepared according to the method of Gyurik et al. (1981) with the exception of methyl [ 5( 6)hydroxy-6( S)-propylthiobenzimidazol-2yl] carbamate (50H-ABZ) and its corresponding sulphoxide (50H-ABZ-SO) and sulphone ( 50H-ABZ-S02), which were prepared using benzyloxy ether instead of propyloxy ether as cited in the method. The benzyloxy ether was hydrolysed using trifluoroacetic acid (Bhatt and Kulkami, 1983). All compounds synthesised were character&d by proton nuclear magnetic spectroscopy, mass spectrometry and melting point determination. All compounds were assayed by high performance liquid chromatography or by thin layer chromatography to ensure the absence of related derivatives before use. Egg collection and isolation Approximately 50 g of faeces were collected from the rectum of sheep infected with H. contortus (McMaster strain) and gently blended with water. The slurry was coarsely sieved through a No. 60 Mesh grid and the filtrate passed through a 25 p nylon mesh, retaining the eggs which were washed with water into a centrifuge tube and spun at 2000 X g for 10 min at room temperature. The sediment was resuspended with saturated sugar solution (1.5 kg 1-l) and centrifuged at 3000 X g for 10 min. The supernatant was diluted with 2 parts of water and recentrifuged. The combined sediments were then resuspended in water and reprecipitated to yield the eggs, which were resuspended in water to a concentration of approximately 200 eggs ml-‘.

Inhibition assay Inhibitors were dissolved in dimethyl sulphoxide (DMSO) to obtain stock solutions of 5 X 10m3 M and diluted to achieve a series of concentrations of

108

1 X 10e3 M, 5 X 10m4, 1 X 10e4 and 2.5 X 10e5 M. The standard assay consisted of adding 25 ~1 of each concentration to a suspension of 100 eggs in a total volume of 0.5 ml in a tissue culture plate (Well size 3 ml, Flow Laboratories, Australia). The eggs were incubated at 24°C for 48 h then stained with Lugol’s iodine solution and counted in duplicate. Concentrations showing no hatch and complete hatch were used as the extreme concentrations for the EDso determination. Four to six concentrations were interpolated between these concentrations and the assay repeated in duplicate. The normalised percentage hatch at each concentration was obtained according to the following equation: Normalised Hatch =

no. larvae/no. larvae + eggs (inhib)

x 1oo

no. larvae/no. larvae + eggs (control)

The concentration required to inhibit egg hatch by 50% (ED,,) was obtained by interpolation at the 50% value from the computed linear regression of the % normalised hatch versus drug concentrations. In all quoted data the correlation coefficient (r) was greater than 0.95 (with the exception of EDs0 of MBZ at 200 h, where r = 0.93). Each ED5,, determination was repeated within 2-3 weeks, with reproducibility in the range of + 15% when performed using the same infection. The effect of variation of DMSO concentration, time of incubation and number of eggs incubated was tested by appropriate modification of the above procedure. Tubulin polymerisation assay Methods used for the isolation of tubulin from sheep brain and inhibition studies have been previously reported (Lacey and Watson, 1985a). The concentrations required to inhibit the rate of polymerisation by 50% (ICsO) quoted in Table I were obtained from that study. RESULTS

Effect of DMSO The poor aqueous solubility of BZCs necessitates the use of co-solvents DMSO was observed to be a superior co-solvent to ethanol, methanol and dimethylformamide and showed no effect on hatching at 5% v/v; however, higher concentrations reduced hatching in a concentration-dependent manner, giving an EDSo value of 1.22 M (Fig. 2). Effect of variation in biomass The effect of the biomass (eggs plus isolated debris) was assessed by determination of the ED5,, of MBZ over a g-fold variation of egg numbers. The

109

5

80

;ii7 g6 ED w= 1.22M P:, In

l /. DMSO 0

0.64

I.26

1.92

Fig. 2. Inhibition of the hatching points are the mean of two separate

2.56

Cont.(M)

of H. contortus determinations.

eggs by dimethyl

sulphoxide.

Data

observed EDS0 values for the incubation of 58 +_7 (n = 20), 116 f 11, 179 * 14 and 339 f 33 eggs was 5.3, 4.0, 4.9 and 4.7 PM, respectively. The range of these values (1.3 PM) represents a standard deviation of 20.54 PM, slightly less than the normal variation (+15%) experienced with duplicate EDSo determinations using 100 eggs. The results do not establish the presence of any clear dependency on the observed EDS0 value of the biomass.

OL’

I

J

0

6 12 18 Pre-incubation time (h)

Fig. 3. Effect of pre-incubation of H. contortus eggs on the ED,, obtained for mebendazole (MBZ) and parbendazole (PBZ). Values quoted at time = 0 h represents the mean and standard deviation of four separate determinations. ED,, of MBZ at 18 h was obtained by extrapolation of date up to 8 PM.

110 Effect

of pre-incubation

To quantitate the nature of the effect of the developmental stage of the egg on the observed EDSo. MBZ and PBZ were added to H. contortus eggs immediately post-isolation (time = 0) and after 6, 12 and 18 h incubation at 24”C, corresponding to development stages El, E2 to early E3, E3 and E4, respectively (Silverman and Campbell, 1959). At 20-22 h 75% of viable control eggs had hatched, with complete hatch achieved by 36 h. As shown in Fig. 3, the inhibition of egg hatch by MBZ and PBZ is independent of the stage of egg development up to 12 h pre-incubation (E, stage). At 18 h (E4) a substantial reduction in potency (2-fold increase in the EDSO value) was observed for both compounds. These results suggest BZCs exert their action throughout embryogenesis (from El to E4) eliciting a weaker effect only during the E4 to LI transition. Effect

of incubation

time

The dependence on incubation time of the EDS0 value was examined using MBZ and PBZ (Fig. 4). With increasing incubation time, the EDs0 of MBZ increased linearly from 3.70 PM at 36 h to 9.8 PM at 200 h. Based on the computed line of best fit, the calculated rate of increase in the ED5,, was 0.037 PM h-’ of incubation (r = 0.998). Note that the EDso at 200 h was calculated by extrapolation of the data beyond 8 PM (representing 35% inhibition) and as such represents an estimated value. Although data from 9 and 10 PM concentrations were obtained, these results were not utilised in the EDS0 calculation as MBZ precipitated under these conditions.

incubation time(h) Fig. 4. Effect of incubation time (h) on the calculated ED,, parbendazole (PBZ).

of mebendazole (MBZ) and

111

For PBZ, the ED5,, values increased from 1.88 PM at 36 h to 2.70 PM at 200 h; the calculated line of best fit gave a rate of increase of 0.005 PM h-l (r = 0.92) up to 200 h. Inter-assay variability Investigation of the EDs0 values of MBZ, PBZ and other BZCs revealed the presence of substantial inter-assay variation. Over a 12 month period the EDSo value for MBZ was observed to vary from 4.00 to 9.2 PM (12 determinations). The EDSO values for other compounds were observed to be similarly higher or lower with respect to MBZ. The extent of this variability was diminished (less than f 15%) if assays were performed using eggs isolated from the same infection within 2 weeks, while duplicates carried out using different infections showed as much as 50% variation from the mean values. Based on the absence of any factor examined during standardisation of the assay, this effect is considered to relate to an inherent variation in the individual host-parasite interaction. To minimise this effect, comparison of the EDs0 values for various BZSs must be determined using eggs isolated from the same infection. Inhibition of the egg hatch by commercial BZCs and their respective metabolites The mean I&,, and EDso values for the inhibition of the polymerisation of sheep brain tubulin and H. contortus egg hatch by commercially available BZCs and the major metabolites of MBZ, FBZ, OFZ and ABZ are presented in Tables I and II, respectively. TABLE I Comparison of the inhibitory tubulin and egg hatch assays

activity

of commercial

anthelmintics

against mammalian

Drug

R

IC,, (PM)~~

ED,,

Parbendazole Oxibendazole Albendazole Mebendazole Flubendazole Febendazole Oxfendazole

CH,CH,CH,CH,CH,CH,CH,OCH,CH,CH,SC,H,CO(4’-FjC,H,COC,H,-SC,H,-SO-

3.1 2.4 6.9 6.1 3.5 5.4 NIIOOd

2.3 1.0 5.7 7.6 4.6 6.0 N1250

(PM)~

required to inhibit the rate of polymerisation of tubulin by 60%. aB& 1 concentration bIC,, values for these compounds were originally presented in Lacey and Watson, 1985a. required to inhibit the normalised percentage hatch of susceptible ‘ED,, , concentration H. contortus egg by 50%. dNI, no inhibition at the concentration shown (normally the maximum solubility obtainable under assay conditions).

112 TABLE

II

Comparison of the mammalian microtubule inhibitory activity (IC,,) and ovicidal against H. contortus eggs by metabolites of mebendazole, fenbendazole/oxfendazole albendazole

activity and

Ra

R’n

Abbrev.

IC,,

Mebendazole series C,H,COC,H,CH(OH)C,H,CO-

-CO,CH, -CO,CH, -H

MBZ OH-MBZ NH, -MBZ

6.1 70 N12000

7.6 120 N12000

FBZ OFZ FBZ-SO, OH-FBZ OH-OFZ OH-FBZ-SO, NH, -FBZ NH,- OFZ NH,- FBZSO,

5.4 NIlOO NIlOO 2.5 70 N1200 N12000 N12000 N12000

6.0 N1250 160 18 N1250 N1250 N12000 N12000 N12000

ABZ ABZ-SO ABZ-SO, NH, -ABZ NH, -ABZ-SO NH, -ABZ-SO, 2’-OH-ABZ 2’-OH -ABZ-SO 2’-OH -ABZSO, 3’-OH -ABZ 3’-OH -ABZ-SO 3’-OH -ABZ-SO, 50H-ABZ S-OH-ABZSO 5-OH-ABZSO,

6.9 NIlOO NIlOO N1250 N12000 N12000 5oc N1168 N1168 30= N1168 NI168 NIlOO N1168 N1168 _d

5.7 NIlOO NIlOO N1250 N12000 N12000 70 N1168

Fenbendazole/Oxfendazole C,H,SOC,H,SO,(4’-OH)-C,H,S(4’-OH)X,H,SQ(4’-OH)-C,H,SO,C,H,--SC,H,-SOC,H,-SO,Albendazole series CH,CH,CH,SCH,CH,CH,S+ CH,CH,CH,SO,CH,CH,CH,SCH,CH,CH,SOCH,CH,CH,SO,CH,CH(OH)CH,SCH,CH(OH)CH,S* CH,CH(OH)CH,SO,CH,OHCH,CH,SCH,OHCH,CH,SOCH,OHCH,CH,SO,CH,CH,CH,S-(S-OH) CH,CH,CH,SO-(5-OH) CH,CH,CH,SO,-(S-OH) CH,SCH ,SOCH ,SO,-

series -CO&H, -CO,CH, -CO,CH, -CO,CH, -CO,CH, -CO,CH, -H -H -H -CO,CH, --CO,CH, -CO,CH, -H --H -H -CO,CH, -CO,CH, --CO,CH, --CO,CH, -CO,CH, -CO,CH, -+O,CH, -CO,CH, -CO,CH, -CO,CH, -CO,CH, -CO,CH,

MeS-ABZ MeSO-ABZ MeSO, -ABZ

(PM)~

NIlOO NIlOO

ED,,

(PM)~

60% at 160 80 N1168 N1168 119 N1168 N1160 10 125 151

aR,, R, refer to Fig. la. bMean of two separate determinations. CSingle IC,, determination. d(-), not tested.

The bioisosteres of PBZ, OBZ and ABZ, showed the same rank order of potency in both assays for the modification of the methylene (-CH,-) group to -0(OBZ) and -S(ABZ), respectively. The 5(6)-keto-substituted BZCs, mebendazole and flubendazole, showed a similar trend, with

113

comparable orders of potency in both assays. Oxidation of the sulphur of FBZ to its sulphoxide analogue, OFZ, resulted in loss of activity on both assays. The failure of OFZ to inhibit polymerisation of tubulin was considered to be due to the presence of an undesirable polar moiety in the 5(6)-position adjacent to the BZ nucleus and thus the low ovicidal activity of the -SOand -SO?moieties is consistent with a microtubule-dependent mechanism. Although the rank order of potency varied slightly for some analogues in each assay, compounds exhibiting potent activity against mammalian tubulin were generally also potent inhibitors of egg hatch, while OFZ was inactive in both assays. The major routes of metabolism of FBZ, OFZ and ABZ have been shown to involve S-oxidation, hydroxylation and decarbamoylation (Duwel, 1977; Gyruik et al., 1981). Metabolism of FBZ reduced ovicidal activity in all cases. S-oxidation of FBZ to the intermediate sulphoxide, OFZ, resulted in a greater than 40-fold loss of activity, while the sulphone was only a weak ovicide. Hydroxylation of FBZ in the 4’-position (4’-OH-FBZ)resulted in a 3-fold loss of activity against H. contortus eggs, in sharp contrast to the more potent activity of 4’-OH-FBZ against mammalian tubulin. Oxidation of 4’-OH-FBZ to 4’-OH-OFZ and 4’-OH-FBZ-SO2 and decarbamoylation of FBZ to form NH2-FBZ abolished ovicidal and tubulin activity. Similarly, hydrolysis of OFZ and FBZ-SO? to NH2-OFZ and NH2-FBZ-SO1 resulted in total loss of activity. S-oxidation of ABZ to the sulphoxide and sulphone resulted in complete loss of activity in both assays. Hydroxylation of ABZ in the 2’- and 3’-positions reduced potency in both assays, while S’-OH-ABZ was inactive. Oxidation of the hydroxylated derivatives of ABZ and hydrolysis of the carbamates of ABZ, ABZ-SO and ABZ-SO2 resulted in complete loss of activity in both assays. Hence the metabolism of MBZ, FBZ/OFZ and ABZ resulted in the formation of derivatives which in all cases were less active than the parent compound as inhibitors of H. contortus hatching. With the exception of OH-FBZ and OH-OFZ, the activity of these metabolites as inhibitors of the polymer&&ion of mammalian tubulin was similarly reduced with respect to the parent compound. DISCUSSION

Although the use of egg-hatch as an in vitro assay for detection and quantitation of anthelmintic resistance has been routinely used for thiabendazole, its use for other benzimidazoles, particularly the BZCs, is restricted to initial studies by Coles and Simpkins (1977) and Hall et al. (1978a). Little or no information on its standardisation has been published and this has led to wide variations in the reported EDs0 values. These problems have been reviewed by Boersema (1984). In an attempt to standardise the procedure, sources of experimental variation such as the effects of solvent, incubation time, stage of egg development and isolated biomass were exam-

114

ined in this study. In most cases a quantitative comparison with the published literature (Coles and Simpkin, 1978; Kirsch, 1978; Hall et al.,1978a) cannot be drawn, as only anecdotal comments were made. As the present study involved the comparison of a number of BZCs possessing varying molecular weights, the data are reported in molarity units (PM) as distinct from previous studies where pg ml-’ or p.p.m. have been utilised. The use of DMSO as a co-solvent is essential since the aqueous solubility of these compounds is substantially lower than the concentration required for inhibition. While the use of suspensions will result in inhibition of hatching (Hall et al.,1978a), such studies do not provide an adequate measure of the actual concentration of drug available to the eggs since only drug in solution is adsorbed. The concentration of DMSO used for all inhibitor studies was 4.8%, this concentration providing maximal solubility for the BZCs without evidence of deleterious effects on the hatching process. The independence of biomass (eggs plus debris) and EDs0 over a g-fold range, demonstrated that sufficient MBZ was present to ensure equilibrium with the entire incubation mass. The ED5,, is therefore also independent of the viability of the eggs (as infertile eggs can be regarded as constituting an increased proportion of the debris) and the purity of the isolation within this range. Published reports of the crucial importance of rapid collection and isolation of nematode eggs suggest that TBZ exerts its activity only at the initial stages of embryonation, although evidence for this hypothesis is largely anecdotal (Coles and Simpkin, 1977). In this study, however, MBZ and PBZ demonstrated no significant change in potency up to 12 h (E3) pre-incubation. Although the possibility that the mechanism of action of TBZ on the egg is different from that of PBZ and MBZ cannot be dismissed, it is more probable that this discrepancy is due to other factors inherent in the method of isolation of eggs. Such a phenomenon could manifest its effect in vitro as a reduction in potency. After > 18 h pre-incubation, significant reduction in potency for PBZ and MBZ was observed, thereby indicating that a change in the effect of the drugs on the hatching process had occurred. The egg hatch assay is generally referred to as an ‘ovicidal’ assay, implying that the process is independent of incubation time before the EDs0 is determined, as eggs failing to hatch are considered dead. Based on the observed time dependence of the EDs0 of MBZ, and to a lesser extent PBZ, the action appears to be a mixed ovistatic/ovicidal one. The balance of this action appeared to be compound specific, with the EDs0 of MBZ showing a linear relationship to time representing principally an ovistatic effect, while PBZ exhibited primarily an ovicidal action with a minor ovistatic component. The balance possibly relates to the individual drug-egg tubulin equilibrium (association constant, drug availability, amount bound etc.). The presence of an ovistatic component in the inhibition of egg hatch has also been recognised for FBZ (Kirsch, 1978). Hence, it is probable that the primary manifestation of BZC inhibition of the hatching process is to reduce the rate or turnover of cellular processes involved in embryo development.

116

Investigation of the morphological changes in eggs produced from established infections by H. contortus, Ostertagia circumcinctu and Trichostrongylus colubriformis on treatment with fenbendazole showed that inhibition of egg development could be detected at an early stage (even in unlaid eggs) resulting in the formation of atypical blastomeres (Kirsch and Schleich, 1982). These observations are in agreement with the proposed mechanism of action of BZCs as microtubule inhibitors, manifesting their effect in rapidly dividing cells, such as developing eggs, via the inhibition of formation of the mitotic spindle with resultant failure of normal cellular division (Dustin, 1978). This hypothesis is further supported by the potent inhibitory action of MBZ and PBZ during El-E3 development, where cell division and cellular differentiation could be considered to be major components of embryogenesis. The reduction in potency at the E4 stage may demonstrate a shift in the mechanism of action possibly relating to the inhibition of other tubulin-related processes such as the inhibition of transport of substances crucial to the hatching process (Friedman et al., 1980). Since only unembryonated eggs could be detected after long incubation periods (200 h), this action is not considered to be a primary determinant in the assay. The inhibitory activity of the commercial BZCs at 48 h closely paralleled the I& obtained from studies of the inhibition of polymerisation of mammalian tubulin (Table I). These results and the results of a previous comparison of the inhibitory activity of BZCs on the polymerisation of mammalian tubulin and on the growth of L1210 mouse leukaemia cells (Lacey and Watson, 1985b) which gave a high quantitative correlation between the two assays, suggest that these compounds exert their effect by inbibition of cell division in the developing eggs. These results also support the recent observations of Dawson et al. (1984) on the effect of BCZs on Ascuridia tubulin. The authors reported that for MBZ, ABZ, FBZ, PBZ and OBZ, little difference existed between inhibitory activity against mammalian or nematode tubulin. However, substantial selectivity existed in the case of OFZ, which was found to be a non-inhibitor of the polymerisation of mammalian tubulin but was equipotent to the other BZCs against Ascaridia tubulin. Although the commercial BZCs have been shown to undergo extensive metabolism in mammalian species, little is known of the intrinsic activity of their respective metabolites. To date, no published literature has unequivocally defined whether the intrinsic activity is due to the parent BZC or to the metabolites using metabolically stable systems. For example, published literature on the pharmacokinetics of ABZ and FBZ suggests that the sulphoxide metabolites, ABZ-SO and OFZ, are the active moieties based on plasma profiles and in vivo efficacy (Marriner and Bogan, 1981). Since the sulphoxides are known to exist in a reversible equilibrium with the parent thioethers, it is’feasible that the sulphoxides are merely acting to alter the pharmacokinetics of the parent drug in a favourable manner. To

116

further examine the relationship between the egg hatch assay and microtubule function, it was considered necessary to examine the activity of known (or postulated) metabolites of a number of commercial BCZs (Table II). MBZ undergoes rapid first-pass metabolism in vivo (Dawson et al., 1982), as do FBZ and ABZ. Reduction of MBZ to the benzhydrol (OH-MBZ) resulted in a substantial reduction in activity both as an ovicide and as a microtubule inhibitor. Hydrolysis of the carbamate (NH2-MBZ) resulted in complete loss in activity. These observations are consistent with the observed reduction in in vivo efficacy of these metabolites as anthelmintics (Meuldermans et al., 1976). While the results qualitatively support the hypothesis that the egg hatch assay is an index of the inhibition of the polymerisation of nematode egg tubulin during egg development, it is necessary to point out possible limitations which may inherently bias this conclusion. The I& values are derived from an assay where the drug is capable of direct equilibrium to the binding site (postulated to be within the tubulin structure). With the EDso, the drug can only establish equilibrium at the binding site after passive partitioning from aqueous solution into the shell and subsequently into the egg lumen. For polar compounds, such as ABZ-SO, ABZ-S02, OFZ and FBZ-S02, where the partition coefficients due to sulphoxidation are lOOfold less than the parent thioether (ABZ and FBZ), the possibility exists that the failure to inhibit is due to lack of absorption rather than to inherent lack of activity. While no evidence exists that this is indeed the case, there is a clear need to establish the relationship between the physico-chemical properties of the inhibitors and their ability to partition into the egg and to the binding site. Such a study is currently underway in this laboratory. The data clearly suggest that little or no species specificity exists between the mammalian tubulin and nematode egg models. While this may in part be artifactual, it is feasible that other factors may determine adult nematode toxicity. Friedman et al. (1980) have noted that colchicine binding to egg tubulin extracts of Ascaris suum alters depending on the stage of embryogenesis. Since BZCs are competitive inhibitors of colchicine binding to mammalian tubulin, the nature of the binding site may undergo some alteration during development of the egg to the adult nematode. While no direct evidence of such a change exists, the reduction in potency of MBZ and PBZ during embryogenesis (Fig. 3) supports this hypothesis. Further, the action of BZCs on actively dividing cells and in vitro polymerisation assays represents a specific index of inhibition of formation of microtubules. While this represents an essential component of the role of tubulin in the cell, it is by no means its only function. Hence, in adult nematodes where active cell division is minimal (Hyman, 1951), the biochemical manifestation of the BZC-tubulin interaction may relate to other functions, which the ‘polymerisation’ models do not adequately describe. This would imply that while the correlation may exist between egg hatch assay and polymerisation of mammalian tubulin, the situation in E4, larvae

117

or adults may be altered. This could in part be an explanation for the differences between the resistance factors obtained using the egg hatch assay and in vivo efficacy assays with nematode strains showing varying degrees of BZC susceptibility. To substantiate this hypothesis it would be necesary to develop methods sensitive enough to detect changes in the binding characteristics of BZs at all stages of the nematode life cycle. CONCLUSIONS

The egg hatch assay has been routinely used for investigating the resistance status of nematodes to benzimidazoles. As an in vitro assay it possesses the advantage of eliminating the effect of host drug metabolism on activity. In this study, the activity of commercial BZC anthelmintics against hatching of H. contortus eggs was shown to be consistent with their ability to inhibit the polymer&&ion of mammalian tubulin. For the bioisosteres, PBZ, OBZ and ABZ and MBZ, FBZ and ABZ, the rank order of potency is the same in both assays. The metabolites of MBZ, FBZ and ABZ also conform to this trend. Compounds exhibiting tubulin inhibitory activity inhibited egg hatch, while compounds which did not inhibit tubulin polymerisation exhibited marginal or no ovicidal activity. The data support the hypothesis that the principal mode of action of BZCs as inhibitors of egg hatch is via an interaction with tubulin, inhibiting the formation of microtubules. ACKNOWLEDGEMENT

This work was supported by Australian Wool Board Grant Number L/9/921.

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