Pharmacokinetic disposition of benzimidazole drugs in the ruminant gastrointestinal tract

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Pharmacokinetic Dispositio n of Benzimidazole Drugs ,n the Ruminant Gastrointestinal Tract D.R. Hennessy Orally administered benzimidazole (BZ) drugs are ideally deposited in the rumen where they associate extensively with particulate digesta material, the residence time of this drug-digesta complex being a major influence on the subsequent rate and duration of BZ availability. This duration is shortened if the dose should bypass the rumen due to oesophageal groove closure. Benzimidazole metabolites flow from the rumen primarily in association with particulate digesta. In the abomasum, the majority of soluble metabolites result from gastric secretions. These metabolites flow into the small intestine where they are absorbed into the systemic circulation. Depending on the chemical structure a significant portion are secreted in bile either in a free (ie. unconjugated) or conjugated form. Free biliary metabolites are absorbedfrom the upper small intestine whereas bacteria in the large intestine hydrolyse the conjugated biliary metabolites to promote further absorption. Biliary derived metabolites are enterohepatically recycled but contribute little to the peripheral plasma metabolite pool. In this review, Des Hennessy discusses these issues in relation to the pharmacology of BZ drugs in the gastrointestinal tract of ruminants. Introduction of the benzimidazole (BZ) chemical class of anthelmintic drugs over 30 years ago heralded a major advance in the treatment of nematode parasites of ruminants. Subsequent modifications to the BZ molecular structure during the 1960s and 1970s produced improved compounds that were safe and that possessed a wide spectrum of activity. In a comprehensive review of their mode of action, Lacey 3 described how BZ drugs cause the disintegration of parasite cellular microtubule complex, the efficacy of which is dependent on the kinetics of the tubulin-BZ interaction and parasite expulsion. Time is a crucial element of BZ action, particularly if the mechanism(s) of removal of the parasite by the host (eg. inhibited or resistant strains) requires a longer period than the residence time of the anthelmintic drug. It is therefore evident that effective chemotherapy requires exposure of the parasite to the drug for as long as is practicable to reduce the selection for drug-resistant strains. The duration of BZ availability, as assessed by the concentrations of active metabolites in the bloodstream and, in some cases, the abomasal fluid, has been extensively examined in ruminants. As important as these findings might be, they provide little detailed information of BZ disposition throughout the reminder of the gastrointestinal tract (GIT). While BZ exposure to parasites residing outside the GIT occurs via the bloodstream, significant contact to parasites Des Hennessy is at CSIRO Division of Animal Health, HcNlaster Laboratory, Private Bag No. I, Glebe, NSW 2037, Australia. © 1993, Elsevier $oence Pubhshers I td, (UK)

inhabiting the gut lumen is obtained from metabolites flowing with digesta. With the escalation of resistance to BZ drugs, particularly by GI parasites, a more detailed pharmacokinetic understanding of BZ disposition in the ruminant GIT is vital if improved delivery, and therefore efficacy, is to be obtained. Distribution in and absorption from the rumen The low aqueous solubility of BZ compounds ideally requires their formulation as oral suspensions that deposit the drug directly and wholly within the rumen. Due to the strong reducing conditions in the rumen, BZ metabolites exist largely as the reduced moiety, in particular the sulphides fenbendazole (FBZ) and albendazole (ABZ). The pro-BZ drugs febantel and netobimin are rapidly reduced in the rumen to their respective FBZ and ABZ parent. Even a significant portion of an oxfendazole (OFZ) dose is reduced to FBZ, this reaction providing virtually the only source of FBZ that occurs in the bloodstream after ORZ administration 2. From this point onwards the pharmacokinetic behaviour of the compound throughout all compartments will be profoundly influenced by the dynamics of the rumen environment. Occupying up to 70% of the animal's abdominal cavity volume, the rumen is filled with digesta fluid and particulate material, the outflow of these digesta phases relying on mixing and propulsive movements that integrate with the opening of the reticulo-omasal orifice3 in response to chemical and tactile stimuli 4,5. These stimuli are, in turn, governed by dietary factors including digesta particle size 6, density 7, chemical and physical characteristics 8 and the quantity of feed consumed 6 resulting in the half-life of digesta fluid and particulate material being about 12 and 18 h, respectively9. Increasing the surface area by reducing the particle size will assist dissolution and absorption 1° but, within two hours of administration, BZ drugs are almost completely associated with digesta particulate material 9 (Fig. la). Such association, together with the reduction of OFZ to FBZ, would contribute to the low concentrations of OFZ in the rumen fluid of sheep, which has been attributed only to the low solubility of OFZ 2. The physicochemical nature of the association of BZ with particulate digesta is unclear, but the attraction is probably by physical adsorption rather than specific chemical bindinglL The rapidity and magnitude of particulate association is undoubtedly due to the large availability of binding sites of digesta cellulose. To determine the fate of OFZ in rumen digesta, the kinetic disposition of OFZ and its metabolites was examined; (1) after intraruminal (IR) injection, and (2) when the equivalent OFZ dose was presented to the rumen totally contained in either rumen fluid or

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330

Dose

~

estafluid

i ~ f~~(c)

" (f)

Rumen Particulate~/ ~ (d) ~ digesta ' r " - ~ ~.

Abomasum

a~_ P,asm

(e ! ~ ~ = ~ _ ~ • (-~

Intestine

I

Faeces

~

(i)

~

Urine

~ ] ~

~

~ 1 ~ Liver

)

(rn)

Fig. I. Schematic representation of the disposition of benzimidazole drugsin the ruminant. See text for details.

particulate material9. The latter administration regimen was achieved by collecting rumen digesta three hours after IR injection of OFZ to a donor sheep, and dividing the digesta into fluid and particulate phases based on the size of particulates that naturally flow through the omasum 12. Each digesta phase, containing OFZ and its metabolites, was mixed with the alternate metabolite-free phase obtained from the rumen of an untreated recipient sheep and the reconstituted rumen digesta then returned to the recipient animal. Within two hours, over 90% of the OFZ metabolites that were transferred in rumen fluid associated with rumen particulate digesta. During this period, some unassociated OFZ a n d / o r metabolites were absorbed from digesta fluid into the bloodstream, and some flowed from the rumen with digesta fluid contributing to the initial metabolite appearance in subsequent compartments. An equilibrium of OFZ and its metabolites then established between the particulate and fluid digesta (Fig. lb), the rate of exchange of drug between these phases being governed by desorption from the particulate material in response to decreasing metabolite concentration in the fluid following direct absorption (Fig. ld) and outflow (Fig. lc). Thereafter, the kinetic disposition of the transferred OFZ and metabolites was generally similar to that observed in the IR-treated sheep indicating that the association of OFZ with rumen particulate material contributed to drug residence time. This indication was supported by experiments involving with the administration of OFZ and its metabolites when they were completely associated with rumen particulate material. The period necessary for the desorption of metabolites from particulate to fluid digesta, followed by adsorption, emphasizes that the extended presentation of drug from the rumen 'reservoir' is a primary determinant of the broad metabolite concentration-with-time profile of this anthelmintic in the bloodstream and GIT 2,13,14. In practice, this response might be reduced by full or partial closure of the oesophageal groove. Before weaning, when rumen microflora have yet to develop fully, optimum nutritional value of colostrum is

obtained if it deposits in the abomasum. The physical action of suckling, together with continued introduction of fluid into the buccal cavity, stimulates a muscular contraction along the lesser curvature of the rumen to form a groove that directs colostrum past the rumen and into the abomasum. The oesophagealgroove reflex, originally considered to cease operating after weaning, continues at varying degrees throughout the ruminant's life. Oral administration of anthelmintic has been demonstrated to stimulate closure of the groove, with a proportion of the dose being directed to the abomasum of cattle 15 and goats 16. Complete rumen bypass also occurred in about 25% of one-year-old sheep treated orally with OFZ, while partial bypass was evident in a further 30% 17. Rapid absorption of OFZ following rumen bypass slightly increased plasma OFZ concentrations, but the absence of the ruman reservoir effect reduced OFZ residence time and dramatically lowered anthelmintic availability.

Feedeffects Residence time of drug in the rumen is also a function of the rate of flow of digesta. While ruminants continuously graze, rumen digesta flow is relatively steady but varies greatly with changes in dietary intake 6. Since the rumen volume is essentially constant, an inverse relationship exists between the quantity of feed consumed and digesta residence time 18. The level of feed intake, then, will affect drug residence time. Following oral administration to sheep fed a daily ration of 400 or 800 g lucerne/wheaten chaff, OFZ availability in sheep on high compared with low intake showed a more rapid rate of drug absorption, an earlier maximum concentration in plasma, a faster rate of elimination and overall lower systemic metabolite availability 19. In explaining these results, the sheep on a higher feed intake had a more rapid rate of rumen particulate digesta outflow than those sheep fed the reduced ration. Since OFZ and its metabolites were almost completely associated with particulate material 9, the metabolite half-life in the rumen was identical to the half-life of particulate digesta; its faster passage in sheep on high feed intake reducing the duration for drug-digesta exchange, absorption and recycling. Recent reports show that the disposition of BZ drugs and their metabolites in sheep, cattle and buffalo is affected by the feed type. As the proportion of the fresh green feed component of the daily intake increased, so the systemic availability of FBZ and OFZ decreased 13,14,2°. The earlier appearance of a digesta fluid marker in the faeces of fresh pasture fed compared with dry fed animals 13 suggests that the lower availability of FBZ in the pasture-fed animals was due to a reduced duration for drug absorption. The results confirm the concept that a shorter gastric transit time in sheep on high feed intake, particularly of fresh feed with high water content, decreases the time for drug absorption and recycling which contributes to reduced drug availability.

Dispositionintheabomasum

Benzimidazole metabolites enter the abomasum from two directions: those in digesta that flows from the rumen (Fig. lc), and those in gastric secretion

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(Fig. lf). There have been no reports of BZ disposition in the omasum but since this organ functions as a digesta filter, entry of metabolites to the abomasum by this route can be regarded as equivalent to exit from the rumen. It goes without saying that the flow characteristics of rumen digesta directly influence the disposition of metabolites in subsequent compartments, particularly the abomasum. Following oral administration of the FBZ to cattle 2~, ~30% of the dose flowed into the abomasum in fluid and digesta particles which were less than 50 ~m in dL~meter. The presence of FBZ in larger particulate digesta was not determined, the authors concluding that the unaccounted balance of FBZ dose had been absorbed from the rumen. However, further studies on the disposition of FBZ in the sheep intestine showed that a considerably lower proportion of the dose had been adsorbed, and had probably flowed into the abomasum associated with the large particulate digesta 22. Much of the drug in the abomasum appears to remain predominantly associated with particulate material 9,19. This is a little surprising when one considers that the solubility of BZ drugs increases significantly at the low pH of the abomasum 23,24. The greater fluid content of abomasal digesta compared with the rumen would also be expected to facilitate the exchange of metabolites from the particulate to fluid digesta phases (Fig. le); and the extent to which gastric secretions (Fig. lf) contribute to soluble abomasal metabolites must be considered. Studies with isolated gastric pouch preparations in sheep revealed that about 12% of an orally administered OFZ dose was contained in abomasal secretions (J.W. Steel et al., abstract)t. The metabolite concentration-with-time profile in abomasal fluid was qualitatively similar and followed the same time course as that in the bloodstream, indicating that most of the metabolites in abomasal fluid were not desorbed from particulate digesta flowing from the rumen but arose from the plasma pool via gastric secretions. Indeed, the outflow of 10-14% of the OFZ dose in abomasal fluid 9,19 compares well with the 12% in gastric secretion (J.W. Steel et al., abstract)t. Furthermore the contribution of gastric secreted metabolites to abomasal fluid was evident when the sheep's feed intake was reduced 19. Gastric secretions responded quickly to feed intake 25, the abomasal fluid flow rate was reduced by some 60% when feed intake was halved. Although the same OFZ dose was intraruminally administered, a lower proportion of the dose was secreted into the abomasum contributing to only 4% of the dose in abomasal fluid of sheep on low feed intake, compared to 10% on high feed intake. The exchange of BZ metabolites between abomasal particulate and fluid digesta therefore appears to be influenced by the influx of metabolites contained in gastric secretions. Notwithstanding that the surface area and availability of binding sites on particulate material may be greater in the abomasum than in the rumen, the combination of low pH and significant gastric secreted metabolites retains a larger proportion of the dose in the fluid phase of the abomasum than

t Parasitology - Quo Vadit? VI International Congress of ParasitologyHand

book (Howell, M.J.,ed.), p. 234, AustralianAcademy of Science

Table I. Comparative disposition of BZ compounds in bile, urine and faecesa

Drug

Biliary

Dose (%) Faecal

Urinary

5(6) Aromatic substituted BZ FBZ OFZ

38 63

87 86

I0 17

M BZ

-

85

I0

TCBZ

46

91

7

19 20

80 69

5(6) Aliphatic substituted BZ PBZ ABZ

24 14

Data from Refs 21, 28-31. Abbreviations: FBZ, fenbendazole; OFZ, oxfendazole; MBZ, mebendazole;TCBZ, triclabendazole; PBZ, parben-

dazole; ABZ, albendazole; BZ, benzimidazole.

rumen. Because only dissolved drug is anthelmintically active 2, the presence of such large quantities of soluble metabolites provides significant contact and therefore activity, against parasites of the abomasum. S e c r e t i o n a n d r e - a b s o r p t i o n in the i n t e s t i n e

On entry to the duodenum, the proportion of digesta-associated xenobiotics appears to decrease11; the particulate association of OFZ and its metabolites was as low as 60% of total metabolites present (S. Tremain, pers. commun.) Studies in cattle showed that 27% of an FBZ dose flowed through the pylorus in digesta fluid and fine particles (Fig. lh) whereas 52% of the dose traversed the ileo-caecal junction 21 clearly implicating the biliary route as a major contributor of BZ metabolites to the ruminant GIT. The presence of biliary metabolites helps to explain the lower particulate bound proportion in the duodenum. Large quantities of BZ metabolites are excreted in faeces 26, the initial inference of this observation was that BZ compounds were poorly absorbed; those that were absorbed were only excreted in urine. While some metabolites may indeed pass through the GIT unabsorbed, the majority of the dose excreted in faeces will have undergone some degree of absorption and re-secretion. A major route of secretion of xenobiotics into the GIT is biliary, and the presence of many compounds have been examined in rodent and human bile 27. There has been qualitative examination of mebendazole (MBZ) in rats 28 and humans 29 but the quantitative biliary secretion of other BZ drugs in ruminants has been neglected. This was undoubtedly due to the difficulty in obtaining sequential bile samples from a live animal. With the subsequent development of a specifically designed pump mounted on the sheep's side and attached to a re-entrant bile cannula 22,3°,31bile production was continuously monitored revealing that 14% of an ABZ dose was secreted in this fluid (Fig. li). Following initial absorption, the 5(6) aliphatic substituted ABZ is oxidized at a rate which is consistent with 'first pass' metabolism (Fig. lg) with ABZ sulphoxide (ABZ.SO) being the predominant metabolite in plasma and urine 32. Due to the polarity and low molecular weight of ABZ.SO, it is largely excreted in urine without the need for significant further metabolism; biliary secretion is thus a relatively minor

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pathway in the clearance of ABZ (Table 1). For the 5(6) aromatic substituted OFZ, FBZ and triclabendazole (TCBZ), biliary secretion plays a more significant role in metabolism and elimination22, 31. To increase solubility and facilitate excretion these compounds are sulphoxidized, hydroxylated at the 4' position and between 40% and 63% of the FBZ, OFZ or TCBZ dose is secreted in bile (Table 1). Of the total biliary metabolites, between 10% and 20% are secreted 'free' with 80-90% conjugated as a glucuronide or sulphate ester. On entry to the intestine a proportion of biliary metabolites are re-absorbed and enterohepatically recycled. This process increases the apparent volume of distribution and extends the half-life, the more extensive the recycling, the longer the residence of the drug. To quantify the enterohepatic recycling of OFZ, unconjugated (or 'free') and conjugated biliary metabolites of OFZ were infused into the small intestine of sheep 22. The immediate appearance of 'free' biliary metabolites in plasma and their re-secretion in bile, together with the rapid disappearance of the metabolites in these pools after cessation of the infusion identified the upper small intestine as a major site of re-absorption of unconjugated biliary metabolites (Fig. lk). It is not unreasonable to suggest that similar metabolites (eg. OFZ, ABZ.SO) that flow from the abomasum in digesta fluid, or those which may exchange from particulate material in the intestine, might be similarly absorbed from this site. Since the mucosal surface of the upper small intestine is the habitat of pathogenic nematodes such as Trichostrongylus colubriformis, this extended metabolite exchange contributes significantly to anthelmintic action. The appearance of metabolites in bile and plasma for several hours after cessation of the infusion of conjugated biliary metabolites indicated that these were deconjugated by bacteria and reabsorbed from the large intestine (Fig. lm). As much as 17% of an equivalent oral OFZ dose traversed the large intestinal wall but minimal exchange with peripheral plasma occurred, the metabolites almost exclusively transported via the portal circulation and re-secreted in bile (Fig. ln). These processes of detoxication and excretion are, of course, time dependent and this is reflected in the broad metabolite concentration-withtime profiles of aromatic compared with aliphatic substituted BZ compounds 2,17,33,34. It should be pointed out, however, that the aliphatic ABZ metabolites (ie. ABZ.SO) are intrinsically more potent than equivalent aromatic substituted compounds 35. Metabolites that occur in proximal regions of the intestine and that passage the GIT associated with digesta material are present in the large intestine 33,34. However, the reduction of OFZ to FBZ at this site provides a likely explanation for low levels of faecal OFZ, an observation that prompted Short et al. 33 to question the significance of this putative active metabolite and suggest the involvement of hydroxy-FBZ. Biliaryderived hydroxy-OFZ was not measured 33,34, probably accounting for their underestimation of total faecal clearance of FBZ, but hydroxy-metabolites are anthelmintically active3s and the extended exposure of these metabolites as they enterohepatically recycle undoubtedly contributes to the high efficacy against parasites of the large intestine.

While biliary secretion has been identified as a major pathway for intestinal metabolite secretion, entero-enteric recycling may also contribute to the disposition of BZ drugs in the intestine (Fig. 11). The mechanism(s) of intestinal secretion (IS) are not fully understood, but the extent of IS depends on the solubility of the metabolite, the adsorptive capacity of digesta within the intestine and the performance of other processes of metabolite elimination 36. As IS involves passive transfer of metabolites from plasma to the intestine, the quantity and type of metabolites transferred is a reflection of their systemic concentrations. Quantitation of the IS of BZ drugs has not been defined specifically but in the goat it has been estimated at 3 -5% of an FBZ dose 34. The greater systemic availability of similar metabolites that follow OFZ administration 23 could contribute to an even greater IS than that reported from FBZ. An indication of how IS is influenced by other mechanisms can be obtained indirectly from biliary investigations. A characteristic action of the now-superseded BZ parbendazole (PBZ), is a temporary reduction in bile secretion37 and when PBZ was co-administered with OFZ, the proportion of the OFZ dose secreted in bile was reduced from 62% to 49% but excretion in faeces was reduced only from 86% to 81% 22. The apparent increase of about 8% of the dose that entered the intestine via a non-biliary route was attributed to an increased IS. The greater anthelmintic response of the PBZ/OFZ combination emphasizes the importance of this kinetic pathway in parasite-drug interaction. Concluding c o m m e n t s

Resistance by parasites of ruminants to the existing drug arsenal will soon be upon us. While the potential profits are significant, the huge costs and risks associated with developing innovative chemical classes have dramatically reduced the introduction of new compounds. In effect, those drugs on which we so heavily rely will continue to be the only available drugs at least for the immediate future. Coupled with a potential loss of anthelmintic activity is society's concern over the use of chemicals in agriculture which may lead to residues in edible tissues and in the environment. It therefore behoves us to use existing compounds more efficiently. With greater understanding of the kinetic disposition of BZ drugs in the GIT, procedures that increase availability can be promoted. Most importantly, the drug must be wholly administered over the tongue to reduce oesophageal-groove effects and maximize the reservoir action of the rumen. Broadening the BZ concentration-with-time profile by multiple dosing has advantages: by dividing a 10 mg kg 1 OFZ dose into three treatments (ie. 5, 2.5 and 2.5 mg kg 1) separated by the approximate half-life of OFZ in the rumen (about 12 h) increased OFZ activity against BZ-resistant nematodes from 53% to 81% 18. The inconvenience of multiple dosing may be overcome with the development of appropriate slowrelease formulations or short-term pulse release intraruminal devices. Ruminants that are consuming large quantities of feed, particularly fresh green pasture which induce rapid gastric transit, reduce the duration for drug absorption and recycling. This has the potential for

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sub-therapeutic anthelmintic action and should be avoided. Slowing digesta flow rate by feeding smaller quantities of a dry, bulkier, ration will very effectively extend BZ availability. Indeed, halving the feed intake of sheep for 36 h before treatment significantly increased the activity of OFZ or ABZ against BZresistant Haemonchus contortus and T. colubriformis. It is hoped that the information in this review will demonstrate that treatment procedures that are based on physiological/pharmacological aspects of the ruminant GIT can be used to increase the availability of BZ, and other compounds such as closantel and ivermectin. Not only might this prolong their effective therapeutic life, but reduced dose rates will contribute significantly in lowering residues in edible tissues and the pasture environment. References 1 Lacey, E. (1988) Int. J. Parasitol. 18, 885--936 2 Marriner, S.E. and Bogan, J.A. (1981) Am. J. Vet. Res. 42, 1143--1145 3 Reid, C.S.W. (1984) in Ruminant Physiology - Concepts and Consequences (Baker, S.K. et al., eds), pp 79-84, University of Western Australia 4 Leek, B.F. and Harding, R.H. (1975) in Digestion and Metabolism in the Ruminant (McDonald, I.W. and Warner, A.C.I., eds), pp 60-76, University of New England 5 Crichlow, E.C. et al. (1980) Fed. Proc. 39, 890 6 Faichney, G.J. (1986) in Control of Digestion and Metabolism in Ruminants (Milligan, L.P., Grovum, W.L. and Dobson, A., eds), p. 173, Prentice Hall 7 Koritz, G.D. (1983) in Veterinary Pharmacology and Toxicology (Ruckebusch, Y., Toutain, P-L. and Koritz, G.D., eds), pp

More on Unravelling the Cytokine Network in Malaria In response to the article entitled 'Unravelling the Cytokine Network in Malaria' by J. Tavem@, we believe that our recent results are a step in the right direction toward unravelling the cytokine network in blood-stage malaria. Using inbred strains of mice which are either resistant or susceptible to infection with the rodent malaria species, Plosmodium chdbdudi AS, we have characterized the profile of cytokine production by spleen cells during the course of infection in response to parasite antigen and the mitogen, ConA. Initially, we determined the levels of interferon gamma (IFN-~/) and interteukin S (IL-5) in spleen cell supematants by ELISA~. fflore recently, we have determined IL-4 levels (NI.M. Stevenson, et all., unpublished). Spleen cells from resistant C57BL/6 mice, which experience moderate and transient parasitemia with clearance of the parasite by 28 days post infection, and immunity to re-infection, produce high levels of IFN-~/ within the first week followed by production of IL-4 and IL-5 between two and four weeks post infection. In contrast, spleen cells from susceptible A/J mice, which experience severe infection characterized by fulminant parasitemia and high mortality

151-163, MTP Press Hendricksen, R.E. et al. (1981) Aust. J. Agric. Res. 32, 389-398 Hennessy, D.R. et al. Int. J. ParasitoI. (in press) Shastri, S. et al. (1980) Am. J. Vet. Res. 41, 2095-2101 Lees, P., et al. (1988) Res. Vet. Sci. 44:50-56 Van Soest, P.J. (1982) Nutritional Ecology of the Ruminant, O&B Books 13 Taylor, S.M. et al. (1992) Vet. Rec. 130, 264-268 14 Ali, D.N. and Chick, B.F. (1992) Res. Vet. Sci. 52, 382-383 15 McEwan, A.D. and Oakley, G.A. (1978) Vet. Rec. 102, 314-315 16 Sangster, N.C. et al. (1991) Res. Vet. Sci. 51,258-263 17 Prichard, R.K. and Hennessy, D.R. (1981) Res. Vet. Sci. 30, 22-27 18 Kay, R.N.B. (1986) in Physiological and Pharmacological Aspects of the Reticulo-rumen (Ooms, L.A.A., Degryse, A.D. and Van Miert, A.S.J.P.A.M., eds), pp 115-170, Nijhoff 19 Ali, D.N. and Hennessy, D.R. Int. J. Parasitol. (in press) 20 Sanyal, P.K. et al. Int. J. Parasitol. (in press) 21 Prichard, R.K. et al. (1981) J. Vet. Pharmacol. Ther. 4, 295-304 22 Hennessy, D.R. et al. J. Vet. Pharmacol. Ther. (in press) 23 Ngomuo, A.J. et al. (1984) Vet. Res. Commun. 8, 187-193 24 Marriner, S.E. et al. (1985) Vet. Parasitol. 17, 239-249 25 McLeay, L.M. and Titchen, D.A. (1974) Br. J. Nutr. 32, 375-387 26 Gottschall, D.W. et al. (1990) Parasitology Today 6, 115-124 27 Levine, W.G. (1981) Prog. Drug Res. 25, 361-420 28 Allan, R.J. and Watson, T.R. (1982) Eur. J. Drug Metab. Pharmacokinet. 7, 131-136 29 Witassek, F. et al. (1983) Eur. J. Clin. Pharmacol. 25, 81-84 30 Hennessy, D.R. et al. (1989) J. Vet. Pharmacol. Ther. 12, 421-429 31 Hennessy, D.R. et al. (1987) J. Vet. Pharmacol. Ther. 10, 64-72 32 Gyurik, R.J. et al. (1981) Drug Metab. Dispos. 9, 503-508 33 Short, C.R. (1987) Am. J. Vet. Res. 48, 811-815 34 Short, C.R. et al. (1987) Am. J. Vet. Res. 48, 958-961 35 Lacey, E. et al. (1987) Vet. Parasitol. 23, 105-119 36 Israili, Z.H. and Dayton, P.G. (1984) Drug Metab. Rev. 15, 1123-1159 37 Hennessy, D.R. et al. (1992) J. Vet. Pharmacol. Ther. 15, 10-18 8 9 10 11 12

with death between I 0 and 12 days post infection, produce significantly lower (background) levels of IFN-~ and high levels of IL-4 and IL-5 within the first week post infection. Results of preliminary studies suggest that susceptible A/J mice also produce high levels of IL- 10 within the first week of infection (M.iVl.Stevenson et ol., unpublished). These results confirm previous observations by Langhome and colleagues34 that resolution of blood-stage P. chobaudi infection in CS7BL/6 mice requires sequential activation ofTHI cells followed by activation of Tm2 cells and that transfer of protective immunity to severe combined immunodeficient mice requires CD4 + T cells and antibody. Based on these observations, Langhome 3 proposed that, during the eady or acute phase, THI cells mediate the rapid decrease in peak parasitemia to low or sub-patent levels by antibody-independent, cell-mediated mechanisms. These mechanisms presumably involve activated macrophages, the hallmark of a TH I-type response. Langhome further proposed that TH2 cells, which provide help for antibody production, mediate control of the late or chronic stage of infection via an antibody-dependent mechanism. Taken together, these results argue for the role of both cell-mediated and antibody-mediated mechanisms in the development of immunity to primary blood-stage infection. However, these mechanisms appear to be

required at discrete times during infection. We believe more importantly that our results demonstrate for the first time that activation of the TH2 subset of CD44 T cells early in the course of blood-stage malaria, as occurs in susceptible hosts, leads to a severe and lethal outcome. We have hypothesized that inappropriate activation of Tm2 cells may mediate the immunosuppression and hypergammaglobulinemia which occur during malaria in humans and in experimental animals, and which contribute to the severity of malaria in humans. Indeed, the ability of the TH2 derived cytokines, IL-4 and IL-10 to regulate cell-mediated responses negatively, particularly the effector functions of macrophages has recently received attention. As pointed out by Taverne, the ability of human recombinant IL-4 to inhibit macrophage-mediated killing of P. falciporum in vitro was demonstrated by Kumaratilake and Ferrante5. Recent results fi-om other parasite systems demonstrate the abilityof IL-4 to inhibit the microbicidal functions of IFN-~/activated macrophages in vitro 6,7. The ability of IL- I 0 to down regulate cell mediated immunity by its de-activating effects on macrophages has also been established in other parasite models but the role of IL- I 0 in malaria is as yet unknown7 ~. We would agree with the concept proposed by Kumaratilake and Ferrante l0