References. I Piekarski, G. (1954) Lehrbuch tier Parasitologie,. Spdnger-Verlag. 2 Martinez-Palomo, A. (1982) The Biology of. Entamoeba histolytica, Research ...
Parasitology Today, voL 9. no. /, / 993
Acknowledgements We thank M. Hasegawa and T. CavalierSmith for preprints, M. M011er and P. Gogarten for useful information and H. Scholze for drawing our attention to Retl I. Work from our lab has been supported by the Deutsche Forschungsgemeinschaft (SFB 171 project C2) and by the Fonds der Chemischen Industrie. References I Piekarski,G. (1954) Lehrbuch tier Parasitologie, Spdnger-Verlag 2 Martinez-Palomo, A. (1982) The Biology of Entamoeba histolytica, Research Studies Press 3 Reeves,R.E.(1984)Adv. Parasitol. 23, 105 142 4 Cavalier-Smith,T. (1983) in Endocytobiology II (Schenk H.E.A and Schwemmler,W., eds), pp 1027-1034, de Gruyter 5 Sogin,Mi. (I 989) Am. ZooL 29, 487499 6 Johnson, A.M. and Baverstock, PR. (1989) Parasitology Today 5, 102-105 7 W6stmann, C., Tannich, E. and Bakker Grunwald, T. (1992) FEBSLett. 308, 54-58 8 Hasegawa,M. et al.]. Mol. Evol. (in press) 9 SiddaEM,E, Hang, H. and Desser,S.S.(1992) ]. Protozool. 39, 361- 367
31 10 MOiler, M. (1988) Annu. Rev. Microbial. 42, 465488 II Bhattacharya,S. et of. (1989)J. Protozool. 36, 455458 12 Adam, R.D. (1991) Microbial. Rev. 55, 70(~732 13 Cavalier-Smith,T. ( 1991) BioSystems25, 25-38 14 de Duve, C. (1991) Blueprint for a Cell: The Nature and Origin of Life, Neil Patterson 15 Cavalie~Smith,T. ( 1991) in Evolution of Life (Osawa, S. and Honjo, T., eds), pp 271 304, Spnnger-Vertag 16 Bakker-Grunwald, T. (199I) in Biochemical Protozoology (Coombs, G. and North, M., eds), pp 367 376, Taylor & Francis 17 Gillin, F.D., Reiner, D.S. and McCaffery, M. ( 1991) Parasitology Today 7, I 13 I 16 18 Damell,J. and Lodish, H. (1990) Molecular Cell Biology, ScientificAmerican Books 19 Maloney, P.C. and Wilson, T.H. (1985) BioScience 35, 4348 20 Fahey,R.C.et al. (1984) Science 224, 70- 72 21 Clark, C.G. and Diamond, LS. (1991) Mol. Biochem. Parasitol. 49, 297 302 22 Tannich, E. et al. (1991) J. Biol. Chem. 266, 47984803 23 Cavalier-Smith,T. Endocytobiology V (Ishikawa, H., Ishida,M. and Sato, S., eds),T0bingen Uni versityPress(in press)
24 Li, W-H. and Graur, D. ( 199I) Fundamentals of Molecular Evolution, SinauerAssociates 25 Wheelis, M.L, Kandler, O. and Woese, C.R. (1992) Proc NatJ Acad Sci. USA 89, 2930~2934 26 Jentsch,S.,Seufert,W. and Hauser,H.P. ( 199I) Biochim. Biophys.Acta 1089, 127 139 27 Scholze,H. et aL Arch. Meal Res. (in press) 28 Aley, S.B.,Cohn, Z.A. and Scott W.A. (1984) J. Exp.Meat 160,724-737 29 L6hden, U. and Bakker-Grunwald,T. (1989) Anal. Biochem. 182,77 83 30 McLaughlin,J.and Aley,S.(1985)J.ProtozooL32, 221 240 31 Bakker-Grunwald,T. (1992) j. Exp Biol. 172, 31 I 322 32 Huber, M. et aL (I 987) Mol. Biochem. ParositoL 24, 227 234 33 Torres-Guerrero,H., Peattie,D.A. and Meza, I. ( 199t) Mol. Biochem. Parasitol. 45, 121 130 34 Li, E., Kunz-Jenkins,C. and Stanley,S.L. (1992) Mol. Biochem. Parasitol. 50, 355-358 35 Tannich,E. et al. ( 199I) Mol. Biochem. Parasitol. 49, 61 72 Tilly Bakker-Grunwald and Claudia W6stmann are at the Department of Microbiology, University of Osnabrdck Bdrborostrasse I I, W-4500 Osnabr~ck Germany.
Mechanistic Approaches to Quantitate Anthelmintic Absorption by Gastrointestinal Nematodes D.P. Thompson, N.F.H. Ha, S.M. Sims and T.G. Geary In this article, David Thompson, Norman Ha, Sandra Sims and Timothy Geary look at the problem o f quantitating drug absorption by gastrointestinal nematodes.
The utility of compounds as drugs is dependent on two general factors: the avidity (and selectivity) with which they interact with the desired target (receptor), and the transport properties that allow the d e l i v e r / o f effective concen trations of the compound to the receptor in sufficient time to cause the therapeutic effect. These two factors, while not governed by mutually exclusive structural features, are nevertheless independent; the structural properties that confer maximum affinity for the receptor may or may not be those that result in optimal pharmacokinetics. The structural features that govem receptor binding vary with the receptor and chemical series of interest. In contrast, the physicochemical properties of molecules that govern absorption and distribution can be characterized in more general terms. This principle has been exploited to © 1993. ElsevierScience Publishers Ltd, (UK)
improve the performance of a wide range of therapeutic agents q, However, drug delivery science has been applied to anthelmintics in only a few cases, and restricted to achieving sustained release or more convenient application forms of existing drugs 2. Factors T h a t Affect A n t h e l m i n t i c Delivery Factors that influence the bioavailability of anthelmintics include host biology (eg. site and kinetics of absorption, drug metabolism, disease state), parasite biology (eg. site of predilection, absorption kinetics), dosage form and physicochemical properties of the drug (eg. lipophilicity, pK~, molecular size). Although the principles that govern the absorption and distribution of drugs in mammals are well established, little is known about the mechanisms by which drugs are absorbed by gastrointestinal (GI) nematodes. We do not know, for instance, if useful anthelmintics are
absorbed following oral ingestion (from host blood or intestinal contents) or via their extemal surface (cuticle) or some combination of both routes. We do not know if the physicochemical factors that favor absorption by the intestine of mammals also favor absorption by helminths. Since these organisms occupy a wide range of ecological niches in mammals and employ a variety of feeding strategies, optimal deliver/of anthelmintics is a multifactorial concept. Previous research on the absorption of drugs by GI nematodes has focused primarily on the large parasite of swine, Ascaris suum. Factors contributing to this include its wide distribution, the importance of Ascaris spp in both human and veterinary medicine and, most importantly, the fact that it is large enough to study using a variety of techniques. Trim 3 showed that the rates of absorption of nicotine, chloroform and homologs of resorcinol by A. suum were unaffected by mechanical ligation of the mouth and anus, and that absorption paralleled changes in lipid partitioning. Fetterer~
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Parasitology Today, vol. 9. no. I. 1993
showed that absorption of a steroid series in isolated segments of A. suurn cuticle and by third-stage, ie. nonfeeding, larvae of Haemonchus contortus, a stomach parasite of sheep and cattle, correlated well with lipid partitioning. Though few studies have examined the rates or mechanisms by which modem anthelmintics are absorbed by nematodes, Verhoeven et al. s showed that accumulation of levamisole by A. suum can be accounted for solely by transcuticular diffusion. These studies indicate the importance of lipoidal barriers and suggest that the cuticle is an important site for drug absorption. To provide quantitative and mechanistic insight into the drug transport properties of GI nematodes, we designed mass transport studies using isolated A. suum cuticle and a wide range of model permeants and drugs. A biophysical model for drug absorption by nematodes was developed and evaluated using intact A. suum and a closed perfusion system. These studies have been further extended to other GI nematodes, including H. contortus and Trichostrongylus colubriformis. The factors that regulate drug absorption by A. suum appear to extend to the above species and probably to others. Isolated Cuticle Segments
Standard two-chamber diffusion cell systems can be used to quantitate the rates at which drugs diffuse across isolated membranes. Among GI nematodes this approach is restricted to A. suum, from which segments of cuticle as large as 2 x 2 cm can be prepared 6. Segments ofA. suum cuticle (muscle tissue scraped away) are mounted between a matched pair of diffusion cells, and medium is added to each side. The diffusion cells exposed to the epicuticle and the hypodermis serve as the 'donor' and 'receiver' sides, respectively. The transport study is initiated by adding permeant to the donor cell. Samples are obtained from both chambers immediately thereafter and at selected timepoints over a 2-8 h period. The amount of permeant added, the duration of the study and the volume of the samples collected depend on the permeant(s) used. For radiolabeled permeants, it is possible to use trace levels ( 1000 Da will cross
the cuticle slowly; measuring their diffusion rates may require high initial concentrations or extended incubations. When appropriate source: sink conditions are maintained, ie. when the donor cell contains at least 90% of the permeant, the transport of permeants across segments of A. suum cuticle remains linear for at least 24 h (Fig. I). The general equation for deriving drug permeability coefficients using isolated cuticle segments is shown in Box I. The system should be evaluated for edge effects or perforations in the tissue, either of which would invalidate the study, at the end of each experiment. Residual medium on the donor side is replaced with fresh medium containing a high concentration of an impermeant (molecular mass >3000 Da) marker, such as blue dextran or Nile blue A. It is useful to include model permeants with known physicochemical properties in studies to delineate the permeability coefficients of anthelmintics, because they provide both standards for comparison and a measure of the quality of the data. Unlike absorption across mammalian intestine, which is strictly lipid in nature, absorption of drugs and model permeants across the cuticle ofA. suum and other nematodes is restricted by lipid barriers in the hypodermis and possibly other regions within the cuticle (where lipid and collagen interdigitate) and also by the collagen matrix 6. Restrictions imposed by the lipid barriers are probably similar to those of standard membranes, ie. the rate of penetration of a drug will depend mainly on its lipophilicity (approximated by the log of its partition coefficient in an organic/aqueous
solvent system, such as octanol/water). In the case of an acidic or basic drug, absorption will also depend on the pK~ of the drug and on the pH of the aqueous environment within the cuticle, which will determine the fraction of drug in unionized, ie. lipid-permeable, form. Drug penetration through the collagen barrier of the cuticle is restricted by the size of the negatively charged, aqueousfilled pores within the collagen matrix 6. All other factors (eg. lipophilicity, pK a, charge) being equal, the net effect of size restriction imposed by the collagen barrier is that large molecules penetrate more slowly than do smaller ones. It is thus helpful to normalize the data on a size basis (usingthe Renkin factor, F(r/R) 7) when comparing the permeability coefficients of a set of compounds according to their apparent partition coefficients. This requires knowledge of the radius of the aqueous pore barrier (R, 1.5 nm in A. suum) and the molecular radius of the drug or model permeant (r), which may be estimated using the Stokes-Einstein relationship8. The effect of size normalization on the permeability coefficients for a set of drugs and model permeants is shown in Fig. 2. Isolated tissue offers several advantages over intact organisms. Most important, anatomical openings ( % mouth, anus, genital or excretory openings) can be avoided, allowing unambiguous assessment of the permeability properties of the cuticle itself. Other factors that affect transport kinetics, including pH, protein concentration, temperature and the unstirred water layer at both surfaces of the preparation, can be better controlled. Also, the cuticle surface area
0.25
b 0.2
0.15 -
0.1-
0.05-
0
2
4
6
8
0
2
4
6
6
Time (hours) Fig. I. Transport kinetics of iverrnectin (a) and urea (b) across isolated segments of A. suum cuticle (lipid-extracted, solid curves;unextracted, broken curves) at pH 7.4 and 3 7~C.
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Parasitology Today, vol. 9, no. I, t993
The closed perfusion system we use to measure drug absorption by intact A. suurn has been described previously9. A single A. suum is perfused with medium containing a known amount of permeant. Samples of medium are collected at selected timepoints for either scintillation counting or high pressure liquid chromatography analysis. In general, when designing experimental protocols, the same principles apply to the experiment duration, drug concentrations and sample volumes that apply to studies using isolated cuticle segments. The general equation for deriving drug permeability and partition coefficients using intact nematodes is shown in Box 2. The quality of data used to derive permeability and partition coefficients (Peand K, respectively) from studies using intact onganisms is augmented by measuring drug levels in the nematode as a function of time, ie. tissue appearance kinetics, along with external medium levels. When there is no evidence of either drug metabolism or significant loss of compound to the walls of the perfusion system, tissue levels can be determined at one timepoint only, as long as it occurs after the equilibrium phase is reached in the disappearance kinetic plot (Fig. 3). Other factors may require determination of tissue drug levels at several timepoints, including (I) use of very small helminths for which it is
Box I. Drug Transport Across Isolated Cuticle Segments Plots of the fraction of the initial donor concentration of ivermectin and the model permeant, urea, appearing in the receiver cell as a function of time show typical zero order kinetics (Fig. I). Using the linear slope of the fraction of permeant appearing in the receiver cell as a function of time, the permeability coefficient (Pe, cm min-I) for each permeant is derived by the equation:
p,_ AC0(0)\ At ) where Co (0) = initial concentration of drug in the donor cell (~mol ml-I)
VR= volume of receiver cell (ml) A = area of exposed cuticle (cm 2) CR= concentration of drug in the receiver cell (Hmol ml i) L~CR/L~t ----linear rate of appearance of drug available for drug transport is known precisely and drug levels can be measured on each side of the preparation, facilitating quantitative assessments.
ligated and nonligated nematodes and a closed perfusion system9. This approach can also be extended to smaller species of nematodes. To quantitatively follow the absorption kinetics of drugs by intact helminths, a closed perfusion system that maximizes the ratio of the parasite surface areato-volume of incubation medium is required. Small perfusion chambers can be used for studies involving A. suum, and small culture tubes with minimal volumes of culture medium are adequate for studies involving other species, ie. adequate surface area-to-volume ratios are also achieved using 25 adult H. contortus and 0.5 ml medium.
Intact Organisms
The major disadvantages of the isolated cuticle approach are that it addresses neither the relative contributions of the transcuticular and oral pathways nor the influence that underlying tissues may have on drug absorption. These issues can be addressed directly by measuring the disappearance kinetics and tissue distribution of drugs using intact,
1o-
a
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-4
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-1
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2
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Fig. 2. Correlation between permeability coefficients (P) for selected permeants across the A. suum cuticle and the apparent partition coefficients
of the permeants in n-octanol/water at pH 7.4 and 37°C before (a) and after (b) correction for molecular size restriction.
Parasitology Today, voL 9, no. I, 1993
34
Box 2. Drug Absorption and Partitioning in Intact Nematodes The rate of transcuticular permeation followed by distribution in the body tissues of intact nematodes is derived from the change in drug concentrations in both the external solution and the nematode, leading to equilibrium (Fig. 3). These processes are described by the equation:
where C = drug concentration in incubation medium (~mol ml-~) at time t A, = nematode surface area (cm 2) P~ = permeability coefficient of the drug (cm min-I) V = volume of incubation medium (ml) Cn = drug concentration in nematode (pmol ml-I) at time t K = partition coefficient of the drug between the nematode and the incubation medium (dimensionless) When equilibrium is attained, ie. at the plateau phase of the disappearance curves in Fig. 3, the net flux is equal to zero. Hence, the effective partition coefficient is obtained by:
K- C.(~)
c(oo)
where C, (oo) = concentration of drug in the nematode at equilibrium C (oo) = concentration of drug in the medium at equilibrium Due to the lipophilic nature of modern anthelmintics, K may greatly exceed unity, indicating that drug accumulates within the parasites to concentrations that exceed those in the incubation medium. High K values occur when intermolecular forces attracting lipophilic drugs to the organic, ie. lipid-containing, components of the parasite exceed the potentially opposing forces of the diffusional gradiend L
kinetics nor nematode-to-incubation medium partition coefficients are affected by mechanical ligation of the intestine, indicating that drug absorption in A. suum occurs primarily via the transcuticular pathway. This concept also appears to extend to smaller nematodes, including H. contortus and T. colubriformis, which can be chemically ligated using low concentrations (< I riM) of ivermectin.
impossible to achieve an adequate ratio of worm surface area-to-volume of incubation medium, and (2) use ofpermeants with low specific activity. In each of these cases, the tendency for lipophilic drugs to concentrate in parasite tissue facilitates quantitative detection. To determine the relative contribution oftranscuticular absorption versus ingestion, separate studies can be performed using A. suum ligated with surgical thread. Ligation patency can be assessed by measuring accumulation by the intestine of Nile blue A or blue dextran. Among the model permeants and anthelmintics we have examined, neither absorption
Helminth
Metabolism
The presence of metabolizing tissues in intact nematodes may complicate
1(
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0.9
0.8
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0.7
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I
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I
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Time (hours)
l
20
efforts to delineate Pe and K if the drug itself is metabolized. Though the capacity for drug metabolism by helminths is generally thought to be extremely low, based mainly on studies using A. suum, significant metabolism of some permeants occurs. When drug metabolism occurs, Pe may be calculated using data from the initial period of the absorption kinetic experiment. This is accomplished by the equation: C
In--C(0)
AnPet
V
where C, A, P and V are explained in Box l. Note that no term for K appears in this expression. When metabolism occurs, the concentration of parent drug in the carcass does not reach an equilibrium with that in the incubation medium, ie. the nematode acts as an infinite 'sink' for the drug. Drug metabolism by the parasites will often lead to effiux of metabolites. Metabolism should be suspected when mass balance of the parent molecule is not achieved. Metabolizing tissue may also affect the rate of absorption of acidic and basic drugs by affecting the pH of incubation medium at the host-parasite interface. That is, the cuticle is an important site for excretion of organic acid end-products of energy metabolism, principally the volatile fatty acids, in nematodes ~°. This process results in the formation of a buffered microenvironment within the aqueous pores of the collagen matrix, which is maintained at pH -5.0, near the collective pK~ of the organic acids excreted. The pH within the aqueous pore microenvironment will dictate the ratio of unionized to ionized forms of acidic and basic compounds, according to the pH/pK~ relationship for each drug. Since only the unionized fraction will readily diffuse across the lipoidal barriers
b
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I
26
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0
~
5
i 10
Time
i
16
2
i0
215
(hours)
Fig. 3. Disappearance Idnetics of hydrocortisone in ligated (a) and non-ligated (b) A. suum. Closed and open circles represent separate experiments.
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Parasitology Today, vol. 9, no. I, 1993
3
10-
b
t~ T
X
f
-
v
2
o O
I
I
/
1
2
3
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7
8
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I
I
I
I
I
I
r
1
2
3
4
5
6
7
8
log K(n-octanol/pH 7.5)
log K (n-octanol/pH 7.5)
Fig. 4. Relationshipbetween permeability coefficient(Pe) (a) or nematode to medium partition coefficient(K) (b) and lipophilicity, expressedas log
K (n-octanol/buffer at pH 7.5). Permeants include aniline (open triangle), hydrocortisone (closed circles), levamisole (open diamonds), thiabendazole (open circles), dosantel (closed squares) and two analogs of closantel (open, inverted triangles and open squares), which are less lipophilic. Similar relationshipsare obtained in studies usingintact A. suum. of the cuticle, the microenvironment pH may profoundly influence P, (Ref. 10). Neutral compounds will not be affected by this process. Applications and F u t u r e Directions
Knowledge of the mechanisms by which anthelmintics are absorbed and the physicochemical factors that favor their absorption by pathogenic helminths should facilitate the discovery of anthelmintics and support the design of more effective delivery systems. Based on information already derived from the approaches described, the cuticle appears to be the principal site for anthelmintic absorption. The rate-determining barrier for absorption of most drugs (molecular radii _5, and weak bases of pK~
