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Comparative assessment of the access of albendazole,fenbendazole and triclabendazole to Fasciola hepatica :effect of bile in the incubation medium L. I. ALVAREZ1, M. L. MOTTIER2 and C. E. LANUSSE1* 1 Laboratorio de Farmacologi'a, Departamento de Fisiopatologi'a, Facultad de Ciencias Veterinarias,Universidad Nacional del Centro de la Provincia de Buenos Aires, Campus Universitario, 7000, Tandil, Argentina2 Consejo Nacional de Investigaciones Cientificas y Te'cnicas (CONICET), Argentina (Received 28 May 2003; revised 6 August 2003; accepted 6 August 2003) The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole(TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical com-position of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusionkinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for thebenzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificiallyinfected sheep. The flukes were incubated (37 xC) over 60 and 90 min in incubation media (pH 7.4) prepared with dif-ferent proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0 % of bile) containing eitherABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material waschemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrationswithin the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as anindicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica atboth incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic moleculesABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ bythe liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound.
This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drugconcentrations measured within the parasite were lower in the incubations containing the highest bile proportions. Thehighest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of bile. Altogether,these findings demonstrated that the entry of the drug into a target parasite may not only depend on a concentrationgradient, the lipophilicity of the molecule and absorption surface, but also on the physicochemical composition of theparasite's surrounding environment.
Key words : trans-tegumental drug diffusion, benzimidazole anthelmintics, Fasciola hepatica, bile acids, albendazole,fenbendazole, triclabendazole.
kinetic properties that allow the delivery of effectivedrug concentrations to the receptor inside the para- The economic importance of helminth infections in site, in sufficient time to cause the therapeutic effect livestock has long been recognized and it is probably (Thompson et al. 1993). Anthelmintic drugs can for this reason that the most relevant advances in the reach target helminth parasites by either oral inges- chemotherapy of helminthiasis have come from the tion or by diffusion through the external surface animal health area (Horton, 1990). Chemotherapy of the parasite, or some combination of both routes still remains the most widely used method to control (Thompson & Geary, 1995). The accumulated parasitism in livestock (Zajac, Sangster & Geary, data show that the main route of acquisition of 2000) and human health (Quellette, 2001). The ac- broad-spectrum anthelmintics by target parasites tivity of most anthelmintic molecules is based on appears to be by passive diffusion through their their affinity for a specific receptor, and on the tegument (cestodes/trematodes) (Alvarez, Sa'nchez& Lanusse, 1999 ; Alvarez et al. 2000, 2001) orcuticle (nematodes) (Ho et al. 1990 ; Sims et al. 1996 ; * Corresponding author : Laboratorio de Farmacologi'a, Cross, Renz & Trees, 1998). Consequently, the Departamento de Fisiopatologi'a, Facultad de Ciencias rate of penetration of a drug will mainly depend on Veterinarias, Universidad Nacional del Centro de la Pro- the intrinsic lipid-to-water partition coefficient of vincia de Buenos Aires, Campus Universitario, 7000, the molecule (Mottier et al. 2003), pH/pK relation- Tandil, Argentina. E-mail : clanusse@vet.unicen.edu.ar1,2 ship, molecular size, concentration gradient and the Both authors have equally contributed to the work surface area of contact between drug and parasite.
L. I. Alvarez, M. L. Mottier and C. E. Lanusse Benzimidazoles (BZD) are broad-spectrum an- metabolite found in plasma after ABZ adminis- thelmintic compounds widely used in human and tration to sheep, has been recovered at higher con- veterinary medicine to control nematode, cestode centrations compared with the parent drug in and trematode infections (McKellar & Scott, 1990).
abomasal and intestinal fluids of treated sheep (Alvarez, Sa'nchez & Lanusse, 1999 ; Alvarez et al.
anthelmintics can be grouped as BZD thiazolyls, 2000). However, in specimens of Moniezia spp.
BZD methylcarbamates, pro-BZD and halogenated (Alvarez et al. 1999) and Haemonchus contortus BZD thiols (Lanusse & Prichard, 1993). Only a few (Alvarez et al. 2000) collected from the same ABZ- molecules within the BZD chemical family demon- treated animals, the availability of ABZ parent drug strated activity against the trematode, Fasciola was greater than that of its sulphoxide metabolite.
hepatica. Albendazole (ABZ) is the only BZD The in vivo uptake studies carried out in Moniezia methylcarbamate recommended to control fascio- spp. and H. contortus demonstrated that ABZ has the liasis in domestic animals, despite its activity being capability to concentrate in the parasite. Such a restricted to flukes older than 12 weeks (McKellar & pattern was not observed in liver flukes (Alvarez et al.
Scott, 1990). Fenbendazole (FBZ), a similar BZD 2000), where the ABZ ratio of area under the con- methylcarbamate widely used in veterinary medicine as a nematodicidal drug, is not as effective as ABZ F. hepatica and bile was 0.33, which clearly demon- against F. hepatica, but a single treatment of 5 mg/kg strated a lower drug accumulative process in the adult reduced F. gigantica infection in sheep by up to 95 % trematode parasite. These findings suggest that the (Roberson & Courtney, 1995). Unlike other BZD drug-partitioning phenomenon between gastroin- compounds, the halogenated derivative triclabenda- testinal fluid and parasite tissues might be different zole (TCBZ) has been shown to have an excellent from that occurring between the surrounding bile efficacy against the adult and juvenile stages of F. hepatica (Boray et al. 1983). However, TCBZ ac- The current experiments were designed to inves- tivity appears to be restricted to the liver fluke and tigate the comparative ability of ABZ, FBZ and the lung fluke, Paragonimus spp. (Weber, Buscher & TCBZ to diffuse into mature F. hepatica and to Buttner, 1988 ; Calvopina et al. 1998), because the assess the influence of the physico-chemical com- drug does not show clinical efficacy against nema- position of the incubation medium on the drug todes, cestodes and other trematode (Dicrocoelium diffusion process. The results obtained from the dendriticum, Paramphistomun spp. and Schistosoma trans-tegumental diffusion kinetic studies were mansoni) parasites. BZD nematodicidal activity is complemented with the determination of lipid- based on its binding to parasite b-tubulin (Borgers & to-water partition coefficients (octanol-water) of the De Nollin, 1975 ; Lacey, 1988 ; Lubega & Prichard, anthelmintic drugs assayed, as an indicator of drug 1991), which inhibits polymerization into micro- tubules (Friedman & Platzer, 1980). Thus, all thefunctions ascribed to microtubules at the cellularlevel are altered (cell division, maintenance of cell shape, cell motility, cellular secretion, nutrient ab- sorption and intracellular transport) (Lacey, 1988).
BZD methylcarbamate molecules such as ABZ or Eight (8) parasite-free Corriedale sheep were in- FBZ act upon nematode microtubules at the tubulin fected with 200 metacercariae of F. hepatica each, colchicine binding site (Lacey, 1988). It is likely that given in a gelatine capsule by the oral route. Sixteen a different site of action is involved on the flukicidal weeks after infection the animals were killed by activity of TCBZ, which could also explain its lack of captive bolt plus exsanguination, following inter- efficacy against other helminth parasites (Stitt & nationally accepted animal welfare guidelines. To Fairweather, 1994). However, differences in the recover adult specimens of F. hepatica from the ability of ABZ, FBZ and TCBZ to penetrate liver, common bile ducts and the gall-bladder of through the F. hepatica external tegument may help each sheep were removed and opened. The speci- to explain the observed differences in clinical efficacy mens were rinsed extensively with saline solution among those chemically related drugs. Comprehen- (NaCl 0.9 %) (37 xC) to remove bile and/or adhering sion of the patterns of drug diffusion into target parasites, in conjunction with the available pharma-codynamic information on drug-receptor interac- tions, may substantially contribute to elucidation ofthe mechanisms of drug action and enhancement of The collected flukes were maintained for 2 h before starting the incubation in a Krebs' Ringer Tris BZD anthelmintics are extensively metabolized in (KRT) buffer (pH 7.4) at 37 xC (McCraken & all mammalian species studied (Lanusse & Prichard, Lipkowitz, 1990). Two flukes (approximately 0.2 g) 1993). Albendazole sulphoxide (ABZSO), the main were incubated at 37 xC for 60 and 90 min in 2 ml of Effect of bile on drug diffusion into F. hepatica an incubation medium (pH 7.4) prepared with bile of each analyte were quantified by comparison of the and KRT buffer in different proportions (100/0, 75/ chromatographic peak area of each analyte with that 25, 50/50, 25/75 and 0/100), containing either ABZ, obtained for the internal standard, using the Class FBZ or TCBZ at a final concentration of 5 nmol/ml.
LC 10 Software (Shimadzu, Kyoto, Japan) on an This is a pharmacologically relevant concentration IBM 486-AT computer. The final concentration obtained from previously reported work where the values for the different drugs assayed are expressed BZD concentrations in bile were measured after as nmol/100 mg protein. The determination of conventional treatments in ruminants (Hennessy parasite protein concentrations was carried out ac- et al. 1987 ; Alvarez et al. 2000). Ovine bile was col- cording to the methodology described by Smith lected from the gall-bladder of non-infected un- treated sheep killed at the local abattoir at the sametime as the infected animals. There were 6 replicateincubation assays for each drug at each incubation Octanol-water partition coefficients (PC) time. Blank samples containing parasite material The octanol-water PC (Log P) was used as an indi- and incubation medium without drug, and drug- cator of lipid solubility of the BZD molecules used in spiked medium without parasite material were in- the current experiment. The methodology used to cubated during the same time-intervals. Once the calculate this parameter was adapted from that re- incubation time had elapsed, the flukes were rinsed ported by Pe'hourcq, Thomas & Jarry (2000). The thoroughly with saline solution, blotted on coarse- octanol-water PC was estimated by the combination filter paper and stored at x20 xC until their prep- of the traditional shake-flask technique and HPLC aration for high performance liquid chromatography (HPLC) to measure drug concentrations. The para- Germany) and desionized ultrapure water (pH 7.4) site material was processed shortly after the incu- (Simplicity1, Water purification system, Millipore, Brazil) as a biphasic liquid system. Samples of 20 mlof either ABZ, FBZ or TCBZ (from 1 mM stock solutions) were added to 1980 ml of desionized ultra-pure water previously saturated with n-octanol.
The parasite material (0.2 g) was homogenized using Under the chromatographic conditions described an Ultraturrax1 homogeniser (T 25, Ika Works above, 200 ml of the aqueous phase were collected, Inc., Labortechnik, USA) and spiked with oxiben- evaporated to dryness and re-suspended in 150 ml of dazole (OBZ) used as internal standard. The parasite the HPLC mobile phase (27 % acetonitrile, 73 % material homogenate was mixed with 1.5 ml of water) to calculate the peak area of the analyte before methanol and shaken for 5 min to extract the drug partitioning (W0). In screw-capped glass tubes, the analyte/s present in the fluke sample. The collected remaining 1800 ml of the aqueous phase (Vaq) were methanol phase was evaporated to dryness. The supplemented with 200 ml of an octanol phase (Voc), residue obtained was dissolved in 1 ml of a meth- previously saturated with desionized water (pH 7.4).
anol/water solution (20/80) and prepared for HPLC The mixture was shaken for 90 min in a mechanical analysis using the extraction procedure described by shaker (Cole Parmer1, Vernon Hills, Illinois, USA) Alvarez et al. (1999). All the solvents and reagents at 15 xC. The mixture was then centrifuged (1500 g, used during the extraction and drug analysis pro- 5 min) and 1 ml of the lower aqueous phase was Experimental and spiked liver fluke samples were in 150 ml of mobile phase and injected into the analysed to measure the concentrations of each drug HPLC system to determine drug concentration by HPLC using a model 10 A system (Shimadzu, in the aqueous phase after partitioning (W1). The Kyoto, Japan). The extraction efficiency of the dif- partitioning of the drug between both phases ferent analytes from parasite material samples, ex- (P value) was calculated using the following equation pressed as absolute recovery, ranged between 85 and 97.5 % with a coefficient of variation (CV) off15.5%. The quantification limits of the HPLC 0.27 nmol/100 mg protein. The molecules understudy were identified by comparison with the reten- The partition coefficient (Log P) was calculated as tion times of pure drug standards, which were used the logarithm of the obtained experimental P value.
to prepare standard solutions to construct the cali-bration lines for each analyte in the parasite material analysed. The linear regression lines for each analytein the range between 0.27 and 27.2 nmol/100 mg pro- The individual concentration values are reported as tein (triplicate determinations) showed correlation mean¡S.D. The statistical analysis of the data was coefficients greater than 0.995. The concentrations performed as follows : (a) the comparison of the L. I. Alvarez, M. L. Mottier and C. E. Lanusse concentrations achieved in F. hepatica in the dif-ferent assayed incubation media, for each drug(ABZ, FBZ or TCBZ) and at each incubation time(60 and 90 min), was performed by analysis of vari-ance (ANOVA) ; (b) Student's t-test was used tocompare drug concentrations obtained at 60 and90 min of incubation in the different incubation asciola hepatica
media. The statistical analysis was performed using F
the Instat 3.0 Software (Graph Pad Software, SanDiego, California). When ANOVA was employedand a significant F value was obtained, Tukey'srange test was performed to indicate order of sig- (nmol/100 mg protein)
Drug concentrations in
The 3 molecules investigated were detected in F.
hepatica after their ex vivo incubation. The amountsof ABZ, FBZ and TCBZ recovered from F. hepaticaincubated in the absence of bile were significantlygreater than those obtained with media containingbile (100, 75, or 50 %). The comparison of the drugconcentration profiles recovered in liver flukes in- asciola hepatica
cubated with ABZ and FBZ in media with different F
composition is shown in Fig. 1. The highest con-centration values for ABZ (20.1¡8.15 nmol/100 mgprotein) and FBZ (13.5¡4.06 nmol/100 mg protein)were measured in flukes incubated in the absence of (nmol/100 mg protein)
bile. In the presence of bile in the incubation me-dium (100 % bile), TCBZ concentrations recovered Drug concentrations in
from the flukes ranged between 0.32¡0.07 and0.47¡0.17 nmol/100 mg protein. Those TCBZ con-centrations have a significant enhancement in theincubations without bile, reaching values up to7.48¡2.62 (60 min) and 8.76¡3.16 (90 min) nmol/ Fig. 1. Comparison (mean¡S.D.) between albendazole 100 mg protein. There was a positive correlation (ABZ) and fenbendazole (FBZ) concentrations (nmol/ between the percentage of KRT buffer in the 100 mg protein) measured in Fasciola hepatica incubated incubation medium and the drug concentrations with different proportions of ovine bile.
measured in F. hepatica, with high correlation coef- concentrations are significantly different from those ficients obtained for ABZ (>0.81), for FBZ (>0.88) Although all drugs penetrated the trematode's tegument, the rates of penetration were different. In slightly greater FBZ lipid solubility (compared to all cases, the concentrations of the most lipophilic ABZ) may have to be taken into account to explain BZD methylcarbamates (FBZ, ABZ) recovered in why the extension of the incubation time up to F. hepatica were higher than those of TCBZ. The 90 min allowed its recovery at higher concentrations partition coefficients (Log P) obtained for FBZ, ABZ in flukes incubated in the presence of bile at 25, 50 and TCBZ were 3.99, 3.82 and 3.48, respectively.
and 75 % of the total medium composition. Inter- The relative ability of ABZ and FBZ to penetrate estingly, the amount of ABZ recovered from into the liver flukes incubated in different media F. hepatica incubated exclusively in ovine bile after 60 and 90 min is presented in Fig. 1. After (100 %) was between 32 % (90 min) and 220 % 60 min of incubation, the amount of ABZ recovered (60 min) higher than that measured for FBZ.
from the parasite was significantly greater than that Despite the differences in the amount of FBZ re- of FBZ, regardless of the composition of the incu- covered in F. hepatica between 60 and 90 min of bation medium. However, the length of drug incu- incubation, the length of the incubation period did bation for the flukes seems to play a role, as some not significantly affect the drug concentration pro- differences in the uptake pattern between ABZ and files of both ABZ and TCBZ recovered within the FBZ were observed after 90 min of incubation. The Effect of bile on drug diffusion into F. hepatica asciola hepatica
F
asciola hepatica
F
(nmol/100 mg protein)
(nmol/100 mg protein)
Drug concentrations in
Drug concentrations in
asciola hepatica
F
Fig. 3. Comparison of fenbendazole (FBZ) andtriclabendazole (TCBZ) (mean¡S.D.) concentrations(nmol/100 mg protein) measured in Fasciola hepaticaincubated without bile. The insert shows the octanol- (nmol/100 mg protein)
water partition coefficients (PC) for both molecules.
The diffusion of FBZ into F. hepatica was between 50and 80 % higher than that of TCBZ. The mean Drug concentrations in
concentration values obtained at 60 min of incubation arestatistically different at P<0.05.
Fig. 2. Diffusion of albendazole (ABZ) and triclabendazole (TCBZ) into Fasciola hepatica. Resultsexpress drug concentrations (mean¡S.D.) (nmol/100 mg The relationship between F. hepatica and its sur- protein) in flukes after 60 and 90 min of incubation with rounding environment occurs both across its exter- (A) and without (B) bile in the incubation media.
nal (tegument) and internal (gastrodermal cavity) concentration values are significantly lower than those surfaces (Thompson & Geary, 1995). The relative importance of these 2 available routes for druguptake in F. hepatica is still unclear. However, the The relative diffusion of ABZ and TCBZ into higher absorption surface of the tegument probably F. hepatica after 60 and 90 min of incubation with determines its major relevance in drug diffusion (100 %) or without (0 %) bile is shown in Fig. 2. The from the surrounding medium. This statement is diffusion of ABZ was significantly greater than that supported by the fact that F. hepatica can survive observed for TCBZ in all the incubation conditions long periods under in vitro conditions, in the absence under investigation. The diffusion of ABZ into of detectable nutrient absorption across the intestine F. hepatica incubated in ovine bile was between (Smith & Clegg, 1981). Additionally, the higher 281 % (60 min) and 434 % (90 min) higher than that concentrations of the lipophilic ABZ parent drug measured for TCBZ. In the absence of bile, ABZ recovered in F. hepatica, compared to the more polar diffusion was between 129 % (90 min) and 151 % sulphoxide metabolite under ex vivo conditions (Alvarez et al. 2000) may also contribute to demon- strate the relevance of the trans-tegumental drug (mean¡S.D.) measured in F. hepatica incubated passage. A large number of experiments have shown without bile during 60 and 90 min, and the octanol- that different chemical substances, as well as an- water partition coefficients for both molecules are thelmintic drugs, are mainly taken up through the compared in Fig. 3. The diffusion of FBZ into the external surface, as opposed to oral ingestion, in trematode parasite was between 50 and 80 % higher H. contortus (Rothwell & Sangster, 1997 ; Alvarez et al. 2000), Ascaris suum (Ho et al. 1990 ; Alvarez L. I. Alvarez, M. L. Mottier and C. E. Lanusse et al. 2001), Moniezia spp. (Alvarez et al. 1999 ; Mottier et al. 2003), F. hepatica (Fetterer & Rew, tegumental rate of ABZ diffusion into F. hepatica 1984 ; Alvarez et al. 2000, 2001), Onchocerca ochengi reported in the current work, and the higher avail- (Cross, Renz & Trees, 1998) among other helminth ability of its active metabolite in bile may account for the advantageous flukicidal activity of ABZ com- The accumulated data show that anthelmintic pared to FBZ. However, other factors such as a drugs move across the external surface of helminth differential portal blood concentration profile and parasites by passive diffusion. In this process, the differences in affinity for fluke b-tubulin should be membrane behaves as an inert lipid-pore boundary, considered in order to understand the low flukicidal and drug molecules traverse this barrier either by activity of FBZ, a compound that is chemically diffusion through the lipoprotein region or, alterna- closely related to ABZ and shows an equivalent tively, filtering through aqueous pores (channels) spectrum of activity against nematode parasites.
without the cellular expenditure of energy if they are of sufficiently small size (Baggot, 1982). The rate of used as flukicidal drugs in domestic animals. While diffusion is proportional to the area of diffusion ABZ is recommended for flukes older than 12 weeks, surface, the concentration gradient across the mem- TCBZ is active against both mature and immature brane and to the lipid-to-water partitioning of the stages of F. hepatica (Boray et al. 1983), being the drug (Baggot, 1982), and it is inverse to the medium most extensively used flukicidal drug in veterinary viscosity of the drug-containing medium (Ho¨rter & medicine (Coles & Stafford, 2001). The intensive use Dressman, 2001). Lipid solubility is a major factor of TCBZ in endemic areas of fascioliasis has resulted determining drug penetration across nematode in the development of liver flukes resistant to this cuticle (Alvarez et al. 2000, 2001) as well as through compound (Overend & Bowen, 1995 ; Mitchell, the tegument of cestodes (Alvarez et al. 1999 ; Maris & Bonniwell, 1998 ; Moll et al. 2000 ; Thomas, Mottier et al. 2003) and trematodes (Fetterer & Rew, Coles & Duffus, 2000), which is considered a major 1984 ; Alvarez et al. 2000, 2001) Although, there are problem for veterinary therapeutics. A recent study relevant structural differences between cuticle and has shown that ABZ is active against TCBZ- tegument, the mechanism of drug entry to both type resistant isolates of F. hepatica (Coles & Stafford, of structures seems to be equally dependent on 2001). If it is assumed that TCBZ and ABZ may act lipophilicity as a major physicochemical determinant on tubulin in F. hepatica, then differences in uptake of drug capability to reach therapeutic concen- or metabolism of these 2 drugs could explain their trations within the target parasite. The logarithm of differential efficacy against TCBZ-resistant flukes the octanol-water PC (Log P) was chosen as an in- (Robinson et al. 2002). The drug biotransformation dicator of drug lipophilicity since it is the most fre- capacity of the liver fluke, recently characterized by quently used parameter for defining the lipophilic Solana, Rodri'guez & Lanusse (2001), could have character of a given drug molecule (Pe'hourcq et al.
potential involvement in the development of resist- 2000). This coefficient represents the fraction of ance to BZD compounds. It is possible that TCBZ molecules that distribute in an organic phase may target a molecule other than b-tubulin, which (octanol) versus an aqueous phase (water), and pro- would explain why ABZ continues to act against vides an estimate of how readily a molecule will TCBZ-resistant flukes. However, the comparative penetrate a lipoidal membrane such as the trematode ability of ABZ and TCBZ to penetrate through tegument. In all cases, the most lipophilic BZD the tegument of susceptible liver flukes shown here methylcarbamates (FBZ, ABZ) were recovered at provides some useful information. The diffusion of higher concentrations, as compared to TCBZ, in the ABZ was significantly greater than that observed for TCBZ in all the incubation conditions under Regardless of the time of incubation, the avail- investigation similar results were observed for FBZ ability of ABZ in liver flukes incubated in the ab- regardless of its lower flukicidal activity. This would sence and presence of bile was significantly higher suggest that the lower PC value obtained for TCBZ than those measured for FBZ. It has been suggested could play against its trans-tegumental diffusion that the sulphoxide metabolites of both ABZ and ability. Finally, the greater trans-tegumental dif- FBZ may contribute substantially to the nemato- fusion capability of ABZ compared to TCBZ may dicidal (Lanusse & Prichard, 1993) and flukicidal account for its efficacy pattern against TCBZ- (Fetterer, Rew & Knight, 1982) activities of the resistant flukes, which is a relevant finding to be parent compounds. A series of free and conjugated ABZ and FBZ metabolites have been recovered in Lipid solubility is a relevant factor to determine the bile of treated sheep (Hennessy et al. 1989 ; drug diffusion into a target parasite. However, Hennessy, Prichard & Steel, 1993). However, the although lipophilicity is an important condition to concentration profiles of the anthelmintically active define drug diffusivity through lipoidal tissues, it unconjugated ABZ sulphoxide metabolite measured does not account for all factors that control this in bile were higher than those of FBZ sulphoxide process. The results presented here demonstrated Effect of bile on drug diffusion into F. hepatica that the presence of bile in the incubation medium parasite location and the lipid solubility of the an- modified the diffusion of ABZ, FBZ and TCBZ into thelmintic molecule are not the only parameters to F. hepatica. The higher the proportion of the KRT consider when drug kinetics is evaluated. The buffer in the incubation medium, the greater the physicochemical characteristics of the tissues and concentrations of the 3 molecules recovered within fluids surrounding the parasite may play a relevant the flukes. Why did bile modify drug diffusion into role in drug diffusion into the parasite. The findings the parasite ? Bile is an hepatic aqueous secretion described here, together with those previously composed of biliary acids and pigments, lipids, reported from in vivo drug uptake studies, indicate amino acids and glucose, amongst others. Biliary that availability is dependent upon features of the secretion has different functions, such as providing environment where the parasite is located. For in- an excretory route for metabolic detoxification prod- stance, a given ABZ concentration (e.g. 5 nmol/ml or ucts, including metabolites and drugs ; neutralize g) may not represent the same in the gastrointestinal the H+ in the duodenum ; and providing a source of fluid content, in a mucosal tissue or in the bile. The bile acids that are necessary for fat digestion and partitioning of the active drug/metabolites between absorption. Bile acids are surfactants and they re- an aqueous gastrointestinal fluid and the lipoidal duce the surface tension of water. This enables water tissue of the target parasite may facilitate the ac- to wet surfaces that are normally water-repellent, cumulation of the drug within the parasite, as has dissolving substances that are normally insoluble in been shown for H. contortus (Alvarez et al. 2000).
water and emulsifying substances that do not nor- This drug partitioning phenomenon may be differ- mally mix with water. Consequently, bile acids act as ent for other sites of parasite location such as the detergents and bring water-insoluble material into biliary tract, where the bile-induced micelle for- solution by forming a negatively charged aggregate mation may affect the diffusion of the active drug/ called a micelle. This increases the surface area metabolite into the target parasite (e.g. liver fluke).
enormously and facilitates the diffusion across the These findings seem to indicate that the physico- lipid membrane into the cell. Solubilization into chemical features of the environment where the tar- simple bile salt micelles has been reported for many get parasite is immersed play a pivotal role in the poorly water-soluble drugs, which has been corre- process of drug access, indicating that some hel- lated with higher intestinal drug absorption (Del minths may be protected from the deleterious effect Estal et al. 1993 ; Virkel et al. 2003). Drug solutions of an anthelmintic drug when living in their pre- in micellar media consist of 2 separate phases : an dilective location tissues. This phenomenon may aqueous phase with a fraction of the drug free in also explain many therapeutic failures observed in solution, and a micellar phase with the remaining parasite control in both human and veterinary fraction of the drug solubilized in micelles (Poelma medicine, which in some cases have contributed to et al. 1991). The inclusion of lipophilic drugs into exposure of target parasites to subtherapeutic drug micelles increases their solubility in an aqueous en- vironment (Ho¨rter & Dressman, 2001). However,the concentration of the free drug in solution is The authors would like to acknowledge Dr Juan Sallesfrom Instituto DILAVE ' Miguel C. Rubino ', Monte- considered as the only driving force for diffusion.
video, Uruguay, who provided the metacercariae of Under our experimental conditions, the presence of F. hepatica. Lourdes Mottier is a recipient of a fellowship amphiphilic bile components in the incubation me- from the Consejo Nacional de Investigaciones Cienti'ficas y dium may have induced the micellar solubilization Te'cnicas (CONICET), Argentina. This research was of ABZ, FBZ and TCBZ reducing the proportion of partially supported by the Agencia Nacional de Promocio'nCienti'fica y Tecnolo'gica (PICT 08-07277) (Argentina), free drug in solution and thus decreasing drug dif- Universidad Nacional del Centro de la Provincia de fusibility through the parasite tegument, which Buenos Aires (Argentina) and Consejo Nacional de In- would explain the reduced drug penetration ob- vestigaciones Cienti'ficas y Te'cnicas (Argentina).
served in the medium containing the higher bileproportions.
The knowledge of anthelmintic drug concen- trations achieved in tissues/fluids of parasite location ' NCHEZ, S. & LANUSSE, C. (1999). In vivo and contributes to an understanding of differences in ex vivo uptake of albendazole and its sulphoxide clinical efficacy. Furthermore, the comparative metabolite by cestode parasites : relationship with their ex vivo diffusion studies provide relevant infor- kinetic behaviour in sheep. Journal of VeterinaryPharmacology and Therapeutics 22, 77-86.
mation on drug capability to reach its specific re- ceptors inside the target parasite. Understanding the LANUSSE, C. (2000). Uptake of albendazole and mechanisms involved in drug access to the target albendazole sulphoxide by Haemonchus contortus and parasite, together with drug pharmacodynamics, Fasciola hepatica in sheep. Veterinary Parasitology 94, will enhance overall comprehension of anthelmintic drug activity. However, results reported here dem- ' NCHEZ, S. & LANUSSE, C. (2001).
onstrate that drug concentrations at the site of Ex vivo diffusion of albendazole and its sulphoxide L. I. Alvarez, M. L. Mottier and C. E. Lanusse metabolite into Ascaris suum and Fasciola hepatica.
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