Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de Valencia, Avda Vte Andrés Estellés s/n. 46100 Burjassot, Valencia and Instituto de Investigación Citológicas (FVIB) Amadeo de Saboya 4, 46010, Valencia, Spain
Received 25 October 1999; in revised form 3 February 2000; accepted 10 February 2000
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ABSTRACT |
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INTRODUCTION |
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In recent years, research into the mechanism of acamprosate action has flourished, although it is as yet unclear. On the contrary, few studies have focused on the intestinal absorption mechanisms of the drug. Chabenat et al. (1988) concluded that, due to its physicochemical properties, the drug presumably crosses the biological barriers with the help of a transporter. However, recently some authors have postulated that the acamprosate absorption pathway is predominantly by the paracellular route (Saivin et al., 1998). Additionally, the kinetic parameters of drug transport are not known. Related compounds, such as GABA or taurine, can permeate the intestinal membrane via a mediated transport system plus passive diffusion in the rat (Munck et al., 1994
; Nácher et al., 1994
; Munck and Munck, 1994
).
As we have, however, demonstrated previously, chronic ethanol intake can modify intestinal drug absorption depending on the mechanism involved. Using seven compounds belonging to a homologous series (ciprofloxacin derivatives), we have shown that the effect of chronic alcohol treatment on passive diffusion depends on the physicochemical properties of the drug. Accordingly, chronic ethanol intake modifies only the absorption rate of highly and markedly hydrophilic homologues (Merino et al., 1997). If mediated transport is involved in the absorption, chronic ethanol treatment can decrease the absorption rate, as in the case of methionine (Polache et al., 1996
). When both mechanisms are operating, the global effect of ethanol will depend on how much each mechanism participates as well as on the physicochemical characteristics of the drug. When we performed experiments with taurine, a homologue of acamprosate that can permeate the intestinal membrane by both mediated transport and passive diffusion, the results showed that chronic ethanol intake enhances the influx of taurine across the brush border membrane. In fact, taurine is a hydrophilic compound and its passive diffusion is more important than mediated transport (Martín-Algarra et al., 1998
).
In order to gain the advantage of maximal oral drug bioavailability, it is first necessary to identify the barriers to oral absorption and so investigate the intrinsic absorption mechanisms of acamprosate. The pharmacotherapy of alcohol dependence with acamprosate could therefore be improved if we had a better understanding of the intestinal absorption mechanisms of this drug and knew more about its kinetic parameters and possible interactions with different compounds present in the intestinal lumen (other drugs or food components). The main aim of this work was therefore to gain an insight into the intestinal absorption mechanism of acamprosate, using an in vitro technique and characterizing its kinetic parameters, as well as the possible influence of chronic ethanol intake on the process. The second aim was to investigate whether the presence of some structurally related compounds in the intestinal lumen inhibit the transport of the drug.
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MATERIALS AND METHODS |
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The animals were distributed randomly into three groups: liquid pair-fed control (control group), alcohol (alcohol group), and solid control (solid group).
After treatment, rats were anaesthetized with ether, the abdomen was opened and the mid-small intestine was removed and rinsed briefly in ice-cold buffer solution. Only the central segment of the small intestine (mid-jejunum) (20 cm) was used for monitoring in the influx chamber.
Acamprosate absorption studies
To characterize acamprosate absorption we used an in vitro technique (Schultz et al., 1967) which allows the unidirectional influx of acamprosate across the brush border membrane of the rat mid-jejunum (JAca) to be measured. Acamprosate influx rate was measured using [14C]acamprosate from LIPHA (Lyon, France) and [3H]polyethyleneglycol (M = 4000; [3H]PEG-4000) from American Radiolabeled Chemicals (ARC; St Louis, MO, USA). The structure of [14C]acamprosate is: CH3CONH14CH2CH2CH2SO3CaSO3CH2CH2 14CH2NHCOCH3.
The experiments were carried out at 37°C under 100% O2. In all experiments, we maintained an osmotic balance by adding mannitol to keep the total concentration of acamprosate and mannitol constant (Munck et al., 1994). All solutions were made with a HEPES buffer (pH 7.4) with the following composition (mM): 140 Na+, 8 K+, 2.6 Ca2+, 1 Mg2+, 140 Cl, 16 HEPES, 1 SO24 , 5 D-glucose. Eleven solutions of acamprosate, at different concentrations, were used in the experiments with the control pair-fed group: 105, 104, 6 x 104, 103, 2.5 x 103, 5 x 103, 102, 1.5 x 102, 2.5 x 102, 4 x 102, and 8 x 102 M; 10 different concentrations of acamprosate in the alcohol group: 105, 104, 103, 2.5 x 103, 5 x 103, 102, 1.5 x 102, 2.5 x 102, 4 x 102, and 8 x 102 M; and 11 concentrations in the solid group: 105, 2.5 x 105, 104, 6 x 104, 2.5 x 103, 5 x 103, 102, 1.5 x 102, 2.5 x 102, 4 x 102 and 8 x 102 M. If a mediated transport system is involved in acamprosate absorption, it would be important to examine a possible sodium-dependent transport in order to characterize the carrier. For that reason, additional experiments, using acamprosate at a concentration of 104 M, were carried out, in the control pair-fed and alcohol groups, in these sodium chloride was substituted with choline chloride in order to maintain the osmotic balance. Thus, it is possible to calculate drug influx in the presence and in the absence of Na+.
Unidirectional influx across the brush border membrane of the rat jejunum was measured essentially as described for the rabbit and rat intestines (Munck, 1985, Munck et al., 1994
). For each rat, the mid 20 cm of the total small intestine were excised, opened along the mesenteric attachment, cut in half, and rinsed in ice-cold buffer. Each segment was mounted on a Lucite plate with the mucosal surface facing upwards and a Lucite block was clamped on top of the plate. In this way, four mucosal areas each of 0.35 cm2 were exposed at the bottom of the wells, where the solution was oxygenated and stirred by high rates of O2 flow. By using two blocks, eight measurements were possible for each rat.
The tissues were incubated for 15 min with an acamprosate-free solution containing 5 mM glucose, which was then withdrawn. The well and mucosal surface were gently wiped with soft paper to remove the adhered incubation fluid before the test solution was injected. The 0.5-min incubation period was terminated by aspiration of the incubation fluid and flushing of the well with an ice-cold 300 mM mannitol solution. The exposed tissues were then punched out, briefly rinsed in ice-cold mannitol solution, blotted on hard filter paper, and extracted for 18 h in 0.1 M HNO3. Analysis by liquid scintillation spectrometry was performed with equal quench in all samples. The amount of [3H]PEG-4000 was used to correct for extracellular contamination assuming that this compound cannot cross the intestinal membrane and remains extracellularly, so that it is possible to quantify exactly the percentage of extracellular contamination and to transform it for [14C]acamprosate; thus corrected, the [14C]-labelled acamprosate activity was used to calculate the rate of influx across the brush border membrane.
Inhibition studies
Several inhibition studies were carried out to check whether mediated transport across biological membranes took place, as suggested by some authors (Chabenat et al., 1988). Three series of experiments were performed using several compounds as inhibitors. In the first series, paired measurement of the flux across the brush border membrane, JAca, were made at 103 M acamprosate with 40 mM glycine, proline, GABA or taurine, in both the control and alcohol groups. Additionally, in a second series of assays, paired measurements of JAca were performed at 104 M acamprosate with 40 mM GABA or taurine, in both the control and alcohol groups. Finally, a third series of studies were performed in the control group, measuring JAca at 104 M acamprosate in the presence of 0, 10, 20 or 80 mM taurine, or a mixture of 40 mM GABA and 40 mM taurine.
Mathematical and statistical methods
Given that no saturation was detected in the absorption process, we assumed that the unidirectional influx of acamprosate could be described as a passive diffusion [equation (1)] or as the sum of a saturable process and a passive diffusion [equation (2)].
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where Jp is the flux caused by a passive process and Js is the transport by a saturable process. Substituting these fluxes by the corresponding equations, we have:
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where JAca and its maximum rate, Jmax, are given in nmol/cm2/h (serosal area), Km is the MichaelisMenten constant in µM, P is the diffusive permeability in cm/h and A is the concentration of acamprosate at the absorption site.
The fits of equations to the experimental data have been performed using a non-linear least-square regression (Sigmaplot 4.0) or a linear least-squares procedure (Statgraphics 7.0).
Statistical comparisons among P-values were carried out using the ANOVA test for linear regression analysis. When significant differences were found, Dunnet's multiple range test was applied in order to detect significantly different means. A probability level of less than 0.05 was considered to be statistically significant.
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RESULTS |
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DISCUSSION |
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The results of the kinetic analysis of JAca data clearly indicate that passive diffusion [equation (3)] is the main mechanism involved in acamprosate intestinal absorption. This conclusion was drawn because it was impossible to reach satisfactory convergence during the fitting procedures of equation (4) to the data. Therefore, the diffusive permeability (P) value was calculated as 0.213 ± 0.003 cm/h for the control pair-fed group and 0.206 ± 0.001 for the alcohol group. The statistical comparison shows that they are significantly different although can be considered as identical for practical purposes. From these data, we can conclude that chronic alcohol intake does not alter the absorption of acamprosate across the rat jejunum. This result is supported by our previous studies (Merino et al., 1997; Martín-Algarra et al., 1998
), which demonstrated that ethanol treatment only affects the passive intestinal transport of very hydrophilic compounds, such as taurine. Accordingly, acamprosate, a homologue of taurine with an acetyl group, is expected to be more lipophilic than taurine and therefore its transport is unlikely to be influenced by ethanol treatment. Our data are in accord with those obtained by Saivin et al. (1998), who have also demonstrated that acamprosate pharmacokinetics after oral administration are not influenced by alcohol.
However, we can also conclude that the diet type (liquid vs solid) does not modify acamprosate absorption, so in this type of experiment it is possible to use animals fed a solid diet as a control group.
It is noteworthy that the P-value obtained for acamprosate is not as low as we expected considering the low bioavailability of the drug in humans. In fact, this value is similar to that obtained for antipyrine, a compound with good bioavailability (Torres-Molina et al., 1992), using the same technique (Polache et al., 1998
). Thus, on the basis of these results we cannot conclude that the magnitude of acamprosate absorption is low. This discordance between our results and those in humans could be explained by the fact that we used an in vitro technique with intact intestine and that acamprosate influx was measured for only 30 s. Under these conditions, it is difficult to detect some processes that could occur in vivo and could markedly reduce the extent of drug absorption (e.g. intestinal secretory transport). The intestinal epithelial membrane has some specialized transport systems which can secrete drugs from the serosal side to the mucosal side, functioning as a barrier to absorption and being an unrecognized cause of low bioavailability (Arimori and Nakano, 1998
). A typical secretory system is P-glycoprotein (P-gp) (Lennernäs and Regardh, 1993
; Arimori and Nakano, 1998
). P-gp functions as an efflux transport pump and can be a factor that limits bioavailability of some hydrophobic drugs and peptides (Gramatte et al., 1996
). Moreover, it has recently been reported that DMP728, a cyclic peptide fibrinogen antagonist, has an oral absorption limited by its hydrophilicity and by the dominance of secretory transport, probably by P-gp (Aungst and Saitoh, 1996
). Acamprosate may also be a substrate for an intestinal secretory system. This possibility has yet to be investigated.
Although our results suggest strongly that passive diffusion is the main mechanism involved in acamprosate intestinal transport, we cannot discard the existence of a simultaneous minor saturable process. To test this hypothesis, several inhibition studies were developed. The first (Fig. 3) showed that 40 mM of glycine, proline, GABA or taurine does not inhibit acamprosate influx at 103 M. The possible explanation of this result could be the high concentration of acamprosate used. At this concentration the drug is probably transported mainly by diffusion, being unaffected by the compounds tested. When a second study was carried out using a lower acamprosate concentration (104 M), the drug influx was significantly reduced in the presence of GABA and taurine (Fig. 4
). These results suggest that the carrier of imino acids could be involved in the acamprosate specialized transport in the rat intestine. To study this possibility, a third assay was performed in the control group. The results (Fig. 5
) showed that acamprosate transport was significantly inhibited by taurine and a mixture of taurine and GABA. Maximum inhibition was reached at 20 mM taurine and is very similar to that obtained in the presence of both amino acids tested. Although no kinetic inhibition study has been carried out, we assume, considering the structural similarities among the compounds, that the inhibition process is a competitive one.
In view of these results, we can conclude that the imino-acid carrier of the rat intestine is probably responsible, at least in part, for the acamprosate-mediated transport. Even so, and surprisingly, we did not detect Na+-dependence in acamprosate transport at 104 M (data not shown).
From these results, it is difficult to predict the practical implications of acamprosate absorption inhibition by taurine, since this amino acid is an unusual component in the normal diet. On the other hand, it is important to bear in mind that, according to the present results, a high percentage of acamprosate is always transported by passive diffusion even though inhibitors are present in the biological medium.
The present study demonstrates that acamprosate is transported in the rat mid-jejunum predominantly by passive diffusion and also by a carrier system, probably the imino-acid carrier. Assuming that the acamprosate absorption mechanism is mainly passive, there are several potential ways of enhancing its transport. If diffusion takes place by the transcellular route (across the intestinal membrane), it could be possible to use surfactants as promoters (Bermejo et al., 1991), whereas if transport occurs by the paracellular route, some compounds such as chitosans can be utilized as enhancers since they are effective in increasing membrane permeability (Shipper et al., 1996
; Kotzé et al., 1997
).
Moreover, we postulate that acamprosate is probably secreted in a serosal-to-mucosal direction by an intestinal exsorption mechanism which could be responsible for its low oral bioavailability. It will be of clinical interest to investigate whether, in acamprosate absorption, a specialized secretion transport such as P-gp is involved and whether this transport could be inhibited to enhance acamprosate absorption. Enhancing acamprosate absorption would not only benefit the patient by reducing side-effects, but also lead to a saving in drug use effects.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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