PharmacologicalResearch,Vol.26, No. 2, 1992
VIGABATRIN ABSORPTION
201
DOES NOT AFFECT
OF PHENYTOIN
THE INTESTINAL
IN RAT DUODENO-JEJUNAL
LOOPS IN SITU M. TONINI, G. GATTI, L. MANZO, G. OLIBET, T. COCCINI* and E. PERUCCA Department of Internal Medicine and Therapeutics, Division of Pharmacology and Toxicology, University of Pavia, Piazza Botta 10, 27100 Pavia; *Siegmar Research Institute, 27100 Pavia, Italy Receivedinfinalform 10 March1992 SUMMARY
The antiepileptic drug vigabatrin (GVG) is known to decrease significantly the serum concentration of concurrently administered phenytoin (PHT) in epileptic patients. To assess a possible mechanism for this interaction, the effect of GVG on the intestinal absorption of PHT was investigated by means of circulation experiments in an in situ rat duodeno-jejunal loop. GVG did not affect the rate of disappearance of PHT from the loop perfusing medium, providing evidence against occurrence of GVG-induced impairment of PHT absorption. KEY WORDS:intestinal absorption, phenytoin, vigabatrim drug-drug interaction, rat intestine in
situ.
INTRODUCTION Vigabatrin (gamma-vinyl-GABA, GVG) is an irreversible enzyme-activated inhibitor of GABA transaminase which exerts anticonvulsant activity by increasing brain GABA levels and by enhancing GABA-ergic transmission in animals and in man [1]. In clinical studies, addition of GVG to the therapeutic regimen of phenytoin (PHT)-treated patients has been found to produce a significant fall in serum PHT levels, by 30% on average [2-5]. The mechanism of the interaction is unknown, but it is unlikely to involve plasma protein binding changes [5,6] or induction of the hepatic drug metabolizing enzymes [6]. The possibility that GVG may impair the gastrointestinal absorption of PHT has not been explored. The purpose of the present study was to evaluate the effect of GVG on the intestinal absorption of PHT using an in situ rat intestinal loop model, which allows circulation and sampling of intraluminal solution [7]. Correspondenceto: M. Tonim. 1043-6618/92/060201-05/$08.00/0
9 1992The ItalianPharmacologicalSociety
202
Pharmacological Research, Vol. 26, No. 2, 1992
MATERIALS AND METHODS
Male Sprague-Dawley albino rats weighing 280-320 g and fed on a standard diet with tap water freely available were used. The animals were anaesthetized approximately 30 min prior to surgery with an i.p. injection of sodium pentobarbitone (45 mg/kg) and then placed on a thermostatically controlled heated (37 ~ pad to maintain the body temperature within the physiological range. The small intestine was exposed by a midline abdominal incision, and two polyethylene cannulae were inserted through small slits at the duodenal and jejunal ends and secured by thread. The selected intestinal segments were approximately 30 cm in length, and represent the site in which maximum absorption of PHT occurs [8]. After the duodeno-jejunal loops were cleared with warm (37 ~ Tyrode solution (composition in mM: NaC1 136.9, KC1 2.7, CaC12 1.8, MgC12 1.04, NaH2PO4 0.4, NaHCO3 11.9, glucose 5.5) and emptied by means of air pumped through from a syringe, the two ends of the intestinal loop were attached to the circulation apparatus [7]. This consisted of a jacketed (37 ~ reservoir (20 ml) connected by two small glass columns to the loop. The fluid contained in the reservoir was circulated through the intestine by means of air (delivered by a peristaltic pump) lift. Under operative conditions, all the system (circulation apparatus and intestine) contained 35-40 ml of perfusion solution, approximately one-third of which was located intraluminally. PHT sodium (60 /2M, as free acid) was dissolved in Tyrode solution, either alone or in the presence of 1.55 or 15.5 mM GVG respectively, pH being adjusted at 7.4 with 5 M HCI. At this pH value and at 37 ~ PHT and vigabatrin [9,10] are freely soluble at the concentrations tested. The solution containing PHT (with or without GVG) was perfused through the loop at a rate of 7.5 ml/min. At regular time intervals over a 120-min period, 110 /21 samples were collected from the reservoir and stored at -20 ~ until PHT analysis by enzyme immunoassay (Emit, Syva, Palo Alto, CA, USA). After 120 min circulation, the volume of perfusing solution decreased by approximately 10% (4-5 ml of solution), indicating that under our experimental conditions, water absorption does not have a major effect on solute concentration. Preliminary experiments showed that the disappearance of phenytoin from the perfusion medium follows a monoexponential process. Least square regression analysis of the log-linear decline in phenytoin concentration over time allowed calculation of the first order rate constant which reflects the passage (absorption) of phenytoin across the intestinal wall. Statistical analysis of PHT kinetic constants in the presence or in the absence of GVG was made by analysis of variance.
RESULTS The time course profiles of disappearance of PHT (60 /2M) from the perfusion medium are shown in Fig. 1. In control experiments, PHT concentration in the medium declined monoexponentially with a first order rate constant of 0.0107 +0.0011 min-1 and an half life of 68.9+17.5 min (means+sD). Addition of GVG in
Pharmacological Research, Vol. 26, No. 2, 1992
203
80A
40 o
c
20
10
I ....
0
i ....
i ....
I ....
30
i ....
I ....
i ....
i ....
60 Time (min)
i ....
i ....
90
I ....
i ....
i
120
Fig. 1. Concentration-time curves illustrating the decline of PHT in the duodeno-jejunal loop perfusing fluid in the absence (0) and in the presence of GVG (O,1.55 mM; [Z,15.5 mM). Values are means +SD of duplicate determinations in seven animals for each experimental condition.
the perfusion medium, at both concentration tested (1.55 or 15.5 mM), did not affect the intestinal absorption of PHT, the rate of decline of the drug in the perfusion medium being virtually superimposable to that observed under control conditions (Fig. 1). The first order rate constant of PHT decline and corresponding PHT half life were 0.0117+0.0034 min -1 and 63.3+16.7 min in the presence of 1.55 mM GVG, and 0.0123+0.0042 min -1 and 62.3+20.7 min respectively in the presence of 15.5 mM GVG (NS vs controls).
DISCUSSION A decrease in steady-state serum PHT concentration following addition of GVG could be theoretically ascribed to several possible mechanisms: (1) displacement from plasma protein binding sites, (2) induction of metabolism, (3) impaired gastrointestinal absorption and (4) increased tissue binding. Displacement of PHT from its plasma protein binding sites has been excluded both in vitro and in vivo [5,6]. Induction of PHT metabolism is most unlikely because there is no evidence that GVG, a renally eliminated drug, may act as an enzyme inducer [6]. Moreover, enzyme induction usually results in enhanced metabolism of several drugs [11], whereas the lowering effect of GVG on serum PHT levels appears to be rather specific and does not extend to other anticonvulsants cleared by oxidation [12], with the possible exception of phenobarbitone for which a marginal decrease in concentration has been occasionally reported [5,13]. Vigabatrin-induced enhancement of tissue binding of PHT has not been formally investigated, but this possibility remains speculative and unsupported by current knowledge on the mode of action of the drug. Based on the above considerations, the possibility of GVG interfering with PHT absorption needs to be taken into account. Impaired gastrointestinal absorption is a recognized mechanism of drug-drug interactions in epileptic patients, examples
204
Pharmacological Research, Vol. 26, No. 2, 1992
being represeaated by the decrease in griseofulvin and frusemide bioavailability by phenobarbitone and P H T respectively [11], The in situ rat intestinal loop technique is a well established model for the evaluation of factors affecting absorption of drugs in the intestinal tract [8,14] and therefore it was a reasonable choice for a preliminary investigation of the potential G V G - P H T interaction at this level. The observation that G V G at high concentrations (with a G V G - P H T molar concentration ratio up to about 260) did not affect the rate of disappearance of PHT from the medium perfusing the duodeno-jejunal loop can be considered as evidence against the occurrence of an absorption interaction between the two drugs. Caution should be taken when attempting to extrapolate the present findings to the clinical situation. First, there may be species differences in susceptibility to the interaction. Second, in our study GVG was administered acutely whereas the interaction is normally observed in patients receiving chronic treatment with the two drugs. In a recent study [6], the decrease in P H T level after addition of GVG in epileptic patients occurred only after a latency of about 4 weeks. Therefore, the possibility of slowly developing gastrointestinal changes during chronic GVG therapy can not be excluded as a possible cause for the interaction observed in the patients.
ACKNOWLEDGEMENTS We wish to thank Dr John Mumford (Marion-Merrell-Dow Research Centre, Winnersh, UK) for a generous gift of vigabatrin.
REFERENCES 1. Grant SM, Heel RC. Vigabatrin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in epilepsy and disorders of motor control. Drugs 1991; 41: 889-926. 2. Rimmer EM, Richens A. Double-blind study of gamma-vinyl-GABA in patients with refractory epilepsy. Lancet 1984; 1: 189-90. 3. Tartara A, Manni R, Galimberti CA, Hardenberg J, Orwin J, Perucca E. Vigabatrin in the treatment of epilepsy: A double-blind placebo controlled study. Epilepsia 1986; 26: 713-23. 4. Tassinari CA, Michelucci R, Ambrosetto G, Salvi F. Double study of vigabatrin in the treatment of drug-resistant epilepsy. Arch Neurol 1987: 44: 907-10. 5. Browne TR, Mattson RH, Penry JK, et al. Vigabatrin for refractory complex partial seizures: Multicentre single blind study with long-term follow-up. Neurology 1987; 37: 184-9. 6. Rimmer EM, Richens A. Interaction between vigabatrin and phenytoin. Br J Clin Pharmacol 1989; 27: 27-33. 7. Jervis EL, Johnson FR, Sheff MF, Smyth DH. The effect of phlorhizin on intestinal absorption and intestinal phosphatase. J Physiol 1956 134: 675-88. 8. Woodbury DM. Phenytoin. Absorption, distribution, and excretion. In: Levy R, Mattson R, Meldrum B, Penry JK, Dreifuss, FE, eds. Antiepileptic Drugs, 3rd Ed. New York: Raven Press, 1989: 177-95.
Pharmacological Research, Vol. 26, No. 2, 1992
205
9. Schwartz PA, Rhodes CT, Cooper JW. Solubility and ionization characteristics of phenytoin. J Pharm Sci 1977; 66: 994--7. 10. Dollery C. Therapeutic drugs. Vol. 2, Edinburgh: Churchill Livingstone, 1991: V23-V26. 11. Perucca E. Pharmacokinetic interactions with antiepileptic drugs. Clin Pharmacokinet 1982; 7: 57-84. l 2. Perucca E, Pisani F. Pharmacokinetics and interactions of the new antiepileptic drugs. In: Pisani F, Perucca E, Avanzini G, Richens A, eds. New antiepileptic drugs. Amsterdam: Elsevier, 1991: 79-88. 13. Italian Collaborative Study Group on Vigabatrin. Simple-blind, placebo controlled multicentre trial of vigabatrin in the treatment of epilepsy. Ital J Neurol (in press). 14. Binks SP, Dobrota M. Kinetics and mechanism of uptake of platinum-based pharmaceuticals by the rat small intestine. Biochem Pharmacol 1990; 40: 1329-36.
VIGABATRIN ABSORPTION
201
DOES NOT AFFECT
OF PHENYTOIN
THE INTESTINAL
IN RAT DUODENO-JEJUNAL
LOOPS IN SITU M. TONINI, G. GATTI, L. MANZO, G. OLIBET, T. COCCINI* and E. PERUCCA Department of Internal Medicine and Therapeutics, Division of Pharmacology and Toxicology, University of Pavia, Piazza Botta 10, 27100 Pavia; *Siegmar Research Institute, 27100 Pavia, Italy Receivedinfinalform 10 March1992 SUMMARY
The antiepileptic drug vigabatrin (GVG) is known to decrease significantly the serum concentration of concurrently administered phenytoin (PHT) in epileptic patients. To assess a possible mechanism for this interaction, the effect of GVG on the intestinal absorption of PHT was investigated by means of circulation experiments in an in situ rat duodeno-jejunal loop. GVG did not affect the rate of disappearance of PHT from the loop perfusing medium, providing evidence against occurrence of GVG-induced impairment of PHT absorption. KEY WORDS:intestinal absorption, phenytoin, vigabatrim drug-drug interaction, rat intestine in
situ.
INTRODUCTION Vigabatrin (gamma-vinyl-GABA, GVG) is an irreversible enzyme-activated inhibitor of GABA transaminase which exerts anticonvulsant activity by increasing brain GABA levels and by enhancing GABA-ergic transmission in animals and in man [1]. In clinical studies, addition of GVG to the therapeutic regimen of phenytoin (PHT)-treated patients has been found to produce a significant fall in serum PHT levels, by 30% on average [2-5]. The mechanism of the interaction is unknown, but it is unlikely to involve plasma protein binding changes [5,6] or induction of the hepatic drug metabolizing enzymes [6]. The possibility that GVG may impair the gastrointestinal absorption of PHT has not been explored. The purpose of the present study was to evaluate the effect of GVG on the intestinal absorption of PHT using an in situ rat intestinal loop model, which allows circulation and sampling of intraluminal solution [7]. Correspondenceto: M. Tonim. 1043-6618/92/060201-05/$08.00/0
9 1992The ItalianPharmacologicalSociety
202
Pharmacological Research, Vol. 26, No. 2, 1992
MATERIALS AND METHODS
Male Sprague-Dawley albino rats weighing 280-320 g and fed on a standard diet with tap water freely available were used. The animals were anaesthetized approximately 30 min prior to surgery with an i.p. injection of sodium pentobarbitone (45 mg/kg) and then placed on a thermostatically controlled heated (37 ~ pad to maintain the body temperature within the physiological range. The small intestine was exposed by a midline abdominal incision, and two polyethylene cannulae were inserted through small slits at the duodenal and jejunal ends and secured by thread. The selected intestinal segments were approximately 30 cm in length, and represent the site in which maximum absorption of PHT occurs [8]. After the duodeno-jejunal loops were cleared with warm (37 ~ Tyrode solution (composition in mM: NaC1 136.9, KC1 2.7, CaC12 1.8, MgC12 1.04, NaH2PO4 0.4, NaHCO3 11.9, glucose 5.5) and emptied by means of air pumped through from a syringe, the two ends of the intestinal loop were attached to the circulation apparatus [7]. This consisted of a jacketed (37 ~ reservoir (20 ml) connected by two small glass columns to the loop. The fluid contained in the reservoir was circulated through the intestine by means of air (delivered by a peristaltic pump) lift. Under operative conditions, all the system (circulation apparatus and intestine) contained 35-40 ml of perfusion solution, approximately one-third of which was located intraluminally. PHT sodium (60 /2M, as free acid) was dissolved in Tyrode solution, either alone or in the presence of 1.55 or 15.5 mM GVG respectively, pH being adjusted at 7.4 with 5 M HCI. At this pH value and at 37 ~ PHT and vigabatrin [9,10] are freely soluble at the concentrations tested. The solution containing PHT (with or without GVG) was perfused through the loop at a rate of 7.5 ml/min. At regular time intervals over a 120-min period, 110 /21 samples were collected from the reservoir and stored at -20 ~ until PHT analysis by enzyme immunoassay (Emit, Syva, Palo Alto, CA, USA). After 120 min circulation, the volume of perfusing solution decreased by approximately 10% (4-5 ml of solution), indicating that under our experimental conditions, water absorption does not have a major effect on solute concentration. Preliminary experiments showed that the disappearance of phenytoin from the perfusion medium follows a monoexponential process. Least square regression analysis of the log-linear decline in phenytoin concentration over time allowed calculation of the first order rate constant which reflects the passage (absorption) of phenytoin across the intestinal wall. Statistical analysis of PHT kinetic constants in the presence or in the absence of GVG was made by analysis of variance.
RESULTS The time course profiles of disappearance of PHT (60 /2M) from the perfusion medium are shown in Fig. 1. In control experiments, PHT concentration in the medium declined monoexponentially with a first order rate constant of 0.0107 +0.0011 min-1 and an half life of 68.9+17.5 min (means+sD). Addition of GVG in
Pharmacological Research, Vol. 26, No. 2, 1992
203
80A
40 o
c
20
10
I ....
0
i ....
i ....
I ....
30
i ....
I ....
i ....
i ....
60 Time (min)
i ....
i ....
90
I ....
i ....
i
120
Fig. 1. Concentration-time curves illustrating the decline of PHT in the duodeno-jejunal loop perfusing fluid in the absence (0) and in the presence of GVG (O,1.55 mM; [Z,15.5 mM). Values are means +SD of duplicate determinations in seven animals for each experimental condition.
the perfusion medium, at both concentration tested (1.55 or 15.5 mM), did not affect the intestinal absorption of PHT, the rate of decline of the drug in the perfusion medium being virtually superimposable to that observed under control conditions (Fig. 1). The first order rate constant of PHT decline and corresponding PHT half life were 0.0117+0.0034 min -1 and 63.3+16.7 min in the presence of 1.55 mM GVG, and 0.0123+0.0042 min -1 and 62.3+20.7 min respectively in the presence of 15.5 mM GVG (NS vs controls).
DISCUSSION A decrease in steady-state serum PHT concentration following addition of GVG could be theoretically ascribed to several possible mechanisms: (1) displacement from plasma protein binding sites, (2) induction of metabolism, (3) impaired gastrointestinal absorption and (4) increased tissue binding. Displacement of PHT from its plasma protein binding sites has been excluded both in vitro and in vivo [5,6]. Induction of PHT metabolism is most unlikely because there is no evidence that GVG, a renally eliminated drug, may act as an enzyme inducer [6]. Moreover, enzyme induction usually results in enhanced metabolism of several drugs [11], whereas the lowering effect of GVG on serum PHT levels appears to be rather specific and does not extend to other anticonvulsants cleared by oxidation [12], with the possible exception of phenobarbitone for which a marginal decrease in concentration has been occasionally reported [5,13]. Vigabatrin-induced enhancement of tissue binding of PHT has not been formally investigated, but this possibility remains speculative and unsupported by current knowledge on the mode of action of the drug. Based on the above considerations, the possibility of GVG interfering with PHT absorption needs to be taken into account. Impaired gastrointestinal absorption is a recognized mechanism of drug-drug interactions in epileptic patients, examples
204
Pharmacological Research, Vol. 26, No. 2, 1992
being represeaated by the decrease in griseofulvin and frusemide bioavailability by phenobarbitone and P H T respectively [11], The in situ rat intestinal loop technique is a well established model for the evaluation of factors affecting absorption of drugs in the intestinal tract [8,14] and therefore it was a reasonable choice for a preliminary investigation of the potential G V G - P H T interaction at this level. The observation that G V G at high concentrations (with a G V G - P H T molar concentration ratio up to about 260) did not affect the rate of disappearance of PHT from the medium perfusing the duodeno-jejunal loop can be considered as evidence against the occurrence of an absorption interaction between the two drugs. Caution should be taken when attempting to extrapolate the present findings to the clinical situation. First, there may be species differences in susceptibility to the interaction. Second, in our study GVG was administered acutely whereas the interaction is normally observed in patients receiving chronic treatment with the two drugs. In a recent study [6], the decrease in P H T level after addition of GVG in epileptic patients occurred only after a latency of about 4 weeks. Therefore, the possibility of slowly developing gastrointestinal changes during chronic GVG therapy can not be excluded as a possible cause for the interaction observed in the patients.
ACKNOWLEDGEMENTS We wish to thank Dr John Mumford (Marion-Merrell-Dow Research Centre, Winnersh, UK) for a generous gift of vigabatrin.
REFERENCES 1. Grant SM, Heel RC. Vigabatrin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in epilepsy and disorders of motor control. Drugs 1991; 41: 889-926. 2. Rimmer EM, Richens A. Double-blind study of gamma-vinyl-GABA in patients with refractory epilepsy. Lancet 1984; 1: 189-90. 3. Tartara A, Manni R, Galimberti CA, Hardenberg J, Orwin J, Perucca E. Vigabatrin in the treatment of epilepsy: A double-blind placebo controlled study. Epilepsia 1986; 26: 713-23. 4. Tassinari CA, Michelucci R, Ambrosetto G, Salvi F. Double study of vigabatrin in the treatment of drug-resistant epilepsy. Arch Neurol 1987: 44: 907-10. 5. Browne TR, Mattson RH, Penry JK, et al. Vigabatrin for refractory complex partial seizures: Multicentre single blind study with long-term follow-up. Neurology 1987; 37: 184-9. 6. Rimmer EM, Richens A. Interaction between vigabatrin and phenytoin. Br J Clin Pharmacol 1989; 27: 27-33. 7. Jervis EL, Johnson FR, Sheff MF, Smyth DH. The effect of phlorhizin on intestinal absorption and intestinal phosphatase. J Physiol 1956 134: 675-88. 8. Woodbury DM. Phenytoin. Absorption, distribution, and excretion. In: Levy R, Mattson R, Meldrum B, Penry JK, Dreifuss, FE, eds. Antiepileptic Drugs, 3rd Ed. New York: Raven Press, 1989: 177-95.
Pharmacological Research, Vol. 26, No. 2, 1992
205
9. Schwartz PA, Rhodes CT, Cooper JW. Solubility and ionization characteristics of phenytoin. J Pharm Sci 1977; 66: 994--7. 10. Dollery C. Therapeutic drugs. Vol. 2, Edinburgh: Churchill Livingstone, 1991: V23-V26. 11. Perucca E. Pharmacokinetic interactions with antiepileptic drugs. Clin Pharmacokinet 1982; 7: 57-84. l 2. Perucca E, Pisani F. Pharmacokinetics and interactions of the new antiepileptic drugs. In: Pisani F, Perucca E, Avanzini G, Richens A, eds. New antiepileptic drugs. Amsterdam: Elsevier, 1991: 79-88. 13. Italian Collaborative Study Group on Vigabatrin. Simple-blind, placebo controlled multicentre trial of vigabatrin in the treatment of epilepsy. Ital J Neurol (in press). 14. Binks SP, Dobrota M. Kinetics and mechanism of uptake of platinum-based pharmaceuticals by the rat small intestine. Biochem Pharmacol 1990; 40: 1329-36.
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