- Email: [email protected]
Bioavailability and Anticonvulsant Activity of a Monoglyceride‐Derived Prodrug of Phenytoin after Oral Administration to Rats
Bioavailability and Anticonvulsant Activity of a Monoglyceride-Derived Prodrug of Phenytoin after Oral Administration to Rats GERHARD K. E. SCRIBA*~, DIDIERM. LAMBERT~, AND JACQUES H. POUPAERT~ Received September 19, 1994, from the *Department of Pharmaceutical Chemistry, School of Pharmacy, University of Miinster, ffittorfstrasse 58-62, 48149 Munster, Germany, and #Laboratory of Pharmaceutical Chemistry, School of Pharmacy, Catholic University of Accepted for publication December 20, 19949 Louvain, CMFA 73.40, Avenue E. Mounier 73, 1200 Brussels, Belgium. Abstract 0 The plasma levels of phenytoin after oral administration of phenytoin and phenytoin 2-monoglyceride, a phenytoin prodrug, to rats were determined by gas chromatography. Compared to the application of the parent drug, administration of the prodrug resulted in a 3-fold ,, and a 4-fold increase of the AUC. This correlated with increase of C an earlier onset and peaking of the anticonvulsant activity determined in the maximal electroshock (MES) test. The peak effect was reached 1 h after dosing the monoglyceride compared to 2 h after application of phenytoin itself. On the basis of the median effective dose, the prodrug was 3 times more effective antagonizing MES-induced seizures than the parent drug. It is concluded that phenytoin 2-monoglyceride might be a useful prodrug for the oral delivery of phenytoin.
Introduction Phenytoin is a slightly water-soluble, slightly lipid-soluble drug that has shown erratic bioavailability upon oral administration.1,2 Superior availability was obtained by coadministration of lipids3or by ester prodrugs of 3-(hydroxymethyl)p h e n y t ~ i n . ~The . ~ phosphate ester of 3-(hydroxymethyl)phenytoin, fosphenytoin (ACC-96531,has been developed as an alternative to sodium phenytoin preparations for parental administration in the emergency treatment of the status epilepticus6-8and is currently under clinical investigation in the United States. Dihydropyridine derivatives of 3-(hydroxymethy1)phenytoinas redox delivery systems have been described r e ~ e n t l y . ~ In the course of the investigation of lipid-derived prodrugs of phenytoin, phenytoin 2-monoglyceride was synthesized by covalent binding of 3-(hydroxymethyl)phenytoin to position 2 of glycerol via a succinic acid spacer (Figure l).Io Rapid release of phenytoin from the monoglyceride derivative by plasma esterases and other hydrolytic enzymes was observed in uitro.ll Moreover, the compound displayed anticonvulsant activity and a pharmacological profile similar to that of the parent drug after ip administration to mice.12 Therefore, it was concluded that the monoglyceride might act as a prodrug of phenytoin. Oral administration is generally considered the route of choice for a drug. Therefore, the present study was conducted in order to evaluate the bioavailability and the anticonvulsant activity of phenytoin 2-monoglyceridein comparison to phenytoin after oral administration to rats.
Materials and Methods Chemicals-Phenytoin was obtained from Caelo (Hilden, Germany), 5-@-Methylphenyl)-5-phenylhydantoinwas purchased from Sigma (Deisenhofen, Germany); methyl cellulose, phenyltrimethylammonium hydroxide (0.1 M solution in methanol), and tetramethylammonium hydroxide (25% in water) were from Fluka (Neu-Ulm, @Abstractpublished in Advance ACS Abstracts, February 1, 1995.
300 / Journal of Pharmaceutical Sciences Vol. 84,No. 3, March 1995
HNKNH HO 0 phenytoin
V
0
phenytoin 2-monoglyceride
Figure 1-Structures of the compounds. Germany), and heparin solution (25 000 IWmL) was from Ratiopharm
(Ulm, Germany). Phenytoin 2-monoglyceride was synthesized as described.*O All other chemicals were obtained from commercial sources at the highest purity available. Solutions were prepared in double-distilled, deionized water. Pharmacokinetics-Male Wistar rats (bred at the animal facilities at UCL), weighing 220-260 g, were housed individually with a 12 h light-dark cycle and free access to commercial rodent chow and water. The animals were fasted overnight and during the experiment but were allowed water ad libitum. Doses of 119 ymollkg of phenytoin and phenytoin 2-monoglyceride (30 m g k g equivalents of phenytoin) were administered as suspensions in 0.5% methyl cellulose by oral intubation in a volume of 2 m U g . Approximately 300 yL of blood was collected via the tail clip method into Eppendorf tubes containing 20 yL of the heparin solution. The samples were centrifuged at 4 "C at 4000g, the plasma was immediately separated, frozen a t -80 "C, and stored frozen until analyzed. Analysis of Plamsa Samples-The analysis of the plasma samples was carried out as described13 with minor modifications. Briefly, 100-150 yL of plasma was placed in 10 mL stoppered centrifuge tubes, acidified with 50 yL of 3 M NaHzP04, pH 3, and extracted by vortexing with 2 mL of toluene containing 1pg of 5-(pmethylphenyl)5-phenylhydantoin as internal standard. After centifugation at 2500g for 10 min a t room temperature, the organic layer was transferred to a clean centrifuge tube and extracted with 25 fiL of 0.01 M phenyltrimethylammonium hydroxidedo.1M tetramethylammonium hydroxide in methanoVwater ( l : l , v/v) by vortexing for 1 min. Following centrifugation at 2500g for 5 min at room temperature, the toluene phase was discarded and approximately 1y L of the aqueous methanol layer was injected into a Shimadzu GC-14A gas chromatograph equipped with a flame ionization detector (Shimadzu, Duisburg, Germany). The separation of the compounds was obtained on a 25 m HP-1column (Hewlett-Packard,Diisseldorf, Germany) with helium as the carrier gas at a flow rate of 0.75 mumin. The column temperature was maintained at 225 "C. The detector temperature was set to 300 "C and the injector temperature to 270 "C. The injector was operated in the splitless mode during the injection and for the first 10 s of each analysis. A split of 1 : l O was applied throughout the remainder of the analysis. The retention times of phenytoin and 5-(p-methylphenyl)-5-phenylhydantoin were 9.0 and 11.7 min, respectively. Phenytoin concentrations were calculated by the peak area ratio method using a calibration curve obtained from spiked plasma samples. Data Analysis-Noncompartmental analysis of the data was performed. The maximum plasma concentration (Cmm)and the time to reach this concentration (t,& were obtained directly from the plasma concentration uersus time profiles. The area under the curve (AUC) was calculated by the log-linear trapezoidal method for the observed values and by extrapolation to infinity. The elimination half-life (tuz) was estimated from the final segment of the plasma concentration
0022-3549/95/3184-0300$09.00/0
0 1995, American Chemical Socieiy and American Pharmaceutical Association
Table 1-Pharmacokinetic Parameters for Pheytoin Obtained from Phenytoin Plasma Levels and Median Effective Dose Determined in the MES Test after Oral Administration of Phenytoin and Phenytoin 2-Monoglyceride to Rats Phenytoin Pharmacokinetic Parametersa Compound
AUC bghlmL)
Phenytoin Phenytoin 2-monoglyceride
MES Test
Cmax luglmL)
Lax (h)
tin (h)
10.0 1.I 40.5 7.3
1.90 2 0.1 1 6.42 2 1.51
1.25 f 0.5 1.oc
3.48 0.46 4.16 f 0.90
P < 0.001d
P < 0.001
nse
ns
+ +
Time of Test (h)
EDmbkmollkg)
2 1
91.9 (84.8-100.7) 34.5 (30.0-42.2)
+
P < 0.001
a Mean f SD, n = W. ED% calculated from five doses with eight animals per dose. One hour in all experiments. f-test for unpaired observations. ns, statistically not significant.
curve. Statistical comparison was performed using the t-test for unpaired observations. P < 0.05 was considered statistically significant. PharmacoZogy-Articonvulsant testing was provided by the Antiepileptic Drug Development Program, Epilepsy Branch, Division of Convulsive, Developmental and Neuromuscular Disorders, National Institutes of Health, according to standard procedure^'^^'^ and included the maximal electroshock (MES) test and the seizure threshold tests with subcutaneouspentetrazol (scMet test). The acute neurological toxicity was determined in the rotorod test. For all these evaluations the compounds were dissolved or suspended in 0.5% aqueous methyl cellulose.
6
2
E m
2
.c
:
4
Q)
s
n 2
Results The structures of the compounds are shown in Figure 1. The plasma concentrations of phenytoin were determined by gas chromatography with flame ionization detection. The assay was linear in the range between 0.5 and 25 yg/mL. The precision was 7.1%at 0.5 yg/mL and better than 2.6% at the other concentrations used. The detection limit was approximately 0.2 yglmL plasma. Preliminary experiments verified that measurable quantities of the prodrug were not present in the samples. For these initial studies the samples were divided. One aliquot was analyzed immediately, the other aliquot was left at room temperature for 1 h before ' analysis in order t o ensure complete hydrolysis of the prodrug. No differences in the plasma concentrations of phenytoin between the two aliquots could be detected. The plasma concentration uersus time profile after oral administration of phenytoin and phenytoin 2-monoglyceride, respectively, is shown in Figure 2. The pharmacokinetic parameters obtained by noncompartmental analysis of the plasma data are summarized in Table 1. Compared to the application of the parent drug, administration of the prodrug resulted in a higher bioavailability of phenytoin. An approximate %fold increase of C,, and a 4-fold increase of the AUC was observed. No significant differences regarding t,, and t l l z were found. The time course of the anticonvulsant activity determined in the MES test after oral administration of phenytoin 2-monoglyceride and phenytoin is shown in Figure 3. The monoglyceride derivative antagonized seizures 15 min after oral application of a dose of 66 pmol/kg. The anticonvulsant effect peaked around 1 h and lasted for a t least 6 h. In contrast, oral administration of almost twice the dose of phenytoin (119 ymoykg) did not yield any anticonvulsant activity after 15 min. The maximal activity occurred at 2 h postdose. Four hours after the administration no anticonvulsant effect was observed (Figure 3). The median effective doses (EDSO)of the compounds are summarized in Table 1. Upon oral administration to rats, phenytoin 2-monoglyceride did not exhibit any effect in the rotorod test at doses of up to 660 ymoVkg and was inactive in the seizure threshold test with subcutaneous pentetrazol (scMet) at doses up to 330 pmolkg.
0
0
10
5
15
20
time [h]
Figure 2-Mean (fSD)plasma concentration of phenytoin versus time profile following oral administration of equimolar doses of phenytoin (0)and phenytoin 2-monoglyceride (0)to rats (119 pmollkg corresponding to 30 mg/kg phenytoin equivalents; n = W).
o
o
l
0
2
4
6
time (h]
Figure 3-Time course of the MES activity after oral administration of 119 pmoll kg phenytoin (0)and 66 pmollkg of phenytoin 2-monoglyceride (0)to rats. The values are expressed as percentage of the animals protected (n = 4).
Discussion Phenytoin plasma levels and the anticonvulsant activity were evaluated after oral administration of phenytoin and phenytoin 2-monoglyceride, a phenytoin prodrug (Figure 11, to rats. Compared to the parent drug, administration of the monoglyceride resulted in substantially increased plasma levels of phenytoin (Figure 2). Noncompartmental pharmacokinetic analysis of the data showed an approximate 3.4-fold C and a 4-fold increase of the AUC (Table 1). increase of , The relativey low bioavailability obtained after dosing of Journal of Pharmaceutical Sciences / 301 Vo/. 84, No. 3, March 1995
phenytoin might be attributed to the slow and incomplete dissolution of the compound due to its poor aqueous solubility. Prodrug formation resulted in an approximate 40-fold increase of the water solubility,ll however, this increase is only moderated compared to the ca. 4000-5000-fold increase of the aqueous solubility obtained with other prodrugs such as fo~phenytoin.~ Therefore, physicochemical parameters other than the aqueous solubility appear to influence the absorption of the monoglyceride. Solubilization of the monoglyceride by bile acids and/or efficient enzymatic hydrolysis of the prodrug might contribute to the rapid absorption process. Compared to the oral administration of phenytoin, application of the monoglyceride prodrug did not significantly alter t,, and t112 (Table 1).A shift in t,, has been observed after coadministration of phenytoin with lipids3 or after administration of a lipophilic prodrug of 3-(hydroxymethy1)phenytoin dissolved in triglycerides.5 Thus, the present data confirm a rapid in vivo hydrolysis of the monoglyceride prodrug. The slight increase of the elimination half-life (tllz) was not statistically significant (Table 1). It is known that phenytoin displays nonlinear, dose-dependent pharmacokinetics in the rat.17-19 Thus, the higher plasma levels obtained after administration of the prodrug might be responsible for a slower metabolism and/or elimination of phenytoin. The pharmacokinetic data correlated with the anticonvulsant activity in the MES test (Figure 3). Administration of phenytoin 2-monoglyceride resulted in a faster onset and peaking of the MES activity than the administration of phenytoin itself. Moreover, the prodrug was still active after 6 h while the activity of phenytoin ceased aRer 4 h. On the basis of the ED50 values, the monoglyceridewas about 3 times more effective in antagonizing MES-induced seizures than the phenytoin itself (Table 1). These observations can be attributed to the increased bioavailability of phenytoin after administration of the monoglyceride derivative. At any time the prodrug produced higher plasma levels of phenytoin than the parent drug (Figure 2). Only phenytoin but no measurable levels of the prodrug could be detected in the plasma samples. The exact site of the hydrolysis of the monoglyceride prodrug is unknown. However, the high plasma levels of phenytoin after 30 min (Figure 2) as well as the early peaking of the anticonvulsant activity after oral administration (1h) compared to a maximal activity 4 h aRer intraperitoneal administration12suggest that the prodrug may be hydrolyzed during the absorption process. This might be accomplished by pancreatic lipase and other hydrolytic enzymes in the intestinal lumen or by esterases in the intestinal cells. Pancreatic lipase cleaves with high positional specificity the ester bonds in position 1 and 3 of glycerides. Thus, phenytoin 2-monoglyceride should not be a good substrate for the enzyme. However, rapid release of phenytoin from the monoglyceride by pancreatic lipase has been demonstrated in uitro." Intestinal hydrolysis is further supported by the fact that the isomeric phenytoin l-monoglyceride, which was equipotent to phenytoin 2-monoglyceride following intraperitoneal dosing to mice, did not exhibit significant anticonvulsant activity after oral administration to rats.12 On the other hand, rat plasma esterases hydrolyzed the monoglyceride extremely rapidly in vitro with a half-life of less than 4 s.ll Thus, it cannot be totally excluded that the prodrug is, a t least in part, absorbed intact and instantly hydrolyzed by plasma esterases. Upon oral administration, phenytoin 2-monoglyceride did not exhibit any significant activity in the scMet test or any neurological toxicity in the rotorod test at the doses tested. No activity in the scMet test nor acute toxicity have been demonstrated for phenytoin.14J5 Thus, coupling to glycerol did not alter the pharmacological profile of the drug as has 302 / Journal of Pharmaceutical Sciences Vol. 84, No. 3, March 1995
been described for dihydropyridine esters of 3-(hydroxymethy1)phenytoin. Although developed for intravenous use, fosphenytoin was found to be slightly more active than phenytoin in the MES test after intravenous, intraperitoneal, or oral administration to m i ~ e . The ~ , ~dihydropyridine esters of 3-(hydroxymethyl)phenytoin displayed a higher anticonvulsant activity than p h e n y t ~ i n .However, ~ a major drawback of the dihydropyridine derivatives is the fact that they are only active when administered intravenously. Phenytoin 2-monoglyceride displayed an anticonvulsant activity comparable to that of the drug itself upon intraperitoneal administration to mice12but was more active than phenytoin after oral dosing to rats (Table 1). Due to the different animals and dosing used in these studies, a direct comparison of the three types of prodrugs appears to be difficult. However, the present results suggest that the monoglyceride derivative might be a useful prodrug for the oral delivery of phenytoin. In conclusion, the oral administration of phenytoin 2-monoglyceride resulted in a higher bioavailability and anticonvulsant efficacy than the application of the phenytoin itself while the pharmacological profile of the drug was unaltered. Monoglyceride-derived prodrugs might represent useful prodrugs for the oral delivery of poorly water-soluble compounds.
References and Notes 1. Arnold, K.; Gerber, N.; Levy, G. Can. J . Pharm. Sci. 1970,5, 89-92. 2. Suzuki, T.; Saitoh, Y.; Nishihara, K. Chem. Pharm. Bull. 1984, 18,405-411. 3. Chakrabarti, S.;Belpaire, F. M. J . Pharm. Pharmacol. 1978, 30,330-331. 4. Varia, S. A.;Schuller, S.; Sloan, K. B.; Stella, V. J. J . Pharm. Sci. 1984,73,1068-1073. 5. Yamaoka, Y.;Roberts, R. D.; Stella, V. J. J . Pharm. Sci. 1983, 72,400-405. 6. Leppik, I. E.; Boucher, R.; Wilder, B. J.; Murthy, V. S.; Rask, C. A.; Watridge, C.; Graves, N. M.; Rangel, R. J.; Turlapaty, P. Epilepsia 1989,30,S22-S26. 7. Smith, R. D.; Brown, B. S.; Maher, R. W.; Matier, W. L. Epilepsia 1989,30,S15-S21. 8. Uthman, B. M.; Wilder, B. J. Epilepsia 1969,30,S33-S37. 9. Shek, E.;Murakami, T.; Nath, C.; Pop, E.; Bodor, N. S. J . Pharm. Sci. 1989,78,837-843. 10. Scriba, G. K.E.Arch. Pharm. 1993,327,477-481. 11. Scriba, G. K.E.Pharm. Res. 1993,10,1181-1186. 12. Scriba, G. K. E.; Lambert, D. M.; Poupaert, J. H. J . Pharm. Pharmacol. In press. 13. Stella, V. J. J . Pharm. Sci. 1977,66,1510-1511. 14. Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.; Swinyard, E. A. Epilepsia 1978,19,409-428. 15. Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B. J.; Kupferberg, H. J.; Scoville, B.; White, B. G. Cleveland Clin. Q . 1984,51,293-305. 16. Varia, S. A.; Stella, V. J. J . Pharm. Sci. 1984,73, 1080-1087. 17. Gerber, N.; Wagner, J. G. Res. Comm. Ckem. Pathol. Pharmacol. 1972,3,455-466. 18. Ashley, J. J.; Levy, G. J . Pharmacokin. Biopharm. 1973,1,99102. 19. Vicuna, A.;Lalka, D.; dusouich, P.; Vicuna, N.; Ludden, T. M.; McLean, A. J. Res. Comm. Chem.Pathol. Pharmacol. 1980,28, 3-11.
Acknowledgments The technical assistance of Ms. Isabelle de Zurpele as well as the helpful discussions of Dr. Hans-Gunther Schafer are gratefully acknowledged. The authors are indepted to Dr. James P. Stables and his staff at the Epilepsy Branch, Division of Convulsive, Developmental and Neuromuscular Disorders, National Institutes of Health, Bethesda, MD.
JS940571N
Introduction Phenytoin is a slightly water-soluble, slightly lipid-soluble drug that has shown erratic bioavailability upon oral administration.1,2 Superior availability was obtained by coadministration of lipids3or by ester prodrugs of 3-(hydroxymethyl)p h e n y t ~ i n . ~The . ~ phosphate ester of 3-(hydroxymethyl)phenytoin, fosphenytoin (ACC-96531,has been developed as an alternative to sodium phenytoin preparations for parental administration in the emergency treatment of the status epilepticus6-8and is currently under clinical investigation in the United States. Dihydropyridine derivatives of 3-(hydroxymethy1)phenytoinas redox delivery systems have been described r e ~ e n t l y . ~ In the course of the investigation of lipid-derived prodrugs of phenytoin, phenytoin 2-monoglyceride was synthesized by covalent binding of 3-(hydroxymethyl)phenytoin to position 2 of glycerol via a succinic acid spacer (Figure l).Io Rapid release of phenytoin from the monoglyceride derivative by plasma esterases and other hydrolytic enzymes was observed in uitro.ll Moreover, the compound displayed anticonvulsant activity and a pharmacological profile similar to that of the parent drug after ip administration to mice.12 Therefore, it was concluded that the monoglyceride might act as a prodrug of phenytoin. Oral administration is generally considered the route of choice for a drug. Therefore, the present study was conducted in order to evaluate the bioavailability and the anticonvulsant activity of phenytoin 2-monoglyceridein comparison to phenytoin after oral administration to rats.
Materials and Methods Chemicals-Phenytoin was obtained from Caelo (Hilden, Germany), 5-@-Methylphenyl)-5-phenylhydantoinwas purchased from Sigma (Deisenhofen, Germany); methyl cellulose, phenyltrimethylammonium hydroxide (0.1 M solution in methanol), and tetramethylammonium hydroxide (25% in water) were from Fluka (Neu-Ulm, @Abstractpublished in Advance ACS Abstracts, February 1, 1995.
300 / Journal of Pharmaceutical Sciences Vol. 84,No. 3, March 1995
HNKNH HO 0 phenytoin
V
0
phenytoin 2-monoglyceride
Figure 1-Structures of the compounds. Germany), and heparin solution (25 000 IWmL) was from Ratiopharm
(Ulm, Germany). Phenytoin 2-monoglyceride was synthesized as described.*O All other chemicals were obtained from commercial sources at the highest purity available. Solutions were prepared in double-distilled, deionized water. Pharmacokinetics-Male Wistar rats (bred at the animal facilities at UCL), weighing 220-260 g, were housed individually with a 12 h light-dark cycle and free access to commercial rodent chow and water. The animals were fasted overnight and during the experiment but were allowed water ad libitum. Doses of 119 ymollkg of phenytoin and phenytoin 2-monoglyceride (30 m g k g equivalents of phenytoin) were administered as suspensions in 0.5% methyl cellulose by oral intubation in a volume of 2 m U g . Approximately 300 yL of blood was collected via the tail clip method into Eppendorf tubes containing 20 yL of the heparin solution. The samples were centrifuged at 4 "C at 4000g, the plasma was immediately separated, frozen a t -80 "C, and stored frozen until analyzed. Analysis of Plamsa Samples-The analysis of the plasma samples was carried out as described13 with minor modifications. Briefly, 100-150 yL of plasma was placed in 10 mL stoppered centrifuge tubes, acidified with 50 yL of 3 M NaHzP04, pH 3, and extracted by vortexing with 2 mL of toluene containing 1pg of 5-(pmethylphenyl)5-phenylhydantoin as internal standard. After centifugation at 2500g for 10 min a t room temperature, the organic layer was transferred to a clean centrifuge tube and extracted with 25 fiL of 0.01 M phenyltrimethylammonium hydroxidedo.1M tetramethylammonium hydroxide in methanoVwater ( l : l , v/v) by vortexing for 1 min. Following centrifugation at 2500g for 5 min at room temperature, the toluene phase was discarded and approximately 1y L of the aqueous methanol layer was injected into a Shimadzu GC-14A gas chromatograph equipped with a flame ionization detector (Shimadzu, Duisburg, Germany). The separation of the compounds was obtained on a 25 m HP-1column (Hewlett-Packard,Diisseldorf, Germany) with helium as the carrier gas at a flow rate of 0.75 mumin. The column temperature was maintained at 225 "C. The detector temperature was set to 300 "C and the injector temperature to 270 "C. The injector was operated in the splitless mode during the injection and for the first 10 s of each analysis. A split of 1 : l O was applied throughout the remainder of the analysis. The retention times of phenytoin and 5-(p-methylphenyl)-5-phenylhydantoin were 9.0 and 11.7 min, respectively. Phenytoin concentrations were calculated by the peak area ratio method using a calibration curve obtained from spiked plasma samples. Data Analysis-Noncompartmental analysis of the data was performed. The maximum plasma concentration (Cmm)and the time to reach this concentration (t,& were obtained directly from the plasma concentration uersus time profiles. The area under the curve (AUC) was calculated by the log-linear trapezoidal method for the observed values and by extrapolation to infinity. The elimination half-life (tuz) was estimated from the final segment of the plasma concentration
0022-3549/95/3184-0300$09.00/0
0 1995, American Chemical Socieiy and American Pharmaceutical Association
Table 1-Pharmacokinetic Parameters for Pheytoin Obtained from Phenytoin Plasma Levels and Median Effective Dose Determined in the MES Test after Oral Administration of Phenytoin and Phenytoin 2-Monoglyceride to Rats Phenytoin Pharmacokinetic Parametersa Compound
AUC bghlmL)
Phenytoin Phenytoin 2-monoglyceride
MES Test
Cmax luglmL)
Lax (h)
tin (h)
10.0 1.I 40.5 7.3
1.90 2 0.1 1 6.42 2 1.51
1.25 f 0.5 1.oc
3.48 0.46 4.16 f 0.90
P < 0.001d
P < 0.001
nse
ns
+ +
Time of Test (h)
EDmbkmollkg)
2 1
91.9 (84.8-100.7) 34.5 (30.0-42.2)
+
P < 0.001
a Mean f SD, n = W. ED% calculated from five doses with eight animals per dose. One hour in all experiments. f-test for unpaired observations. ns, statistically not significant.
curve. Statistical comparison was performed using the t-test for unpaired observations. P < 0.05 was considered statistically significant. PharmacoZogy-Articonvulsant testing was provided by the Antiepileptic Drug Development Program, Epilepsy Branch, Division of Convulsive, Developmental and Neuromuscular Disorders, National Institutes of Health, according to standard procedure^'^^'^ and included the maximal electroshock (MES) test and the seizure threshold tests with subcutaneouspentetrazol (scMet test). The acute neurological toxicity was determined in the rotorod test. For all these evaluations the compounds were dissolved or suspended in 0.5% aqueous methyl cellulose.
6
2
E m
2
.c
:
4
Q)
s
n 2
Results The structures of the compounds are shown in Figure 1. The plasma concentrations of phenytoin were determined by gas chromatography with flame ionization detection. The assay was linear in the range between 0.5 and 25 yg/mL. The precision was 7.1%at 0.5 yg/mL and better than 2.6% at the other concentrations used. The detection limit was approximately 0.2 yglmL plasma. Preliminary experiments verified that measurable quantities of the prodrug were not present in the samples. For these initial studies the samples were divided. One aliquot was analyzed immediately, the other aliquot was left at room temperature for 1 h before ' analysis in order t o ensure complete hydrolysis of the prodrug. No differences in the plasma concentrations of phenytoin between the two aliquots could be detected. The plasma concentration uersus time profile after oral administration of phenytoin and phenytoin 2-monoglyceride, respectively, is shown in Figure 2. The pharmacokinetic parameters obtained by noncompartmental analysis of the plasma data are summarized in Table 1. Compared to the application of the parent drug, administration of the prodrug resulted in a higher bioavailability of phenytoin. An approximate %fold increase of C,, and a 4-fold increase of the AUC was observed. No significant differences regarding t,, and t l l z were found. The time course of the anticonvulsant activity determined in the MES test after oral administration of phenytoin 2-monoglyceride and phenytoin is shown in Figure 3. The monoglyceride derivative antagonized seizures 15 min after oral application of a dose of 66 pmol/kg. The anticonvulsant effect peaked around 1 h and lasted for a t least 6 h. In contrast, oral administration of almost twice the dose of phenytoin (119 ymoykg) did not yield any anticonvulsant activity after 15 min. The maximal activity occurred at 2 h postdose. Four hours after the administration no anticonvulsant effect was observed (Figure 3). The median effective doses (EDSO)of the compounds are summarized in Table 1. Upon oral administration to rats, phenytoin 2-monoglyceride did not exhibit any effect in the rotorod test at doses of up to 660 ymoVkg and was inactive in the seizure threshold test with subcutaneous pentetrazol (scMet) at doses up to 330 pmolkg.
0
0
10
5
15
20
time [h]
Figure 2-Mean (fSD)plasma concentration of phenytoin versus time profile following oral administration of equimolar doses of phenytoin (0)and phenytoin 2-monoglyceride (0)to rats (119 pmollkg corresponding to 30 mg/kg phenytoin equivalents; n = W).
o
o
l
0
2
4
6
time (h]
Figure 3-Time course of the MES activity after oral administration of 119 pmoll kg phenytoin (0)and 66 pmollkg of phenytoin 2-monoglyceride (0)to rats. The values are expressed as percentage of the animals protected (n = 4).
Discussion Phenytoin plasma levels and the anticonvulsant activity were evaluated after oral administration of phenytoin and phenytoin 2-monoglyceride, a phenytoin prodrug (Figure 11, to rats. Compared to the parent drug, administration of the monoglyceride resulted in substantially increased plasma levels of phenytoin (Figure 2). Noncompartmental pharmacokinetic analysis of the data showed an approximate 3.4-fold C and a 4-fold increase of the AUC (Table 1). increase of , The relativey low bioavailability obtained after dosing of Journal of Pharmaceutical Sciences / 301 Vo/. 84, No. 3, March 1995
phenytoin might be attributed to the slow and incomplete dissolution of the compound due to its poor aqueous solubility. Prodrug formation resulted in an approximate 40-fold increase of the water solubility,ll however, this increase is only moderated compared to the ca. 4000-5000-fold increase of the aqueous solubility obtained with other prodrugs such as fo~phenytoin.~ Therefore, physicochemical parameters other than the aqueous solubility appear to influence the absorption of the monoglyceride. Solubilization of the monoglyceride by bile acids and/or efficient enzymatic hydrolysis of the prodrug might contribute to the rapid absorption process. Compared to the oral administration of phenytoin, application of the monoglyceride prodrug did not significantly alter t,, and t112 (Table 1).A shift in t,, has been observed after coadministration of phenytoin with lipids3 or after administration of a lipophilic prodrug of 3-(hydroxymethy1)phenytoin dissolved in triglycerides.5 Thus, the present data confirm a rapid in vivo hydrolysis of the monoglyceride prodrug. The slight increase of the elimination half-life (tllz) was not statistically significant (Table 1). It is known that phenytoin displays nonlinear, dose-dependent pharmacokinetics in the rat.17-19 Thus, the higher plasma levels obtained after administration of the prodrug might be responsible for a slower metabolism and/or elimination of phenytoin. The pharmacokinetic data correlated with the anticonvulsant activity in the MES test (Figure 3). Administration of phenytoin 2-monoglyceride resulted in a faster onset and peaking of the MES activity than the administration of phenytoin itself. Moreover, the prodrug was still active after 6 h while the activity of phenytoin ceased aRer 4 h. On the basis of the ED50 values, the monoglyceridewas about 3 times more effective in antagonizing MES-induced seizures than the phenytoin itself (Table 1). These observations can be attributed to the increased bioavailability of phenytoin after administration of the monoglyceride derivative. At any time the prodrug produced higher plasma levels of phenytoin than the parent drug (Figure 2). Only phenytoin but no measurable levels of the prodrug could be detected in the plasma samples. The exact site of the hydrolysis of the monoglyceride prodrug is unknown. However, the high plasma levels of phenytoin after 30 min (Figure 2) as well as the early peaking of the anticonvulsant activity after oral administration (1h) compared to a maximal activity 4 h aRer intraperitoneal administration12suggest that the prodrug may be hydrolyzed during the absorption process. This might be accomplished by pancreatic lipase and other hydrolytic enzymes in the intestinal lumen or by esterases in the intestinal cells. Pancreatic lipase cleaves with high positional specificity the ester bonds in position 1 and 3 of glycerides. Thus, phenytoin 2-monoglyceride should not be a good substrate for the enzyme. However, rapid release of phenytoin from the monoglyceride by pancreatic lipase has been demonstrated in uitro." Intestinal hydrolysis is further supported by the fact that the isomeric phenytoin l-monoglyceride, which was equipotent to phenytoin 2-monoglyceride following intraperitoneal dosing to mice, did not exhibit significant anticonvulsant activity after oral administration to rats.12 On the other hand, rat plasma esterases hydrolyzed the monoglyceride extremely rapidly in vitro with a half-life of less than 4 s.ll Thus, it cannot be totally excluded that the prodrug is, a t least in part, absorbed intact and instantly hydrolyzed by plasma esterases. Upon oral administration, phenytoin 2-monoglyceride did not exhibit any significant activity in the scMet test or any neurological toxicity in the rotorod test at the doses tested. No activity in the scMet test nor acute toxicity have been demonstrated for phenytoin.14J5 Thus, coupling to glycerol did not alter the pharmacological profile of the drug as has 302 / Journal of Pharmaceutical Sciences Vol. 84, No. 3, March 1995
been described for dihydropyridine esters of 3-(hydroxymethy1)phenytoin. Although developed for intravenous use, fosphenytoin was found to be slightly more active than phenytoin in the MES test after intravenous, intraperitoneal, or oral administration to m i ~ e . The ~ , ~dihydropyridine esters of 3-(hydroxymethyl)phenytoin displayed a higher anticonvulsant activity than p h e n y t ~ i n .However, ~ a major drawback of the dihydropyridine derivatives is the fact that they are only active when administered intravenously. Phenytoin 2-monoglyceride displayed an anticonvulsant activity comparable to that of the drug itself upon intraperitoneal administration to mice12but was more active than phenytoin after oral dosing to rats (Table 1). Due to the different animals and dosing used in these studies, a direct comparison of the three types of prodrugs appears to be difficult. However, the present results suggest that the monoglyceride derivative might be a useful prodrug for the oral delivery of phenytoin. In conclusion, the oral administration of phenytoin 2-monoglyceride resulted in a higher bioavailability and anticonvulsant efficacy than the application of the phenytoin itself while the pharmacological profile of the drug was unaltered. Monoglyceride-derived prodrugs might represent useful prodrugs for the oral delivery of poorly water-soluble compounds.
References and Notes 1. Arnold, K.; Gerber, N.; Levy, G. Can. J . Pharm. Sci. 1970,5, 89-92. 2. Suzuki, T.; Saitoh, Y.; Nishihara, K. Chem. Pharm. Bull. 1984, 18,405-411. 3. Chakrabarti, S.;Belpaire, F. M. J . Pharm. Pharmacol. 1978, 30,330-331. 4. Varia, S. A.;Schuller, S.; Sloan, K. B.; Stella, V. J. J . Pharm. Sci. 1984,73,1068-1073. 5. Yamaoka, Y.;Roberts, R. D.; Stella, V. J. J . Pharm. Sci. 1983, 72,400-405. 6. Leppik, I. E.; Boucher, R.; Wilder, B. J.; Murthy, V. S.; Rask, C. A.; Watridge, C.; Graves, N. M.; Rangel, R. J.; Turlapaty, P. Epilepsia 1989,30,S22-S26. 7. Smith, R. D.; Brown, B. S.; Maher, R. W.; Matier, W. L. Epilepsia 1989,30,S15-S21. 8. Uthman, B. M.; Wilder, B. J. Epilepsia 1969,30,S33-S37. 9. Shek, E.;Murakami, T.; Nath, C.; Pop, E.; Bodor, N. S. J . Pharm. Sci. 1989,78,837-843. 10. Scriba, G. K.E.Arch. Pharm. 1993,327,477-481. 11. Scriba, G. K.E.Pharm. Res. 1993,10,1181-1186. 12. Scriba, G. K. E.; Lambert, D. M.; Poupaert, J. H. J . Pharm. Pharmacol. In press. 13. Stella, V. J. J . Pharm. Sci. 1977,66,1510-1511. 14. Krall, R. L.; Penry, J. K.; White, B. G.; Kupferberg, H. J.; Swinyard, E. A. Epilepsia 1978,19,409-428. 15. Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B. J.; Kupferberg, H. J.; Scoville, B.; White, B. G. Cleveland Clin. Q . 1984,51,293-305. 16. Varia, S. A.; Stella, V. J. J . Pharm. Sci. 1984,73, 1080-1087. 17. Gerber, N.; Wagner, J. G. Res. Comm. Ckem. Pathol. Pharmacol. 1972,3,455-466. 18. Ashley, J. J.; Levy, G. J . Pharmacokin. Biopharm. 1973,1,99102. 19. Vicuna, A.;Lalka, D.; dusouich, P.; Vicuna, N.; Ludden, T. M.; McLean, A. J. Res. Comm. Chem.Pathol. Pharmacol. 1980,28, 3-11.
Acknowledgments The technical assistance of Ms. Isabelle de Zurpele as well as the helpful discussions of Dr. Hans-Gunther Schafer are gratefully acknowledged. The authors are indepted to Dr. James P. Stables and his staff at the Epilepsy Branch, Division of Convulsive, Developmental and Neuromuscular Disorders, National Institutes of Health, Bethesda, MD.
JS940571N