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Pharmacokinetic Analysis of Ester Prodrugs of Valproic Acid
Pharmacokinetic Analysis of Ester Prodrugs of Valproic Acid SALlM
HADAD’,TOMB. vREE*,
EPPO VAN DER KLElJN*, AND
MEIR BIALER*’
Received April 4, 1991, from the ‘Depament of Pharmacy, School of Pharmacy, Hebrew Univesity of Jerusalem, P.O. Box 12065, Jerusalem, Israel, and the *Department of Clinical Pharmacy, St. Raboud Hospital, Catholic University of NQmegen, Postbus 9107, 6800 HB NJmegen, The Netherlands. Accepted for publication January 16, 1992. Abstract 0 The pharmacokinetics of five monoester prodrugs of Val-
proic acid (VPA) were investigated: propyl valproate (P-VPA), butyl valproate (B-VPA), isobutyl valproate (18-VPA), isoamyl valproate (IAVPA), and hexyl valproate (H-VPA). In addition, the anticonvulsant activity of these compounds was evaluated and compared with that of VPA and valpromide (VPD). The pharmacokinetics of VPA and its five ester derivatives were determined after intravenous administration of equivalent doses (400mg of VPA) to six dogs. The five ester prodrugs of VPA were biotransformed to VPA; the biotransformationwas complete for P-VPA, B-VPA, and H-VPA but was only partial for IB-VPA and IA-VPA. Because of the rapid conversion of the prodrugs to the parent drug, levels of VPA in plasma after administration of the prodrugs peaked at 6-26 min after dosing and did not yield an in vivo sustained-release dosage profile. Of the five ester prodrugs of VPA, only P-VPA demonstrated anticonvulsantactivity. P-VPA also was less neurotoxicthan VPA and VPD; therefore, it has a better protective index.
Valproic acid (VPA), one of the major antiepileptic drugs, has a broad spectrum of activity against both convulsive and nonconvulsive generalized epilepsies. As a short, branched fatty acid, VPA differs from other major antiepileptics in that it contains neither a nitrogen atom nor a cyclic moiety in its chemical structure.1-4 Pharmacokinetically, VPA has the shortest half-life of all of the existing antiepileptics. As a result of this short half-life, VPA has to be administered several times a day. To minimize fluctuations in levels of drug in plasma and to reduce the dosing regimen or frequency, two approaches are generally taken: pharmaceutical and chemical, In the pharmaceutical approach, one develops a sustained-release dosage form by which sustained release or prolonged absorption minimizes fluctuations in levels of drug in plasma. This approach has been applied quite successfully with VPA.6 In the chemical approach, a prodrug is designed, and the rate of biotransformation of the prodrug to the parent drug is used to obtain sustained levels of VPA in plasma. The chemical approach is mainly used for oral administration, although it has implications for other modes of administration, including the intravenous (iv) route. For VPA, prodrugs can actually minimize side effects, such as gastric irritation (after oral administration), and assist in different pharmaceutical problems arising with VPA (a liquid) or sodium valproate (a hygroscopic material). There are several reports in the literature about the primary amide of VPA, valpromide (VPD), which is a prodrug of VPA for oral and iv administration in humans.s9 A combined (pharmaceutical and chemical)approach also has quite successfullybeen applied to VPA.10 In this combined approach, the VPA prodrug (VPD)was placed in a sustained-release carrier. Except for VPD, only two brief reports about ester prodrugs of VPA exist: glyceryl trivalproatell and valproyl valproate.12 However, as yet, no pharmacokinetic analysis of these prodrugs has been reported. We recently explored the pharmacokinetics of three monoalkyl esters of VPA: ethyl valproate, trichloroethyl valproate, and valproyl valproate.13These three ester derivatives 0022-3!549/92/1 OOO-1O47$O2.50/0 Q 1992, American Pharmaceutical Association
underwent rapid biotransformation to VPA and did not demonstrate pharmaceutical or pharmacological advantages over the parent compound. Taillandier et al.14 investigated a series of analogues of VPA, including ester derivatives. However, in these ester derivatives, 2-propylpentanol (the alcoholic congener of VPA) was esterified by acetic, isobutyric, or benzylic acid. This paper describes a pharmacokinetic analysis of five monoester prodrugs of VPA after iv administration to dogs: propyl valproate (P-VPA), butyl valproate (B-VPA), isobutyl valproate (IB-VPA), isoamyl valproate (IA-VPA), and hexyl valproate (H-VPA).There are reports in the literature about structureactivity relationships of VPA derivatives.lk19 Reports about structure-pharmacokineticrelationships of VPD analogues20.21 can also be found. However, none of these reports refers to any investigation of ester derivatives of VPA. This paper is a continuation of a previous study13 and completes the investigation of the pharmacokinetic and anticonvulsant activity of a series of monoalkyl esters of VPA. To evaluate and analyze the pharmacokinetics of the investigated prodrugs in comparison with VPA, the five prodrugs and the parent drug were administered iv to dogs. The antiepileptic activities of the five prodrugs in comparison with VPA and VPD were also tested by using the anticonvulsant screening project of the National Institutes of Health (NIH) Epilepsy Branch.22
Experimental Section Materials-VPA and VPD were supplied by Sanofi (Paris, France). P-VPA, B-VPA, IB-VPA, and IA-VPA were supplied by Chemische Fabriek Katwijk (Katwijk, The Netherlands). AnirnalsThe experiments were carried out in six dogs (mongrels), three males and three females and ranging in weight from 17 to 21 kg. Although mice and rats are usually used for anticonvulaant screening,= these animals are too small to be used in pharmacokinetic studies with a crossover design. In addition, drug disposition in dogs is more similar to that in humans than is drug disposition in rodents.= In a randomized crossover design, each dog was injected iv (into one of the cephalic veins) with P-VPA, B-VPA, IB-VPA, (?3MfiH2
\
(N-COOH 013cH2a2’
VALPROIC ACID - VPA
R=PROPYL - P-VPA R-BUl’YLB-VPA R= ISOBUTYL- IB - VPA R- ISOAMLYL-IA- VF’A R=HEXYL- H-VPA
Journal of Pharmaceutical Sciences I 1047 Vol. 81, No. 10, October 1992
IA-VPA, and H-VPA (in an amount equivalent to 400 mg of VPA) and a parenteral preparation of sodium valproate in saline (50mg/mL). Protocol-Venous blood samples (6 mL) were collected via an indwelling catheter (from the other cephalic vein) at specified intervals aRer drug injection (0,2,5,10,15,20,30,40, and 50 min and 1, 1.25, 1.5,2, 2.5,3,3.5,4,5, and 6 h). The plasma was immediately separated by centrifugation at 7000 rpm (3000 x g ) for 15 min and stored at -20 "C. Before each assay, the plasma was allowed to reach mom temperature, mixed in a vortex mixer, and centrifuged. The residual clot was removed. Levels of VPA and its prodrug in plasma were assayed by gaa-liquid chromatography as described earlier.% The minimum quantifiable concentration of the assay was 0.2 mgL, with an interday coefficient of variation among replicate samples of 4%. In preliminary studies, we verified that the prodrugs can be detected and monitored simultaneously with VPA in our GLC!assay. Anticonvulsant Activity-The following compounds were screened in mice for their anticonvulsant activity and neurotoxicity by the NIH Epilepay Branch? VPA, VPD, P-VPA, B-VPA, IB-VPA, IAVPA, and H-VPA. The screening procedure involved the following: (1) the maximal electroshock (MEW test, which measures seizure spread; (2) the subcutaneous pentylenetetrazol test (sc Met test), which measures seizure threshold; and (3) the rotorod ataxia test, which assews neurotoxicity.22 Pharmacokinetic A n a l y s i e T h e linear terminal slope (p) of log C (VPA concentration in plasma) versus t (time) was calculated by the method of least squares. The terminal half-life of VPA (t,,z p) was calculated from the quotient 0.69/p, where 0 is the terminal slope. The peak concentration in plasma (Cmm)and the time to reach,,C (t-) were determined by inspection. The area under the curve of C versua t (AUC) was calculated by using the trapezoidal rule with extrapolation to infinity (i.e., dividing the last experimental value of C by p). The total body clearance (CL)of VPA was calculated &r iv administration of VPA by using the quotient of the iv dose (D)and the AUC. The volume of distribution (Vdhof VPA was calculated by using the quotient of CL and p. The volume of distribution at steady state (Vd,) and the mean residence time (MRT) were calculated by using eq 1 and 2%28:
Vd,
=D
*
AUMC/(AUCI2
MRT = AUMClAUC
(1) (2)
In eqs 1 and 2,AUMC is the area under the curve of C * t versus t, from t = 0 to t = m. The AUMC was calculated by the trapezoidal rule with extrapolation to infinity.26
The relative bioavailability (F)of VPA after administration of the prodrug was determined from the ratio of the AUCs aRer iv administration of the prodrug and the parent drug. The pharmacokinetic parameters were calculated in a noncompartmental manner b d on the statistical moment the0ry.m-m Stability S t u d i e e A blood stability study of P-VPA, B-WA, IB-VPA, IA-VPA, and H-VPA was carried out by incubating 400 pg of each compound in 20 mL of dog blood (placed in heparhized test tubes) at 37 "C with continuous shaking. Blood samples (2 mL) were then collected at the following times: 0, 1, 2, 3, 4,5, 6,7, and 8 h. Plasma was immediately separated, and the concentration of compound in the plasma was then assayed by GLC.
Results f i r iv administration, the level of the prodrugs in plasma was nil. The prodrugs were rapidly biotransformed to VPA. Figure 1 shows the mean levels of VPA in plasma after iv administration of VPA and the prodrugs, and Table I summarizes the mean pharmacokinetic parametem of VPA obtained after iv administration of the prodruge and VPA to dogs. Stability studies showed that the prodrugs were stable in dog blood for 8 h under physiological conditions. In phase 1 of the anticonvulsant screening project of the NIH Epilepsy Branch, of the five ester derivatives of VPA investigated, only P-VPA demonstrated qualitative anticonvulsant activity. Therefore, we decided to test P-VPA in phase II of the anticonvulsant screening project to determine the doses that are effective and toxic in 50% of the animals (ED, and TD, values, respectively), as well as ita protective indices (PI; the TD,,:ED, ratios). The anticonvulsant activity of P-VPA, in comparison with those of VPA and VPD, is shown in Table 11.
Unlike VPD, which undergoes a relatively slow and partial biotransformation to VPA in d o g ~ , s the ~ . ~five ~ monoester prodrugs of VPA were rapidly and completely converted to VPA after iv administration. No prodrugs could be detected in plasma (i.e., the levels in plasma were below the minimum quantifiable concentration of the assay) aRer their iv administration, perhaps because of rapid biotransformation of the
TIYE(m1n)
Figure l-Mean levels of VPA in plasma obtained following iv administratlon of VPA, P-VPA, B-VPA, IB-VPA, IA-VPA, and H-VPA to six dogs. 1048 / Journal of Pharmaceutical Sciences Vol. 81, No. 10, October 1992
Tabk I-Comparlson of the Pharmacoklnetlc Parameters of VPA OMalned atter Iv Admlnlstratlon of VPA, P-VPA, EVPA, IB-VPA, IA-VPA, and KVPA to Dogs' ~
Parameter
4, Pt h
AUC, mg/L * h C-,
mg/L
k,min MRT, h
F. Ohc CL, mUmin Vd L L
~~
P-VPA
8-VPA
IB-VPA
IA-VPA
H-VPA
1.3 f 0.2 4 5 2 11
1.4 t- 0.1 48 f 5 29 f 5 1922 17 f 0.1 1122 25
1.0 t- 0.2 42 2 7 33211 1624 1.4 2 0.1 101 2 4 0
1.1 It 0.4 32 f 14 27 f 6 622 1.4 f 0.5 73 f 25
1.6 ? 0.4 26 f 5 31 f 11 20 2 12 2.1 f 0.3 61 f 22
1.1 2 0.3 59 2 12 25 f 7 262 12 1.9 2 0.1 140 f 50
-b 1.6 f 0.2 -
-
158 f 44 1725 15 f 3
vd,
~~
VPA
-
'Values are exmssed as mean t standard deviation: dose. 400 ma" . n
=
6. '-,
-
-
-
-
Not determined. 'Bioavailability relative to the iv administration
of VPA.
prodrugs to VPA and large Vd values caused by high lipophilicity. The fastest biotransformation was noted with IB-VPA, and the slowest was with H-VPA. Thus, the rate of the biotransformation from ester to acid as the alcohol decreases moiety in this homologous series of VPA ester prodrugs becomes larger. The value of F for VPA after iv administration of P-VPA, B-VPA, and H-VPA (relative to iv administration of VPA) was -100%. However, the branched esters of VPA (IB-VPA and IA-VPA) were only partially available. Although the mean AUC value of H-VPA was higher than that of VPA, the two values were not significantly different. Table I1 shows that P-VPA was active (only in the sc Met test) and less toxic than VPA or VPD. Also, because of its lower neurotoxicity, it has a better PI. Because of the different rates and/or metabolic pathways, it may be generally better to investigate the pharmacokinetics and anticonvulsant activity of drugs in the same animal species. However, because the pharmacokinetics of VPA in mice and dogs are quite similar,32 the pharmacokinetics of VPA prodrugs was studied in dogs, and their anticonvulsant activity was tested in mice.22 In addition, dogs are the common animal model in pharmacokinetic studies, and mice are the common animal model for anticonvulsant testings and preclinical evaluation of new antiepileptic drugs. The anticonvulsant testing was carried out in mice after intraperitoneal administration. The rate of delivery of VPA may have been slower than its rate of elimination. VPA has a short t,,, of -50 min in mice.22 I t is plausible that the rate of absorption of the VPA prodrugs (after intraperitoneal administration to mice) anJ!or their rate of biotransformation was slower than the r. ..& of elimination of VPA. This difference in rates leads to low levels of VPA in plasma, similar to the situation in a formation-rate-limited metabolism. This tentative explanation, in addition to the different species used in this study, may explain the lack of a pharmacokinetic-pharmacodynamic correlation for B-VPA and H-VPA or in the differences in the anticonvulsant activities of P-VPA and the other four ester prodrugs of VPA. For IB-VPA and IA-VPA, the lack of anticonvulsant activity may
be explained by a partial biotransformation of these two prodrugs to the active entity, VPA. In conclusion, of the five investigated ester derivatives of VPA. onlv P-VPA showed a better marein between activitv (in the sc"Met test) and neurotoxicity thvan the known dru& VPA and VPD.
References and Notes 1. Lev , R. H.; Shen, D. D. In Antie ile tic Drugs, 3rd ed.; Levy, R. Dreifuss, F. E.;Mattaon, Meldrum, B. S.; Penry, J. K., Eds.; Raven: New York, 1989; pp 583-600. 2. Levy, R. H. Epilepsiu 1984,25, Suppl. 1, S l S 7 7 . 3. Zaccara, G.; Messori, A.; Moroni, F. Clin. Pharmacokinet. 1989, 15,367-389. 4. Gugler, R.; von Unruh, E.Clin. Pharmacokinet. 1980,5, 67-83. 5. Bialer, M.; Friedman, M.; Dubrovsky, J.; Raz, I.; Abramsky, 0. BioDharm. D r w Diems. 1985.6.401411. 6. Pisani, F.; Fa&, A.: Oteri, G.; Di Perri, A. Ther. Drug Monit. 1981.3. 297-301. 7. Pisani, F.; Di Perri, R. Ital. J . Neurol. Sci. 1980,4, 245-249. 8. Bialer, M.; Rubinstein, A.; Raz,I.; Abramsky, 0. Eur. J . Clin. Pharmacol. 1984,27,501403. 9. Bialer, M.; Rubinstein, A.; Dubrovsky, J.; Raz,I.; Abramsky, 0. Znt. J . Pharm. 1985,23, 25-33. 10. Rubinstein, A.; Bialer, M.; Friedman, M.; Raz, I.; Abramsky, 0. J . Controlled Release 1986.4, 33-38. 11. Mei'er J. W. A.; Kalff, R. Clinical Pharmacology of AntiEpijepiic Drugs; Springer Verlag: Berlin, 1975; pp 222-228. 12. Reekers-Ketting, J. J.; van der Kleijn, E.; Leliveld, B. A.; Schobben, A. F. A. M.; Vree, T. B. Pharm. Week. 1975, 110, 1233-1236. 13. Badir, K.; Hqj-Yehia,A.; Vree, T. B.; van der Kleun, E.;Bialer, M. Pharm. Res. 1991,8,750-754. 14. Taillandier, G.; Benoit-Gu od, J. L.; Boucherle, A.; Bmll, M.; Eymard, P. Eur. J . Med. C L m . 1975,10,453-462. 15. Kupferberg, H. J. In Antie ileptic Dru s. Mechanism of Action; Glasser, G. H.; Penry, J. and W d u r y , D. M. Eds.; Raven: New York, 1980; pp 643-654. 16. Keane, P. E.; Simiand, J.; Mendes, E.;Santucci, V.; Mom , M. Neurophurmacology 1983,22,875-879. 17. Cha man, A. G.; Meldrum, B. S.;Mendes, E. Life Sci. 1983,32, 202L2031. 18. Loscher, W.; Nau, H. Neurophurmacology 1988,27,287-294. 19. Abbott, F . S.;Acheampong, A. A. Neuropharmacology 1988,27, 287-294. 20. Ha-Yehia, A.; Bialer, M. Pharm. Res. 1989, 6, 683-684. 21. Haj-Yehia, A.; Bialer, M. J . Pharm. Sci. 1990, 79, 719-724. 22. Porter, R. J.; Cere 'no, J. J.; Gladdin G. D.; Hessie, B. J.; Kupferberg, H. J.; goville, B.; White, G. Cliu. Cin. Q. 1984, 51,293-305. 23. Crouthamel, W.; Sarapu, A. C. Anitnul Models for Oral Drug Delivery in Man; American Pharmaceutical Association: Washington, D.C., 1983. 24. Bialet, M.; Friedman, M.; Rubinstein, J. J . Phurm. Sci. 1984,73, 991-993. 25. Gibaldi, M.; Perrier, D. Phurmacokinetics, 2nd ed.; Marcel Dekker: New York, 1982; pp 445449. 26. Gibaldi, M.; Perrier, D. Phurmacokinetics, 2nd ed.; Marcel Dekker: New York, 1982; pp 409-417. 27. Benet, L. Z.; Galeazzi, R. L. J . Pharm. Sci. 1979,68, 1071-1074.
d.;
k:
8;
If
Table ICResults of Phaw II Study of the NIH Antlconvulmnt Screenlng Project
Test MES test: ED,, m g k g sc Met test: ED,, mglkg
Neurotoxicity:TD ,, PI, MES PI, sc Met
mglkg
VPA
VPD
P-VPA
200
56 55 81 1.o 1.5
2500 287
146 21 3 1.4
1.9
>a00 >1.6 >2.8
Journal of Pharmaceutical Sciences I 1049 Vol. 81, No. 10, October 7992
28. Yamaoka, K.; Nakagawa, T.; Uno, T. J. Phurmucokinet. Biophnrm. 1977,6,547-558. 29’ Yamaoka9K’Methods forPhnrmucokinetic forPersonaz Computers, 2nd ed.; Nanko-D: Tokyo, 1986; pp 145-175. 30. Bialer, M.; Rubinstein, A. J. Phurm. Phur-01. 198% 35, 607-609. 31. Bialer, M.; Rubinstein, A. Biopharm. Drug Dispos. 1984, 5, 177-183. 32. Loscher, W.; Esenwein, H. Anneim.-Forsch. 1978,28,782-787.
1050 I Journal of Pharmaceutical Sciences Vol. 81, No. 70, October 1992
Acknowledgments This project was supporbd by the E. D. Bergmann Fund. We thank Dr. Harvey J. Kupferberg and Mr. James Stables of the NIH Epilepey Branch for screening the compounds in their anticonvulsant screening project. Our thanks to Mr. Abu Salach Omar for hie skillful technical assistance. This work is abstracted in part from the doctoral dissertation of Mr. Salim Hadad, which was presented in partial fulfillment of the Ph.D. degree requirements of the Hebrew University of Jerusalem.
HADAD’,TOMB. vREE*,
EPPO VAN DER KLElJN*, AND
MEIR BIALER*’
Received April 4, 1991, from the ‘Depament of Pharmacy, School of Pharmacy, Hebrew Univesity of Jerusalem, P.O. Box 12065, Jerusalem, Israel, and the *Department of Clinical Pharmacy, St. Raboud Hospital, Catholic University of NQmegen, Postbus 9107, 6800 HB NJmegen, The Netherlands. Accepted for publication January 16, 1992. Abstract 0 The pharmacokinetics of five monoester prodrugs of Val-
proic acid (VPA) were investigated: propyl valproate (P-VPA), butyl valproate (B-VPA), isobutyl valproate (18-VPA), isoamyl valproate (IAVPA), and hexyl valproate (H-VPA). In addition, the anticonvulsant activity of these compounds was evaluated and compared with that of VPA and valpromide (VPD). The pharmacokinetics of VPA and its five ester derivatives were determined after intravenous administration of equivalent doses (400mg of VPA) to six dogs. The five ester prodrugs of VPA were biotransformed to VPA; the biotransformationwas complete for P-VPA, B-VPA, and H-VPA but was only partial for IB-VPA and IA-VPA. Because of the rapid conversion of the prodrugs to the parent drug, levels of VPA in plasma after administration of the prodrugs peaked at 6-26 min after dosing and did not yield an in vivo sustained-release dosage profile. Of the five ester prodrugs of VPA, only P-VPA demonstrated anticonvulsantactivity. P-VPA also was less neurotoxicthan VPA and VPD; therefore, it has a better protective index.
Valproic acid (VPA), one of the major antiepileptic drugs, has a broad spectrum of activity against both convulsive and nonconvulsive generalized epilepsies. As a short, branched fatty acid, VPA differs from other major antiepileptics in that it contains neither a nitrogen atom nor a cyclic moiety in its chemical structure.1-4 Pharmacokinetically, VPA has the shortest half-life of all of the existing antiepileptics. As a result of this short half-life, VPA has to be administered several times a day. To minimize fluctuations in levels of drug in plasma and to reduce the dosing regimen or frequency, two approaches are generally taken: pharmaceutical and chemical, In the pharmaceutical approach, one develops a sustained-release dosage form by which sustained release or prolonged absorption minimizes fluctuations in levels of drug in plasma. This approach has been applied quite successfully with VPA.6 In the chemical approach, a prodrug is designed, and the rate of biotransformation of the prodrug to the parent drug is used to obtain sustained levels of VPA in plasma. The chemical approach is mainly used for oral administration, although it has implications for other modes of administration, including the intravenous (iv) route. For VPA, prodrugs can actually minimize side effects, such as gastric irritation (after oral administration), and assist in different pharmaceutical problems arising with VPA (a liquid) or sodium valproate (a hygroscopic material). There are several reports in the literature about the primary amide of VPA, valpromide (VPD), which is a prodrug of VPA for oral and iv administration in humans.s9 A combined (pharmaceutical and chemical)approach also has quite successfullybeen applied to VPA.10 In this combined approach, the VPA prodrug (VPD)was placed in a sustained-release carrier. Except for VPD, only two brief reports about ester prodrugs of VPA exist: glyceryl trivalproatell and valproyl valproate.12 However, as yet, no pharmacokinetic analysis of these prodrugs has been reported. We recently explored the pharmacokinetics of three monoalkyl esters of VPA: ethyl valproate, trichloroethyl valproate, and valproyl valproate.13These three ester derivatives 0022-3!549/92/1 OOO-1O47$O2.50/0 Q 1992, American Pharmaceutical Association
underwent rapid biotransformation to VPA and did not demonstrate pharmaceutical or pharmacological advantages over the parent compound. Taillandier et al.14 investigated a series of analogues of VPA, including ester derivatives. However, in these ester derivatives, 2-propylpentanol (the alcoholic congener of VPA) was esterified by acetic, isobutyric, or benzylic acid. This paper describes a pharmacokinetic analysis of five monoester prodrugs of VPA after iv administration to dogs: propyl valproate (P-VPA), butyl valproate (B-VPA), isobutyl valproate (IB-VPA), isoamyl valproate (IA-VPA), and hexyl valproate (H-VPA).There are reports in the literature about structureactivity relationships of VPA derivatives.lk19 Reports about structure-pharmacokineticrelationships of VPD analogues20.21 can also be found. However, none of these reports refers to any investigation of ester derivatives of VPA. This paper is a continuation of a previous study13 and completes the investigation of the pharmacokinetic and anticonvulsant activity of a series of monoalkyl esters of VPA. To evaluate and analyze the pharmacokinetics of the investigated prodrugs in comparison with VPA, the five prodrugs and the parent drug were administered iv to dogs. The antiepileptic activities of the five prodrugs in comparison with VPA and VPD were also tested by using the anticonvulsant screening project of the National Institutes of Health (NIH) Epilepsy Branch.22
Experimental Section Materials-VPA and VPD were supplied by Sanofi (Paris, France). P-VPA, B-VPA, IB-VPA, and IA-VPA were supplied by Chemische Fabriek Katwijk (Katwijk, The Netherlands). AnirnalsThe experiments were carried out in six dogs (mongrels), three males and three females and ranging in weight from 17 to 21 kg. Although mice and rats are usually used for anticonvulaant screening,= these animals are too small to be used in pharmacokinetic studies with a crossover design. In addition, drug disposition in dogs is more similar to that in humans than is drug disposition in rodents.= In a randomized crossover design, each dog was injected iv (into one of the cephalic veins) with P-VPA, B-VPA, IB-VPA, (?3MfiH2
\
(N-COOH 013cH2a2’
VALPROIC ACID - VPA
R=PROPYL - P-VPA R-BUl’YLB-VPA R= ISOBUTYL- IB - VPA R- ISOAMLYL-IA- VF’A R=HEXYL- H-VPA
Journal of Pharmaceutical Sciences I 1047 Vol. 81, No. 10, October 1992
IA-VPA, and H-VPA (in an amount equivalent to 400 mg of VPA) and a parenteral preparation of sodium valproate in saline (50mg/mL). Protocol-Venous blood samples (6 mL) were collected via an indwelling catheter (from the other cephalic vein) at specified intervals aRer drug injection (0,2,5,10,15,20,30,40, and 50 min and 1, 1.25, 1.5,2, 2.5,3,3.5,4,5, and 6 h). The plasma was immediately separated by centrifugation at 7000 rpm (3000 x g ) for 15 min and stored at -20 "C. Before each assay, the plasma was allowed to reach mom temperature, mixed in a vortex mixer, and centrifuged. The residual clot was removed. Levels of VPA and its prodrug in plasma were assayed by gaa-liquid chromatography as described earlier.% The minimum quantifiable concentration of the assay was 0.2 mgL, with an interday coefficient of variation among replicate samples of 4%. In preliminary studies, we verified that the prodrugs can be detected and monitored simultaneously with VPA in our GLC!assay. Anticonvulsant Activity-The following compounds were screened in mice for their anticonvulsant activity and neurotoxicity by the NIH Epilepay Branch? VPA, VPD, P-VPA, B-VPA, IB-VPA, IAVPA, and H-VPA. The screening procedure involved the following: (1) the maximal electroshock (MEW test, which measures seizure spread; (2) the subcutaneous pentylenetetrazol test (sc Met test), which measures seizure threshold; and (3) the rotorod ataxia test, which assews neurotoxicity.22 Pharmacokinetic A n a l y s i e T h e linear terminal slope (p) of log C (VPA concentration in plasma) versus t (time) was calculated by the method of least squares. The terminal half-life of VPA (t,,z p) was calculated from the quotient 0.69/p, where 0 is the terminal slope. The peak concentration in plasma (Cmm)and the time to reach,,C (t-) were determined by inspection. The area under the curve of C versua t (AUC) was calculated by using the trapezoidal rule with extrapolation to infinity (i.e., dividing the last experimental value of C by p). The total body clearance (CL)of VPA was calculated &r iv administration of VPA by using the quotient of the iv dose (D)and the AUC. The volume of distribution (Vdhof VPA was calculated by using the quotient of CL and p. The volume of distribution at steady state (Vd,) and the mean residence time (MRT) were calculated by using eq 1 and 2%28:
Vd,
=D
*
AUMC/(AUCI2
MRT = AUMClAUC
(1) (2)
In eqs 1 and 2,AUMC is the area under the curve of C * t versus t, from t = 0 to t = m. The AUMC was calculated by the trapezoidal rule with extrapolation to infinity.26
The relative bioavailability (F)of VPA after administration of the prodrug was determined from the ratio of the AUCs aRer iv administration of the prodrug and the parent drug. The pharmacokinetic parameters were calculated in a noncompartmental manner b d on the statistical moment the0ry.m-m Stability S t u d i e e A blood stability study of P-VPA, B-WA, IB-VPA, IA-VPA, and H-VPA was carried out by incubating 400 pg of each compound in 20 mL of dog blood (placed in heparhized test tubes) at 37 "C with continuous shaking. Blood samples (2 mL) were then collected at the following times: 0, 1, 2, 3, 4,5, 6,7, and 8 h. Plasma was immediately separated, and the concentration of compound in the plasma was then assayed by GLC.
Results f i r iv administration, the level of the prodrugs in plasma was nil. The prodrugs were rapidly biotransformed to VPA. Figure 1 shows the mean levels of VPA in plasma after iv administration of VPA and the prodrugs, and Table I summarizes the mean pharmacokinetic parametem of VPA obtained after iv administration of the prodruge and VPA to dogs. Stability studies showed that the prodrugs were stable in dog blood for 8 h under physiological conditions. In phase 1 of the anticonvulsant screening project of the NIH Epilepsy Branch, of the five ester derivatives of VPA investigated, only P-VPA demonstrated qualitative anticonvulsant activity. Therefore, we decided to test P-VPA in phase II of the anticonvulsant screening project to determine the doses that are effective and toxic in 50% of the animals (ED, and TD, values, respectively), as well as ita protective indices (PI; the TD,,:ED, ratios). The anticonvulsant activity of P-VPA, in comparison with those of VPA and VPD, is shown in Table 11.
Unlike VPD, which undergoes a relatively slow and partial biotransformation to VPA in d o g ~ , s the ~ . ~five ~ monoester prodrugs of VPA were rapidly and completely converted to VPA after iv administration. No prodrugs could be detected in plasma (i.e., the levels in plasma were below the minimum quantifiable concentration of the assay) aRer their iv administration, perhaps because of rapid biotransformation of the
TIYE(m1n)
Figure l-Mean levels of VPA in plasma obtained following iv administratlon of VPA, P-VPA, B-VPA, IB-VPA, IA-VPA, and H-VPA to six dogs. 1048 / Journal of Pharmaceutical Sciences Vol. 81, No. 10, October 1992
Tabk I-Comparlson of the Pharmacoklnetlc Parameters of VPA OMalned atter Iv Admlnlstratlon of VPA, P-VPA, EVPA, IB-VPA, IA-VPA, and KVPA to Dogs' ~
Parameter
4, Pt h
AUC, mg/L * h C-,
mg/L
k,min MRT, h
F. Ohc CL, mUmin Vd L L
~~
P-VPA
8-VPA
IB-VPA
IA-VPA
H-VPA
1.3 f 0.2 4 5 2 11
1.4 t- 0.1 48 f 5 29 f 5 1922 17 f 0.1 1122 25
1.0 t- 0.2 42 2 7 33211 1624 1.4 2 0.1 101 2 4 0
1.1 It 0.4 32 f 14 27 f 6 622 1.4 f 0.5 73 f 25
1.6 ? 0.4 26 f 5 31 f 11 20 2 12 2.1 f 0.3 61 f 22
1.1 2 0.3 59 2 12 25 f 7 262 12 1.9 2 0.1 140 f 50
-b 1.6 f 0.2 -
-
158 f 44 1725 15 f 3
vd,
~~
VPA
-
'Values are exmssed as mean t standard deviation: dose. 400 ma" . n
=
6. '-,
-
-
-
-
Not determined. 'Bioavailability relative to the iv administration
of VPA.
prodrugs to VPA and large Vd values caused by high lipophilicity. The fastest biotransformation was noted with IB-VPA, and the slowest was with H-VPA. Thus, the rate of the biotransformation from ester to acid as the alcohol decreases moiety in this homologous series of VPA ester prodrugs becomes larger. The value of F for VPA after iv administration of P-VPA, B-VPA, and H-VPA (relative to iv administration of VPA) was -100%. However, the branched esters of VPA (IB-VPA and IA-VPA) were only partially available. Although the mean AUC value of H-VPA was higher than that of VPA, the two values were not significantly different. Table I1 shows that P-VPA was active (only in the sc Met test) and less toxic than VPA or VPD. Also, because of its lower neurotoxicity, it has a better PI. Because of the different rates and/or metabolic pathways, it may be generally better to investigate the pharmacokinetics and anticonvulsant activity of drugs in the same animal species. However, because the pharmacokinetics of VPA in mice and dogs are quite similar,32 the pharmacokinetics of VPA prodrugs was studied in dogs, and their anticonvulsant activity was tested in mice.22 In addition, dogs are the common animal model in pharmacokinetic studies, and mice are the common animal model for anticonvulsant testings and preclinical evaluation of new antiepileptic drugs. The anticonvulsant testing was carried out in mice after intraperitoneal administration. The rate of delivery of VPA may have been slower than its rate of elimination. VPA has a short t,,, of -50 min in mice.22 I t is plausible that the rate of absorption of the VPA prodrugs (after intraperitoneal administration to mice) anJ!or their rate of biotransformation was slower than the r. ..& of elimination of VPA. This difference in rates leads to low levels of VPA in plasma, similar to the situation in a formation-rate-limited metabolism. This tentative explanation, in addition to the different species used in this study, may explain the lack of a pharmacokinetic-pharmacodynamic correlation for B-VPA and H-VPA or in the differences in the anticonvulsant activities of P-VPA and the other four ester prodrugs of VPA. For IB-VPA and IA-VPA, the lack of anticonvulsant activity may
be explained by a partial biotransformation of these two prodrugs to the active entity, VPA. In conclusion, of the five investigated ester derivatives of VPA. onlv P-VPA showed a better marein between activitv (in the sc"Met test) and neurotoxicity thvan the known dru& VPA and VPD.
References and Notes 1. Lev , R. H.; Shen, D. D. In Antie ile tic Drugs, 3rd ed.; Levy, R. Dreifuss, F. E.;Mattaon, Meldrum, B. S.; Penry, J. K., Eds.; Raven: New York, 1989; pp 583-600. 2. Levy, R. H. Epilepsiu 1984,25, Suppl. 1, S l S 7 7 . 3. Zaccara, G.; Messori, A.; Moroni, F. Clin. Pharmacokinet. 1989, 15,367-389. 4. Gugler, R.; von Unruh, E.Clin. Pharmacokinet. 1980,5, 67-83. 5. Bialer, M.; Friedman, M.; Dubrovsky, J.; Raz, I.; Abramsky, 0. BioDharm. D r w Diems. 1985.6.401411. 6. Pisani, F.; Fa&, A.: Oteri, G.; Di Perri, A. Ther. Drug Monit. 1981.3. 297-301. 7. Pisani, F.; Di Perri, R. Ital. J . Neurol. Sci. 1980,4, 245-249. 8. Bialer, M.; Rubinstein, A.; Raz,I.; Abramsky, 0. Eur. J . Clin. Pharmacol. 1984,27,501403. 9. Bialer, M.; Rubinstein, A.; Dubrovsky, J.; Raz,I.; Abramsky, 0. Znt. J . Pharm. 1985,23, 25-33. 10. Rubinstein, A.; Bialer, M.; Friedman, M.; Raz, I.; Abramsky, 0. J . Controlled Release 1986.4, 33-38. 11. Mei'er J. W. A.; Kalff, R. Clinical Pharmacology of AntiEpijepiic Drugs; Springer Verlag: Berlin, 1975; pp 222-228. 12. Reekers-Ketting, J. J.; van der Kleijn, E.; Leliveld, B. A.; Schobben, A. F. A. M.; Vree, T. B. Pharm. Week. 1975, 110, 1233-1236. 13. Badir, K.; Hqj-Yehia,A.; Vree, T. B.; van der Kleun, E.;Bialer, M. Pharm. Res. 1991,8,750-754. 14. Taillandier, G.; Benoit-Gu od, J. L.; Boucherle, A.; Bmll, M.; Eymard, P. Eur. J . Med. C L m . 1975,10,453-462. 15. Kupferberg, H. J. In Antie ileptic Dru s. Mechanism of Action; Glasser, G. H.; Penry, J. and W d u r y , D. M. Eds.; Raven: New York, 1980; pp 643-654. 16. Keane, P. E.; Simiand, J.; Mendes, E.;Santucci, V.; Mom , M. Neurophurmacology 1983,22,875-879. 17. Cha man, A. G.; Meldrum, B. S.;Mendes, E. Life Sci. 1983,32, 202L2031. 18. Loscher, W.; Nau, H. Neurophurmacology 1988,27,287-294. 19. Abbott, F . S.;Acheampong, A. A. Neuropharmacology 1988,27, 287-294. 20. Ha-Yehia, A.; Bialer, M. Pharm. Res. 1989, 6, 683-684. 21. Haj-Yehia, A.; Bialer, M. J . Pharm. Sci. 1990, 79, 719-724. 22. Porter, R. J.; Cere 'no, J. J.; Gladdin G. D.; Hessie, B. J.; Kupferberg, H. J.; goville, B.; White, G. Cliu. Cin. Q. 1984, 51,293-305. 23. Crouthamel, W.; Sarapu, A. C. Anitnul Models for Oral Drug Delivery in Man; American Pharmaceutical Association: Washington, D.C., 1983. 24. Bialet, M.; Friedman, M.; Rubinstein, J. J . Phurm. Sci. 1984,73, 991-993. 25. Gibaldi, M.; Perrier, D. Phurmacokinetics, 2nd ed.; Marcel Dekker: New York, 1982; pp 445449. 26. Gibaldi, M.; Perrier, D. Phurmacokinetics, 2nd ed.; Marcel Dekker: New York, 1982; pp 409-417. 27. Benet, L. Z.; Galeazzi, R. L. J . Pharm. Sci. 1979,68, 1071-1074.
d.;
k:
8;
If
Table ICResults of Phaw II Study of the NIH Antlconvulmnt Screenlng Project
Test MES test: ED,, m g k g sc Met test: ED,, mglkg
Neurotoxicity:TD ,, PI, MES PI, sc Met
mglkg
VPA
VPD
P-VPA
200
56 55 81 1.o 1.5
2500 287
146 21 3 1.4
1.9
>a00 >1.6 >2.8
Journal of Pharmaceutical Sciences I 1049 Vol. 81, No. 10, October 7992
28. Yamaoka, K.; Nakagawa, T.; Uno, T. J. Phurmucokinet. Biophnrm. 1977,6,547-558. 29’ Yamaoka9K’Methods forPhnrmucokinetic forPersonaz Computers, 2nd ed.; Nanko-D: Tokyo, 1986; pp 145-175. 30. Bialer, M.; Rubinstein, A. J. Phurm. Phur-01. 198% 35, 607-609. 31. Bialer, M.; Rubinstein, A. Biopharm. Drug Dispos. 1984, 5, 177-183. 32. Loscher, W.; Esenwein, H. Anneim.-Forsch. 1978,28,782-787.
1050 I Journal of Pharmaceutical Sciences Vol. 81, No. 70, October 1992
Acknowledgments This project was supporbd by the E. D. Bergmann Fund. We thank Dr. Harvey J. Kupferberg and Mr. James Stables of the NIH Epilepey Branch for screening the compounds in their anticonvulsant screening project. Our thanks to Mr. Abu Salach Omar for hie skillful technical assistance. This work is abstracted in part from the doctoral dissertation of Mr. Salim Hadad, which was presented in partial fulfillment of the Ph.D. degree requirements of the Hebrew University of Jerusalem.