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Transdermal absorption of l -dopa from hydrogel in rats
European Journal of Pharmaceutical Sciences, 7 (1998) 67–71
Transdermal absorption of L-dopa from hydrogel in rats a, b a a a Jun-ichi Sudo *, Hiroaki Iwase , Jun Terui , Katsuhiko Kakuno , Momoko Soyama , Kozo Takayama b , Tsuneji Nagai b a
Department of Clinical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, IshikariTobetsu, Hokkaido 061 -02, Japan b Department of Pharmaceutics, Hoshi University, Ebara 2 -4 -41, Shinagawa-ku, Tokyo 142, Japan Received 6 May 1997; accepted 9 January 1998
Abstract To improve compliance in administration of L-dopa, transdermal absorption of the agent was investigated in rats in vitro employing two-chamber diffusion cells in which the excised rat abdominal skin was mounted, and in vivo using an alcoholic hydrogel containing L-menthol. The in vitro study revealed that in presence of L-menthol (2%, W/ W), ethanol (20 and 40%, V/ V) accelerated transdermal penetration of L-dopa with an increase of its percentages. The in vivo study showed that when the L-dopa-hydrogel containing 2% L-menthol and 40% ethanol was attached on the skin, plasma levels of L-dopa and norepinephrine increased with the time elapsed; the level of dopamine increased and reached a plateau thereafter; and the level of epinephrine was unchanged. These in vitro and in vivo findings indicated that the hydrogel formulation of L-dopa provides new direction in treating Parkinson’s disease. 1998 Elsevier Science B.V. All rights reserved. Keywords: L-Dopa; Hydrogel; Ethanol; L-Menthol; Transdermal absorption; Rat
1. Introduction
2. Experimental procedures
L-Dopa is a therapeutic agent used for Parkinson’s disease, and the agent is generally administered orally or intravenously (Shoulson et al., 1975; Quinn, 1984). The patients of Parkinson’s disease are occasionally geriatric and tend to have dementia and dysphagia. These types of patients cannot be expected to comply with oral administration; thus, intravenous administration is employed. However, patients with dementia sometimes pull out the needle for the injection. Therefore, we considered a transdermal delivery system would be a better route for the administration. So, we attempted to make a hydrogel formulation of L-dopa, using ethanol and menthol as representative cutaneous absorption enhancers (Levison et al., 1994). We then examined the possible transdermal absorption of L-dopa from the hydrogel in vitro and in vivo in rats.
2.1. Animals and chemicals
*Corresponding author: Tel.: 181 1332 22921; fax: 181 1332 21820. 0928-0987 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 98 )00007-4
Male Wistar strain rats (Hokudo Co., Abuta, Hokkaido, Japan) weighing 200610 g were housed in ordinary cages and allowed free access to water and a standard pellet diet (CE-2; Clea Japan Co., Tokyo, Japan) prior to the study. L-Dopa ( L-3-4-dihydroxyphenylalanine) was purchased from Sigma Chemicals Co. (St Louis, MO, USA), and other chemicals used were of the highest grade available.
2.2. Procedures 2.2.1. In vitro cutaneous permeation study Cutaneous permeation of L-dopa was investigated in vitro in the same way as previously reported (Ohara et al., 1994). Full-thickness abdominal skin was excised from rats, whose hair had been removed beforehand by an electric clipper. The skin excised was used as a permeation membrane. Two-chamber diffusion cells (available diffusion area, 0.785 cm 2 ; volume of each half-cell, 3.0 ml) with a water jacket were employed. L-Dopa was suspended
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J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
with excess amounts in the following solutions: a water; a 20% (V/ V) ethanol and 2% (W/ V) L-menthol solution; and a 40% ethanol and 2% L-menthol solution. L-Dopa suspended in each of the three solutions was applied to the donor cell, and the receiver cell was filled with phosphatebuffered saline (pH 7.4). Both cells were warmed at 408C and stirred by magnetic stirrers. At 0- (before), 30-, 60-, 120-, and 180-min after the L-dopa suspension was applied to the donor cell, 0.5 ml aliquots were drawn from the receiver cell. Thereafter, an equivalent volume of the phosphate-buffered saline was supplied to the receiver cell.
2.2.2. In vivo intravenous administration study Rats were anesthetized with ether, and the left jugular vein was catheterized with a polyethylene tube (PE-50). Through this route, saline was continuously infused at 0.1 ml / kg body weight / min, and urethane (500 mg / kg body weight) and a-chloralose (70 mg / kg body weight) were given for deeper anesthesia and immobilization (Terui et al., 1994). When necessary, L-dopa dissolved in saline at a concentration of 2.5 mg / ml was injected at a dose of 2.5 mg / kg body weight through this route. Then, the animal was intubated for free respiration. Thirty min before collection of blood, the left femoral artery was catheterized with a polyethylene tube (PE-50) that had been filled with 0.2 M EGTA dissolved in saline (Sudo et al., 1995). Blood was collected at 0- (before), 5-, 15-, 25-, 37.5-, and 52.5-min after the intravenous bolus injection of L-dopa and then put into chilled tubes containing 40 ml of a solution containing 0.2 M EGTA and 0.2 M reduced glutathione (Eriksson and Persson, 1982). The blood volume collected was restricted to within 2 ml to avoid possible physiological alterations in concentrations of the amines induced by the loss of blood; blood was sampled once per rat. 2.2.3. In vivo cutaneous absorption study A hydrogel containing L-dopa was prepared, composition of which was as follows (100 g, in total): 5 g of L-dopa, 10 g of propylene glycol, 2 g of L-menthol, 2 g of diisopropyl adipate, 1 g of diisopropanolamine, 1 g of carboxyvinyl polymer, 40 ml of ethanol, and water. The hydrogel was saturated with L-dopa in this formula. Rats were anesthetized, infused, and intubated in the same manner as described in the above intravenous administration study. Then, the abdominal hairs were gently removed by an electric clipper. A glass cell (inner diameter, 1.13 cm; area adhered by the hydrogel, 1 cm 2 ; height, 1 cm) was attached on the shaved abdominal skin by an adhesive (cyanoacrylate type; Alon-Alpha A, Sankyo Co., Tokyo, Japan). Then, the glass tube was filled with either saline (control) or the hydrogel, and covered with a parafilm. Before (0-), and 30-, 60-, and 180-min after the initiation of filling the hydrogel into the glass tube, blood was collected as described in the above intravenous administration study. Blood was sampled once per rat.
2.3. Determination of L-dopa and amines Blood samples in the in vivo studies were centrifuged (1 7003g, 10 min, 48C) to obtain plasma. Aliquots from the in vitro study as well as the plasma samples from the in vivo studies were pretreated by the method of Eriksson and Persson (1982). L-Dopa, dopamine, norepinephrine, and epinephrine in the samples were determined electrochemically by the high-performance liquid chromatographic method of Sudo et al. (1995).
2.4. Pharmacokinetic analysis and statistics In the intravenous administration study, 1 group constituted 5 rats for 5 time-points including 5-, 15-, 25-, 37.5-, and 52.5-min after the intravenous bolus injection of Ldopa (one time-point per one rat). In the cutaneous absorption study in vivo, 4 rats for 4 time-points, including 0-, 30-, 60-, and 180-min after the cutaneous attachment of the L-dopa-hydrogel (one time-point per one rat), constituted 1 group. Both studies were carried out in 5 groups of rats. In the intravenous administration study in vivo, plasma concentration at 0 time (C0 ), elimination rate constant (k e ), half-life time (T 1 / 2 ), distribution volume (Vd ), total body clearance (CL ), and area under plasma concentration-time curve (AUC) were estimated by the one-compartment open model with the least-squares method (Yamaoka et al., 1981). Then, in the cutaneous absorption study in vivo, apparent cutaneous penetration rate (R p ) of L-dopa was estimated by the above parameters of Vd and k e and by the below formula based on the simple model on the assumption of a constant permeation through the skin (Ohara et al., 1994). R p (1 2 e k e ?t ) ]]]] Ct 5 k e ?Vd In the formula, Ct was the plasma concentration of L-dopa at time t, and t was time point during drug input. We applied the in vitro cutaneous permeation data to the Fickian diffusion equation, and explored its promoting mechanism by estimation of parameters in diffusion (D9) and partition (K9). The permeation profiles of L-dopa were analyzed by the method described by Okamoto et al. (1988), based on the following diffusion model.
F
1 2 Q t 5 AK9C0 D9t 2 ] 2 ]2 6 p
(21) O ]] exp(2D9n p t) G n `
n
2
2
2
1
D9 5 D/L 2 K9 5 KL D is diffusion constant; L, effective diffusion length of membrane; K, partition coefficient of the penetrant between the membrane and the donor phase; Q t , cumulative
J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
69
Fig. 1. Cumulative amount of L-dopa through rat abdominal skin in in vitro study employing two-chamber diffusion cells. Points and bars: means 6SEM in five experiments.
Fig. 2. Plasma levels of L-dopa after intravenous bolus administration of L-dopa in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the values at 0-min.
amount of penetrant in the receiver fluid at time t; A, area of application; C0 , solubility of L-dopa in the donor phase. In the steady-state, the penetration rate (J) of L-dopa was estimated as follows.
observed in 40% ethanol and 2% L-menthol solution. The K9 value was somewhat enhanced in 20% ethanol and 2% L-menthol solution, compared with water, while no further enhancement of the K9 value was observed at higher concentration of ethanol (40%). The penetration rate (J) of 2 L-dopa (ng / cm / min) was 146 in water, 436 in 20% ethanol and 2% L-menthol solution, and 1375 in 40% ethanol and 2% L-menthol solution.
J 5 AC0 KD/L 5 AC0 K9D9 The data were expressed as means 6SEM and were statistically analyzed using Student’s unpaired t-test; P values of less than 0.05 were considered significant.
3.2. In vivo intravenous administration study 3. Results
3.1. In vitro cutaneous permeation study In the in vitro study employing two-chamber diffusion cells in which the rat abdominal skin was mounted, cumulative amounts of L-dopa that permeated through the skin into the receiver cell were determined (Fig. 1). LDopa suspension in water revealed that L-dopa had cutaneously penetrated through the rat skin into the receiver cell with the time elapsed. L-Dopa suspension in 20% ethanol and 2% L-menthol solution indicated that the cutaneous permeation of L-dopa had been accelerated, and furthermore with increase of percentages of ethanol to 40. To find out how ethanol and L-menthol combination promoted permeation, the profiles of L-dopa permeation through the skin, were analyzed (Fig. 1 and Table 1). An impressive elevation in the D9 value of L-dopa was
L-Dopa and the amines in the plasma were determined after the intravenous injection of L-dopa (2.5 mg / kg body weight) (Figs. 2 and 3). L-Dopa, dopamine, and norepinephrine reached the highest concentrations (ng / ml, n55) at 5-min after the L-dopa injection: 664.0659.7 in L-dopa, 21.363.5 in dopamine, and 2.060.1 in norepinephrine. Thereafter, they fell toward the control levels with elapsing time. Epinephrine showed no significant tendency throughout the experimental period.
Table 1 Effect of donor solutions on diffusion parameter (D9 ) and partition parameter (K9 ) of L-dopa Donor solution
D9 (h 21 )
K9 (cm)
Water 20% Ethanol12% L-menthol 40% Ethanol12% L-menthol
0.01160.006 0.03360.017 0.14460.054
0.08760.029 0.12360.095 0.14960.034
Values: means 6SEM in five experiments.
Fig. 3. Plasma levels of dopamine, norepinephrine, and epinephrine after intravenous bolus administration of L-dopa in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the values at 0-min.
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J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
Fig. 4. Plasma level of L-dopa during cutaneous attachment of L-dopahydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (a), P,0.05; (b), P,0.01, compared with the control values at each time period.
Fig. 6. Plasma level of norepinephrine during cutaneous attachment of L-dopa-hydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the control values at each time period.
3.3. In vivo cutaneous absorption study L-Dopa and the amines in the plasma were determined after the L-dopa-hydrogel was attached on the skin. L-Dopa levels rose until 180-min Fig. 4). Dopamine reached a plateau level at 30-min, and this level was maintained from 30- to 180-min (Fig. 5). Norepinephrine levels also rose until 180-min (Fig. 6. Epinephrine did not show any marked changes (Fig. 7).
3.4. Pharmacokinetic analysis Table 2 shows the kinetic parameters concerning the data in the intravenous administration of L-dopa in vivo. When apparent cutaneous penetration rate (R p ) of L-dopa in the cutaneous absorption study in vivo was estimated on the basis of the parameters of Vd and k e , it was 1236662 ng / cm 2 / min in five experiments. Furthermore, when the amount of L-dopa that had penetrated through the skin was estimated by the R p values, it was 1.11260.056 mg / kg body weight for 180-min.
Fig. 7. Plasma level of epinephrine during cutaneous attachment of L-dopa-hydrogel in vivo. Points and bars: means 6SEM in five rats. There were no significant differences between the control and the L-dopahydrogel-administered groups at each time period.
4. Discussion First, we employed two-chamber diffusion cells using the excised rat abdominal skin as a permeation membrane, and investigated effectiveness of L-menthol and ethanol as absorption enhancers concerned with a possible transdermal absorption of L-dopa. This in vitro study proved that Table 2 Kinetic parameters of plasma concentration of L-dopa and time curves based on one-compartment open model, in intravenous bolus administration of L-dopa in vivo C0 (ng / ml) k e (min 21 ) T 1 / 2 (min) Vd (ml / kg body weight) CL (ml / min / kg body weight) AUC (ng?min / ml)
Fig. 5. Plasma level of dopamine during cutaneous attachment of L-dopahydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (a), P,0.05, compared with the control values at each time period.
559634 0.039260.0023 18.061.1 909651 35.362.2 144446896
C0 , plasma concentration at 0 time; k e , elimination rate constant; T 1 / 2 , half-life time; Vd , distribution volume; CL , total body clearance; AUC, area under plasma concentration-time curve. Values: means6SEM in five experiments.
J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
ethanol and L-menthol had possessed a very strong activity on the transdermal absorption of the agent. Pronounced increase of the D9 value in 40% ethanol and 2% L-menthol (Table 1) suggested that the combination of 40% ethanol and 2% L-menthol had effectively changed the dense barrier structure of the stratum corneum (Ohara et al., 1994), and that, as its result, the diffusivity of L-dopa through the skin had been increased. Although a little enhancement of the K9 value was seen in 20% ethanol and 2% L-menthol, no further enhancement of the K9 value was observed at higher concentration of ethanol (40%). Therefore, the partitioning of L-dopa from the donor solution to the skin is not greatly improved with the solution containing 40% ethanol and 2% L-menthol. Based on these results, we prepared L-dopa in a hydrogel form which contained 40% ethanol and 2% L-menthol. When this L-dopa-hydrogel was attached on the rat skin in vivo, L-dopa was detected in the plasma to the same extent as in the in vitro study: cutaneous permeation rate was 1375 ng / cm 2 / min in vitro and 1236 ng / cm 2 / min in vivo. Our in vivo study with the intravenous administration of L-dopa revealed that dopamine and norepinephrine appeared promptly at 5-min following the injection. This prompt appearance of dopamine and norepinephrine indicated that the injected L-dopa had been converted to dopamine by L-amino acid decarboxylase, and further, dopamine to norepinephrine by dopamine b-hydroxylase in the plasma rather than in the organs (Kato et al., 1978; Rahman et al., 1981). On the other hand, our in vivo study with the transdermal administration of the L-dopa-hydrogel showed that dopamine had reached a plateau level from 30- to 180-min, while norepinephrine level had risen gradually until 180min. Aromatic L-amino acid decarboxylase and dopamine b-hydroxylase were found to distribute in some organs (Kato et al., 1978; Sperk et al., 1980; Rahman et al., 1981). Therefore, we considered that the enzymes in the organs as well as in the plasma had been involved with the decarboxylation of L-dopa and b-hydroxylation of dopamine, since L-dopa and dopamine resided in the body long enough for the enzymatic reactions in the organs. Elevated levels of dopamine and norepinephrine in plasma exert digestive, circulatory, psychiatric, and other effects (Quinn, 1984; Ellenhorn and Barceloux, 1988). Thus, the continuous elevation in the plasma levels of dopamine and norepinephrine in the transdermal administration of the L-dopa-hydrogel, as well, might manifest the above adverse effects on the body. In fact, in the in vivo study with the transdermal administration of the L-dopahydrogel, all rats were alive during 0- to 180-min, whereas some rats died later than 240-min. We considered that the in vivo data were valid during 0- to 180-min, and this was the reason why the experimental period was restricted to 180 min in the in vivo and in vitro studies. In addition,
71
408C was employed as the incubation temperature of the diffusion cells in the in vitro cutaneous permeation study. This temperature was higher than the average temperature (328C) in human skin. Also, 40% was employed as the concentration of ethanol in the in vitro and the in vivo studies. This percentage of ethanol was considered it to be possible to give any adverse effects to the rat skin (Ohara et al., 1994). Therefore, further improvement will be required not only to minimize these adverse effects on the body (Reimherr et al., 1980) but also to clinically apply the transdermal delivery system of L-dopa to humans. Nevertheless, this transdermal delivery system using the hydrogel formulation of L-dopa provides new direction in treating Parkinson’s disease.
References Ellenhorn, M.J., Barceloux, D.G., 1988. Medical Toxicology: Diagnosis and Treatment of Human Poisoning, Elsevier, New York, pp. 16. Eriksson, B.-M., Persson, B.-A., 1982. Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detection. J. Chromatogr. 228, 143–154. Kato, T., Wakui, Y., Nagatsu, T., Ohnishi, T., 1978. An improved dual-wavelength spectrophotometric assay for dopamine-b-hydroxylase. Biochem. Pharmacol. 27, 829–831. Levison, K.K., Takayama, K., Isowa, K., Okabe, K., Nagai, T., 1994. Formulation optimization of indomethacin gels containing a combination of three kinds of cyclic monoterpenes as percutaneous penetration enhancers. J. Pharm. Sci. 83, 1367–1372. Ohara, N., Takayama, K., Machida, Y., Nagai, T., 1994. Combined effect of D-limonene and temperature on the skin permeation of ketoprofen. Int. J. Pharm. 105, 31–38. Okamoto, H., Hashida, M., Sezaki, H., 1988. Structure-activity relationship of 1-alkyl- or 1-alkenylazacycloalkanone derivatives as percutaneous penetration enhancers. J. Pharm. Sci. 774, 418–424. Quinn, N.P., 1984. Anti-parkinsonian drugs today. Drugs 28, 236–262. Rahman, M.K., Nagatsu, T., Kato, T., 1981. Aromatic L-amino acid decarboxylase activity in central and peripheral tissues and serum of rats with L-dopa and L-5-hydroxytryptophan as substrates. Biochem. Pharmacol. 30, 645–649. Reimherr, F.W., Wood, D.R., Wender, P.H., 1980. An open clinical trial of L-dopa and calbidopa in adults with minimal brain dysfunction. Am. J. Psychiatry 137, 73–75. Shoulson, I., Glaubiger, G.A., Chase, T.N., 1975. On-off response. Clinical and biochemical correlations during oral and intravenous levodopa administration in parkinsonian patients. Neurology 25, 1144–1148. ¨ E., Hortnagl, ¨ Sperk, G., Galhaup, I., Schlogl, H., Hornykiewicz, O., 1980. A sensitive and reliable assay for dopamine b-hydroxylase in tissue. J. Neurochem. 35, 972–976. Sudo, J., Iwase, H., Terui, J., Hayashi, T., Soyama, M., 1995. Higher dopamine level in lymph from the cervical lymph trunk than in plasma following intravenous bolus injection of L-dopa in rats. Biol. Pharm. Bull. 18, 610–614. Terui, J., Tamoto, K., Sudo, J., 1994. Proteinuric potentials of angiotensin II, and [des-Asp 1 ]- angiotensin II, and [des-Asp 1 , des-Arg 2 ]-angiotensin II in rats. Biol. Pharm. Bull. 17, 1516–1518. Yamaoka, K., Tanigawara, Y., Nakagawa, T., Uno, T., 1981. A pharmacokinetic analysis program (MULTI) for micro-computer. J. Pharmacobio. Dyn. 4J. Pharmacobio. Dyn. 4, 879–885.
Transdermal absorption of L-dopa from hydrogel in rats a, b a a a Jun-ichi Sudo *, Hiroaki Iwase , Jun Terui , Katsuhiko Kakuno , Momoko Soyama , Kozo Takayama b , Tsuneji Nagai b a
Department of Clinical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, IshikariTobetsu, Hokkaido 061 -02, Japan b Department of Pharmaceutics, Hoshi University, Ebara 2 -4 -41, Shinagawa-ku, Tokyo 142, Japan Received 6 May 1997; accepted 9 January 1998
Abstract To improve compliance in administration of L-dopa, transdermal absorption of the agent was investigated in rats in vitro employing two-chamber diffusion cells in which the excised rat abdominal skin was mounted, and in vivo using an alcoholic hydrogel containing L-menthol. The in vitro study revealed that in presence of L-menthol (2%, W/ W), ethanol (20 and 40%, V/ V) accelerated transdermal penetration of L-dopa with an increase of its percentages. The in vivo study showed that when the L-dopa-hydrogel containing 2% L-menthol and 40% ethanol was attached on the skin, plasma levels of L-dopa and norepinephrine increased with the time elapsed; the level of dopamine increased and reached a plateau thereafter; and the level of epinephrine was unchanged. These in vitro and in vivo findings indicated that the hydrogel formulation of L-dopa provides new direction in treating Parkinson’s disease. 1998 Elsevier Science B.V. All rights reserved. Keywords: L-Dopa; Hydrogel; Ethanol; L-Menthol; Transdermal absorption; Rat
1. Introduction
2. Experimental procedures
L-Dopa is a therapeutic agent used for Parkinson’s disease, and the agent is generally administered orally or intravenously (Shoulson et al., 1975; Quinn, 1984). The patients of Parkinson’s disease are occasionally geriatric and tend to have dementia and dysphagia. These types of patients cannot be expected to comply with oral administration; thus, intravenous administration is employed. However, patients with dementia sometimes pull out the needle for the injection. Therefore, we considered a transdermal delivery system would be a better route for the administration. So, we attempted to make a hydrogel formulation of L-dopa, using ethanol and menthol as representative cutaneous absorption enhancers (Levison et al., 1994). We then examined the possible transdermal absorption of L-dopa from the hydrogel in vitro and in vivo in rats.
2.1. Animals and chemicals
*Corresponding author: Tel.: 181 1332 22921; fax: 181 1332 21820. 0928-0987 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 98 )00007-4
Male Wistar strain rats (Hokudo Co., Abuta, Hokkaido, Japan) weighing 200610 g were housed in ordinary cages and allowed free access to water and a standard pellet diet (CE-2; Clea Japan Co., Tokyo, Japan) prior to the study. L-Dopa ( L-3-4-dihydroxyphenylalanine) was purchased from Sigma Chemicals Co. (St Louis, MO, USA), and other chemicals used were of the highest grade available.
2.2. Procedures 2.2.1. In vitro cutaneous permeation study Cutaneous permeation of L-dopa was investigated in vitro in the same way as previously reported (Ohara et al., 1994). Full-thickness abdominal skin was excised from rats, whose hair had been removed beforehand by an electric clipper. The skin excised was used as a permeation membrane. Two-chamber diffusion cells (available diffusion area, 0.785 cm 2 ; volume of each half-cell, 3.0 ml) with a water jacket were employed. L-Dopa was suspended
68
J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
with excess amounts in the following solutions: a water; a 20% (V/ V) ethanol and 2% (W/ V) L-menthol solution; and a 40% ethanol and 2% L-menthol solution. L-Dopa suspended in each of the three solutions was applied to the donor cell, and the receiver cell was filled with phosphatebuffered saline (pH 7.4). Both cells were warmed at 408C and stirred by magnetic stirrers. At 0- (before), 30-, 60-, 120-, and 180-min after the L-dopa suspension was applied to the donor cell, 0.5 ml aliquots were drawn from the receiver cell. Thereafter, an equivalent volume of the phosphate-buffered saline was supplied to the receiver cell.
2.2.2. In vivo intravenous administration study Rats were anesthetized with ether, and the left jugular vein was catheterized with a polyethylene tube (PE-50). Through this route, saline was continuously infused at 0.1 ml / kg body weight / min, and urethane (500 mg / kg body weight) and a-chloralose (70 mg / kg body weight) were given for deeper anesthesia and immobilization (Terui et al., 1994). When necessary, L-dopa dissolved in saline at a concentration of 2.5 mg / ml was injected at a dose of 2.5 mg / kg body weight through this route. Then, the animal was intubated for free respiration. Thirty min before collection of blood, the left femoral artery was catheterized with a polyethylene tube (PE-50) that had been filled with 0.2 M EGTA dissolved in saline (Sudo et al., 1995). Blood was collected at 0- (before), 5-, 15-, 25-, 37.5-, and 52.5-min after the intravenous bolus injection of L-dopa and then put into chilled tubes containing 40 ml of a solution containing 0.2 M EGTA and 0.2 M reduced glutathione (Eriksson and Persson, 1982). The blood volume collected was restricted to within 2 ml to avoid possible physiological alterations in concentrations of the amines induced by the loss of blood; blood was sampled once per rat. 2.2.3. In vivo cutaneous absorption study A hydrogel containing L-dopa was prepared, composition of which was as follows (100 g, in total): 5 g of L-dopa, 10 g of propylene glycol, 2 g of L-menthol, 2 g of diisopropyl adipate, 1 g of diisopropanolamine, 1 g of carboxyvinyl polymer, 40 ml of ethanol, and water. The hydrogel was saturated with L-dopa in this formula. Rats were anesthetized, infused, and intubated in the same manner as described in the above intravenous administration study. Then, the abdominal hairs were gently removed by an electric clipper. A glass cell (inner diameter, 1.13 cm; area adhered by the hydrogel, 1 cm 2 ; height, 1 cm) was attached on the shaved abdominal skin by an adhesive (cyanoacrylate type; Alon-Alpha A, Sankyo Co., Tokyo, Japan). Then, the glass tube was filled with either saline (control) or the hydrogel, and covered with a parafilm. Before (0-), and 30-, 60-, and 180-min after the initiation of filling the hydrogel into the glass tube, blood was collected as described in the above intravenous administration study. Blood was sampled once per rat.
2.3. Determination of L-dopa and amines Blood samples in the in vivo studies were centrifuged (1 7003g, 10 min, 48C) to obtain plasma. Aliquots from the in vitro study as well as the plasma samples from the in vivo studies were pretreated by the method of Eriksson and Persson (1982). L-Dopa, dopamine, norepinephrine, and epinephrine in the samples were determined electrochemically by the high-performance liquid chromatographic method of Sudo et al. (1995).
2.4. Pharmacokinetic analysis and statistics In the intravenous administration study, 1 group constituted 5 rats for 5 time-points including 5-, 15-, 25-, 37.5-, and 52.5-min after the intravenous bolus injection of Ldopa (one time-point per one rat). In the cutaneous absorption study in vivo, 4 rats for 4 time-points, including 0-, 30-, 60-, and 180-min after the cutaneous attachment of the L-dopa-hydrogel (one time-point per one rat), constituted 1 group. Both studies were carried out in 5 groups of rats. In the intravenous administration study in vivo, plasma concentration at 0 time (C0 ), elimination rate constant (k e ), half-life time (T 1 / 2 ), distribution volume (Vd ), total body clearance (CL ), and area under plasma concentration-time curve (AUC) were estimated by the one-compartment open model with the least-squares method (Yamaoka et al., 1981). Then, in the cutaneous absorption study in vivo, apparent cutaneous penetration rate (R p ) of L-dopa was estimated by the above parameters of Vd and k e and by the below formula based on the simple model on the assumption of a constant permeation through the skin (Ohara et al., 1994). R p (1 2 e k e ?t ) ]]]] Ct 5 k e ?Vd In the formula, Ct was the plasma concentration of L-dopa at time t, and t was time point during drug input. We applied the in vitro cutaneous permeation data to the Fickian diffusion equation, and explored its promoting mechanism by estimation of parameters in diffusion (D9) and partition (K9). The permeation profiles of L-dopa were analyzed by the method described by Okamoto et al. (1988), based on the following diffusion model.
F
1 2 Q t 5 AK9C0 D9t 2 ] 2 ]2 6 p
(21) O ]] exp(2D9n p t) G n `
n
2
2
2
1
D9 5 D/L 2 K9 5 KL D is diffusion constant; L, effective diffusion length of membrane; K, partition coefficient of the penetrant between the membrane and the donor phase; Q t , cumulative
J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
69
Fig. 1. Cumulative amount of L-dopa through rat abdominal skin in in vitro study employing two-chamber diffusion cells. Points and bars: means 6SEM in five experiments.
Fig. 2. Plasma levels of L-dopa after intravenous bolus administration of L-dopa in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the values at 0-min.
amount of penetrant in the receiver fluid at time t; A, area of application; C0 , solubility of L-dopa in the donor phase. In the steady-state, the penetration rate (J) of L-dopa was estimated as follows.
observed in 40% ethanol and 2% L-menthol solution. The K9 value was somewhat enhanced in 20% ethanol and 2% L-menthol solution, compared with water, while no further enhancement of the K9 value was observed at higher concentration of ethanol (40%). The penetration rate (J) of 2 L-dopa (ng / cm / min) was 146 in water, 436 in 20% ethanol and 2% L-menthol solution, and 1375 in 40% ethanol and 2% L-menthol solution.
J 5 AC0 KD/L 5 AC0 K9D9 The data were expressed as means 6SEM and were statistically analyzed using Student’s unpaired t-test; P values of less than 0.05 were considered significant.
3.2. In vivo intravenous administration study 3. Results
3.1. In vitro cutaneous permeation study In the in vitro study employing two-chamber diffusion cells in which the rat abdominal skin was mounted, cumulative amounts of L-dopa that permeated through the skin into the receiver cell were determined (Fig. 1). LDopa suspension in water revealed that L-dopa had cutaneously penetrated through the rat skin into the receiver cell with the time elapsed. L-Dopa suspension in 20% ethanol and 2% L-menthol solution indicated that the cutaneous permeation of L-dopa had been accelerated, and furthermore with increase of percentages of ethanol to 40. To find out how ethanol and L-menthol combination promoted permeation, the profiles of L-dopa permeation through the skin, were analyzed (Fig. 1 and Table 1). An impressive elevation in the D9 value of L-dopa was
L-Dopa and the amines in the plasma were determined after the intravenous injection of L-dopa (2.5 mg / kg body weight) (Figs. 2 and 3). L-Dopa, dopamine, and norepinephrine reached the highest concentrations (ng / ml, n55) at 5-min after the L-dopa injection: 664.0659.7 in L-dopa, 21.363.5 in dopamine, and 2.060.1 in norepinephrine. Thereafter, they fell toward the control levels with elapsing time. Epinephrine showed no significant tendency throughout the experimental period.
Table 1 Effect of donor solutions on diffusion parameter (D9 ) and partition parameter (K9 ) of L-dopa Donor solution
D9 (h 21 )
K9 (cm)
Water 20% Ethanol12% L-menthol 40% Ethanol12% L-menthol
0.01160.006 0.03360.017 0.14460.054
0.08760.029 0.12360.095 0.14960.034
Values: means 6SEM in five experiments.
Fig. 3. Plasma levels of dopamine, norepinephrine, and epinephrine after intravenous bolus administration of L-dopa in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the values at 0-min.
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Fig. 4. Plasma level of L-dopa during cutaneous attachment of L-dopahydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (a), P,0.05; (b), P,0.01, compared with the control values at each time period.
Fig. 6. Plasma level of norepinephrine during cutaneous attachment of L-dopa-hydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (b), P,0.01, compared with the control values at each time period.
3.3. In vivo cutaneous absorption study L-Dopa and the amines in the plasma were determined after the L-dopa-hydrogel was attached on the skin. L-Dopa levels rose until 180-min Fig. 4). Dopamine reached a plateau level at 30-min, and this level was maintained from 30- to 180-min (Fig. 5). Norepinephrine levels also rose until 180-min (Fig. 6. Epinephrine did not show any marked changes (Fig. 7).
3.4. Pharmacokinetic analysis Table 2 shows the kinetic parameters concerning the data in the intravenous administration of L-dopa in vivo. When apparent cutaneous penetration rate (R p ) of L-dopa in the cutaneous absorption study in vivo was estimated on the basis of the parameters of Vd and k e , it was 1236662 ng / cm 2 / min in five experiments. Furthermore, when the amount of L-dopa that had penetrated through the skin was estimated by the R p values, it was 1.11260.056 mg / kg body weight for 180-min.
Fig. 7. Plasma level of epinephrine during cutaneous attachment of L-dopa-hydrogel in vivo. Points and bars: means 6SEM in five rats. There were no significant differences between the control and the L-dopahydrogel-administered groups at each time period.
4. Discussion First, we employed two-chamber diffusion cells using the excised rat abdominal skin as a permeation membrane, and investigated effectiveness of L-menthol and ethanol as absorption enhancers concerned with a possible transdermal absorption of L-dopa. This in vitro study proved that Table 2 Kinetic parameters of plasma concentration of L-dopa and time curves based on one-compartment open model, in intravenous bolus administration of L-dopa in vivo C0 (ng / ml) k e (min 21 ) T 1 / 2 (min) Vd (ml / kg body weight) CL (ml / min / kg body weight) AUC (ng?min / ml)
Fig. 5. Plasma level of dopamine during cutaneous attachment of L-dopahydrogel in vivo. Points and bars: means 6SEM in five rats. Statistics: (a), P,0.05, compared with the control values at each time period.
559634 0.039260.0023 18.061.1 909651 35.362.2 144446896
C0 , plasma concentration at 0 time; k e , elimination rate constant; T 1 / 2 , half-life time; Vd , distribution volume; CL , total body clearance; AUC, area under plasma concentration-time curve. Values: means6SEM in five experiments.
J. Sudo et al. / European Journal of Pharmaceutical Sciences 7 (1998) 67 – 71
ethanol and L-menthol had possessed a very strong activity on the transdermal absorption of the agent. Pronounced increase of the D9 value in 40% ethanol and 2% L-menthol (Table 1) suggested that the combination of 40% ethanol and 2% L-menthol had effectively changed the dense barrier structure of the stratum corneum (Ohara et al., 1994), and that, as its result, the diffusivity of L-dopa through the skin had been increased. Although a little enhancement of the K9 value was seen in 20% ethanol and 2% L-menthol, no further enhancement of the K9 value was observed at higher concentration of ethanol (40%). Therefore, the partitioning of L-dopa from the donor solution to the skin is not greatly improved with the solution containing 40% ethanol and 2% L-menthol. Based on these results, we prepared L-dopa in a hydrogel form which contained 40% ethanol and 2% L-menthol. When this L-dopa-hydrogel was attached on the rat skin in vivo, L-dopa was detected in the plasma to the same extent as in the in vitro study: cutaneous permeation rate was 1375 ng / cm 2 / min in vitro and 1236 ng / cm 2 / min in vivo. Our in vivo study with the intravenous administration of L-dopa revealed that dopamine and norepinephrine appeared promptly at 5-min following the injection. This prompt appearance of dopamine and norepinephrine indicated that the injected L-dopa had been converted to dopamine by L-amino acid decarboxylase, and further, dopamine to norepinephrine by dopamine b-hydroxylase in the plasma rather than in the organs (Kato et al., 1978; Rahman et al., 1981). On the other hand, our in vivo study with the transdermal administration of the L-dopa-hydrogel showed that dopamine had reached a plateau level from 30- to 180-min, while norepinephrine level had risen gradually until 180min. Aromatic L-amino acid decarboxylase and dopamine b-hydroxylase were found to distribute in some organs (Kato et al., 1978; Sperk et al., 1980; Rahman et al., 1981). Therefore, we considered that the enzymes in the organs as well as in the plasma had been involved with the decarboxylation of L-dopa and b-hydroxylation of dopamine, since L-dopa and dopamine resided in the body long enough for the enzymatic reactions in the organs. Elevated levels of dopamine and norepinephrine in plasma exert digestive, circulatory, psychiatric, and other effects (Quinn, 1984; Ellenhorn and Barceloux, 1988). Thus, the continuous elevation in the plasma levels of dopamine and norepinephrine in the transdermal administration of the L-dopa-hydrogel, as well, might manifest the above adverse effects on the body. In fact, in the in vivo study with the transdermal administration of the L-dopahydrogel, all rats were alive during 0- to 180-min, whereas some rats died later than 240-min. We considered that the in vivo data were valid during 0- to 180-min, and this was the reason why the experimental period was restricted to 180 min in the in vivo and in vitro studies. In addition,
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408C was employed as the incubation temperature of the diffusion cells in the in vitro cutaneous permeation study. This temperature was higher than the average temperature (328C) in human skin. Also, 40% was employed as the concentration of ethanol in the in vitro and the in vivo studies. This percentage of ethanol was considered it to be possible to give any adverse effects to the rat skin (Ohara et al., 1994). Therefore, further improvement will be required not only to minimize these adverse effects on the body (Reimherr et al., 1980) but also to clinically apply the transdermal delivery system of L-dopa to humans. Nevertheless, this transdermal delivery system using the hydrogel formulation of L-dopa provides new direction in treating Parkinson’s disease.
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