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Pharmacokinetics of Amitriptyline and its Demethylated Metabolite in Serum and Specific Brain Regions of Rats After Acute and Chronic Administration of Amitriptyline
Pharmacokinetics of Amitriptyline and Its Demethylated Metabolite in Serum and Specific Brain Regions of Rats after Acute and Chronic Administration of Amitriptyline KATSUSHI MIYAKE'", HIROSHIFUKUCHI', TERUAKI KITAURA', MASAHIKOKIMURA', KEISUKE SARAIS, AND TOSHIONAKAHARA* Received April 7,1989, from the 'Department of Pharmacsutical Sen&%, Himhima University Hosjntal, and the *hjoartment of Neumloy and Accepted or publicahon July 2 , 1989. Psy&iatry, Hiroshima Univemty School of Medicine, 1-2-3,Kasumi, Mnami-ku, Hiroshima 734, Japan. Abstract 0 The concentrations of amitriptyline (AMT) and its demethylated metabolite nortriptyline (NRT) in the serum and in specific brain regions were determined periodically after acute or chronic administration of 20 rng/kg of AMT in rats. Both AMT and NRT declined from the serum in a biexponential manner and were eliminated monoexponentially from the brain regions, with no significant difference in elimination among the eight brain regions examined. In the brain, both AMT and NRT were unevenly distributed after chronic administration, whereas an even distribution was observed after acute administration. The AUCb,B,,:AUC~,U,ratio of AMT was higher than that of NRT, indicating greater transport of AMT into the brain regions. The AUC,,, value in the serum increased 1.6 times after chronic administration, whereas no significant changes were observed in the brain regions. The AUCNRTvalues increased 9.0 times in the serum and 6.8 times in the brain, with the increase in the serum being greater. These results suggest inhibited distribution of the drugs into the tissues, including the brain regions, and enhanced metabolism of AMT.
Amitriptyline (AMT), a tricyclic antidepressant, is a dibenmycloheputazine derivative whose clinical effect is to inhibit the neuronal u p t a k e of 5-hydroxytryptamine. Its demethylated metabolite, nortriptyline (NRT), also has a clinical efficacy of inhibiting the uptake of noradrenaline. Various efforts h a v e been m a d e to ascertain the relationship between the plasma concentrations of tricyclic antidepressants a n d their therapeutic effects. In spite of the extensive use of AMT, this relationship has not y e t been fully elucidated.14 Furthermore, efforts have been limited to the detection of the d r u g i n the blood, with little effort being made in the brain, the site of action of the tricyclic antidepressant agents. Although a n u m b e r of pharmacokinetic studies h a v e been m a d e with imipramine ( I M P P and chlorimipramine (CIM)loJ1 in rats, little has been reported for AMT.12 The present study was carried out to ascertain the pharmacokinetic relationship between serum and regional b r a i n levels of AMT and its metabolite NRT after acute a n d chronic intraperitoneal administration of AMT.
Experimental Section MaterialsAmitriptyline hydrochloride (lot B63901), nortriptyline hydrochloride (lot 58057), and trimipramine maleate (lot CA8433100) were obtained from Banyu Pharmaceutical Company Ltd. (Tokyo, Japan), Dainippon Pharmaceutical Company, Ltd. (Osaka, Japan), and Shionogi Pharmaceutical Company, Ltd. (Osaka, Japan), respectively. Acetonitrile was of HPLC! grade. All the other reagents were of analytical grade and were used without further purification. Animal Study-Male Sprague-Dawley rats weighing between 230 and 300 g were housed four or five per cage with free access to food and water under a 12-h lightJl2-h dark cycle. Animals were fasted for -20 h before administration of the drug. In the acute experiments, rats received a single intraperitoneal injection of 20 mgkg of AMT. 288 I Journal of Pharmaceutical Sciences Vol. 79,No. 4, April 1990
In the chronic experiments, rats received daily intraperitoneal injections of 20 m g k g of AMT at the same time of the day for 10 days. Blood samples were collected once from the heart a t various times, and then rats were sacrificed by decapitation to remove the brain. The brain was dissected into eight regions according to the technique of Glowinski and Iversen.13 These regions were the frontal cortex (FC), striatum (ST),cerebellum (CE), midbrain (MB), hippocampus (HI), pons and medulla (MO), thalamus (TH), and hypothalamus (HY). Each brain region was weighed and homogenated for 15 s (Polytron, KINEMATICA GmbH, Switzerland) after adding 15-50 volumes of distilled water. Serum and homogenate samples were stored at -20 "C until analysis. Analytical Method for Amitriptyline and Nortriptyline in Serum and B r a i n S e r u m (1.0 mL) or brain homogenate (1.0 mL) samples were mixed with internal standard solution (20 p L of 10 pg/mL trimipramine maleate). f i r adding 0.5 mL of 2 M NaOH and 5 mL of a n-hexane:isoamyl alcohol mixture (99:l) to each sample containing internal standard, the resulting mixture was shaken for 15 min and centrifuged for 15 min at 3000 rpm. The organic phase of the supernatant (4 mL) was transferred to another tube containing 0.2 mL of 0.2 M phosphate buffer (pH 2.0). The mixture was vortexed for 1.5 min and then centrifuged for 15 min at 3000 rpm. The organic layer was discarded and a 100-pL aqueous layer of this sample was injected into the HPLC system. The HPLC system consisted of a solvent delivery pump (Shimadzu LC-3A, Kyoto, Japan), a variable wavelength UV detector (Shimadzu spectrophotometer SPD-6A, 250 nm), an integrator (Shimadzu chromatopac C-MA), and a reversedphase column (Shim-pack CLC-ODs, 6 x 150 mm). The mobile phase consisted of a 40:60 mixture of acetonitrile and 2% sodium perchlorate in 0.1 M phosphate buffer (pH 3.0) and was pumped at a rate of 1.2 mUrnin. The column was kept at 40 "C. The minimum detectable concentration of AMT and NRT was 3 nglmL. Determination of Partition Coeflicient-The partition coefficients of AMT and NRT were determined in a n octano1:water system at room temperature.14 The AMT or NRT was dissolved in 0.1 M phosphate buffer (pH 7.4) a t a concentration of 20 &mL. The volume ratio of octanol to water used was 1:50, since the partition coefficients of drugs were very high. The concentration of drugs in the aqueous layer was determined by a spectrophotometer (Shimadzu UV-240) at a wavelength of 240 nm. Data Analysis-Pharmacokinetic parameters were computed by use of nonlinear least squares regression program (MULTU.15 The area under the concentration curve (AUC) in the serum and brain was calculated by the trapezoidal rule from time 0 to 8 h, and the extrapolated residual area from the final concentration point to infinity. Statistical Analysis-The statistical significance of the difference was determined by the t test or paired t test. A p value of c0.05was considered to be statistically significant.
Results Pharmacokinetics of A m i t r i p t y l i n e (AMT) and Nortriptyline (NRT) after Acute Administration-The time courses of AMT a n d NRT i n the serum a n d three brain regions (FC, CE, and HY)after intraperitoneal administration of AMT at a dose of 20 mg/kg are shown in Figure 1. The pharmacokiOOZZ-3~9/90/04OO-02BB$o 1.00/0 0 1990, American Pharmaceutical Association
1
0
NRT
A A 0
0
1
4
2
8
0
1
2
Time ( hr )
Frontal cortex Hippocampus Hypothalamus Serum
8
4
Time ( hr )
Flgure 1-Time course of amitriptylineand nortriptyline concentrations in serum and brain regions after acute administration of amitriptyline (20mg/kg, ip). Each point represents the mean 2 SEM of four or five rats. netic parameters are summarized in Table I. The time required to achieve the maximum concentration Urn-) in the serum was 5 min for both AMT and its metabolite. Large differences in the t,,, values were not observed among various brain regions; t,, was 15 and 30 min for AMT and NRT, respectively. The maximum concentrations (C,,,J of AMT and NRT in various brain regions were much higher than those in the serum (Table I). Both drugs declined from the serum in a biexponential manner and were eliminated from the brain regions monoexponentially. No difference in the rate of elimination was observed among the eight examined brain regions, and the rate was almost of the same extent
as that of the serum. The C, value of FC appeared to be higher than that of other regions, although no significant difference was observed. The AUC,,,,,:AUC,,,, ratio of AMT was higher than that of NRT (Table 11).The AUCNRT:AUCAMTratio in the serum was higher than that in the brain regions (Table 111). Pharmacokinetics of Amitriptyline (AMT) and Nortriptyline (NRT) after Chronic Administration-Figure 2 shows the time profiles of AMT and NRT in the serum and various brain regions (FC, CE, and HY)after chronic administration of AMT. The parameters are listed in Table I. The t,, values of AMT were delayed in brain regions and those of NRT were
Table CPharmacokinetic Parameters of Amitrlptyline and Nortriptyline In Serum and Braln after Acute and Chronic Admlnlstratlon of Amitriptyline'
C'
Amitriptyline Serum FCd STe CE' MBQ HI" MO Ti+ HYk
A
C
A
C
A
C
A
C
597.1 (202.2) 8870.8 (1836.7) 8613.3 (2099.8) 6351.O (1 262.7) 8070.0(1 204.6) 7483.8(1565.5) 6956.8(832.9) 8014.5(1441.4) 6649.0(1415.3)
882.4(86.5) 12 194.0(1445.9) 12 343.0(2671.2) 9454.3(1242.2) 1 1 729.3(1161.2) 10 715.5(1259.4) 9822.8(829.3) 1 1 387.8(1024.2) 9795.5(915.2)
0.08 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.08 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.249 0.288 0.282 0.284 0.291 0.254 0.247 0.266 0.263
0.343 0.419 0.406 0.401 0.413 0.365 0.367 0.407 0.384
2.78 2.41 2.46 2.44 2.38 2.73 2.81 2.61 2.63
2.02 1.65 1.71 1.73 1.68 1.90 1 .89 1.70 1.80
977.0 33 052.5 31 649.5 23 889.8 28 773.4 33 092.0 31 721.8 34 450.8 27 646.3
1593.8 33 467.5 32 956.2 26 1 1 1.6 30 757.3 35 337.1 30 624.0 34 175.7 27 344.1
165.9(38.2) 950.3(131.5) 856.7(116.9) 779.7 (143.7) 864.3 (163.3) 812.3(134.9) 850.3(169.1) 915.3(171.1) 731.3(122.3)
640.5(93.6) 5247.5(277.1) 5163.5(839.8) 4936.5(599.7) 5429.3 (649.1) 4473.0(443.4) 4794.3(438.5) 5085.0(524.7) 4556.8(423.4)
0.08 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.25 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.309 0.368 0.359 0.430 0.396 0.307 0.331 0.342 0.358
0.303 0.383 0.378 0.413 0.389 0.314 0.349 0.384 0.376
2.24 1.88 1.93 1.61 1.75 2.26 2.09 2.03 1.94
2.29 1.81 1.83 1.68 1.78 2.21 1.99 1 .80 1.84
184.5 2729.7 2560.8 1974.3 2378.3 2708.5 2650.7 2744.6 21 00.0
1660.4 17 839.5 17251.7 14 048.2 16 710.3 19 120.3 16 944.1 17 999.4 14 906.2
Nortriptyline Serum
FC
ST CE MB HI MO
TH HY
* AMT was administered at a dose of 20 mg/kg intraperitoneally;each value representsthe mean (SD) of four or five rats. Acute. Chronic. Frontal cortex. Striatum. ' Cerebellum. g Midbrain. Hippocampus. ' Pons and medulla. Thalamus. Hypothalamus. Journal of Pharmaceutical Sciences / 289 Vol. 79,No. 4, April 7990
Table ICBra1n:Serum Ratlos of Amltriptyllne and Nortriptyllne after Acute and Chronic Admlnlstratlon of Amltrlptyllne' C m a , brem:Cserum
Brain Region
A
C
A
C
37.7 36.7 27.0 34.3 31.8 29.6 34.1 28.3
23.6 23.9 18.3 22.7 20.8 19.0 22.1 19.0
33.8 32.4 24.5 29.5 33.9 32.5 35.3 28.3
21 .o 20.7 16.4 19.3 22.2 19.2 21.4 17.2
19.6 17.7 16.1 17.9 16.8 17.6 18.9 15.1
9.7 9.5 9.1 10.0 8.2 8.8 9.4 8.4
14.8 13.9 10.7 12.9 14.7 14.4 14.9 11.4
10.7 10.4 8.5 10.1 11.5 10.2 10.8 9.0
Amitriptyline FC ST CE
MB HI
MO TH HY
Nortriptyline FC ST CE MB HI MO
TH HY
a See Table I for key to abbreviations;each value represents the mean of four or five rats.
Table IICNortr1ptyllne:AmltrlptyllneRatios in Serum and Brain Regions after Acute and Chronic Admlnlstratlon of AmRrlptylineL %ax,
Sample Serum FC ST CE
MB HI
MO TH HY a
NRT:Cmax. AMT
AUCNRT:AUCA,
A
C
A
C
0.278 0.107 0.099 0.123 0.107 0.109 0.122 0.114 0.110
0.726 0.430 0.418 0.522 0.463 0.417 0.488 0.447 0.465
0.189 0.083 0.081 0.083 0.083 0.082 0.084 0.086 0.076
1.042 0.533 0.523 0.538 0.543 0.541 0.553 0.527 0.545
See Table I for key to abbreviations; each value represents the mean
of four or five rats.
delayed in the serum compared with acute administration. The C,,, values of the two drugs in the brain regions were also higher than that in the serum (Table I). The AUCAXT value in the serum was increased 1.6 times by chronic administration, although no significant changes in brain regions could be observed. On the other hand, the AUCNRT values both in the brain regions and serum showed marked increases. The t,,, values of AMT in the serum and brain regions were lower than those obtained after acute administration, although no marked difference in the tl,, value of NRT was observed in the serum and brain regions. The rates of elimination among the eight examined brain regions showed no significant difference and were similar to that of the serum. The C,,, value of FC after chronic administration was significantly higher than that of CE, MO, and HY for AMT and that of HI and HY for NRT. The AUCb,,i,:AUC,,,, ratio after chronic administration decreased significantly for both drugs compared with acute administration (Table 11). The AUCN,:AUCAMT ratio increased 5.5-fold in the serum and 6.6-fold in the brain regions (Table 111).
Discussion The brain is the target organ of antidepressants such as AMT, so that its clinical efficacy cannot be attained without 290 I Journal of Pharmaceutical Sciences Vol. 79, No. 4, April 1990
penetration of the blood-brain barrier (BBB). Factors affecting the BBB penetration of drugs are lipid-water partition coefficient, pK,, and protein binding.lG18 In this study, rapid distribution into the brain regions after acute intraperitoneal administration of AMT was observed, and the C,, values of the drugs in the brain regions were much higher than that in the serum, although AMT and NRT with high PK, values (AMT, 9.4;NRT, 12.6) are mostly ionized in the serum and have high protein binding. The facts that the AUC,,,,,:AUC,,, ratio of AMT was 2.3-fold higher than that of NRT and that the AUCNH+AUCAMT ratio in the serum was 2.3-fold higher than that in the brain regions in acute administration suggest that AMT is superior to NRT for penetration into the brain regions. Therefore, the partition coefficient was investigated and both drugs showed a high lipophilicity (AMT, 1373; NRT, 56). The present study, in accordance with this view, showed that the more lipophilic AMT penetrates the BBB faster than the corresponding NRT. Our findings are in a good agreement with the results reported by JZrgensen e t a1.18 Because of the lipophilic nature of the BBB, it is considered that the lipophilicity of a compound plays a role in penetration into the brain in the case of high lipophilic compounds such as AMT and NRT. In the present study, a n even distribution pattern of AMT and NRT in the brain was observed following acute administration. On the other hand, an uneven distribution pattern of the drugs in the brain was observed after chronic administration. An uneven distribution pattern has also been reported for IMP19 and CIM.10 An uneven distribution is considered to be attributed to different rate of distribution into the brain and of elimination from the brain and the presence of specific binding sites of the drug. In fact, Sherman et al.19 have reported slow diffusion of IMP into HI from the central spinal fluid, and Friedman e t a1.10have reported delay of CIM in t1,2values of septum, ST, and HY. Furthermore, a number of reports have suggested the presence of specific high-affinity binding sites regarding IMP in the rat brain.6.7.20-22 In this study, however, neither t,,, nor k,, indicated a significant difference among the various brain regions. The binding of a tricyclic drug to its binding sites is considered to be a high affinity phenomenon and, therefore, the drug concentration accumulated under the present treatment schedule hardly seemed to indicate a specific binding phenomenon. Yufu12 has demonstrated a differential distribution of AMT in the brain regions after acute administration. In this study, we could not determine the causes for the different distribution pattern observed in brain regions. The discrepancies between our findings and the previous work by Yufu are considered to be inconclusive. The AUCAMT value in the serum increased 1.6 times after chronic administration, whereas the tIl2 value decreased slightly. Since AMT is a highly lipophilic compound, it would be accumulated in lipophilic tissue such as adipose. Our measurements of the drugs, however, are limited to the detection of both in the serum and the brain and we have no available data after the last sampling time of 8 h. Although we could not determine the causes of the enhanced AUCAMT value, i t might be attributed to the presence of the slower elimination phase andlor inhibited drug distribution into the tissues. The AUC, value in the serum also increased 9.0 times after chronic administration. In addition, the AUCN,:AUC,T ratio was increased markedly in the serum.The enhanced metabolism and the decreased tIl2 of IMP23 and CIMlO aRer chronic adrmnistration has been demonstrated. Furthermore, Yufu12 has also reported a similar result after chronic administration of AMT. These results suggest that the increased AUCN, value is not only due to the same mechanism as seen in AMT but also due to the enhanced metabolism of AMT and formation of NRT by liver
NRT
AMT
-
0 A
5000-
A 0
4 \ B
Frontal c o r t e x Hippocampus Hypothalamus Serum
01
e
:1000-, \
ol
\
C
A
c 0
4 CI
:cm:
100-
50V
20
-
I l l
0
I
1
4
2
Time
8 (
hr )
I 1 1
1
1
0
1
2
8
4
Time
(
hr )
Flour8 2-Time course of amitriplyline and nortriptylineconcentrations in serum and brain regions after chronic administration of amitriptyline mgkg, ip). Each point represents ihe mean t SEM of four or five rats.
microsomal enzymes. It seems, however, that further detailed study should be made to clarifythe enhanced metabolism of AMT and formation of NRT. The AUC,, values in various brain regions showed no significant change by chronic administration, suggesting that drug distributions into the brain might be inhibited. The concentrations obtained are several-fold higher than those of therapeutic concentrations. Under this condition, the binding sites in the brain might be occupied. Therefore, the observed inhibition of drug penetration into the brain might be due to the saturated binding sites of the drugs. By chronic administration, the AUC,, values in the brain increased 6.8-fold, showing less increase compared with that in the serum. These results also suggest the suppressed distribution of the drug into the brain as seen in AMT. In addition, the AUC,, values in the brain increased several times compared with acute administration, whereas those of AMT showed no changes, suggesting that the binding affinities andlor the extent of the accumulation of the drugs may differ. Corona et al.24 have suggested that changes in membrane permeability induced by chronic administration can determine the different distribution patterns of AMT and metabolite in the brain of rabbits. It seems, however, that further detailed study should be made to clarify these mechanisms. In conclusion, the present experiments systematically investigated the regional brain and serum pharmacokinetics of AMT and its demethylated metabolite NRT after acute and chronic drug administration. The results demonstrate the following: (I) superiority of AMT over NRT in penetration into the brain; (2)regional brain difference in the maximum concentration of AMT and NRT in chronic administration; (3) marked increases of demethylated metabolite in the serum and brain regions after chronic administration; and (4) inhibition of drugs in penetrating the brain regions after chronic administration.
References and Notes 1. Watanabe, S. Jap. J . Neuropsychophnrmacol. 1985,10,661-669.
2. Asano, Y.; Yamashita, I. Jap. J . Clin. Psychiatry 1979, 8, 787-797. 3. Barrows, G . D.; Davis, B.; Scoggins, B. A. Lancet 1972,2, 619623. 4. Jungkunz, G.; Kurs, H. J. Lancet 1978,2, 1263-1264. 5. Braithwait, R. A.;Goulding, R;Theano, G.;Bailey, J.;Coppen, A. Lancet 1972,1, 1297-1300. 6. Masada. M.: Suzuki. K.: Kikuta. S.: Yamashita. S.: Nakanishi. K.; Nadai, T:; Igarashi, Y . ;Noguchi, T . Chern. Phhrm. Bull. 1986; 34.927-930. 7. Maaada, M.; Suzuki, K.; Kikuta, S.; Yamashita, S.; Nakanishi, K.; Nadai, T.;Igarashi, Y.;Noguchi, T. Chern.Pharrn.Bull. 1986, 34, 2173-2177. 8. Daniel, W.; Adamus, A.; Melzacka, M.; Szymura, J.; Vetulani, J. NaunynSchrniederberg's Arch. Phnrmacol. 1981,317,209-213. 9. Daniel, W.; Adamus, A,; Melzacka, M.; Szymura, J. J . Phurrn. Phurmacol. 1982.34, 676-660. 10. Friedman, E.; Cooper, T. B. J . Phnrmacol. Exp. Ther. 1983,225, 387-390. 11. Nagy, A. J . Phnrm. Phnrmacol. 1977,29, 104-107. 12. Yuf'u, N. Jap. J . Neuropsychophnrmacol. 1987,89, 22-42. 13. Glowinsky, J.; Iversen, L. L. J . Neurochern. 1966,13,655-669. 14. Hogen, C. A. M.; Tocco, D. J.; Brodie, B. B.; Shanker, L. S. J. Phnrmacol. Exp. Ther. 1959,125,275-282. 15. Yamaoka, K.; Nakagawa, T. J . PhurmacobkDyn. 1983, 6, 595-606. 16. Brodie, B. B.; Kurtz, H.;Schanker, L. S. J . Phnrmacol. Exp. Ther. 1960,130,20-25. 17. Mayer, S.; Maickel, R. P.; Brodie, B. B. J . P h u r m o l . Exp. Ther. 1959,127,205-211. 18. Jflrgensen,A.; Hansen, V.; Over@,K. F. ActnPharmacol. Toxicol. 19'73.33.81-91. 19. Sherman, A. D.; Allers, G. L. Neurophnrmacology 1980, 19, 159-162. 20. Raiseman, R.; Briley, M. S.; Langer, S. Z. Eur. J . Phurmacol. 1980,61,373-380. 21. Palkovita, M.; Raiseman, R.; Briley, M.; Langer, S. 2. Brain Res. 1981,210,493-498. 22. Sette, M.; Raiseman, R.; Briley, M. S.; Langer, S. 2. J . Neurochern. 1981,37,4042. 23. Breyer, U. Naunyn-Schrnideberg's Arch. Pharmacol. 1972,272, 277-288. 24. Corona, G . L.; Facino, R. M.; Santagostino, G . Biochern. Phnrmacol. 1971,20, 2765-2771.
Journal of Pharmaceutical Sciences I 291 Vol. 79, No. 4, April 1990
Amitriptyline (AMT), a tricyclic antidepressant, is a dibenmycloheputazine derivative whose clinical effect is to inhibit the neuronal u p t a k e of 5-hydroxytryptamine. Its demethylated metabolite, nortriptyline (NRT), also has a clinical efficacy of inhibiting the uptake of noradrenaline. Various efforts h a v e been m a d e to ascertain the relationship between the plasma concentrations of tricyclic antidepressants a n d their therapeutic effects. In spite of the extensive use of AMT, this relationship has not y e t been fully elucidated.14 Furthermore, efforts have been limited to the detection of the d r u g i n the blood, with little effort being made in the brain, the site of action of the tricyclic antidepressant agents. Although a n u m b e r of pharmacokinetic studies h a v e been m a d e with imipramine ( I M P P and chlorimipramine (CIM)loJ1 in rats, little has been reported for AMT.12 The present study was carried out to ascertain the pharmacokinetic relationship between serum and regional b r a i n levels of AMT and its metabolite NRT after acute a n d chronic intraperitoneal administration of AMT.
Experimental Section MaterialsAmitriptyline hydrochloride (lot B63901), nortriptyline hydrochloride (lot 58057), and trimipramine maleate (lot CA8433100) were obtained from Banyu Pharmaceutical Company Ltd. (Tokyo, Japan), Dainippon Pharmaceutical Company, Ltd. (Osaka, Japan), and Shionogi Pharmaceutical Company, Ltd. (Osaka, Japan), respectively. Acetonitrile was of HPLC! grade. All the other reagents were of analytical grade and were used without further purification. Animal Study-Male Sprague-Dawley rats weighing between 230 and 300 g were housed four or five per cage with free access to food and water under a 12-h lightJl2-h dark cycle. Animals were fasted for -20 h before administration of the drug. In the acute experiments, rats received a single intraperitoneal injection of 20 mgkg of AMT. 288 I Journal of Pharmaceutical Sciences Vol. 79,No. 4, April 1990
In the chronic experiments, rats received daily intraperitoneal injections of 20 m g k g of AMT at the same time of the day for 10 days. Blood samples were collected once from the heart a t various times, and then rats were sacrificed by decapitation to remove the brain. The brain was dissected into eight regions according to the technique of Glowinski and Iversen.13 These regions were the frontal cortex (FC), striatum (ST),cerebellum (CE), midbrain (MB), hippocampus (HI), pons and medulla (MO), thalamus (TH), and hypothalamus (HY). Each brain region was weighed and homogenated for 15 s (Polytron, KINEMATICA GmbH, Switzerland) after adding 15-50 volumes of distilled water. Serum and homogenate samples were stored at -20 "C until analysis. Analytical Method for Amitriptyline and Nortriptyline in Serum and B r a i n S e r u m (1.0 mL) or brain homogenate (1.0 mL) samples were mixed with internal standard solution (20 p L of 10 pg/mL trimipramine maleate). f i r adding 0.5 mL of 2 M NaOH and 5 mL of a n-hexane:isoamyl alcohol mixture (99:l) to each sample containing internal standard, the resulting mixture was shaken for 15 min and centrifuged for 15 min at 3000 rpm. The organic phase of the supernatant (4 mL) was transferred to another tube containing 0.2 mL of 0.2 M phosphate buffer (pH 2.0). The mixture was vortexed for 1.5 min and then centrifuged for 15 min at 3000 rpm. The organic layer was discarded and a 100-pL aqueous layer of this sample was injected into the HPLC system. The HPLC system consisted of a solvent delivery pump (Shimadzu LC-3A, Kyoto, Japan), a variable wavelength UV detector (Shimadzu spectrophotometer SPD-6A, 250 nm), an integrator (Shimadzu chromatopac C-MA), and a reversedphase column (Shim-pack CLC-ODs, 6 x 150 mm). The mobile phase consisted of a 40:60 mixture of acetonitrile and 2% sodium perchlorate in 0.1 M phosphate buffer (pH 3.0) and was pumped at a rate of 1.2 mUrnin. The column was kept at 40 "C. The minimum detectable concentration of AMT and NRT was 3 nglmL. Determination of Partition Coeflicient-The partition coefficients of AMT and NRT were determined in a n octano1:water system at room temperature.14 The AMT or NRT was dissolved in 0.1 M phosphate buffer (pH 7.4) a t a concentration of 20 &mL. The volume ratio of octanol to water used was 1:50, since the partition coefficients of drugs were very high. The concentration of drugs in the aqueous layer was determined by a spectrophotometer (Shimadzu UV-240) at a wavelength of 240 nm. Data Analysis-Pharmacokinetic parameters were computed by use of nonlinear least squares regression program (MULTU.15 The area under the concentration curve (AUC) in the serum and brain was calculated by the trapezoidal rule from time 0 to 8 h, and the extrapolated residual area from the final concentration point to infinity. Statistical Analysis-The statistical significance of the difference was determined by the t test or paired t test. A p value of c0.05was considered to be statistically significant.
Results Pharmacokinetics of A m i t r i p t y l i n e (AMT) and Nortriptyline (NRT) after Acute Administration-The time courses of AMT a n d NRT i n the serum a n d three brain regions (FC, CE, and HY)after intraperitoneal administration of AMT at a dose of 20 mg/kg are shown in Figure 1. The pharmacokiOOZZ-3~9/90/04OO-02BB$o 1.00/0 0 1990, American Pharmaceutical Association
1
0
NRT
A A 0
0
1
4
2
8
0
1
2
Time ( hr )
Frontal cortex Hippocampus Hypothalamus Serum
8
4
Time ( hr )
Flgure 1-Time course of amitriptylineand nortriptyline concentrations in serum and brain regions after acute administration of amitriptyline (20mg/kg, ip). Each point represents the mean 2 SEM of four or five rats. netic parameters are summarized in Table I. The time required to achieve the maximum concentration Urn-) in the serum was 5 min for both AMT and its metabolite. Large differences in the t,,, values were not observed among various brain regions; t,, was 15 and 30 min for AMT and NRT, respectively. The maximum concentrations (C,,,J of AMT and NRT in various brain regions were much higher than those in the serum (Table I). Both drugs declined from the serum in a biexponential manner and were eliminated from the brain regions monoexponentially. No difference in the rate of elimination was observed among the eight examined brain regions, and the rate was almost of the same extent
as that of the serum. The C, value of FC appeared to be higher than that of other regions, although no significant difference was observed. The AUC,,,,,:AUC,,,, ratio of AMT was higher than that of NRT (Table 11).The AUCNRT:AUCAMTratio in the serum was higher than that in the brain regions (Table 111). Pharmacokinetics of Amitriptyline (AMT) and Nortriptyline (NRT) after Chronic Administration-Figure 2 shows the time profiles of AMT and NRT in the serum and various brain regions (FC, CE, and HY)after chronic administration of AMT. The parameters are listed in Table I. The t,, values of AMT were delayed in brain regions and those of NRT were
Table CPharmacokinetic Parameters of Amitrlptyline and Nortriptyline In Serum and Braln after Acute and Chronic Admlnlstratlon of Amitriptyline'
C'
Amitriptyline Serum FCd STe CE' MBQ HI" MO Ti+ HYk
A
C
A
C
A
C
A
C
597.1 (202.2) 8870.8 (1836.7) 8613.3 (2099.8) 6351.O (1 262.7) 8070.0(1 204.6) 7483.8(1565.5) 6956.8(832.9) 8014.5(1441.4) 6649.0(1415.3)
882.4(86.5) 12 194.0(1445.9) 12 343.0(2671.2) 9454.3(1242.2) 1 1 729.3(1161.2) 10 715.5(1259.4) 9822.8(829.3) 1 1 387.8(1024.2) 9795.5(915.2)
0.08 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
0.08 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.249 0.288 0.282 0.284 0.291 0.254 0.247 0.266 0.263
0.343 0.419 0.406 0.401 0.413 0.365 0.367 0.407 0.384
2.78 2.41 2.46 2.44 2.38 2.73 2.81 2.61 2.63
2.02 1.65 1.71 1.73 1.68 1.90 1 .89 1.70 1.80
977.0 33 052.5 31 649.5 23 889.8 28 773.4 33 092.0 31 721.8 34 450.8 27 646.3
1593.8 33 467.5 32 956.2 26 1 1 1.6 30 757.3 35 337.1 30 624.0 34 175.7 27 344.1
165.9(38.2) 950.3(131.5) 856.7(116.9) 779.7 (143.7) 864.3 (163.3) 812.3(134.9) 850.3(169.1) 915.3(171.1) 731.3(122.3)
640.5(93.6) 5247.5(277.1) 5163.5(839.8) 4936.5(599.7) 5429.3 (649.1) 4473.0(443.4) 4794.3(438.5) 5085.0(524.7) 4556.8(423.4)
0.08 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.25 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.309 0.368 0.359 0.430 0.396 0.307 0.331 0.342 0.358
0.303 0.383 0.378 0.413 0.389 0.314 0.349 0.384 0.376
2.24 1.88 1.93 1.61 1.75 2.26 2.09 2.03 1.94
2.29 1.81 1.83 1.68 1.78 2.21 1.99 1 .80 1.84
184.5 2729.7 2560.8 1974.3 2378.3 2708.5 2650.7 2744.6 21 00.0
1660.4 17 839.5 17251.7 14 048.2 16 710.3 19 120.3 16 944.1 17 999.4 14 906.2
Nortriptyline Serum
FC
ST CE MB HI MO
TH HY
* AMT was administered at a dose of 20 mg/kg intraperitoneally;each value representsthe mean (SD) of four or five rats. Acute. Chronic. Frontal cortex. Striatum. ' Cerebellum. g Midbrain. Hippocampus. ' Pons and medulla. Thalamus. Hypothalamus. Journal of Pharmaceutical Sciences / 289 Vol. 79,No. 4, April 7990
Table ICBra1n:Serum Ratlos of Amltriptyllne and Nortriptyllne after Acute and Chronic Admlnlstratlon of Amltrlptyllne' C m a , brem:Cserum
Brain Region
A
C
A
C
37.7 36.7 27.0 34.3 31.8 29.6 34.1 28.3
23.6 23.9 18.3 22.7 20.8 19.0 22.1 19.0
33.8 32.4 24.5 29.5 33.9 32.5 35.3 28.3
21 .o 20.7 16.4 19.3 22.2 19.2 21.4 17.2
19.6 17.7 16.1 17.9 16.8 17.6 18.9 15.1
9.7 9.5 9.1 10.0 8.2 8.8 9.4 8.4
14.8 13.9 10.7 12.9 14.7 14.4 14.9 11.4
10.7 10.4 8.5 10.1 11.5 10.2 10.8 9.0
Amitriptyline FC ST CE
MB HI
MO TH HY
Nortriptyline FC ST CE MB HI MO
TH HY
a See Table I for key to abbreviations;each value represents the mean of four or five rats.
Table IICNortr1ptyllne:AmltrlptyllneRatios in Serum and Brain Regions after Acute and Chronic Admlnlstratlon of AmRrlptylineL %ax,
Sample Serum FC ST CE
MB HI
MO TH HY a
NRT:Cmax. AMT
AUCNRT:AUCA,
A
C
A
C
0.278 0.107 0.099 0.123 0.107 0.109 0.122 0.114 0.110
0.726 0.430 0.418 0.522 0.463 0.417 0.488 0.447 0.465
0.189 0.083 0.081 0.083 0.083 0.082 0.084 0.086 0.076
1.042 0.533 0.523 0.538 0.543 0.541 0.553 0.527 0.545
See Table I for key to abbreviations; each value represents the mean
of four or five rats.
delayed in the serum compared with acute administration. The C,,, values of the two drugs in the brain regions were also higher than that in the serum (Table I). The AUCAXT value in the serum was increased 1.6 times by chronic administration, although no significant changes in brain regions could be observed. On the other hand, the AUCNRT values both in the brain regions and serum showed marked increases. The t,,, values of AMT in the serum and brain regions were lower than those obtained after acute administration, although no marked difference in the tl,, value of NRT was observed in the serum and brain regions. The rates of elimination among the eight examined brain regions showed no significant difference and were similar to that of the serum. The C,,, value of FC after chronic administration was significantly higher than that of CE, MO, and HY for AMT and that of HI and HY for NRT. The AUCb,,i,:AUC,,,, ratio after chronic administration decreased significantly for both drugs compared with acute administration (Table 11). The AUCN,:AUCAMT ratio increased 5.5-fold in the serum and 6.6-fold in the brain regions (Table 111).
Discussion The brain is the target organ of antidepressants such as AMT, so that its clinical efficacy cannot be attained without 290 I Journal of Pharmaceutical Sciences Vol. 79, No. 4, April 1990
penetration of the blood-brain barrier (BBB). Factors affecting the BBB penetration of drugs are lipid-water partition coefficient, pK,, and protein binding.lG18 In this study, rapid distribution into the brain regions after acute intraperitoneal administration of AMT was observed, and the C,, values of the drugs in the brain regions were much higher than that in the serum, although AMT and NRT with high PK, values (AMT, 9.4;NRT, 12.6) are mostly ionized in the serum and have high protein binding. The facts that the AUC,,,,,:AUC,,, ratio of AMT was 2.3-fold higher than that of NRT and that the AUCNH+AUCAMT ratio in the serum was 2.3-fold higher than that in the brain regions in acute administration suggest that AMT is superior to NRT for penetration into the brain regions. Therefore, the partition coefficient was investigated and both drugs showed a high lipophilicity (AMT, 1373; NRT, 56). The present study, in accordance with this view, showed that the more lipophilic AMT penetrates the BBB faster than the corresponding NRT. Our findings are in a good agreement with the results reported by JZrgensen e t a1.18 Because of the lipophilic nature of the BBB, it is considered that the lipophilicity of a compound plays a role in penetration into the brain in the case of high lipophilic compounds such as AMT and NRT. In the present study, a n even distribution pattern of AMT and NRT in the brain was observed following acute administration. On the other hand, an uneven distribution pattern of the drugs in the brain was observed after chronic administration. An uneven distribution pattern has also been reported for IMP19 and CIM.10 An uneven distribution is considered to be attributed to different rate of distribution into the brain and of elimination from the brain and the presence of specific binding sites of the drug. In fact, Sherman et al.19 have reported slow diffusion of IMP into HI from the central spinal fluid, and Friedman e t a1.10have reported delay of CIM in t1,2values of septum, ST, and HY. Furthermore, a number of reports have suggested the presence of specific high-affinity binding sites regarding IMP in the rat brain.6.7.20-22 In this study, however, neither t,,, nor k,, indicated a significant difference among the various brain regions. The binding of a tricyclic drug to its binding sites is considered to be a high affinity phenomenon and, therefore, the drug concentration accumulated under the present treatment schedule hardly seemed to indicate a specific binding phenomenon. Yufu12 has demonstrated a differential distribution of AMT in the brain regions after acute administration. In this study, we could not determine the causes for the different distribution pattern observed in brain regions. The discrepancies between our findings and the previous work by Yufu are considered to be inconclusive. The AUCAMT value in the serum increased 1.6 times after chronic administration, whereas the tIl2 value decreased slightly. Since AMT is a highly lipophilic compound, it would be accumulated in lipophilic tissue such as adipose. Our measurements of the drugs, however, are limited to the detection of both in the serum and the brain and we have no available data after the last sampling time of 8 h. Although we could not determine the causes of the enhanced AUCAMT value, i t might be attributed to the presence of the slower elimination phase andlor inhibited drug distribution into the tissues. The AUC, value in the serum also increased 9.0 times after chronic administration. In addition, the AUCN,:AUC,T ratio was increased markedly in the serum.The enhanced metabolism and the decreased tIl2 of IMP23 and CIMlO aRer chronic adrmnistration has been demonstrated. Furthermore, Yufu12 has also reported a similar result after chronic administration of AMT. These results suggest that the increased AUCN, value is not only due to the same mechanism as seen in AMT but also due to the enhanced metabolism of AMT and formation of NRT by liver
NRT
AMT
-
0 A
5000-
A 0
4 \ B
Frontal c o r t e x Hippocampus Hypothalamus Serum
01
e
:1000-, \
ol
\
C
A
c 0
4 CI
:cm:
100-
50V
20
-
I l l
0
I
1
4
2
Time
8 (
hr )
I 1 1
1
1
0
1
2
8
4
Time
(
hr )
Flour8 2-Time course of amitriplyline and nortriptylineconcentrations in serum and brain regions after chronic administration of amitriptyline mgkg, ip). Each point represents ihe mean t SEM of four or five rats.
microsomal enzymes. It seems, however, that further detailed study should be made to clarifythe enhanced metabolism of AMT and formation of NRT. The AUC,, values in various brain regions showed no significant change by chronic administration, suggesting that drug distributions into the brain might be inhibited. The concentrations obtained are several-fold higher than those of therapeutic concentrations. Under this condition, the binding sites in the brain might be occupied. Therefore, the observed inhibition of drug penetration into the brain might be due to the saturated binding sites of the drugs. By chronic administration, the AUC,, values in the brain increased 6.8-fold, showing less increase compared with that in the serum. These results also suggest the suppressed distribution of the drug into the brain as seen in AMT. In addition, the AUC,, values in the brain increased several times compared with acute administration, whereas those of AMT showed no changes, suggesting that the binding affinities andlor the extent of the accumulation of the drugs may differ. Corona et al.24 have suggested that changes in membrane permeability induced by chronic administration can determine the different distribution patterns of AMT and metabolite in the brain of rabbits. It seems, however, that further detailed study should be made to clarify these mechanisms. In conclusion, the present experiments systematically investigated the regional brain and serum pharmacokinetics of AMT and its demethylated metabolite NRT after acute and chronic drug administration. The results demonstrate the following: (I) superiority of AMT over NRT in penetration into the brain; (2)regional brain difference in the maximum concentration of AMT and NRT in chronic administration; (3) marked increases of demethylated metabolite in the serum and brain regions after chronic administration; and (4) inhibition of drugs in penetrating the brain regions after chronic administration.
References and Notes 1. Watanabe, S. Jap. J . Neuropsychophnrmacol. 1985,10,661-669.
2. Asano, Y.; Yamashita, I. Jap. J . Clin. Psychiatry 1979, 8, 787-797. 3. Barrows, G . D.; Davis, B.; Scoggins, B. A. Lancet 1972,2, 619623. 4. Jungkunz, G.; Kurs, H. J. Lancet 1978,2, 1263-1264. 5. Braithwait, R. A.;Goulding, R;Theano, G.;Bailey, J.;Coppen, A. Lancet 1972,1, 1297-1300. 6. Masada. M.: Suzuki. K.: Kikuta. S.: Yamashita. S.: Nakanishi. K.; Nadai, T:; Igarashi, Y . ;Noguchi, T . Chern. Phhrm. Bull. 1986; 34.927-930. 7. Maaada, M.; Suzuki, K.; Kikuta, S.; Yamashita, S.; Nakanishi, K.; Nadai, T.;Igarashi, Y.;Noguchi, T. Chern.Pharrn.Bull. 1986, 34, 2173-2177. 8. Daniel, W.; Adamus, A.; Melzacka, M.; Szymura, J.; Vetulani, J. NaunynSchrniederberg's Arch. Phnrmacol. 1981,317,209-213. 9. Daniel, W.; Adamus, A,; Melzacka, M.; Szymura, J. J . Phurrn. Phurmacol. 1982.34, 676-660. 10. Friedman, E.; Cooper, T. B. J . Phnrmacol. Exp. Ther. 1983,225, 387-390. 11. Nagy, A. J . Phnrm. Phnrmacol. 1977,29, 104-107. 12. Yuf'u, N. Jap. J . Neuropsychophnrmacol. 1987,89, 22-42. 13. Glowinsky, J.; Iversen, L. L. J . Neurochern. 1966,13,655-669. 14. Hogen, C. A. M.; Tocco, D. J.; Brodie, B. B.; Shanker, L. S. J. Phnrmacol. Exp. Ther. 1959,125,275-282. 15. Yamaoka, K.; Nakagawa, T. J . PhurmacobkDyn. 1983, 6, 595-606. 16. Brodie, B. B.; Kurtz, H.;Schanker, L. S. J . Phnrmacol. Exp. Ther. 1960,130,20-25. 17. Mayer, S.; Maickel, R. P.; Brodie, B. B. J . P h u r m o l . Exp. Ther. 1959,127,205-211. 18. Jflrgensen,A.; Hansen, V.; Over@,K. F. ActnPharmacol. Toxicol. 19'73.33.81-91. 19. Sherman, A. D.; Allers, G. L. Neurophnrmacology 1980, 19, 159-162. 20. Raiseman, R.; Briley, M. S.; Langer, S. Z. Eur. J . Phurmacol. 1980,61,373-380. 21. Palkovita, M.; Raiseman, R.; Briley, M.; Langer, S. 2. Brain Res. 1981,210,493-498. 22. Sette, M.; Raiseman, R.; Briley, M. S.; Langer, S. 2. J . Neurochern. 1981,37,4042. 23. Breyer, U. Naunyn-Schrnideberg's Arch. Pharmacol. 1972,272, 277-288. 24. Corona, G . L.; Facino, R. M.; Santagostino, G . Biochern. Phnrmacol. 1971,20, 2765-2771.
Journal of Pharmaceutical Sciences I 291 Vol. 79, No. 4, April 1990