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The concentrations of orphenadrine and its N-demethylated derivatives in rat brain, after intraperitoneal administration of orphenadrine and tofenacine
Int. J. Neuropharmacol., 1968, 7,293-300
Pergamon Press.
Printed in Gt. Britain.
THE CONCENTRATIONS OF ORPHENADRINE A N D ITS N-DEMETHYLATED DERIVATIVES IN RAT BRAIN, AFTER INTRAPERITONEAL ADMINISTRATION OF ORPHENADRINE A N D TOFENACINE R. C. ROOZEMOND*, W. HESPE and W. Th. NAUTA Research Department N.V. Koninklijke Pharmaceutische Fabrieken v/h Brocades-Stheeman en Pharmacia, Looiersgracht 27-39, Amsterdam, The Netherlands
(Accepted 20 November 1967) Summary--As an extension of a previous investigation into the effect of orphenadrine and tofenacine on the concentration and binding of 5-hydroxytryptamine (5HT) in rat brain, the concentrations of orphenadrine and/or its N-demethylated derivatives in rat brain were detertnined at various intervals after intraperitoneal administration of either tritiated orphenadrine hydrochloride or tofenacine hydrochloride. Thin-layer chromatography of brain extracts showed that both drugs attain their maximal concentration in brain within 15 rain after administration. Following orphenadrine dosage, tofenacine was also found in the brain, the maximum concentration occurring after 45-60 rain. A slight amount of N,N-didemethyl orphenadrine was only found after tofenacine had been administered. Apparently both orphenadrine and tofenacine penetrate rapidly into the brain. From comparison of the present data with the previous ones on the effect of these drugs on 5HT brain concentrations it is presumed that in the case of both drugs, a two-pool system is involved. One of the pools would be a slowly equilibrating one, directly linked to the storage of 5HT, whereas the other would be a rapidly equilibrating pool.
INTRODUCTION T u n EFVECTSof o r p h e n a d r i n e and its N-demethylated derivative t o f e n a c i n e t , on the c o n c e n t r a t i o n and b i n d i n g of 5-hydroxytryptamine (5HT) in rat brain, have been discussed in a previous p u b l i c a t i o n by ROOZEMOND a n d NAUTA (1966). The drugs m e n t i o n e d were found to raise the a m o u n t of bound++ 5HT, w i t h o u t affecting that of free~+ 5HT. We interpreted these results as a n increased b i n d i n g of 5 H T by cell particles u n d e r the influence of orphenadrine and tofenacine; furthermore they confirmed previous findings with regard to a direct effect of o r p h e n a d r i n e on m e m b r a n e s . I n order to determine any possible relationship between the biochemical effect on the 5 H T c o n c e n t r a t i o n which is m a x i m a l a b o u t 1 hr after intraperitoneal a d m i n i s t r a t i o n , and the a m o u n t s of o r p h e n a d r i n e and its metabolites in the brain, d e t e r m i n a t i o n s of these c o m p o u n d s were carried out at various intervals after a d m i n i s t r a t i o n of o r p h e n a d r i n e
*Present address: Histologisch Laboratorium, Universiteit van Amsterdam, Jan Swammerdam lnstituut, 1° Constantijn Huygensstraat 20, Amsterdam, The Netherlands. tNonproprietary name, published by the WHO. Brocades' brand names for orphenadrine hydrochloride: DisipalR, BrocadisipalR, BrocasipalR, and MephenaminR. +The term "bound" relates to the fraction of 5HT found in the particulate material after homogenisation of thebrain in isotonic sucrose. The amount of 5HT present in the supernatant is termed "free", 293
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R. C. ROOZEMOND,W. HESPEand W. TH. NAUTA
and tofenacine. The results were accounted for by the hypothesis of a binding of orphenadrine and tofenacine in the brain by means of a double-pool system, one pool being in direct contact with the pool of bound 5HT.
MATERIALS
AND
METHODS
We used N,N-dimethyl-2-[(o-methyl-a-phenylbenzyl)oxylethylamine hydrochloride (orphenadrine hydrochloride) and N-methyl-2-[(o-methyl-a.phenylbenzyl)oxy]ethylamine hydrochloride (tofenacine hydrochloride) tritiated in the side chain as indicated; the specific radioactivities were 57-8 and 61.4 mc/g, respectively. The radiochemical purity was established by thin-layer chromatography and analysis of the radiograms, by means of a Berthold radioactivity scanner LB 2720.
"1"~ t,? • 1
R R ~ CH3; orphenadrine-aH R == H ; tofenacine-3H The drugs were administered as the hydrochlorides, dissolved in 0.5 ml of water per 100 g of body weight, to TNO-Wu albino rats (weight 150-200 g). The animals were sacrificed by elongation of the spinal cord at various intervals after drug administration ; the brains were immediately removed, cleaned with cold saline, blotted on filtering paper, halved, weighed, and homogenised in 0.01 N hydrochloric acid (1 : 5 w/v), using a Potter-Elvehjem tube with a Teflon pestle. Each experiment involved the brains of four rats, and since these were halved, two identical homogenates were obtained for duplicate analyses. A 0-5-ml sample of each homogenate was taken for liquid scintillation counting (TriCarb spectrometer 314EX); to the remaining homogenates were added for every 9 ml: 2 m l of 2N NaOH, 5 g of NaCI, 25 ml of n-heptane (purified by passage through an alumina column), and 0.2 mi of methanol. The mixture was thoroughly shaken for 10 min, and centrifuged. To determine the overall extraction yield 1 ml of the organic layer was taken for liquid scintillation counting. The remaining organic layer was evaporated, and the residue taken up in 0.01 N hydrochloric acid, and finally subjected to thin-layer chromatography on silica gel G with butanol-25 ~ ammonia (98:2). After drying, the chromatoplate was analysed with the aid of the Berthold scanner. Radioactive spots were scraped off, mixed with a scintillation cocktail and counted. The amount of the radioactive products present could be deduced from the ratio of the radioactivities on the chromatoplate to the total amount of the radioactivity in the homogenate. The values obtained have to be corrected for the difference between the partition coeflicients of orphenadrine and tofenacine. Preliminary experiments had shown that after administration of orphenadrine to rats none or only small amounts of N,N-didemethyl orphenadrinc could be detected in the brain and that nearly all radioactivity in the homogenate
The concentrations of orphenadrine and its N-demethylated derivatives in rat brain
295
was attributable to orphenadrine and tofenacine. Further investigations served to determine the exact distribution of orphenadrine and tofenacine between alkaline rat brain homogenate and n-heptane. To rat brain homogenates in 0.01 N HC1 (1:5 w/v) orphenadrine and tofenacine, of the same specific radioactivities, were added in equal amounts corresponding roughly to the total amount found in rat brain 15 min after intraperitoneal administration of 50 mg/kg of orphenadrine hydrochloride. The procedure described above was then applied; TLC-scanning and scintillation-counting of the scraped spots enabled computation of the ratio of the yields of orphenadrine and tofenacine (Table 1). The mean yields TABLE 1.
RECOVERIES OF ORPHENADRINE AND TOFENACINE OBTAINED BY SCINTH.LATION COUNTING ( A ) OR BY' SCANNING OF THIN-LAYER CHROMATOGRAMS ( B )
Method used
Recovery in the heptane-phase (%)
(A)
35 39 48 35
Recovery on the chromatoplate % orphen. % tofen, 13"5 15"2 7"3 9"5 19-9 4"0 I1"1
20"5 18"4 14.6 9"5 23"5 9"! 8'6
R-- recov, tofen. recov, orphen. 1"52 1"21 2"00 1.00 1"18 2"28 0"78 R : 1"42~ 0"21(1"48%)*
(B)
35
1"37 1.04 2.05
39
1.06
48 35
2.66 1.58 1.81 i~, ~ 1.65:t 0"22(13"3%)*
*Average standard deviation calculated as: v' [Z' (x-,~)z] n(n-l) of the radioactivity in the heptane-phase and on the chromatoplate were 39 and 26%, respectively, of the total radioactivity in the homogenate. In the brains of tofenacinetreated rats no orphenadrine and only traces of N,N-didemethyl orphenadrine were detected; thus corrections for difference in partition coefficients were not necessary. RESULTS
a. Levels of orphenadrine and tofenacine in rat brain after i.p. administration of orphenadrine As shown in Table 2, the orphenadrine brain level reached a maximum of (corrected) 44/~g/g tissue, 15 min after administration of 50 mg/kg, i.p., of orphenadrine hydrochloride. At that point, however, the concentration of tofenacine, continued to increase, until a m a x i m u m of 19/zg/g tissue was reached, 45 min after administration. A fair agreement between the scintillation and TLC-data can be observed; the increase in standard deviation from uncorrected to corrected values is due to the standard deviation in the ratio of the recoveries for orphenadrine and tofenacine (Table 1).
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ROOZEMOND,
W. HESPEand W. TH. NAUTA
TABLE2. LEVELSOF ORPHENADRINEANDTOFENACINEIN RATBRAINDETERMINEDBYSCINTILLATIONCOUNTING (A) AND TLC-sCANNING(B) AT VARIOUSINTERVALSOF ADMINISTRATIONOF 50 mg/kg OF TRlTIATEDORPHENADRINEHYDROCHLORIDE Number of expts.
Interval (miD)
/zg orphenadrine/g tissue uncorr, corr.
/~g tofenacine/g tissue uncorr, corr.
(A)
6 6 6 4 4
15 30 45 60 90
40.23:4.2 32.13:2.9 30.63:1.3 27.53:2.0 17.7il.8
43.3±10.9 36.04- 8.7 34.33: 6.6 31.8i 6.7 20.03:4.7
13.0±1.9 19.4±2.5 25.04,3.1 23.34-0.8 11.2±1.5
9.94.1.6 15.44.4.3 20.4±5.7 19.04,3.7 9.74-2.8
(B)
6 6 6 4 4
15 30 45 60 90
39.04-4.7 29.94-3.6 30.3 :k2.2 28.84.1.0 18.14,0.9
43.64-10.6 35.74- 8.8 36.73:7.5 34.7-A 5.8 21.54. 3.8
13.94,1.5 21.54.2-0 24.4+2.3 22.0:f0.5 11.43:0.7
9.63:2.3 15.64.3-2 18.03:4.2 16.1 :i2.6 8.23:1.7
b. Levels of tofenacine and N,N-didemethyl orphenadrine in rat brain after i.p. administration of tofenacine As shown in T a b l e 3, tofenacine reached a m a x i m a l c o n c e n t r a t i o n of 46 vg/g tissue in rat brain, 15 miD after a d m i n i s t r a t i o n of 30 mg/kg, i.p., of tofenacine hydrochloride. The small a m o u n t of N , N - d i d e m e t h y l o r p h e n a d r i n e , which was also demonstrated, showed a tendency to increase with time, b u t did n o t exceed 4 t*g/g tissue d u r i n g the entire experiment. TABLE 3. LEVELS OF TOFENACINE AND N,N-DIDEMETHYL ORPHENADRINE IN RAT BRAIN DETERMINED BY SCINTILLATION COUNTING (A)AND T L C SCANNING (B) AT VARIOUS INTERVALS AFTER ADMINISTRATION OF 30 m g / k g OF TRITIATED TOFENACINE HYDROCHLORIDE
Number of expts.
Interval (miD)
/zg Tofenacine/g tissue
/zg N,N-Didemethyl orphenadrine/g tissue
(A)
4 4 4 4 4
15 30 45 60 90
46.73:3.9 37-73:1 "8 31-94,2"4 21.54-1.1 18.9:t:2.5
2.063:0.18 2.71 ±0"36 2.48±0.15 2.40+0.19 2.62-!-0.20
(B)
4 4 4 4 4
15 30 45 60 90
45.0±4.1 36-8:k 1-7 31.1 4-3.8 20.04-0.9 19.33:2.2
3.71 ::k0.25 3.61 :k0.27 3.254-0.25 3-914-0.25 3-774-0.22
c. The effect of reserpine on the brain levels of orphenadrine and tofenacine Earlier, we d e m o n s t r a t e d that p r e t r e a t m e n t with o r p h e n a d r i n e or tofenacine did n o t inhibit the 5 H T - d e p l e t i n g action of reserpine in rat b r a i n (ROOZEMOND a n d NAUTA, 1966). It seemed worthwhile to determine the exact o r p h e n a d r i n e a n d tofenacine levels in rat brain after a d m i n i s t r a t i o n of 50 mg/kg o f o r p h e n a d r i n e hydrochloride and 5 mg/kg of reserpine. W e did not find any significant effect of reserpine on the levels o f o r p h e n a d r i n e a n d tofenacine (Table 4).
The concentrations of orphenadrine and its N-demethylated derivatives in rat brain
297
TABLE 4. LEVELS OF ORPHENADRINE AND TOFENACINE IN RAT BRAIN DETERMINED BY SCINTILLATION COUNTING ( A ) AND TLC-SCANNING (B), 45 r a i n AFTER ADMINISTRATION OF 50 mg/kg OF TRITIATED ORPHENADRINE HYDROCHLORIDE (I) AND OF 5 mg/kg OF RESERPINE (II).
Number of expts.
/~g orphen./g tissue uncorr, corr.
Treatment
#g tofen./g tissue uncorr, corr.
(A)
6 6
! 1 4_ I!
30.6 -1.3 24.0±3.1
34-3-[-6"6 27.9±7.3
25-04_3.1 24.3-3_2"7
20.4_4-5.7 20.44-5.7
(B)
6 6
l + I + II
30.3_-t-2.2 25-74-2.2
36.74-7.5 31.44-6.6
24.44-2.3 22.24-0.9
18.04-4.2 16.64-2.9
DISCUSSION The fate of orally administered orphenadrine hydrochloride in rats has been investigated by HESPE et al., (1965). Its biotransformation was found to consist for an important part in N-demethylation in the liver, producing N-demethyl and N,N-didemethyl orphenadrine. The latter compound could not be detected in the brain. This is in accordance with the present experiments, showing after intraperitoneal administration of orphenadrine, only the unchanged product and its N-demethyl derivative tofenacine in the brain. However, after administration of tofenacine itself, N,N-didemethyl orphenadrine appeared in the brain at a low concentration, which remained almost constant during the experiment. Thin-layer chromatography permitted the separation of orphenadrine and tofenacine in brain extracts and the accurate determination of their respective values, at various intervals after intraperitoneal administration of radioactive-labelled orphenadrine. This revealed a different course of their respective concentrations in the brain (Table 2), fully accounted for by the fact that tofenacine, prior to its absorption in the brain, has to be formed out of orphenadrine in the liver. If tofenacine hydrochloride is intraperitoneally administered to rats, it is absorbed just as rapidly as orphenadrine hydrochloride administered by the same route (Table 3). The small amounts of N,N-didemethyl orphenadrine, which then slowly appear in the brain, are formed out of tofenacine in the liver (Fig. 2). It is, however, more difficult to explain the relation between the patterns of the brain levels of the drugs and of 5HT (Figs. 1 and 2).
504
4s~
~ 3o '-
Orphenadrine
o~ 10
To f e n o c i n e 15
30
60
90
B o r 5, C/o i n c r e o s e in t o t o t 5 H T
Hin. after administrotion
FIG. 1. Levelsof orphenadrine and of tofenacine, and percentage increase in the total 5HT level in rat brain (see ROOZEMONDand NAtrrA, 1966), at different intervals after i.p. administration of 50 mg/kg of orphenadrine hydrochloride. For greater clarity, standard deviations have been omitted.
298
R. C. ROOZEMOND,W. HESPEand W. TH. NALrTA
t
40%
o, ~10
Tofenacine x ~"-~" ~ - - - ' ~ - - - - : - - - ' ~ - - 15 30 60
.--"0, 90
H i m o f t e r administration
N.N-Didernethy/. orphenodrine Bars-% increase in t o t e r 5HT
FIG. 2. Levels of tofenacine and of N,N-didemethyl orphenadrine, and percentage increase in the total 5HT level in rat brain (see ROOZEMONDand NAUTA,1966), at different intervals after i.p. administration of 30 mg/kg of tofenacine hydrochloride For greater clarity, standard deviations have been omitted. From a comparison of our data on brain levels of orphenadrine and its metabolites, on the one hand, and of 5HT on the other we presume the existence of two separate pools for the drugs, one slowly, and one rapidly equilibrating:
blood
i -~
" cellular extraI__
pool 1 (intracellular)
pool 2
~
f
5HT storage
The first pool would be in equilibrium with the drug supplied by the blood stream, and the second pool would be directly linked to the storage of 5HT. In this connection we recall that both orphenadrine and tofenacine increase only bound 5HT and leave the free 5HT concentration unaffected (RoOZEMOND and NAUTA, 1966). There are an increasing number of indications that biogenic amines in tissues occur in at least two pools, or are bound in two different ways. The most frequently investigated amines in this respect are: noradrenaline in the heart (COSTA and BRODIE, 1964; AXELROD, 1964; NEFF et al., 1965), in the adrenal medulla (KmsHNER, 1962; CARLSSON et al., 1962; CARLSSON, 1964), in the spleen (VoN EULER and LISHAJKO, 1963), and in the brain (CosTA and BRODIE, 1964; CARLSSON, 1964; DE ROBERTIS, 1964; MAYNERTand KURIYAMA, 1964; MIRKIN et al., 1964; GLOWINSKI et al., 1965; SNYDERet al., 1965), and 5HT in the blood platelets (COSTA and BRODIE, 1964; LESS1Net al., 1965; PAASONEN, 1965), in the mast cells (FURANO and GREEN, 1964; CARLINI et al., 1964) and in the brain (COSTA and BRODIE, 1964; CARLSSON, 1964; DE ROBERTIS, 1964; MAYNERT and KUR1YAMA, 1964; GIARMAN et al., 1964; MARCHBANKS et al., 1964: ROmNSON et al., 1965). The ROBERTS group (SANO and ROBERTS, 1963; VARON et al., 1965a,b) concluded from in vitro investigations of brain homogenates and subcellular fractions, that there are two different transport systems for 7-aminobutyric acid, directing to a slowly and a rapidly equilibrating pool, respectively. SCHANKERand MORRISON (1965) established two different absorption processes for guanethidine in the heart. We may consequently interpret Fig. 1 in the following way: the orphenadrine level attains its maximum after about 15 rain; pool 1 then contains the major part, from which the drug is slowly taken up in pool 2 where it is bound. This process continues, even when the concentration in pool l is already decreasing as a result of a rapid exchange with the extracellular spaces. We may presume that the amount bound in pool 2 shows a maximum at 45-60 min after administration; at the same time the effect on bound 5HT is also maximal. As shown in Fig. 1, the tofenacine
'l'hc concentrations of orphenadrine and its N-demethylated derivatives in rat brain
299
curve deviates from that o f orphenadrine; the main reason for this is that tofenacine has first to be formed by demethylation o f orphenadrine in the liver. The supply o f tofenacine to the brain as c o m p a r e d to that o f orphenadrine being m u c h m o r e gradual, therefore, we may presume that the m a x i m u m o f b o u n d tofenacine in pool 2 coincides with that o f the curve shown in Fig. I. With regard to Fig. 2 we might postulate that administered tofenacine behaves just as stated above for orphenadrine. The impression is gained, however, that the flux from pool 1 to pool 2 is faster in the case of tofenacine, since its effects on the 5HT-level are stronger in the early stages. This is in agreement with the above assumption regarding the absorption o f tofenacine by the brain following administration o f orphenadrine (Fig. 1). We cannot explain, however, that at 90 min after administration of tofenacine, the brain concentration is only slightly lower than at 60 rain whereas its effect on the 5 H T level has become hardly observable (Fig. 2); in contrast, the effects show a parallel course after orphenadrine (Fig. 1). Perhaps the flux from pool 2 to pool I is also faster for tofenacine than for orphenadrine. Both from the respective dose-activity relationships for orphenadrine and tofenacine with regard to 5 H T concentration (RoOZEMOND and NAUTA, 1966), as well as from a comparison o f Figs. 1 and 2, taking into account the double-pool system hypothesis, it m a y be concluded that the activity o f tofenacine is considerably stronger than that of orphenadrine. However, without exact information on the respective amounts o f drug b o u n d in pool 2, it is impossible to give a quantitative relationship between activity and a m o u n t o f drug actually present at the site. Acknowledgement--We wish to thank Miss D. C. STAMfor assistance in the experiments.
REFERENCES AXELROD,J. (1964). Biogenic amines. The uptake and release of catecholamines and the effect of drugs. Progr. Brain Res. 8" 81-89 CARUNI, G. R. S., FISCHrR, G. A. and GIARMAN,N. J. (1964). Differences in characteristics of release of serotonin from neoplastic mast cells in culture by reserpine and chlorpromazine, d. Pharmac. exp. Ther. 146" 74-79. CARLSSON,A. (1964). Functional significance of drug-induced changes in brain monoamine levels. Progr. Brain Res. 8: 9-27. CARLSSON,A., HILLARP,N. A. and WALDECK,B. (1962). A Mg ~~-ATP dependant storage mechanism m the amine granules of the adrenal medulla. Meal. exp. 6: 47-53. Cosr^, E. and BRODIE,B. B. (1964). Concept of the neurochemical transducer as an organized molecular unit at sympathetic nerve endings. Progr. Brain Res. 8" 168-185. DE ROBERTm,E. (1964). Electron microscope and chemical study of binding sites of brain biogenic amines. Progr. Brain Res. 8:118-136. FURANO,A. V. and GREEN,J. P. (1964). The uptake of biogenic amines by mast cells of the rat. J. Physiol. 170: 263-271. GIARMAN,N. J., FREEDMAN,D. X. and SCHANBEg¢~,S. M. (1964). Drug-induced changes in the subcellular distribution of serotonin in rat brain with special reference to the action of reserpine. Progr. Brain Res. 8: 72-80. GLOWINSKI,J., KOPIN, I. J. and AXELROD,J. (1965). Metabolism of (3H)-norepinephrine in the brain. J. Neurochem. 12:25-30.
HESPE,W., ROOS,A. M. DEand NAUTA,W. Th. (1965). Investigation into the metabolic fate of orphenadrine hydrochloride after oral administration to male rats. Archs. Int. Pharmacodyn. 156: 180-200. KmSHNER, N. (1962). Uptake of catecholamines by a particulate fraction of the adrenal medulla. J. biol. Chem. 237:2311-2317. L~SIN, A. W., LONG, R. F. and PARKS, M. W. (1965). The effects of ~-alkyl substituted tryptamines on 5-hydroxytryptamine uptake by blood platelets. Br. J. Pharmac. 24: 68-75.
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MARCHBANKS, R. M., ROSENBLATT,F. and O'BRIEN, R. D. (1964). Serotonin binding to nerve-ending particles of the rat brain and its inhibition by lysergic acid diethylamide. Science 144: 1135-1137. MAYNERT, E. W. and KURIYAMA,K. (1964). Some observations on nerve-ending particles and synaptic vesicles. Life Sci. 3: 1067-1087. MIRKIN,B. L., GIARMAN,N. J. and FREEDMAN,O. X. (1964). Factors influencing the uptake of noradrenaline by subcellular particles in homogenates of rat brain. Biochem. Pharmac. 13: 1027-1036. NEFF, N. H., TOZER,T. N., HAMMER,W. and BRODIE,B. B. (1965). Kinetics of release of norepinephrine by tyramine. Life Sci. 4: 1869-1975. PAASONEN, M. K. (1965). Release of 5-hydroxytryptamine from blood platelets. Or. Pharm. Pharmac. 17: 681-697. ROBINSON,J. D., ANDERSON,J. H. and GREEN,J. P. (1965). The uptake of 5-hydroxytryptamine and histamine by particulate fractions of brain. J. Pharmac. exp. Ther. 147: 236-243. ROOZEMOND, R. C. and NAUTA,W. TH. (1966). The effect of orphenadrine and its N-demethyl derivative on the level of 5-hydroxytryptamine in rat brain. Int. J. NeuropharmacoL 5: 413-419. SANO, K. and ROBERTS,E. (1963). Binding of y-aminobutyric acid by mouse brain preparations. Biochem. Pharmac. 12: 489-502. SCHANKER,L. S. and MORmSON,A. S. (1965). Physiological disposition of guanethidine in the rat and its uptake by heart slices. Int. J. Neuropharmacol. 4: 27-39. SNYDER, S. H., GLOWINSKI,J. and AXELROD,J. (1965). The storage of norepinephrine and some of its derivatives in brain synaptosomes. Life Sci. 4: 797-807. VARON,S., WEINSTEIN,H., BAXTER,C. F. and ROaERTS,E. (1965a). Uptake and metabolism of exogenous y-aminobutyric acid by subcellular particles in a sodium-containing medium. Biochem. Pharmac. 14: 1755-1764. VARON,S., WEINSTEIN,H., KAKEFUDA,T. and ROBERTS,E. (1965b). Sodium-dependant binding of y-aminobutyric acid by morphologically characterized subcellular brain particles. Biochem. Pharmacol. 14: 1213-1224. VON EULER,U. S. and LISHAJKO,F. (1963). Catecholamine release and uptake in isolated adrenergic nerve granules. Acta physiol, scand. 57: 468-480.
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THE CONCENTRATIONS OF ORPHENADRINE A N D ITS N-DEMETHYLATED DERIVATIVES IN RAT BRAIN, AFTER INTRAPERITONEAL ADMINISTRATION OF ORPHENADRINE A N D TOFENACINE R. C. ROOZEMOND*, W. HESPE and W. Th. NAUTA Research Department N.V. Koninklijke Pharmaceutische Fabrieken v/h Brocades-Stheeman en Pharmacia, Looiersgracht 27-39, Amsterdam, The Netherlands
(Accepted 20 November 1967) Summary--As an extension of a previous investigation into the effect of orphenadrine and tofenacine on the concentration and binding of 5-hydroxytryptamine (5HT) in rat brain, the concentrations of orphenadrine and/or its N-demethylated derivatives in rat brain were detertnined at various intervals after intraperitoneal administration of either tritiated orphenadrine hydrochloride or tofenacine hydrochloride. Thin-layer chromatography of brain extracts showed that both drugs attain their maximal concentration in brain within 15 rain after administration. Following orphenadrine dosage, tofenacine was also found in the brain, the maximum concentration occurring after 45-60 rain. A slight amount of N,N-didemethyl orphenadrine was only found after tofenacine had been administered. Apparently both orphenadrine and tofenacine penetrate rapidly into the brain. From comparison of the present data with the previous ones on the effect of these drugs on 5HT brain concentrations it is presumed that in the case of both drugs, a two-pool system is involved. One of the pools would be a slowly equilibrating one, directly linked to the storage of 5HT, whereas the other would be a rapidly equilibrating pool.
INTRODUCTION T u n EFVECTSof o r p h e n a d r i n e and its N-demethylated derivative t o f e n a c i n e t , on the c o n c e n t r a t i o n and b i n d i n g of 5-hydroxytryptamine (5HT) in rat brain, have been discussed in a previous p u b l i c a t i o n by ROOZEMOND a n d NAUTA (1966). The drugs m e n t i o n e d were found to raise the a m o u n t of bound++ 5HT, w i t h o u t affecting that of free~+ 5HT. We interpreted these results as a n increased b i n d i n g of 5 H T by cell particles u n d e r the influence of orphenadrine and tofenacine; furthermore they confirmed previous findings with regard to a direct effect of o r p h e n a d r i n e on m e m b r a n e s . I n order to determine any possible relationship between the biochemical effect on the 5 H T c o n c e n t r a t i o n which is m a x i m a l a b o u t 1 hr after intraperitoneal a d m i n i s t r a t i o n , and the a m o u n t s of o r p h e n a d r i n e and its metabolites in the brain, d e t e r m i n a t i o n s of these c o m p o u n d s were carried out at various intervals after a d m i n i s t r a t i o n of o r p h e n a d r i n e
*Present address: Histologisch Laboratorium, Universiteit van Amsterdam, Jan Swammerdam lnstituut, 1° Constantijn Huygensstraat 20, Amsterdam, The Netherlands. tNonproprietary name, published by the WHO. Brocades' brand names for orphenadrine hydrochloride: DisipalR, BrocadisipalR, BrocasipalR, and MephenaminR. +The term "bound" relates to the fraction of 5HT found in the particulate material after homogenisation of thebrain in isotonic sucrose. The amount of 5HT present in the supernatant is termed "free", 293
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R. C. ROOZEMOND,W. HESPEand W. TH. NAUTA
and tofenacine. The results were accounted for by the hypothesis of a binding of orphenadrine and tofenacine in the brain by means of a double-pool system, one pool being in direct contact with the pool of bound 5HT.
MATERIALS
AND
METHODS
We used N,N-dimethyl-2-[(o-methyl-a-phenylbenzyl)oxylethylamine hydrochloride (orphenadrine hydrochloride) and N-methyl-2-[(o-methyl-a.phenylbenzyl)oxy]ethylamine hydrochloride (tofenacine hydrochloride) tritiated in the side chain as indicated; the specific radioactivities were 57-8 and 61.4 mc/g, respectively. The radiochemical purity was established by thin-layer chromatography and analysis of the radiograms, by means of a Berthold radioactivity scanner LB 2720.
"1"~ t,? • 1
R R ~ CH3; orphenadrine-aH R == H ; tofenacine-3H The drugs were administered as the hydrochlorides, dissolved in 0.5 ml of water per 100 g of body weight, to TNO-Wu albino rats (weight 150-200 g). The animals were sacrificed by elongation of the spinal cord at various intervals after drug administration ; the brains were immediately removed, cleaned with cold saline, blotted on filtering paper, halved, weighed, and homogenised in 0.01 N hydrochloric acid (1 : 5 w/v), using a Potter-Elvehjem tube with a Teflon pestle. Each experiment involved the brains of four rats, and since these were halved, two identical homogenates were obtained for duplicate analyses. A 0-5-ml sample of each homogenate was taken for liquid scintillation counting (TriCarb spectrometer 314EX); to the remaining homogenates were added for every 9 ml: 2 m l of 2N NaOH, 5 g of NaCI, 25 ml of n-heptane (purified by passage through an alumina column), and 0.2 mi of methanol. The mixture was thoroughly shaken for 10 min, and centrifuged. To determine the overall extraction yield 1 ml of the organic layer was taken for liquid scintillation counting. The remaining organic layer was evaporated, and the residue taken up in 0.01 N hydrochloric acid, and finally subjected to thin-layer chromatography on silica gel G with butanol-25 ~ ammonia (98:2). After drying, the chromatoplate was analysed with the aid of the Berthold scanner. Radioactive spots were scraped off, mixed with a scintillation cocktail and counted. The amount of the radioactive products present could be deduced from the ratio of the radioactivities on the chromatoplate to the total amount of the radioactivity in the homogenate. The values obtained have to be corrected for the difference between the partition coeflicients of orphenadrine and tofenacine. Preliminary experiments had shown that after administration of orphenadrine to rats none or only small amounts of N,N-didemethyl orphenadrinc could be detected in the brain and that nearly all radioactivity in the homogenate
The concentrations of orphenadrine and its N-demethylated derivatives in rat brain
295
was attributable to orphenadrine and tofenacine. Further investigations served to determine the exact distribution of orphenadrine and tofenacine between alkaline rat brain homogenate and n-heptane. To rat brain homogenates in 0.01 N HC1 (1:5 w/v) orphenadrine and tofenacine, of the same specific radioactivities, were added in equal amounts corresponding roughly to the total amount found in rat brain 15 min after intraperitoneal administration of 50 mg/kg of orphenadrine hydrochloride. The procedure described above was then applied; TLC-scanning and scintillation-counting of the scraped spots enabled computation of the ratio of the yields of orphenadrine and tofenacine (Table 1). The mean yields TABLE 1.
RECOVERIES OF ORPHENADRINE AND TOFENACINE OBTAINED BY SCINTH.LATION COUNTING ( A ) OR BY' SCANNING OF THIN-LAYER CHROMATOGRAMS ( B )
Method used
Recovery in the heptane-phase (%)
(A)
35 39 48 35
Recovery on the chromatoplate % orphen. % tofen, 13"5 15"2 7"3 9"5 19-9 4"0 I1"1
20"5 18"4 14.6 9"5 23"5 9"! 8'6
R-- recov, tofen. recov, orphen. 1"52 1"21 2"00 1.00 1"18 2"28 0"78 R : 1"42~ 0"21(1"48%)*
(B)
35
1"37 1.04 2.05
39
1.06
48 35
2.66 1.58 1.81 i~, ~ 1.65:t 0"22(13"3%)*
*Average standard deviation calculated as: v' [Z' (x-,~)z] n(n-l) of the radioactivity in the heptane-phase and on the chromatoplate were 39 and 26%, respectively, of the total radioactivity in the homogenate. In the brains of tofenacinetreated rats no orphenadrine and only traces of N,N-didemethyl orphenadrine were detected; thus corrections for difference in partition coefficients were not necessary. RESULTS
a. Levels of orphenadrine and tofenacine in rat brain after i.p. administration of orphenadrine As shown in Table 2, the orphenadrine brain level reached a maximum of (corrected) 44/~g/g tissue, 15 min after administration of 50 mg/kg, i.p., of orphenadrine hydrochloride. At that point, however, the concentration of tofenacine, continued to increase, until a m a x i m u m of 19/zg/g tissue was reached, 45 min after administration. A fair agreement between the scintillation and TLC-data can be observed; the increase in standard deviation from uncorrected to corrected values is due to the standard deviation in the ratio of the recoveries for orphenadrine and tofenacine (Table 1).
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W. HESPEand W. TH. NAUTA
TABLE2. LEVELSOF ORPHENADRINEANDTOFENACINEIN RATBRAINDETERMINEDBYSCINTILLATIONCOUNTING (A) AND TLC-sCANNING(B) AT VARIOUSINTERVALSOF ADMINISTRATIONOF 50 mg/kg OF TRlTIATEDORPHENADRINEHYDROCHLORIDE Number of expts.
Interval (miD)
/zg orphenadrine/g tissue uncorr, corr.
/~g tofenacine/g tissue uncorr, corr.
(A)
6 6 6 4 4
15 30 45 60 90
40.23:4.2 32.13:2.9 30.63:1.3 27.53:2.0 17.7il.8
43.3±10.9 36.04- 8.7 34.33: 6.6 31.8i 6.7 20.03:4.7
13.0±1.9 19.4±2.5 25.04,3.1 23.34-0.8 11.2±1.5
9.94.1.6 15.44.4.3 20.4±5.7 19.04,3.7 9.74-2.8
(B)
6 6 6 4 4
15 30 45 60 90
39.04-4.7 29.94-3.6 30.3 :k2.2 28.84.1.0 18.14,0.9
43.64-10.6 35.74- 8.8 36.73:7.5 34.7-A 5.8 21.54. 3.8
13.94,1.5 21.54.2-0 24.4+2.3 22.0:f0.5 11.43:0.7
9.63:2.3 15.64.3-2 18.03:4.2 16.1 :i2.6 8.23:1.7
b. Levels of tofenacine and N,N-didemethyl orphenadrine in rat brain after i.p. administration of tofenacine As shown in T a b l e 3, tofenacine reached a m a x i m a l c o n c e n t r a t i o n of 46 vg/g tissue in rat brain, 15 miD after a d m i n i s t r a t i o n of 30 mg/kg, i.p., of tofenacine hydrochloride. The small a m o u n t of N , N - d i d e m e t h y l o r p h e n a d r i n e , which was also demonstrated, showed a tendency to increase with time, b u t did n o t exceed 4 t*g/g tissue d u r i n g the entire experiment. TABLE 3. LEVELS OF TOFENACINE AND N,N-DIDEMETHYL ORPHENADRINE IN RAT BRAIN DETERMINED BY SCINTILLATION COUNTING (A)AND T L C SCANNING (B) AT VARIOUS INTERVALS AFTER ADMINISTRATION OF 30 m g / k g OF TRITIATED TOFENACINE HYDROCHLORIDE
Number of expts.
Interval (miD)
/zg Tofenacine/g tissue
/zg N,N-Didemethyl orphenadrine/g tissue
(A)
4 4 4 4 4
15 30 45 60 90
46.73:3.9 37-73:1 "8 31-94,2"4 21.54-1.1 18.9:t:2.5
2.063:0.18 2.71 ±0"36 2.48±0.15 2.40+0.19 2.62-!-0.20
(B)
4 4 4 4 4
15 30 45 60 90
45.0±4.1 36-8:k 1-7 31.1 4-3.8 20.04-0.9 19.33:2.2
3.71 ::k0.25 3.61 :k0.27 3.254-0.25 3-914-0.25 3-774-0.22
c. The effect of reserpine on the brain levels of orphenadrine and tofenacine Earlier, we d e m o n s t r a t e d that p r e t r e a t m e n t with o r p h e n a d r i n e or tofenacine did n o t inhibit the 5 H T - d e p l e t i n g action of reserpine in rat b r a i n (ROOZEMOND a n d NAUTA, 1966). It seemed worthwhile to determine the exact o r p h e n a d r i n e a n d tofenacine levels in rat brain after a d m i n i s t r a t i o n of 50 mg/kg o f o r p h e n a d r i n e hydrochloride and 5 mg/kg of reserpine. W e did not find any significant effect of reserpine on the levels o f o r p h e n a d r i n e a n d tofenacine (Table 4).
The concentrations of orphenadrine and its N-demethylated derivatives in rat brain
297
TABLE 4. LEVELS OF ORPHENADRINE AND TOFENACINE IN RAT BRAIN DETERMINED BY SCINTILLATION COUNTING ( A ) AND TLC-SCANNING (B), 45 r a i n AFTER ADMINISTRATION OF 50 mg/kg OF TRITIATED ORPHENADRINE HYDROCHLORIDE (I) AND OF 5 mg/kg OF RESERPINE (II).
Number of expts.
/~g orphen./g tissue uncorr, corr.
Treatment
#g tofen./g tissue uncorr, corr.
(A)
6 6
! 1 4_ I!
30.6 -1.3 24.0±3.1
34-3-[-6"6 27.9±7.3
25-04_3.1 24.3-3_2"7
20.4_4-5.7 20.44-5.7
(B)
6 6
l + I + II
30.3_-t-2.2 25-74-2.2
36.74-7.5 31.44-6.6
24.44-2.3 22.24-0.9
18.04-4.2 16.64-2.9
DISCUSSION The fate of orally administered orphenadrine hydrochloride in rats has been investigated by HESPE et al., (1965). Its biotransformation was found to consist for an important part in N-demethylation in the liver, producing N-demethyl and N,N-didemethyl orphenadrine. The latter compound could not be detected in the brain. This is in accordance with the present experiments, showing after intraperitoneal administration of orphenadrine, only the unchanged product and its N-demethyl derivative tofenacine in the brain. However, after administration of tofenacine itself, N,N-didemethyl orphenadrine appeared in the brain at a low concentration, which remained almost constant during the experiment. Thin-layer chromatography permitted the separation of orphenadrine and tofenacine in brain extracts and the accurate determination of their respective values, at various intervals after intraperitoneal administration of radioactive-labelled orphenadrine. This revealed a different course of their respective concentrations in the brain (Table 2), fully accounted for by the fact that tofenacine, prior to its absorption in the brain, has to be formed out of orphenadrine in the liver. If tofenacine hydrochloride is intraperitoneally administered to rats, it is absorbed just as rapidly as orphenadrine hydrochloride administered by the same route (Table 3). The small amounts of N,N-didemethyl orphenadrine, which then slowly appear in the brain, are formed out of tofenacine in the liver (Fig. 2). It is, however, more difficult to explain the relation between the patterns of the brain levels of the drugs and of 5HT (Figs. 1 and 2).
504
4s~
~ 3o '-
Orphenadrine
o~ 10
To f e n o c i n e 15
30
60
90
B o r 5, C/o i n c r e o s e in t o t o t 5 H T
Hin. after administrotion
FIG. 1. Levelsof orphenadrine and of tofenacine, and percentage increase in the total 5HT level in rat brain (see ROOZEMONDand NAtrrA, 1966), at different intervals after i.p. administration of 50 mg/kg of orphenadrine hydrochloride. For greater clarity, standard deviations have been omitted.
298
R. C. ROOZEMOND,W. HESPEand W. TH. NALrTA
t
40%
o, ~10
Tofenacine x ~"-~" ~ - - - ' ~ - - - - : - - - ' ~ - - 15 30 60
.--"0, 90
H i m o f t e r administration
N.N-Didernethy/. orphenodrine Bars-% increase in t o t e r 5HT
FIG. 2. Levels of tofenacine and of N,N-didemethyl orphenadrine, and percentage increase in the total 5HT level in rat brain (see ROOZEMONDand NAUTA,1966), at different intervals after i.p. administration of 30 mg/kg of tofenacine hydrochloride For greater clarity, standard deviations have been omitted. From a comparison of our data on brain levels of orphenadrine and its metabolites, on the one hand, and of 5HT on the other we presume the existence of two separate pools for the drugs, one slowly, and one rapidly equilibrating:
blood
i -~
" cellular extraI__
pool 1 (intracellular)
pool 2
~
f
5HT storage
The first pool would be in equilibrium with the drug supplied by the blood stream, and the second pool would be directly linked to the storage of 5HT. In this connection we recall that both orphenadrine and tofenacine increase only bound 5HT and leave the free 5HT concentration unaffected (RoOZEMOND and NAUTA, 1966). There are an increasing number of indications that biogenic amines in tissues occur in at least two pools, or are bound in two different ways. The most frequently investigated amines in this respect are: noradrenaline in the heart (COSTA and BRODIE, 1964; AXELROD, 1964; NEFF et al., 1965), in the adrenal medulla (KmsHNER, 1962; CARLSSON et al., 1962; CARLSSON, 1964), in the spleen (VoN EULER and LISHAJKO, 1963), and in the brain (CosTA and BRODIE, 1964; CARLSSON, 1964; DE ROBERTIS, 1964; MAYNERTand KURIYAMA, 1964; MIRKIN et al., 1964; GLOWINSKI et al., 1965; SNYDERet al., 1965), and 5HT in the blood platelets (COSTA and BRODIE, 1964; LESS1Net al., 1965; PAASONEN, 1965), in the mast cells (FURANO and GREEN, 1964; CARLINI et al., 1964) and in the brain (COSTA and BRODIE, 1964; CARLSSON, 1964; DE ROBERTIS, 1964; MAYNERT and KUR1YAMA, 1964; GIARMAN et al., 1964; MARCHBANKS et al., 1964: ROmNSON et al., 1965). The ROBERTS group (SANO and ROBERTS, 1963; VARON et al., 1965a,b) concluded from in vitro investigations of brain homogenates and subcellular fractions, that there are two different transport systems for 7-aminobutyric acid, directing to a slowly and a rapidly equilibrating pool, respectively. SCHANKERand MORRISON (1965) established two different absorption processes for guanethidine in the heart. We may consequently interpret Fig. 1 in the following way: the orphenadrine level attains its maximum after about 15 rain; pool 1 then contains the major part, from which the drug is slowly taken up in pool 2 where it is bound. This process continues, even when the concentration in pool l is already decreasing as a result of a rapid exchange with the extracellular spaces. We may presume that the amount bound in pool 2 shows a maximum at 45-60 min after administration; at the same time the effect on bound 5HT is also maximal. As shown in Fig. 1, the tofenacine
'l'hc concentrations of orphenadrine and its N-demethylated derivatives in rat brain
299
curve deviates from that o f orphenadrine; the main reason for this is that tofenacine has first to be formed by demethylation o f orphenadrine in the liver. The supply o f tofenacine to the brain as c o m p a r e d to that o f orphenadrine being m u c h m o r e gradual, therefore, we may presume that the m a x i m u m o f b o u n d tofenacine in pool 2 coincides with that o f the curve shown in Fig. I. With regard to Fig. 2 we might postulate that administered tofenacine behaves just as stated above for orphenadrine. The impression is gained, however, that the flux from pool 1 to pool 2 is faster in the case of tofenacine, since its effects on the 5HT-level are stronger in the early stages. This is in agreement with the above assumption regarding the absorption o f tofenacine by the brain following administration o f orphenadrine (Fig. 1). We cannot explain, however, that at 90 min after administration of tofenacine, the brain concentration is only slightly lower than at 60 rain whereas its effect on the 5 H T level has become hardly observable (Fig. 2); in contrast, the effects show a parallel course after orphenadrine (Fig. 1). Perhaps the flux from pool 2 to pool I is also faster for tofenacine than for orphenadrine. Both from the respective dose-activity relationships for orphenadrine and tofenacine with regard to 5 H T concentration (RoOZEMOND and NAUTA, 1966), as well as from a comparison o f Figs. 1 and 2, taking into account the double-pool system hypothesis, it m a y be concluded that the activity o f tofenacine is considerably stronger than that of orphenadrine. However, without exact information on the respective amounts o f drug b o u n d in pool 2, it is impossible to give a quantitative relationship between activity and a m o u n t o f drug actually present at the site. Acknowledgement--We wish to thank Miss D. C. STAMfor assistance in the experiments.
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