Accumulation of methadone by the choroid plexus in vitro

ACCUMULATION

OF METHADONE BY THE PLEXUS IN VITRO’

CHOROID

J. T. HUANG and A. E. TAKEMORI Department

of Pharmacology. University of Minnesota. Health Sciences Center. Medical School. Minneapolis. Minnesota 55455 I A~ceprrtl

15 Auyust

I9741

Summary The uptake of methadone by the choroid plexus of rabbits fulfils the usual criteria for an active transport system. Methadone is taken up against an apparent concentration gradient and high concentrations of the narcotic can saturate the transport system. The uptake of methadone can be inhibited by low temperature, metabolic inhibitors. other narcotic drugs and narcotic antagonists. The transport system is competitively inhlbited by morphine and verapamil. The transport system appears to have a higher affinity for methadone than for morphine. However, the system has about four times greater capacity to transport morphine than methadone. Calcium ions may somehow be involved with the narcotic transport system.

Active uptake of morphine by the choroid plexus of rabbits has been demonstrated previously (TAKEMORI and STENWICK, 1966; HUG, 1967). Although the inhibition of this uptake by derivatives of morphine and structurally related morphinan congeners has been shown. the influence of structurally unrelated narcotic analgesics such as methadone was not investigated. In addition, since methadone is the subject of wide current interest due to its use in maintenance programmes, we felt that more information about the central transport characteristics of methadone would be of interest. The data in the present study indicate that methadone is actively accumulated by the choroid plexus by a transport system similar to that for morphine and the transport system has a higher affinity for methadone than for morphine. METHODS Erpc~$nrr~t.s

\lith choroid plexus in vitro

Male albino rabbits. weighing 1.5-2.5 kg (Oak Crest Rabbitry) were decapitated and the choroid plexuses from the lateral ventricles were excised rapidly. Each choroid plexus was cut into approximately three equal pieces and each piece was incubated in separate beakers containing 3 ml of oxygenated incubation medium and radioactive methadone. The procedure for studying the uptake of methadone by choroid plexus was similar to that described to study the uptake of morphine (TAKEMORI and STENWICK, 1966; CRAIG, O’DEA and TAKEMORI, 1971). After incubation, the choroid plexuses were placed in counting vials containing 1 ml of Nuclear Chicago Solubilizer and dissolved. Ten millilitres of a scintillator fluid consisting of 8 g PPO (2.5-diphenyloxazole) and 0.5 g POPOP (1,4-bis[2-(5phenyloxazolyl)]-benzene) per litre in toluene were placed in the vial and the radioactive content of the solution was determined in a liquid scintillation spectrometer. The radioactive content of a portion of the incubation medium was also determined. All radioactive samples were counted long enough to yield less than 2y; error. Since analysis by both thin layer chromatography and mass spectrometry of the extracts of incubated choroid plexuses indicated that methadone was not metabolized by the tissue. the radioactive counts represented only unaltered methadone under these experimental conditions. Most of the results were expressed as T/M ratios [(dis/min per g tissue)/(dis/min per ml medium)]. The data were statistically analysed by the paired r-test and by the Student t-test where applicable. Double reciprocal plots (LINEWEAVER and BURKE, 1934) were analysed by the method of WILKINSON (1961). * This investigation

was supported Trainee

Health Service Postdoctoral

by U.S. Public Health (GM 01 I 17). 241

Service Grant

DA 00289. J. T. Huang is a U.S. Public

Methadone (heptutme-.?- ’ -I-(‘) hydrochloride LV;IS purch;t~ed from Mallinckrodt Chemical Works with a specitic activity of 54 tnC‘i; mmol. Morphine sulphate was bought from Merck and Co. :tnd hexamctlionittrn chloride was purchased from Nutritional Biochemicals. Lcvallorph:tn tartrate. naloxonc hydrochloride. dcxtropropoxqphene hydrochloride and varapamil hydrochloride u et-c‘ gifts from HotYmann La Kochc. Inc.. Endo Laboratories. Inc.. Eli Lilly 6i C’o.. ;tnd Knoll Pharmaceutical (‘0.. rcspcctivcl!.

,~(.c.tr/llulrrtit)l7

f!f rl7c~ll7trdo~7~ /TJ,c~horf~irlp/(~.ui.f(~~

The choroid plexuses from rabbits in rhc prcscncc of 9.3 x 10 7 )I tncthadone accumulated the narcotic against ;tn apparent conccnttxtion gradient (Fig. 1). The T:‘M increased with time and reached a tnasimum of about 35 within Xl min of incubation. When the choroid plexuses wcrc incubated at 0 C’ instead of i7 C’. the uptake of methadone was markedly reduced. Although the T’M of :thout 5 which W;IS observed at 0 ‘C was only oncseventh that seen at i7 C‘. it \\;Is still well abow~ the thcorotical equilibrium value due to passive diffusion.

As the concentration of methadone in the incubation medium was raised, the amount of the narcotic taken up by the choroid plexus tended to approach ;t tnaximum (Fig. 2). The uptake of methadone by the choroid plexus \v;ts not proportional to the concentration in the incubation medium in that increases of 5. IO-. i.i;- ;tnd IOO-fold in the concentration resulted in only about ?-. 5. 15 and X-fold increases in uptake. respectively. Thcsc results indicate that methadone is accumul;ttcd in the choroid plexus by ;I saturable process.

The uptake of inhibitors (Table displayed lesser IO-’ M failed to

mcthadonc b\ the choroid plexus w;~s inhibited by ;I number of tnetabolic I). Iodoacetate wx the best inhibitor and 2.4dinitrophenol and fluoride inhibitor! ef‘ecth. (‘uriously. sodium aTidc at ;I concentration of 3.3 x inhibit the uptake system.

Since the uptake of tnorphinc by the choroid plexus appeared to be dependent on the presence of calcium and magnesium ions in the external medium and be independent of phosphate ions (TA~;I:,ZZORI:tnd S-t-t~uw~c~t~.t 966). the cffcct of omitting these ions from the medium on the uptnkc of mcth:tdonc h! the choroid plexus wx studied. When phosphate ions were omitted from the medium. the bufYcring capacity of the phosphate buffer was rcplaccd bk Tris [tris I hldro\! mcth! I) aminomcthanc] HC’I butYet-.

Methadone

uptake

by choroid

plexus

243

J

Methadone

Concentration

in Medium

(mM

1

Fig. 2. The effect of various concentrations of methadone on the accumulation of methadone by choroid plexuses. The choroid plexuses were incubated for I hr at 37’C. The values and vertical bars represent mean + SE. of four experiments using a different rabbit for each experiment.

The uptake of methadone by the choroid plexus was depressed significantly by omitting either calcium or magnesium ions from the incubation medium (Table 2). An additive inhibition was observed when both of these ions were omitted. Omission of phosphate ions did not alter the uptake of methadone. The inhibition of uptake seen when calcium ions were omitted was further increased with the addition of the Ca” chelator, EGTA [ethyleneglycol-bis-(p-amino-ethyl ether] N,N’-tetra-acetic acid).

Table

1, Effect of various inhibitors

on methadone

uptake

by choroid

plexus Mean Y0 decrease

T./M k S.E.t Inhibitor*

Control

2,4_Dinitrophenol Fluoride Azide Iodoacetate

33.78 35.59 34.20 41.63

i f f k

Of

+ Inhibitors 3.45 4.08 6.00 5.32

23.22 25.78 28.80 24.17

+ i i f

T/M

2,424 2.40: I.29 5.433:

31 28 16 42

* The final concentration of each inhibitor was 3.3 x 10m3 M. Five experiments were performed with each inhibitor and each experiment was conducted with choroid plexuses from different animals. t T/M ratio was based on the uptake of 9.3 x IO-’ M methadone by choroid plexus for 1hr at 37°C under 0,. . 1:P < 0.05.

Table 2. Effect of ions on the uptake Ions omitted from medium

Number of experiments*

Control

of methadone

T/M k SE.+ medium Ion-omitted

Ca’ Mg’ Ca’ + Mg’ Phosphate Ca’ + EGTA, 3.3 x 10e4~ added Ca’ + EGTA, 3.3 x 10m3 M added

8 3 3 6 4

33.15 25 I5 25. I5 33.65 32.02

3

30.66 i 2.78

* Each experiment

was performed

with choroid

: P < 0.05.

k * * & i

2.62 3.27 3.27 2.10 3.62

25.1 1 18.62 II.11 37.87 21.74

plexus

medium i + f f f

2,90$ 5.131 I,S9$ 4.10 4,351

IS.85 * @73$

plexus from different animals. M methadone by choroid

t T/M ratio was based on the uptake of 9.3 x IO-’ 02.

by choroid

Mean y0 change of T/‘M -24 -29 -56 + 14 -33 -39

plexus for 1 hr at 37°C under

744

J. 7. Hr

A\(;

and

A. E.

TAKI

MOKI

Morphine and narcotic antagonists, naloxone and levallorphan, significantly inhibited the uptake of methadone by the choroid plexus (Table 3). Dextropropoxyphene. which hax a closer structural similarity to methadone. appeared to bc the best inhibitor. Other organic basic compounds such as hexamethonium and A’-methylnicotinamide and an organic acidic compound, probenecid, had no influence on the uptake of methadone. Interestingly, verapamil, which has been suggested as an inhibitor of calcium ion transport in cardiac muscle (FLKKENSTEIN. 1971) was a good inhibitor of the uptake of mcth~~~onc.

Fmal cone 01 compound\ (Ml

C‘ontrol

Morphme Vcrapamll

3.3 x IO ’ 3.3 x IO -b

I;,,,,, i

Sb

Ipmol p

tissue prr hi-)*

h,,, t S.l:.(m\f)

6.X I _t OW

0 52 f

6.39* O~hO 7.5 I _t Ial

0.97 + O.l)X I-V

Ii I4

+ 07y

.~ * Determined by using si.x different medium concentrations of methadone. Four clperiments were performed at each medium concentration and the \;~lucs wcrc c\tlmated from double rcciprocnl plots of the data with the and of ;I computcl -I-P < 045.

In order to characterize the nature of the inhibition of methadone uptake I~! morphine and verapamil and to further compare the uptake systems of morphine and methadone in the choroid plexus. the kinetics of methadone uptake was studied in the absence and presence of morphine or verapamil in the external medium. Both morphine and \,crapamil appeared to be competitive inhibitors of methadone uptake ;IS sect1 h\ the incrcasc in apparent K,, of the system without a change in the maximum rate of ;~pt;\k~ (Table 3). The K; of morphine in these experiments was 4.28 mM and that of verapamil was 0.3 I mu.

DISCUSSION

The present data indicate that methadone is taken up by the choroid plexus b! ;I transport system which displays the usual characteristics of an active transport system. Mcth:\done is accumulated in the choroid plexus against an apparent conccntr:lti~~n gradient and

Methadoneuptake by choroid plexus

245

the uptake system becomes saturated at higher concentrations of methadone. The uptake of methadone is inhibited by other narcotic drugs, narcotic antagonists, low temperature and metabolic inhibitors. The uptake system for methadone in the choroid plexus is very similar to that described for morphine (TAKEMORI and STENWICK, 1966). Another similarity besides the active nature of the transport, is the fact that the uptake of neither morphine nor methadone is affected by quaternary amines such as hexamethonium and N-methylnicotinamide. Additionally, both transport systems are dependent on Ca*+ and Mg*+ ions for full uptake capacity while phosphate ions do not appear to be required. Finally, when morphine is included in the medium together with methadone it acts like a competitive inhibitor of methadone transport. The affinity of the transport system for methadone is higher (K, = 5.2 x 10e4 M) than that for morphine (K, = 1.78 x 10e3 M, CRAIG; rt a/., 1971; In a previous paper in Neropharmacology (CRAIG et al., 1971) this value of K, was erroneously reported as 1.78 PM; the units should have been mM. K, = 2.6 x 10-j M, TAKEMORI, 1968). The higher affinity for methadone is also evidenced by the fact that the Ki of morphine to inhibit the uptake of methadone is nearly 2.5 times the K, for the uptake of morphine. However, the maximum capacity for the uptake of morphine of about 2S30 pmol/g tissue per hr (TAKEMORI and STENWICK. 1966; TAKEMORI. 1968; CRAIG et al.. 1971) is considerably higher than that observed for methadone of about 7 pmol/g tissue per hr. It is interesting that the calcium-transport antagonist, verapamil, also acts as a competitive inhibitor of the uptake of methadone by the choroid plexus, especially since the removal of calcium ions from the incubation medium inhibited the uptake of methadone and morphine. One explanation for these findings is that the uptake of methadone and morphine is somehow coupled to that of calcium ions. If such a coupling exists, compounds which inhibit the transport of calcium ions would be expected to inhibit the transport of narcotic drugs and vice versa. However in vice. calcium ions were shown to inhibit the transport of morphine from blood to cerebrospinal fluid (WANG, 1971). Another possible explanation is that a certain amount of calcium is required merely to maintain the integrity of the transport system in the tissue and verapamil acts as a competitive inhibitor because its structure contains an aliphatic tertiary amine portion. Verapamil is 5-[(3.4dimethoxyphenyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile cotipared with methadone which is 6-dimethylamino-4,4-diphenyl-3-heptanone. Although uptake of substances into isolated choroid plexus is generally assumed to mimic the transport of the substances from cerebrospinal fluid to blood, morphine. which is actively transported by the choroid plexus, is cleared from the ventriculocisternal system by a concentration dependent process consistent with simple diffusion (WANG and TAKF.MORI. 1972a). In contrast, the transport from blood to cerebrospinal, fluid is concentration independent and appears to involve a carrier-mediated transport system (WANC; and TAKEMORI, 1972b). If the transport system for methadone is indeed similar to that for morphine in oivo, methadone might also be expected to be actively transported unidirectionally into the cerebrospinal fluid. In view of the above discussion on calcium it is interesting that the flux of calcium from cerebrospinal fluid to plasma is also a simple diffusional process while that from plasma to cerebrospinal fluid is largely a carrier-mediated one (KATZMAN, GRAZIANI and GINSBURG, 1968). In conclusion, the choroid plexus accumulates methadone by an active transport system which appears similar to that which actively takes up morphine. Calcium ions are somehow involved in this transport process but no definite conclusions can be made with the present data. The fact that morphine transport in vivo is inhibited by calcium ions may suggest that similar transport systems may be involved for these two substances. REFERENCES CRAIG. A. L.. O’DEA, R. F. and TAKFMOKI, A. E. (1971). The uptake

of morphine by the choroid plexus and cereslices of animals chronically treated with morphine. Neuropharmucoloy~ 10: 709-711. FLITKENSTEIN,A. (1971). Specific inhibitors and promoters of calcmm action in the excitation-contraction coupling of heart muscle and their role in the prevention or production of myocardial lesions. In: Culciu~ tr~tl t/w Hrcwt(HARRIS, P. and OPIC, L. Eds.) Academic Press, New York. bral cortical

1-W

J. T. Hr AU<; and A. E. TAKI-MORI

HN;. c‘. c‘.. JK. (1’967). Transport of narcotic analgesics by choroid plexus and kidney tissue irl ritro. Biochutn P /Icw,lIrlc. 16: 34s- 259. KAT~MA\. R.. C;I