J. psychiar. Res.. Vol. 15, pp. 77-84. Pergamon Press Ltd. 1979. Printed in Great
0002-3956;79/0601-0073
$02.00/O
Britain.
A BIOCHEMICAL MEASURE OF MONOAMINE OXIDASE TYPE A AND B INHIBITOR EFFECTS IN MAN IAIN C. CAMPBELL, DAVID J. WILING, STEVEN LIPPER, STANLEY SLATER
and Clinical Neuropharmacology (Received
Abstract-A
DENNIS L. MURPHY Branch, NIMH, Bethesda,
MD 20014, U.S.A.
15 March 1978; in revised form 13 September)
method is described for the measurement of thz effects of monoamine oxidase
inhibitors (MAOI’s) in human plasma. The procedure was used to estimate the levels of oargvline and clornvline in ulasma from patients with affective disorders. The results indicate ihalafter an oral &se, par&line is rapidly absorbed into the blood, reaches a maximum level in approximately 1 hr and has a half life of approximately 2 hr. After chronic administration, neither pargyline nor clorgyline attain steady state levels of MAO inhibiting potential which are higher than those seen 12 hr after a single dose of drug. A single 50 mg dose of pargyline causes greater than 90% inhibition of platelet MAO (in agreement with the observation that platelet MAO is MAO type B); this dose of pargyline does not significantly alter plasma amine oxidase. Clorgyline at the dose used (20 mg) does not alter platelet MAO activity.
THE inhibition of monoamine oxidase (MAO) by certain antidepressant drugs (MAOI’s), with subsequent increases in biogenic amines, forms part of the dogma of aminergic hypotheses of affective disorders. le2 MAO has been shown to exist in two forms, designated A and B on the basis of sensitivity to inhibition by clorgyline:3 MAO type A preferentially deaminates 5-hydroxytryptamine (5-HT) and norepinephrine (NE) and is inhibited by clorgyline, whereas MAO type B preferentially deaminates phenylethylamine (PEA) and is inhibited by deprenyl and pargyline. 4 In the rat brain, MAO is approximately 45 ‘A type B and 55 % type A5 whereas human brain is approximately 80 % type B and 20 % type A (Murphy, unpublished). Studies on specific inhibitors of type A and type B MAO may provide insight into which amines are of etiological importance in affective disorders or of significance in the behavioral responses to these drugs, and lead to the development of MAOI’s which have fewer side effects. In a trial of pargyline vs clorgyline in patients with affective disorders, one problem encountered was that of deciding on dose regimens.6 This was of special importance as the apparent specificity of drug effects on MAO A and MAO B is dose dependent, and thus a large excess of drug may cause inhibition of both forms of the enzyme.’ An assessment of the degree of MAO B inhibition in viva can be obtained by measuring platelet MAO*; there is no corresponding easily available source of MAO type A. To measure the inhibitory effects of both pargyline and clorgyline in plasma we developed a bioassay system. METHODS The assay procedure used involves measuring the inhibition of MAO activity in rat brain homogenates produced by the addition of plasma from patients who were being 77
78
IAIN C. CAMPBELL, DAVID J.SHILING,STEVEN LIPPER,STANLEYSLATERand DENNIS L. MURPHY
treated with pargyline or clorgyline. The rat brain MAO assay used was a modification of the method of ROBINSON et ~1.~ Fifty microliter aliquots of the rat brain enzyme preparation (in triplicate) were pre-incubated with 425~1 of 0.08 M phosphate buffer (pH 7.2) and 100 ul aliquots of plasma for 20 min at room temperature. This preparation was then transferred to an ice bath, 25 ul of radioactivity-labelled substrate was added and the tubes shaken and incubated for 20 min at 37°C in a water bath.lO Aliquots of the rat brain enzyme heated to 100°C for 10 min were assayed simultaneously to establish blank values. Ascorbic acid (lo- 4M) and EDTA (10-4M) were added to the buffer to prevent nonenzymatic alteration of substrates. Following incubation, the samples were placed on ice for 5 min and then transferred to Pasteur pipettes containing 0.5 x 2.5 cm Amberlite resin (CG 50, 100-200 mesh) prepared as previously described. l1 The columns were washed twice with 1 ml of distilled deionized water and the entire 2.5 ml collected in vials containing 17.5 ml of Aquasol (New England Nuclear, NEN). The radioactivity of the eluted products was determined by liquid scintillation spectrometry. As clorgyline inhibits MAO type A, [3H]5-HT was used as a substrate ([3H]5-hydroxytryptamine creatinine sulphate; 55 mCi/mole; final concentration in assay, 10V3M; NEN, Boston, MA). As pargyline inhibits MAO type B, P-phenylethylamine [phenylethylamine HCI, P-(ethyl-l-[14C]); 50.98 mCi/mmol; final concentration in medium 2 x 10e5M; NEN] was used: this system was identical to that described above for 5-HT except that in this case the rat brain enzyme preparation was a 1 in 15 dilution of the original preparation: this dilution was necessary to limit deamination of substrate to less than 10%. Sample preparation
Blood obtained by venipuncture was collected into a 10% ACD solution (acid-citratedextrose, NIH formula A) and centrifuged to obtain platelet-rich plasma (PRP), aliquots of which were used to measure platelet and plasma amine oxidase activity and platelet number@. The remaining PRP was centrifuged at 1500 g for 20 min to obtain plateletpoor plasma which was frozen at -70°C until it was assayed in the rat brain preparation. Occasionally plasma was found to contain extraneous MAO type B activity which was probably due to mitochondrial MAO from disintegrated platelets. This possible source of error can be eliminated by centrifuging the platelet-poor plasma at 50,000 g for 20 min. Pooled platelet free plasma prepared in this manner was used in the establishment of standard curves by adding varying concentrations of the inhibitors in vitro. 100 ul of plasma was used in all assays as high concentrations of plasma proteins alter substrate bindins to the Amberlite resin. Assay of platelet
and plasma amine oxidases
Platelet and plasma amine oxidase activity were assayed using [‘“Cl benzylamine (methyICN Pharmaceuticals, Irvine, CA): the lene-[14C]benzylamine HCl; 4.0 mCi/mmole; [14C]benzaldehyde formed in the reaction was isolated by extraction into heptane.12 Rat brain MAO preparation
Rats (Sprague-Dawley, 6-8 weeks old, male, Taconic Farms, MD) were sacrificed by decapitation and their brains rapidly removed and homogenized in 10 volumes of 0.08 M
PLASMA CHANGES AFTER
MAO1
79
phosphate buffer (pH 7.2) using a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenates were centrifuged at 900 g for 10 min and the supernatant were removed and sonicated for 15 sec. This preparation contains 0.247 mg protein per 50 pl aliquot and if frozen as -70°C can be used as an enzyme source for periods of several months without loss of activity. RESULTS Pargyline and clorgyline added in vitro in varying concentrations to pooled control plasma (NIH Blood Bank) and incubated with rat brain homogenates produced a concentration dependent inhibition of 5HT and PEA deamination by rat brain MAO (Fig. 1). With 5-HT as substrate, ciorgyline had an ID,,, of 5.62 x 10-sM. Using PEA as substrate loo-
9080--
70-
40-
30-
FIG. 1. In vim dose response curves of rat brain MAO inhibition: pargyline and clorgyline were added to the system in varying concentrations dissolved in 100 1.11of blood bank plasma. O---O Inhibition of deamination of 13H]5-HT by clorgyline; Q--a inhibition of deamination of [“CIPEA by pargyline; a---n inhibition of deamination of [WIPEA by clorgyline. The IDSo of pargyline with [14C]PEA is 3.16 x lO-*M; the ID,,, of clorgyline with [SH]5-HT is 5.62 x 10W8M and the ID,, of clorgyline with [‘YJPEA is 1.26 x 10m6M. Values are the mean of 6 samples and the S.E.M. in each case was less than 5 %. To increase the accuracy of the measurement of plasma values from these graphs the X-axis can be lengthened and also the values in the range used can be expressed as actual concentrations rather than as logarithms.
80
IAIN C.
CAMPBELL,DAVID J.
SHILING, STEVENLIPPER,STANLEYSLATERand DENNIS L. MURPHY
pargyline
had an ID,, o f 3.16 x 10W8M. In contrast PEA deamination was markedly insensitive to inhibition by clorgyline (ID,, = 1.26 x 10-6M). ., Plasma samples were obtained over a 24-hr period from patients who received a single 50 mg does of pargyline. 1OOl.daliquots of plasma from these patients were incubated with rat brain MAO and [14C]PEA and the resultant inhibition of deamination was measured (Fig. 1). In all cases the inhibition reached a maximum after approximately 1 hr and decreased to 50 y0 of this level after 2 hr and to less than 25 % after 6 hr. There was a large variation in the maximum inhibition produced in the different individuals, with an observed range from approximately 40 to 95 %. This variation is evidently not related to body weight as the two plasma samples causing greatest inhibition came from the two heavier patients.
‘@I
I 2
1 4
I 6
I 8
I
I
I
I
10
12
14
16
Time bufs)
I
18
I
20
I
22
I
24
FIG. 2.
Inhibition of [‘“Cl phenylethylamine deamination in rat brain, produced by the addition of 100 ~1 of platelet poor plasma from 4 patients who had received a single 50 mg dose of pargyline and who had blood drawn at times shown on the graph.
By 24 hr, the levels of induced inhibition are reduced to l&20%. By assuming that the observed MAO inhibition directly reflected plasma concentrations of clorgyline and pargyline, it can be calculated from the dose response data in Fig. 1 that the levels of pargyline in plasma over a 24-hr period follow the curves shown in Fig. 3. Using this data, it can be seen that the maximum estimated pargyline plasma levels range from 25 ng/ml to 160 ng/ml. Comparable data for clorgyline (using 5-HT as substrate) indicates that after a single oral dose (20 mg) the plasma levels of drug are low, reaching a maximum of 10 pg/ml (Fig. 4).
PLASMACHANGES
AFTERMAOI
81
The effects of a single dose of pargyline and plasma amine oxidase activity are shown in Fig. 5. The drug causes rapid and prolonged inhibition of the platelet enzyme even though the data presented above strongly suggests that the drug is rapidly cleared from blood-in keeping with other evidence that this drug is an irreversible inhibitor of mitochondrial MAO activity.
l&
160-
130-
EO( 110-j
loo
,
I I
TIME hours)
Fro. 3. Estimatea pargyline levels in plasma from 4 patients who had received a single oral dose (50 mg) of the drug.
Plasma from patients receiving pargyline (75-l 50 mg/day) and clorgyline (20-40 mg/day) for periods of several weeks was found to not significantly inhibit either phenylethylamine or 5-HT deamination in rat brain homogenates, when plasma was collected at 9 a.m., 11 hr after the last dose of drug.
82
IAIN
C.
CAMPBELL,
J.
DAVID
SHILING, STEVEN LIPPER, STANLEY SLATER and DENNIS L. MURPHY
2
4
6 l-II
8 kw5)
10
12
24
FIG. 4. Inhibition [SH]5-HT deamination in rat brain produced by the addition of 100 ul of plasma from two patients who had received a single oral dose of clorgyline (20 mg) and who had blood drawn at times shown.
I i;
70 -1
F
60 -\
z zg
4
I
I
50 -I 40-I 3020-
I 1 ;
10
’
_________------------------4
o. 1_y__7__; 9
14 TIME
FIG. 5.
19
24
(hours)
Platelet and plasma amine oxidase activity in 4 patients who received a single dose of pargyline (50 mg). Platelet MAO a- - -0; plasma amine oxidase 0-O.
DISCUSSION This study provides data indicating that sequentially drawn plasma from pargylinetreated patients inhibits rat brain MAO activity with a regular time course and with fairly large differences in inhibition between individuals receiving the same dose of the drug. This bioassay system may provide an index of individual differences in plasma levels
kk~m
&4NOeS
hER
ht.401
83
and inhibitory capacities of this drug. In contrast, plasma from clorgyline-treated individuals led to barely detectable inhibition of rat brain MAO. It may be that the higher lipophilicity of clorgyline leads to a more rapid disappearance of the drug from plasma. The rapid rise and fall in the inhibitory potency in the plasma from pargyline-treated patients is in agreement with data from gas chromatograph electron capture assays of another MAOI, tranylcypromine, where a similar pattern was observed.13 There are apparently no other published reports of plasma levels of MAOI. Except in one case where a patient complained of severe side effects, no detectable MAO inhibitor effects were found in plasma of subjects 11 hr after the last dose of drug even after treatment for 14 days. BASELTet al. l4 found no evidence for steady state plasma levels of tranylcypromine in patients after 18 days of treatment: in these patients the average plasma concentrations were 5-10 ng/ml (24 hr after the last dose) which is approximately what we estimated for the pargyline-treated subjects. ROBINSONand NIES (personal communication)15 have recently found comparable low plasma levels of phenelzine (Nardil) 12 hr after a 75 mg dose of drug. These results indicate that unlike tricyclic antidepressant drugs (such as imipramine, which may attain steady state levels of 210 f 110 ng/ml following a 3.5 mg/kg/day regimen)16 MAOI’s do not attain measurable steady state levels in plasma. This finding indicates that physiological measures of MAO inhibition, e.g. changes in platelet MAO activity or in urinary amine metabolite excretion, may be more suitable as possible biochemical correlates of drug-related behavioral changes than the levels of drug in plasma. The difference between the two drug groups in this respect may be related to the fact that MAOI’s (unlike the tricyclic antidepressants) form, covalent bonds with proteins and thus probably have a higher association with tissues. Alternatively it may be related to a more rapid rate of catabolism (eg. by acetylation). The variation in maximum levels of plasma drug concentrations might be related to individual plasma protein binding or other differences. This issue could be addressed by removing an individual protein binding or other plasma during a drug-free period and adding it to a rat brain MAO and pargyline sample in vitro and measured the individual effect on MAO inhibition. In this study, a possible measure of MAO inhibitors in plasma has been described. However, until the actual drug concentrations are determined by a direct assay procedure there are certain constraints which should be considered. The possibility exists that the observed inhibitory effects may be caused in part by some metabolite of the MAOI’s as well as the drugs themselves. This seems unlikely as the inhibitory effects are maximal within 1 hr of oral drug administration and thus most likely represents a direct drug effect. Furthermore, if a metabolite was also a MAOI, then one might have expected a less rapid fall in inhibitory potency or even a bimodal effect. The possibility also exists that the drugs produced the inhibition in rat brain MAO via some indirect mechanism, e.g. from elevation of amines or other substrates and/or inhibitors in plasma. The lowest concentration of substrate used in the assays was 2 x 10e5M with PEA, It is unlikely that endogenous amines such as tyramine would reach concentrations capable of causing competitive inhibition in the assay because plasma levels of such amines are several orders of magnitude lower (lo- ‘-lo-*M). The method described appears to be more suited to the measurement of the effects of
84
LAINC. CAMPBELL, DAVIDJ. SHILIN~,STEVENLIPPER,STANLEYSLATERand DENM~L. MURP~~
pargyline than clorgyline. In attempting studies of other MAOI’s, e.g. tranylcypromine or phenelzine, where both forms of MAO are inhibited, it is suggested that the standard curves be made using phenylethylamine as substrate. It might be possible to use the technique to measure drug effects in other fluids such as C.S.F. Acknowledgement-The authors wish to thank Ms. PHYLLISPINKESTfor technical assistance and Mrs. IRENE BELLE~KYfor typing the manuscript. REFERENCES 1. COPPEN,A. The biochemistry of affective disorders, Br. J. Psych& 113, 1237, 1967. 2. SCHILDKRAUT, J. J. Catecholamine hypothesis of affective disorders: Review of supporting evidence. Am. J. Psychiat. 122, 509, 1965. 3. JOHNSTONE,J. P. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. BZochem. Pharmac. 17, 1285, 1968. 4. KNOLL, J. and MAGYAR-K. Some puzzling pharmacological effects of MAO inhibitors. In: Advances in Biochemical Psychopharmacology, COSTA,E. and GREENOAFW,P. (Editors), Raven Press, New York, 1972. 5. NEFF, N. H., YANG, H.-Y. T. and Goarms, C. Degradation of the transmitter amines by specific types of monoamine oxidases. In: Frontiers in Catecholamine Research, DD. __ 133-137. Pernamon Press, London, 1973. 6. MURPHY, D. L., LIPPER, S., SLATER,S. and SHILING,D. Selectivity of clorgyline and pargyline as inhibitors of monoamine oxidases A and B in vivo in man. Psychopharmacology (in press). 7. CAMPBELL,I. C., ROBINSON,D. S., LOVENBERG,W. M. and MURPHY, D. L. The effects of chronic regimens of clorgyline and pargyline on monoamine metabolism in the rat brain. J. Neurochem. 32, 49.1979. 8. MURPHY,D. L. and DONNELLY,C. H. Monoamine oxidase in man: Enzyme characteristics in platelets, plasma and other human tissues. In: NeuropsychopharmacoZogy of Monoamines and Their Regulatory Enzymes, Raven Press, New York, 1972. 9. ROBINSON,D. S., LOVENBWG,W., KEISER, H. and SJOERDSMA, A.. Effects of drugs on human blood platelet and plasma amine oxidase activity in vitro and in vivo. Biochem. Pharmac. 17, 109, 1968. 10. DONNELLY,C. H. and MURPHY, D. L. Substrate and inhibitor-related characteristics of human platelet monoamine oxidase. Biochem. Pharmac. 26, 853, 1977. 11. DONNELLY,C. H., RICHELSON, E. and MURPHY, D. L. Properties of monoamine oxidase in mouse neuroblastoma NlE-115 cells. Biochem. Pharmac. 25, 1639, 1977. 12. MURPHY, D. L., WRIGHT, C., BUCHSBAUM,M., NICHOLS;A., C?OSTA, 3. L. and WYA~, R. 3. Platelet and plasma amine in 680 normals: Sex and age differences and stability over time. Biochem. Med. 16, 254, 1976. 13. BASELT,R. C., SHAXAN, E. and GROSS,E. M. Tranylcypromine concentrations and monoamine oxidase activity in tissues from a fatal poisoning. J. Analyt. Toxicol. 1, 168, 1977. 14. BASELT,R. C., STEWARD,C. B. and SHASKAN,E. Determination of therapeutic concentrations of tranylcypromine in human serum and urine by electron-captive gas liquid chromatography. J. Analyt. Toxicol. (in mess). 15. ROBINSON,D. S. and NIES. A. Personal communication. 16. PEREL, J. M., SHOSTAK,M., GANN, E., KANTOR, S. J. and GLASSMAN,A. H. Pharmacodynamics of imipramine and clincial outcome in patients. In: Pharmacokinetics of Psychoactive Drugs, GOTTSCHALK,L. A. and MERLIS, S. (Editors), Holsted Press, New York, 1976.
0002-3956;79/0601-0073
$02.00/O
Britain.
A BIOCHEMICAL MEASURE OF MONOAMINE OXIDASE TYPE A AND B INHIBITOR EFFECTS IN MAN IAIN C. CAMPBELL, DAVID J. WILING, STEVEN LIPPER, STANLEY SLATER
and Clinical Neuropharmacology (Received
Abstract-A
DENNIS L. MURPHY Branch, NIMH, Bethesda,
MD 20014, U.S.A.
15 March 1978; in revised form 13 September)
method is described for the measurement of thz effects of monoamine oxidase
inhibitors (MAOI’s) in human plasma. The procedure was used to estimate the levels of oargvline and clornvline in ulasma from patients with affective disorders. The results indicate ihalafter an oral &se, par&line is rapidly absorbed into the blood, reaches a maximum level in approximately 1 hr and has a half life of approximately 2 hr. After chronic administration, neither pargyline nor clorgyline attain steady state levels of MAO inhibiting potential which are higher than those seen 12 hr after a single dose of drug. A single 50 mg dose of pargyline causes greater than 90% inhibition of platelet MAO (in agreement with the observation that platelet MAO is MAO type B); this dose of pargyline does not significantly alter plasma amine oxidase. Clorgyline at the dose used (20 mg) does not alter platelet MAO activity.
THE inhibition of monoamine oxidase (MAO) by certain antidepressant drugs (MAOI’s), with subsequent increases in biogenic amines, forms part of the dogma of aminergic hypotheses of affective disorders. le2 MAO has been shown to exist in two forms, designated A and B on the basis of sensitivity to inhibition by clorgyline:3 MAO type A preferentially deaminates 5-hydroxytryptamine (5-HT) and norepinephrine (NE) and is inhibited by clorgyline, whereas MAO type B preferentially deaminates phenylethylamine (PEA) and is inhibited by deprenyl and pargyline. 4 In the rat brain, MAO is approximately 45 ‘A type B and 55 % type A5 whereas human brain is approximately 80 % type B and 20 % type A (Murphy, unpublished). Studies on specific inhibitors of type A and type B MAO may provide insight into which amines are of etiological importance in affective disorders or of significance in the behavioral responses to these drugs, and lead to the development of MAOI’s which have fewer side effects. In a trial of pargyline vs clorgyline in patients with affective disorders, one problem encountered was that of deciding on dose regimens.6 This was of special importance as the apparent specificity of drug effects on MAO A and MAO B is dose dependent, and thus a large excess of drug may cause inhibition of both forms of the enzyme.’ An assessment of the degree of MAO B inhibition in viva can be obtained by measuring platelet MAO*; there is no corresponding easily available source of MAO type A. To measure the inhibitory effects of both pargyline and clorgyline in plasma we developed a bioassay system. METHODS The assay procedure used involves measuring the inhibition of MAO activity in rat brain homogenates produced by the addition of plasma from patients who were being 77
78
IAIN C. CAMPBELL, DAVID J.SHILING,STEVEN LIPPER,STANLEYSLATERand DENNIS L. MURPHY
treated with pargyline or clorgyline. The rat brain MAO assay used was a modification of the method of ROBINSON et ~1.~ Fifty microliter aliquots of the rat brain enzyme preparation (in triplicate) were pre-incubated with 425~1 of 0.08 M phosphate buffer (pH 7.2) and 100 ul aliquots of plasma for 20 min at room temperature. This preparation was then transferred to an ice bath, 25 ul of radioactivity-labelled substrate was added and the tubes shaken and incubated for 20 min at 37°C in a water bath.lO Aliquots of the rat brain enzyme heated to 100°C for 10 min were assayed simultaneously to establish blank values. Ascorbic acid (lo- 4M) and EDTA (10-4M) were added to the buffer to prevent nonenzymatic alteration of substrates. Following incubation, the samples were placed on ice for 5 min and then transferred to Pasteur pipettes containing 0.5 x 2.5 cm Amberlite resin (CG 50, 100-200 mesh) prepared as previously described. l1 The columns were washed twice with 1 ml of distilled deionized water and the entire 2.5 ml collected in vials containing 17.5 ml of Aquasol (New England Nuclear, NEN). The radioactivity of the eluted products was determined by liquid scintillation spectrometry. As clorgyline inhibits MAO type A, [3H]5-HT was used as a substrate ([3H]5-hydroxytryptamine creatinine sulphate; 55 mCi/mole; final concentration in assay, 10V3M; NEN, Boston, MA). As pargyline inhibits MAO type B, P-phenylethylamine [phenylethylamine HCI, P-(ethyl-l-[14C]); 50.98 mCi/mmol; final concentration in medium 2 x 10e5M; NEN] was used: this system was identical to that described above for 5-HT except that in this case the rat brain enzyme preparation was a 1 in 15 dilution of the original preparation: this dilution was necessary to limit deamination of substrate to less than 10%. Sample preparation
Blood obtained by venipuncture was collected into a 10% ACD solution (acid-citratedextrose, NIH formula A) and centrifuged to obtain platelet-rich plasma (PRP), aliquots of which were used to measure platelet and plasma amine oxidase activity and platelet number@. The remaining PRP was centrifuged at 1500 g for 20 min to obtain plateletpoor plasma which was frozen at -70°C until it was assayed in the rat brain preparation. Occasionally plasma was found to contain extraneous MAO type B activity which was probably due to mitochondrial MAO from disintegrated platelets. This possible source of error can be eliminated by centrifuging the platelet-poor plasma at 50,000 g for 20 min. Pooled platelet free plasma prepared in this manner was used in the establishment of standard curves by adding varying concentrations of the inhibitors in vitro. 100 ul of plasma was used in all assays as high concentrations of plasma proteins alter substrate bindins to the Amberlite resin. Assay of platelet
and plasma amine oxidases
Platelet and plasma amine oxidase activity were assayed using [‘“Cl benzylamine (methyICN Pharmaceuticals, Irvine, CA): the lene-[14C]benzylamine HCl; 4.0 mCi/mmole; [14C]benzaldehyde formed in the reaction was isolated by extraction into heptane.12 Rat brain MAO preparation
Rats (Sprague-Dawley, 6-8 weeks old, male, Taconic Farms, MD) were sacrificed by decapitation and their brains rapidly removed and homogenized in 10 volumes of 0.08 M
PLASMA CHANGES AFTER
MAO1
79
phosphate buffer (pH 7.2) using a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenates were centrifuged at 900 g for 10 min and the supernatant were removed and sonicated for 15 sec. This preparation contains 0.247 mg protein per 50 pl aliquot and if frozen as -70°C can be used as an enzyme source for periods of several months without loss of activity. RESULTS Pargyline and clorgyline added in vitro in varying concentrations to pooled control plasma (NIH Blood Bank) and incubated with rat brain homogenates produced a concentration dependent inhibition of 5HT and PEA deamination by rat brain MAO (Fig. 1). With 5-HT as substrate, ciorgyline had an ID,,, of 5.62 x 10-sM. Using PEA as substrate loo-
9080--
70-
40-
30-
FIG. 1. In vim dose response curves of rat brain MAO inhibition: pargyline and clorgyline were added to the system in varying concentrations dissolved in 100 1.11of blood bank plasma. O---O Inhibition of deamination of 13H]5-HT by clorgyline; Q--a inhibition of deamination of [“CIPEA by pargyline; a---n inhibition of deamination of [WIPEA by clorgyline. The IDSo of pargyline with [14C]PEA is 3.16 x lO-*M; the ID,,, of clorgyline with [SH]5-HT is 5.62 x 10W8M and the ID,, of clorgyline with [‘YJPEA is 1.26 x 10m6M. Values are the mean of 6 samples and the S.E.M. in each case was less than 5 %. To increase the accuracy of the measurement of plasma values from these graphs the X-axis can be lengthened and also the values in the range used can be expressed as actual concentrations rather than as logarithms.
80
IAIN C.
CAMPBELL,DAVID J.
SHILING, STEVENLIPPER,STANLEYSLATERand DENNIS L. MURPHY
pargyline
had an ID,, o f 3.16 x 10W8M. In contrast PEA deamination was markedly insensitive to inhibition by clorgyline (ID,, = 1.26 x 10-6M). ., Plasma samples were obtained over a 24-hr period from patients who received a single 50 mg does of pargyline. 1OOl.daliquots of plasma from these patients were incubated with rat brain MAO and [14C]PEA and the resultant inhibition of deamination was measured (Fig. 1). In all cases the inhibition reached a maximum after approximately 1 hr and decreased to 50 y0 of this level after 2 hr and to less than 25 % after 6 hr. There was a large variation in the maximum inhibition produced in the different individuals, with an observed range from approximately 40 to 95 %. This variation is evidently not related to body weight as the two plasma samples causing greatest inhibition came from the two heavier patients.
‘@I
I 2
1 4
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I 8
I
I
I
I
10
12
14
16
Time bufs)
I
18
I
20
I
22
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24
FIG. 2.
Inhibition of [‘“Cl phenylethylamine deamination in rat brain, produced by the addition of 100 ~1 of platelet poor plasma from 4 patients who had received a single 50 mg dose of pargyline and who had blood drawn at times shown on the graph.
By 24 hr, the levels of induced inhibition are reduced to l&20%. By assuming that the observed MAO inhibition directly reflected plasma concentrations of clorgyline and pargyline, it can be calculated from the dose response data in Fig. 1 that the levels of pargyline in plasma over a 24-hr period follow the curves shown in Fig. 3. Using this data, it can be seen that the maximum estimated pargyline plasma levels range from 25 ng/ml to 160 ng/ml. Comparable data for clorgyline (using 5-HT as substrate) indicates that after a single oral dose (20 mg) the plasma levels of drug are low, reaching a maximum of 10 pg/ml (Fig. 4).
PLASMACHANGES
AFTERMAOI
81
The effects of a single dose of pargyline and plasma amine oxidase activity are shown in Fig. 5. The drug causes rapid and prolonged inhibition of the platelet enzyme even though the data presented above strongly suggests that the drug is rapidly cleared from blood-in keeping with other evidence that this drug is an irreversible inhibitor of mitochondrial MAO activity.
l&
160-
130-
EO( 110-j
loo
,
I I
TIME hours)
Fro. 3. Estimatea pargyline levels in plasma from 4 patients who had received a single oral dose (50 mg) of the drug.
Plasma from patients receiving pargyline (75-l 50 mg/day) and clorgyline (20-40 mg/day) for periods of several weeks was found to not significantly inhibit either phenylethylamine or 5-HT deamination in rat brain homogenates, when plasma was collected at 9 a.m., 11 hr after the last dose of drug.
82
IAIN
C.
CAMPBELL,
J.
DAVID
SHILING, STEVEN LIPPER, STANLEY SLATER and DENNIS L. MURPHY
2
4
6 l-II
8 kw5)
10
12
24
FIG. 4. Inhibition [SH]5-HT deamination in rat brain produced by the addition of 100 ul of plasma from two patients who had received a single oral dose of clorgyline (20 mg) and who had blood drawn at times shown.
I i;
70 -1
F
60 -\
z zg
4
I
I
50 -I 40-I 3020-
I 1 ;
10
’
_________------------------4
o. 1_y__7__; 9
14 TIME
FIG. 5.
19
24
(hours)
Platelet and plasma amine oxidase activity in 4 patients who received a single dose of pargyline (50 mg). Platelet MAO a- - -0; plasma amine oxidase 0-O.
DISCUSSION This study provides data indicating that sequentially drawn plasma from pargylinetreated patients inhibits rat brain MAO activity with a regular time course and with fairly large differences in inhibition between individuals receiving the same dose of the drug. This bioassay system may provide an index of individual differences in plasma levels
kk~m
&4NOeS
hER
ht.401
83
and inhibitory capacities of this drug. In contrast, plasma from clorgyline-treated individuals led to barely detectable inhibition of rat brain MAO. It may be that the higher lipophilicity of clorgyline leads to a more rapid disappearance of the drug from plasma. The rapid rise and fall in the inhibitory potency in the plasma from pargyline-treated patients is in agreement with data from gas chromatograph electron capture assays of another MAOI, tranylcypromine, where a similar pattern was observed.13 There are apparently no other published reports of plasma levels of MAOI. Except in one case where a patient complained of severe side effects, no detectable MAO inhibitor effects were found in plasma of subjects 11 hr after the last dose of drug even after treatment for 14 days. BASELTet al. l4 found no evidence for steady state plasma levels of tranylcypromine in patients after 18 days of treatment: in these patients the average plasma concentrations were 5-10 ng/ml (24 hr after the last dose) which is approximately what we estimated for the pargyline-treated subjects. ROBINSONand NIES (personal communication)15 have recently found comparable low plasma levels of phenelzine (Nardil) 12 hr after a 75 mg dose of drug. These results indicate that unlike tricyclic antidepressant drugs (such as imipramine, which may attain steady state levels of 210 f 110 ng/ml following a 3.5 mg/kg/day regimen)16 MAOI’s do not attain measurable steady state levels in plasma. This finding indicates that physiological measures of MAO inhibition, e.g. changes in platelet MAO activity or in urinary amine metabolite excretion, may be more suitable as possible biochemical correlates of drug-related behavioral changes than the levels of drug in plasma. The difference between the two drug groups in this respect may be related to the fact that MAOI’s (unlike the tricyclic antidepressants) form, covalent bonds with proteins and thus probably have a higher association with tissues. Alternatively it may be related to a more rapid rate of catabolism (eg. by acetylation). The variation in maximum levels of plasma drug concentrations might be related to individual plasma protein binding or other differences. This issue could be addressed by removing an individual protein binding or other plasma during a drug-free period and adding it to a rat brain MAO and pargyline sample in vitro and measured the individual effect on MAO inhibition. In this study, a possible measure of MAO inhibitors in plasma has been described. However, until the actual drug concentrations are determined by a direct assay procedure there are certain constraints which should be considered. The possibility exists that the observed inhibitory effects may be caused in part by some metabolite of the MAOI’s as well as the drugs themselves. This seems unlikely as the inhibitory effects are maximal within 1 hr of oral drug administration and thus most likely represents a direct drug effect. Furthermore, if a metabolite was also a MAOI, then one might have expected a less rapid fall in inhibitory potency or even a bimodal effect. The possibility also exists that the drugs produced the inhibition in rat brain MAO via some indirect mechanism, e.g. from elevation of amines or other substrates and/or inhibitors in plasma. The lowest concentration of substrate used in the assays was 2 x 10e5M with PEA, It is unlikely that endogenous amines such as tyramine would reach concentrations capable of causing competitive inhibition in the assay because plasma levels of such amines are several orders of magnitude lower (lo- ‘-lo-*M). The method described appears to be more suited to the measurement of the effects of
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LAINC. CAMPBELL, DAVIDJ. SHILIN~,STEVENLIPPER,STANLEYSLATERand DENM~L. MURP~~
pargyline than clorgyline. In attempting studies of other MAOI’s, e.g. tranylcypromine or phenelzine, where both forms of MAO are inhibited, it is suggested that the standard curves be made using phenylethylamine as substrate. It might be possible to use the technique to measure drug effects in other fluids such as C.S.F. Acknowledgement-The authors wish to thank Ms. PHYLLISPINKESTfor technical assistance and Mrs. IRENE BELLE~KYfor typing the manuscript. REFERENCES 1. COPPEN,A. The biochemistry of affective disorders, Br. J. Psych& 113, 1237, 1967. 2. SCHILDKRAUT, J. J. Catecholamine hypothesis of affective disorders: Review of supporting evidence. Am. J. Psychiat. 122, 509, 1965. 3. JOHNSTONE,J. P. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. BZochem. Pharmac. 17, 1285, 1968. 4. KNOLL, J. and MAGYAR-K. Some puzzling pharmacological effects of MAO inhibitors. In: Advances in Biochemical Psychopharmacology, COSTA,E. and GREENOAFW,P. (Editors), Raven Press, New York, 1972. 5. NEFF, N. H., YANG, H.-Y. T. and Goarms, C. Degradation of the transmitter amines by specific types of monoamine oxidases. In: Frontiers in Catecholamine Research, DD. __ 133-137. Pernamon Press, London, 1973. 6. MURPHY, D. L., LIPPER, S., SLATER,S. and SHILING,D. Selectivity of clorgyline and pargyline as inhibitors of monoamine oxidases A and B in vivo in man. Psychopharmacology (in press). 7. CAMPBELL,I. C., ROBINSON,D. S., LOVENBERG,W. M. and MURPHY, D. L. The effects of chronic regimens of clorgyline and pargyline on monoamine metabolism in the rat brain. J. Neurochem. 32, 49.1979. 8. MURPHY,D. L. and DONNELLY,C. H. Monoamine oxidase in man: Enzyme characteristics in platelets, plasma and other human tissues. In: NeuropsychopharmacoZogy of Monoamines and Their Regulatory Enzymes, Raven Press, New York, 1972. 9. ROBINSON,D. S., LOVENBWG,W., KEISER, H. and SJOERDSMA, A.. Effects of drugs on human blood platelet and plasma amine oxidase activity in vitro and in vivo. Biochem. Pharmac. 17, 109, 1968. 10. DONNELLY,C. H. and MURPHY, D. L. Substrate and inhibitor-related characteristics of human platelet monoamine oxidase. Biochem. Pharmac. 26, 853, 1977. 11. DONNELLY,C. H., RICHELSON, E. and MURPHY, D. L. Properties of monoamine oxidase in mouse neuroblastoma NlE-115 cells. Biochem. Pharmac. 25, 1639, 1977. 12. MURPHY, D. L., WRIGHT, C., BUCHSBAUM,M., NICHOLS;A., C?OSTA, 3. L. and WYA~, R. 3. Platelet and plasma amine in 680 normals: Sex and age differences and stability over time. Biochem. Med. 16, 254, 1976. 13. BASELT,R. C., SHAXAN, E. and GROSS,E. M. Tranylcypromine concentrations and monoamine oxidase activity in tissues from a fatal poisoning. J. Analyt. Toxicol. 1, 168, 1977. 14. BASELT,R. C., STEWARD,C. B. and SHASKAN,E. Determination of therapeutic concentrations of tranylcypromine in human serum and urine by electron-captive gas liquid chromatography. J. Analyt. Toxicol. (in mess). 15. ROBINSON,D. S. and NIES. A. Personal communication. 16. PEREL, J. M., SHOSTAK,M., GANN, E., KANTOR, S. J. and GLASSMAN,A. H. Pharmacodynamics of imipramine and clincial outcome in patients. In: Pharmacokinetics of Psychoactive Drugs, GOTTSCHALK,L. A. and MERLIS, S. (Editors), Holsted Press, New York, 1976.
