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Metabolic disposition of 2-phenylethylamine and the role of depression in methadone-dependent and detoxified patients
Drug and Alcohol Dependence, 1 (1975/76) @ Elsevier Sequoia S.A., Lausanne - Printed
METABOLIC DISPOSITION OF 2-PHENYLETHYLAMINE ROLE OF DEPRESSION IN METHADONE-DEPENDENT DETOXIFIED PATIENTS*
EDET
Diseases and Mental Health, Friendship Chicago, Illinois 60643, and Addiction Research Street, Brooklyn, New York (U.S.A.)
J. PRIMM
Addiction (U.S.A.) FRANK Illinois
AND THE AND
E. INWANG
Division for the Treatment of Addictive Medical Center, 850 West 103rd Street, and Treatment Corporation,. 22 Chapel BENY
295
295 - 303 in the Netherlands
Research
and Treatment
de L. JONES, State Psychiatric
CYNTHIA
Corporation,
HAROUTUNE Institute,
1601
22 Chapel
DEKIRMENJIAN West Taylor
Street,
Brooklyn,
New
York
and JOHN M. DAVIS
Street,
Chicago,
Illinois
(U.S.A.)
of Medicine,
Chicago,
Illinois
(U.S.A.)
T. HENDERSON
University
of Illinois
(Received
October
Abraham
Lincoln
College
20, 1975)
Summary We have now postulated that differences in the innate capacity of individuals to synthesize, store and utilize biogenic amines may provide the biological basis for human abuse of narcotic and other drugs, and that these drugs are used in an apparent unconscious effort to self-medicate against an inherent affective disorder. In this communication, we attempted a preliminary characterization of the narcotics withdrawal syndrome on biochemical and clinical parameters. Abstinence was found to be characterized by low urinary excretion of 2-phenylethylamine and depression. An indication for use of tricyclic drugs has been discussed.
Introduction If indeed neuropsychotropic drugs modulate behavior by altering central amine@ systems [ 1, 21, then it should be expected that narcotic drugs exert their action, at least in part, through similar systems. It has been reported that in animals acute administration of morphine not only causes release of catecholamines (CA) from adrenal and from central sympathetic centers [ 3 - 51, but it also increases their turnover rate; the amines return to *Presented in part at the 37th Annual Scientific of Sciences, Washington, D. C., May 19 - 21, 1975.
Meeting
of the National
Academy
296
baseline levels during chronic treatment [ 61. Similarly, increase in the turnover rate of serotonin (5-HT) has been reported for morphine-dependent rats and mice [ 61. More recently, even the putative neurotransmitter gamma-aminobutyric acid (GABA) has also been associated with the process of morphine-dependence [7]. Therefore, if biogenic amines play any real role in the process of drug-dependence, the previously studied amines are not likely to be the only candidates. In fact, several chemically functional adrenergic neurons in the CNS are known to exist and help to explain the biphasic stimulant and depressant effects of neurotropic drugs [ 81. Moreover, we have recently elicited a similar biphasic effect with methadone hydrochloride and L-alpha acetylmethadol (LAAM) [ 91. It is highly unlikely, however, that any of these different classes of biogenic amines would individually explain physical dependence. It is more logical to presume that a modulating phenomenon exists involving an interaction between several amines and their metabolites. Our motivation to conduct the present studies was based on two major premises. Firstly, since mood and affect are modulated by adrenergic amines [l, 2, 10 - 121 and narcotic drugs as well as alcohol are known to modulate the levels of these amines [ 3 - 71, we postulated that differences in the baseline capacity of individuals to synthesize biogenic amines may provide the biological basis for human abuse of narcotic and other drugs. This hypothesis implies that those individuals with low capacity to synthesize adequate amounts of ergo tropic adrenergic amines have an innate predisposition to use narcotic drugs in an apparent unconscious effort to self-medicate against an inherent affective disorder. Of course, it is difficult to determine the validity of such a proposition, since there is no way to ascertain the baseline levels of amines in drug-dependent individuals prior to their initial use of the drugs. We, therefore, felt that the study of these amines during withdrawal might give us some clue to the problem. Secondly, there are no reports prior to this one on the metabolic disposition of 2-phenylethylamine (PEA) in narcotic dependent or methadonl maintained patients, nor are there any which attempt to characterize the abstinence syndrome on combined biochemical and clinical parameters. Thus in this paper we report for the first time the possible role of PEA in the modulation of both the affective and probably some non-affective components of the abstinence syndrome.
Experimental Male and female volunteers, between the ages of 22 years and 24 years, presenting with a history of heroin-dependence and, subsequently, stabilized on varying doses of methadone hydrochloride (60 mg - 30 mg) for varying periods between one to two years, were used for these studies. Prospective candidates were screened for their motivation towards detoxification, absence of major organic or psychiatric disorders, in particular psychosis, and
297
any tendencies towards violent behavior. Selected patients were then hospitalized under strict supervision in a metabolic research unit and placed on a VMA (vanillyl mandelic acid) exclusion diet. In the first experimental regimen, twenty-four hour urine specimens were collected daily during (1) a two-week stable methadone maintenance period, (2) a two-week gradual detoxification period and (3) a two-week post detoxification period. Detoxification was achieved by equal and daily decrements of methadone until zero dose was reached for each patient under study. In the second experimental regimen, twenty-four hour urine samples were collected as indicated during a two-week stable methadone administration; subsequent to that, methadone was stopped abruptly (cold turkey detoxification) and daily twenty-four hour urines were collected during the following three weeks. Urine specimens were preserved either by the addition of sodium metabisulfite or by adding sufficient cont. HCl to bring the pH to 1.0. The bottles were stored frozen at a temperature of --16 “C until used. PEA was extracted from 100 ml aliquot portions of urine at pH 12.5 into spectrograde n-hexane and subsequently extracted into 1 ml 1N HCl. The pH of the acid extract was then adjusted to 6.0 with a 0.025 M tris-maleate buffer solution. Urinary content of PEA in the acid extract was determined using a modification of the homogeneous enzyme multiplied immunoassay technique (EMIT) of Rubenstein et al. [ 131, developed by Syva Corporation for the determination of amphetamines in urine. Note that it is essential to carry out the specific extraction procedure for PEA as previously described by Inwang et al. [14] to eliminate other biogenic amines and amphetamines in order to avoid cross reactivity. With the use of a calibrated micropipette apparatus, 50 ~1 of the acid extract of PEA was dispensed into a cuvette containing 250 ~1 of an enzymeactivated antibody solution. PEA competes with the antibody for the enzyme active site resulting in precipitation, and the optical density of the solution was determined at a wavelength of 436 nm using a manual Gilford spectrophotometer thermally regulated at 37 “C. The quantity of PEA in the extract was extrapolated from a standard curve obtained using authentic PEA prepared in the same manner as were the experimental samples. The PEA content of urines of healthy, control subjects within the same age range and sex as the patients was determined in a similar manner for comparison. In addition, daily depression indices using Beck’s depression inventory [ 151 as well as nurses ratings using an incremental 1 - 15 scale were obtained throughout the entire experimental period. Withdrawal symptoms were also recorded daily throughout the entire hospital course. These experiments were double blind so that nurses who scored the symptoms did not know the medication regimen, nor did the analytical team know which urines were being assayed. The codes were broken only after complete biochemical assay of the urines.
298 I IO-
PO--
E70--
s
sQ
so--
30--
15
’
J
I
1.0
2.0
5.0 IO.0 PEA pglml
20.0
50.0
Fig. 1. Standard curves for the determination of PEA in urine. Line A represents pure PEI in buffer without extraction; Line B represents equivalent amounts of PEA after extractio There was a consistent 70% recovery.
Results Figure 1 represents the standard curves used for the calculation of the PEA levels. There was a consistent 70% recovery after extraction in all experi ments. The sensitivity of the immunoassay procedure used here seems to be methyl group dependent-mephentermine, phentermine and methamphetamir being more sensitive than either amphetamine or PEA. We were able to use the procedure for the determination of PEA levels of as low as 1 lg/ml. However, data were often erratic, unreliable and non-reproducible for PEA levels below 5 pg/ml. Twenty-five to 50 ml urines including those from detoxified patients were found adequate to yield reliable results by this procedure, and this was found to be more reliable than the procedure developed by us [16] previously. The urinary excretion of free PEA in control subjects (N = 30) was 0.20 t 0.06 pg/mg creatinine. In methadone maintained patients the twentyfour hour urinary excretion of PEA was approximately 25% higher than in normals. This value decreased by about 50% (P < 0.001) during gradual detoxification; the value on complete detoxification was approximately 25% less than the value during detoxification (Table 1). After acute or cold turkey
299 TABLE
1
Urinary excretion of 2-phenylethylamine during gradual detoxification
(PEA)
in methadone
maintained
patients
and
All values represent means t S.E.M. corrected for 70% recovery. The results for each period represent average of 3 replicates for a total of 5 to 6 days selected on the basis of normal creatinine values and adequate urine volumes Hospital course (2 weeks per period)
Stable period (12)* During detoxification Completely detoxed -__ Controls:
(12) (4)**
N = 30; PEA = 0.20
erg PEA/ mg creatinine
Av. Beck’s depression inventory
Nurses depression ratings
0.24 0.12 0.08
4.0 + 0.0 17.5 * 0.3 11.2 i 0.1
5.8 i 0.3 5.5 + 0.2 5.5 1 0.2
? 0.06
~-
f 0.10 + 0.03 + 0.01
__-.
-.__
pg/mg creatinine.
*Numbers in parentheses represent numbers of patients studied; ratings indicate increased manifestation of depression. **Due to patients signing out of hospital before discharge. TABLE
increased
depression
2
Urinary excretion of 2-phenylethylamine during acute (cold turkey) detoxification
(PEA)
in methadone
maintained
patients
and
All values represent means f S.E.M. corrected for 70% recovery. The results for each period represent average of 3 replicates for a total of 5 to 6 days selected on the basis of normal creatinine values and adequate urine volumes Hospital
course
erg PEA/ mg creatinine
Av. Beck’s depression inventory
Nurses depression ratings
Stable Detoxed
2-week period (12)* - 3-week period (12)
0.14 0.09
5.7 f 0.3 15.5 f 0.0
5.86 5.79
Controls:
N = 30; PEA = 0.20
*Numbers in parentheses ratings indicate increased
* 0.0 * 0.0
? 0.35 + 0.40
+ Mg/mg creatinine.
represent numbers of patients manifestation of depression.
studied;
increased
depression
detoxification, urinary excretion of PEA was also markedly decreased (P < 0.001 -- Table 2). In both detoxification regimens, abstinent patients were found to excrete markedly low quantities of PEA. Concurrently, two weeks after and during detoxification, the average Beck’s depression index increased (Tables 1 and 2). Furthermore, when urinary excretion of PEA in methadone maintained patients was studied in relation to the various drug dosage levels as progressive gradual detoxification proceeded, a clear dose-dependent relationship was obtained (Fig. 2) corresponding to the pattern obtained when all patients were compared during the stable, detoxification and post detoxification periods. The dose of methadone at which urinary excretion of PEA began
300
0.50--
0
040--
.5
z
E 030--
e % 9
020--
0 lO--
000’
60
50
40 me,hodone
30 dose
20 sn mg
10
0
Fig. 2. Relationship between maintenance dose of methadone and urinary excretion of PEA in patients. Numbers at each point on graph represent number of patients studied.
to level off was 20 mg. At a daily dose of 40 mg methadone ingested, the PEA excretion equalled those of normal control non-medicated subjects. Discussion 2-Phenylethylamine (PEA) is an endogenous amine which is structurally and pharmacologically similar to amphetamine. We had identified PEA in human brain and urine and characterized it by mass and i&a-red spectroscop [14, 171. Prior to that, Jackson [18] had demonstrated its presence in humai blood. PEA is ubiquitous in human tissue [ 161. Its amphetamine-like effects are particularly pronounced in animals pretreated with monoamine oxidase inhibitors (MAOI) [19 - 211. Jonsson et al. [22] and Fuxe et al. [ 231 report. ed, respectively, that i.p. administration of PEA resulted in a depletion of norepinephine (NE) and dopamine (DA), so that the excitatory effects of PEA have been attributed in part to the release of catecholamines (CA) in brain. It should be pointed out, however, that Giardina et al. [24] demonstrated by microiontophoretic studies that PEA produced effects opposite to those of CA in some cortical neurons. Furthermore, Feldman and Lebovitz [ 251 demonstrated that although PEA inhibited insulin secretion indirectly through CA release, this action was not terminated by pretreatmen with reserpine, nor was it blocked by phenoxybenzamine, suggesting that either PEA may exert its own direct action in the inhibitory process or that other amines such as DA may contribute to the action. Urinary PEA has been associated, although in no simple 1:l relationship with a number of pathophysiological conditions in man, including abnormall: high excretion in phenylketonurics [ 26, 271 and in schizophrenics [ 281, decreased excretion reported by us in patients suffering from myocardial infarction [17] and in depressed patients [lo, 12, 291. However, the broad
301
peripheral tissue distribution of this adrenergic amine would lead one to suggest that it probably mediates more than CNS functions. The results of these preliminary studies indicate that the withdrawal syndrome may be clearly characterized by (1) 1ow urinary excretion of 2-phenylethylamine and (2) depression. It was interesting to note that while patients showed a significant depression index by the Beck inventory during abstinence, corresponding with progressive decrease in urinary PEA, the levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) reported by us remained, unchanged at 1.03 ? 0.08 pg/mg creatinine (stable); 1.01 + 0.05 pg/mg creatinine (gradual detoxification); and 1.15 * 0.12 pg/mg creatinine (post detoxification) [ 301. Decrease in urinary excretion of MHPG, a compound identified by Axelrod et al. [ 311 and others [32 - 371 as the naturally occurring o-methylated CNS metabolite of norepinephrine, has been associated with endogenous depression in some groups of patients [38 - 401. Furthermore, it was evident that the nurses depression ratings did not seem to be an adequate measure for the type of depression found in these narcotic patients. It should be noted that in a recent study no correlations were obtained between severity of illness as measured by nurses depression ratings and MHPG excretion in patients with diagnosable affective disorders (Jones et al. [40] ). It is, therefore, possible that the self report inventory is indeed a better index of the subjective mood state of the patients, and if this is the case PEA is likely to reflect the biochemical component associated with this peculiar mood alteration in drug-dependent patients. Decreased PEA associated depression may be post amphetamine-type depression [41]. The type of clinical depression observed here clearly characterizes the abstinent state in general in opiate-dependent humans and is identifiable by the Beck’s depression inventory. PEA excretion in patients stabilized on methadone approximately equalled the PEA excretion in healthy normals and was much less after complete withdrawal. Furthermore, the depression indices increased concurrently with decreased PEA excretion. If persistent use of methadone, and for that matter heroin, has not resulted in a permanent physiological lesion in the amine synthesizing systems, then the markedly low excretion of PEA after detoxification may be an index of the original capacity in these patients to synthesize, store and utilize PEA, suggesting that our original proposition that narcotics are consumed in an effort to self-medicate against an inherent affective disorder may be valid and, therefore, deserves further investigation. Results of our preliminary experiments suggest that platelet monoamine oxidase (MAO) in methadonemaintained patients appears to be less than normal indicating that methadone HCl may exhibit MAO-inhibiting properties peripherally as was found to be the case centrally [42]. Thus the excretion of higher levels of PEA in methadone maintained patients should be expected giving more credence to our proposition that abstinent patients may present with the original innate condition of their amine synthesizing pathways prior to narcotic usage. Clinically, these results may have far reaching implications in treatment. In the studies reported here, abstinence was attained with low levels of PEA
302
and depression. Antidepressant drugs increase levels of PEA [ 431 and ameliorate depressive symptomatology. It is common knowledge that post detoxification depression contributes to relapse and re-use of narcotics. Therefore, it is logical for us to further suggest that the studies reported here may provide a biochemical and clinical basis for the use of tricyclic antidepressants in the treatment of post detoxification relapse in some patients. This proposition is particularly interesting in view of the recent findings by Doggett et al. [44] that morphine, the narcotic antagonists cyclazozine and naloxone may each possess some components of clinical antidepressant activity. In fact, a combination of these drugs with methadone at low doses (20 mg’and below) may, indeed, form a rational therapeutic maneuver for the management of some patients during detoxification from opiate drugs.
Acknowledgements This study was supported in part by the Addiction Research and Treatmt Corporation, 22 Chapel Street, Brooklyn, New .York. The amphetamine kits were kindly supplied by Syva Corporation, Palo Alto, California. We wish to thank Joan O’Connell, RN, and the ISPI Hospital staff, Carol Huber, Narendra S. Pate1 and Bertha Wallace, for excellent technical assistance. The preliminary experiments on the levels of MAO in platelets have been the results of collaborative efforts between Dr. G. Pandey of ISPI and the principal author.
References 1 2 3 4 5 6 7 8 9 10 11 12 13
B. B. Brodie and P. A. Shore, Ann. N. Y. Acad. Sci., 66 (1957) 631. B. B. Brodie, M. S. Comer, E. Costa and A. J. Dlabac, J. Pharmacol., 152 (1966) 340. M. Vogt, J. Physiol. (London), 123 (1954) 451. C. R. Rethy, C. B. Smith and J. E. Villarreal, J. Pharmacol. Exptl. Therap., 176 (1971) 472. D. J. Reis, M. Ritkin and A. Corvell, European J. Pharmacol., 9 (1969) 149. F. H. Shen, H. H. Loh and E. L. Way, J. Pharmacol. Exptl. Therap., 175 (1970) 427. I. K. Ho, H. H. Loh and E. Leon-Way, Proc. 36th Ann. Sci. Meeting Natl. Acad. Sci. (Il. S. Comm. Problems Drug Dependence), 1974, p. 474. A. J. Krakowski, Psychosomatics, 9 (1968) 89. E. E. Inwang, N. S. Patel, B. J. Primm, A. McBride and P. E. Bath, Experfentia, 31 (1975) 203. E. E. Inwang, J. H. Sugerman, A. D. Mosnaim and H. C. Sabelli, Proc. Ann. Meeting Sot. Biol. Psychiat., 1972, p. 13. A. D. Mosnaim, E. E. Inwang, J. H. Sugerman, W. J. DeMartini and H. C. Sabelli, Biol. Psychiat., 6 (1973) 235. A. A. Boulton and L. Milward, J. Chromatog., 57 (1971) 287. K. E. Rubenstein, R. S. Schneider and E. F. Uliman, Biochem. Biophys. Res. Commun., 47 (1972) 846.
303 14 E. E. Inwang, A. D. Mosnaim and H. C. Sabelli, J. Neurochem., 20 (1973) 1469. 15 A. T. Beck, Depression: Clinical, Experimental, and Theoretical Aspects, Harper and Row Publ., N. Y., Evanston, London, 1967, p. 333. 16 A. D. Mosnaim and E. E. Inwang, Anal. Biochem., 54 (1973) 561. 17 A. D. Mosnaim, E. E. Inwang, J. H. Sugerman and H. C. Sabelli, Clin. Chim. Acta, 46 (1972) 407. 18 D. M. Jackson, Comp. Gen. Pharmacol., 1 (1970) 263. 19 P. Holtz, K. Credner and F. Heppe, Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 204 (1947) 85. 20 E. Fischer, R. I. Ludmer and H. C. Sabelli, Acta Physiol. Lat. Am., 17 (1967) 15. 21 W. G. Dewhurst, Nature, 218 (1968) 1130. 22 J. Jonsson, H. Grobecker and P. Holtz, Life Sci., 5 (1966) 2235. 23 K. Fuxe, H. Grobecker and J. Jonsson, European J. Pharmacol., 2 (1967) 202. 24 W. J. Giardina, W. A. Pedemonte and H. C. Sabelli, Life Sci., 12 (1973) 153. 25 J. M. Feldman and H. E. Lebovitz, J. Pharmacol. Exptl. Therap., 179 (1971) 56. 26 B. Jepson, W. Lovenberg, P. Zaltman, S. Oates, A. Sjoerdsma and S. Udenfried, Biochem. J., 74 (1960) 5P. 27 J. A. Oates, P. Z. Nirenberg, B. Jepson, A. Sjoerdsma and S. Udenfried, Proc. Sot. Exptl. Biol. Med., 112 (1963) 1078. 28 E. Fischer, H. Spatz, J. M. Saavedra, H. Reggiani, A. Miro and B. Heller, Biol. Psychiat., 5 (1972) 139. 29 E. Fischer, R. I. Ludmer and H. C. Sabelli, Acta Physiol. Latinam., 17 (1967) 15. 30 F. Jones, J. M. Davis, D. Garver, G. Pandey, H. Dekirmenjian, E. E. Inwang and R. Watkins, Proc. Am. Psychiat. Assoc., 127 (N. R. Supp.) (1974) 6. 31 J. Axelrod, I. J. Kopin and J. D. Mann, Biochim. Biophys. Acta, 36 (1959) 576. 32 E. Mannarino, N. Kirshner and B. S. Nashold, J. Neurochem., 10 (1967) 373. 33 C. 0. Rutledge and J. J. Jonason, J. Pharmacol. Exptl. Therap., 157 (1967) 493. 34 J. W. Maas and D. H. Landis, J. Pharmacol. Exptl. Therap., 163 (1968) 147. 35 S. M. Shanberg, G. R. Breese, J. J. Shildraut, E. K. Gordon and I. J. Kopin, Biochem. Pharmacol., 17 (1968) 2006. 36 S. M. Shanberg, G. R. Breese, J. J. Shildkraut and I. J. Kopin, Biochem. Pharmacol., 17 (1968) 247. 37 D. F. Sharmon, Brit. J. Pharmacol., 36 (1969) 523. 38 J. J. Shildkraut and S. S. Kety, Science, 156 (1967) 21. 39 J. W. Maas, J. A. Fawcett and H. Dekirmenjian, Arch. Gen. Psychiat., 26 (1972) 252. 40. F. DeLeon Jones, J. W. Maas, H. Dekirmenjian, J. Sauchez and M. Loren-Maas, Am. J. Psychiat., to be published. 41 J. J. Schildkraut, R. Watson, P. R. Draskoczy and E. Hartmann, Lancet, 2 (1971) 485 42 M. Pfeffer, J. Gaylord and M. Alteras, Toxicol. Appl. Pharmacol., 24 (1973) 333. 43 A. D. Mosnaim, E. E. Inwang and H. C. Sabelli, Biol. Psychiat., 8 (1974) 227. 44 N. S. Doggett, H. Reno and P. S. J. Spencer, Neuropharmacology, 14 (1975) 507.
METABOLIC DISPOSITION OF 2-PHENYLETHYLAMINE ROLE OF DEPRESSION IN METHADONE-DEPENDENT DETOXIFIED PATIENTS*
EDET
Diseases and Mental Health, Friendship Chicago, Illinois 60643, and Addiction Research Street, Brooklyn, New York (U.S.A.)
J. PRIMM
Addiction (U.S.A.) FRANK Illinois
AND THE AND
E. INWANG
Division for the Treatment of Addictive Medical Center, 850 West 103rd Street, and Treatment Corporation,. 22 Chapel BENY
295
295 - 303 in the Netherlands
Research
and Treatment
de L. JONES, State Psychiatric
CYNTHIA
Corporation,
HAROUTUNE Institute,
1601
22 Chapel
DEKIRMENJIAN West Taylor
Street,
Brooklyn,
New
York
and JOHN M. DAVIS
Street,
Chicago,
Illinois
(U.S.A.)
of Medicine,
Chicago,
Illinois
(U.S.A.)
T. HENDERSON
University
of Illinois
(Received
October
Abraham
Lincoln
College
20, 1975)
Summary We have now postulated that differences in the innate capacity of individuals to synthesize, store and utilize biogenic amines may provide the biological basis for human abuse of narcotic and other drugs, and that these drugs are used in an apparent unconscious effort to self-medicate against an inherent affective disorder. In this communication, we attempted a preliminary characterization of the narcotics withdrawal syndrome on biochemical and clinical parameters. Abstinence was found to be characterized by low urinary excretion of 2-phenylethylamine and depression. An indication for use of tricyclic drugs has been discussed.
Introduction If indeed neuropsychotropic drugs modulate behavior by altering central amine@ systems [ 1, 21, then it should be expected that narcotic drugs exert their action, at least in part, through similar systems. It has been reported that in animals acute administration of morphine not only causes release of catecholamines (CA) from adrenal and from central sympathetic centers [ 3 - 51, but it also increases their turnover rate; the amines return to *Presented in part at the 37th Annual Scientific of Sciences, Washington, D. C., May 19 - 21, 1975.
Meeting
of the National
Academy
296
baseline levels during chronic treatment [ 61. Similarly, increase in the turnover rate of serotonin (5-HT) has been reported for morphine-dependent rats and mice [ 61. More recently, even the putative neurotransmitter gamma-aminobutyric acid (GABA) has also been associated with the process of morphine-dependence [7]. Therefore, if biogenic amines play any real role in the process of drug-dependence, the previously studied amines are not likely to be the only candidates. In fact, several chemically functional adrenergic neurons in the CNS are known to exist and help to explain the biphasic stimulant and depressant effects of neurotropic drugs [ 81. Moreover, we have recently elicited a similar biphasic effect with methadone hydrochloride and L-alpha acetylmethadol (LAAM) [ 91. It is highly unlikely, however, that any of these different classes of biogenic amines would individually explain physical dependence. It is more logical to presume that a modulating phenomenon exists involving an interaction between several amines and their metabolites. Our motivation to conduct the present studies was based on two major premises. Firstly, since mood and affect are modulated by adrenergic amines [l, 2, 10 - 121 and narcotic drugs as well as alcohol are known to modulate the levels of these amines [ 3 - 71, we postulated that differences in the baseline capacity of individuals to synthesize biogenic amines may provide the biological basis for human abuse of narcotic and other drugs. This hypothesis implies that those individuals with low capacity to synthesize adequate amounts of ergo tropic adrenergic amines have an innate predisposition to use narcotic drugs in an apparent unconscious effort to self-medicate against an inherent affective disorder. Of course, it is difficult to determine the validity of such a proposition, since there is no way to ascertain the baseline levels of amines in drug-dependent individuals prior to their initial use of the drugs. We, therefore, felt that the study of these amines during withdrawal might give us some clue to the problem. Secondly, there are no reports prior to this one on the metabolic disposition of 2-phenylethylamine (PEA) in narcotic dependent or methadonl maintained patients, nor are there any which attempt to characterize the abstinence syndrome on combined biochemical and clinical parameters. Thus in this paper we report for the first time the possible role of PEA in the modulation of both the affective and probably some non-affective components of the abstinence syndrome.
Experimental Male and female volunteers, between the ages of 22 years and 24 years, presenting with a history of heroin-dependence and, subsequently, stabilized on varying doses of methadone hydrochloride (60 mg - 30 mg) for varying periods between one to two years, were used for these studies. Prospective candidates were screened for their motivation towards detoxification, absence of major organic or psychiatric disorders, in particular psychosis, and
297
any tendencies towards violent behavior. Selected patients were then hospitalized under strict supervision in a metabolic research unit and placed on a VMA (vanillyl mandelic acid) exclusion diet. In the first experimental regimen, twenty-four hour urine specimens were collected daily during (1) a two-week stable methadone maintenance period, (2) a two-week gradual detoxification period and (3) a two-week post detoxification period. Detoxification was achieved by equal and daily decrements of methadone until zero dose was reached for each patient under study. In the second experimental regimen, twenty-four hour urine samples were collected as indicated during a two-week stable methadone administration; subsequent to that, methadone was stopped abruptly (cold turkey detoxification) and daily twenty-four hour urines were collected during the following three weeks. Urine specimens were preserved either by the addition of sodium metabisulfite or by adding sufficient cont. HCl to bring the pH to 1.0. The bottles were stored frozen at a temperature of --16 “C until used. PEA was extracted from 100 ml aliquot portions of urine at pH 12.5 into spectrograde n-hexane and subsequently extracted into 1 ml 1N HCl. The pH of the acid extract was then adjusted to 6.0 with a 0.025 M tris-maleate buffer solution. Urinary content of PEA in the acid extract was determined using a modification of the homogeneous enzyme multiplied immunoassay technique (EMIT) of Rubenstein et al. [ 131, developed by Syva Corporation for the determination of amphetamines in urine. Note that it is essential to carry out the specific extraction procedure for PEA as previously described by Inwang et al. [14] to eliminate other biogenic amines and amphetamines in order to avoid cross reactivity. With the use of a calibrated micropipette apparatus, 50 ~1 of the acid extract of PEA was dispensed into a cuvette containing 250 ~1 of an enzymeactivated antibody solution. PEA competes with the antibody for the enzyme active site resulting in precipitation, and the optical density of the solution was determined at a wavelength of 436 nm using a manual Gilford spectrophotometer thermally regulated at 37 “C. The quantity of PEA in the extract was extrapolated from a standard curve obtained using authentic PEA prepared in the same manner as were the experimental samples. The PEA content of urines of healthy, control subjects within the same age range and sex as the patients was determined in a similar manner for comparison. In addition, daily depression indices using Beck’s depression inventory [ 151 as well as nurses ratings using an incremental 1 - 15 scale were obtained throughout the entire experimental period. Withdrawal symptoms were also recorded daily throughout the entire hospital course. These experiments were double blind so that nurses who scored the symptoms did not know the medication regimen, nor did the analytical team know which urines were being assayed. The codes were broken only after complete biochemical assay of the urines.
298 I IO-
PO--
E70--
s
sQ
so--
30--
15
’
J
I
1.0
2.0
5.0 IO.0 PEA pglml
20.0
50.0
Fig. 1. Standard curves for the determination of PEA in urine. Line A represents pure PEI in buffer without extraction; Line B represents equivalent amounts of PEA after extractio There was a consistent 70% recovery.
Results Figure 1 represents the standard curves used for the calculation of the PEA levels. There was a consistent 70% recovery after extraction in all experi ments. The sensitivity of the immunoassay procedure used here seems to be methyl group dependent-mephentermine, phentermine and methamphetamir being more sensitive than either amphetamine or PEA. We were able to use the procedure for the determination of PEA levels of as low as 1 lg/ml. However, data were often erratic, unreliable and non-reproducible for PEA levels below 5 pg/ml. Twenty-five to 50 ml urines including those from detoxified patients were found adequate to yield reliable results by this procedure, and this was found to be more reliable than the procedure developed by us [16] previously. The urinary excretion of free PEA in control subjects (N = 30) was 0.20 t 0.06 pg/mg creatinine. In methadone maintained patients the twentyfour hour urinary excretion of PEA was approximately 25% higher than in normals. This value decreased by about 50% (P < 0.001) during gradual detoxification; the value on complete detoxification was approximately 25% less than the value during detoxification (Table 1). After acute or cold turkey
299 TABLE
1
Urinary excretion of 2-phenylethylamine during gradual detoxification
(PEA)
in methadone
maintained
patients
and
All values represent means t S.E.M. corrected for 70% recovery. The results for each period represent average of 3 replicates for a total of 5 to 6 days selected on the basis of normal creatinine values and adequate urine volumes Hospital course (2 weeks per period)
Stable period (12)* During detoxification Completely detoxed -__ Controls:
(12) (4)**
N = 30; PEA = 0.20
erg PEA/ mg creatinine
Av. Beck’s depression inventory
Nurses depression ratings
0.24 0.12 0.08
4.0 + 0.0 17.5 * 0.3 11.2 i 0.1
5.8 i 0.3 5.5 + 0.2 5.5 1 0.2
? 0.06
~-
f 0.10 + 0.03 + 0.01
__-.
-.__
pg/mg creatinine.
*Numbers in parentheses represent numbers of patients studied; ratings indicate increased manifestation of depression. **Due to patients signing out of hospital before discharge. TABLE
increased
depression
2
Urinary excretion of 2-phenylethylamine during acute (cold turkey) detoxification
(PEA)
in methadone
maintained
patients
and
All values represent means f S.E.M. corrected for 70% recovery. The results for each period represent average of 3 replicates for a total of 5 to 6 days selected on the basis of normal creatinine values and adequate urine volumes Hospital
course
erg PEA/ mg creatinine
Av. Beck’s depression inventory
Nurses depression ratings
Stable Detoxed
2-week period (12)* - 3-week period (12)
0.14 0.09
5.7 f 0.3 15.5 f 0.0
5.86 5.79
Controls:
N = 30; PEA = 0.20
*Numbers in parentheses ratings indicate increased
* 0.0 * 0.0
? 0.35 + 0.40
+ Mg/mg creatinine.
represent numbers of patients manifestation of depression.
studied;
increased
depression
detoxification, urinary excretion of PEA was also markedly decreased (P < 0.001 -- Table 2). In both detoxification regimens, abstinent patients were found to excrete markedly low quantities of PEA. Concurrently, two weeks after and during detoxification, the average Beck’s depression index increased (Tables 1 and 2). Furthermore, when urinary excretion of PEA in methadone maintained patients was studied in relation to the various drug dosage levels as progressive gradual detoxification proceeded, a clear dose-dependent relationship was obtained (Fig. 2) corresponding to the pattern obtained when all patients were compared during the stable, detoxification and post detoxification periods. The dose of methadone at which urinary excretion of PEA began
300
0.50--
0
040--
.5
z
E 030--
e % 9
020--
0 lO--
000’
60
50
40 me,hodone
30 dose
20 sn mg
10
0
Fig. 2. Relationship between maintenance dose of methadone and urinary excretion of PEA in patients. Numbers at each point on graph represent number of patients studied.
to level off was 20 mg. At a daily dose of 40 mg methadone ingested, the PEA excretion equalled those of normal control non-medicated subjects. Discussion 2-Phenylethylamine (PEA) is an endogenous amine which is structurally and pharmacologically similar to amphetamine. We had identified PEA in human brain and urine and characterized it by mass and i&a-red spectroscop [14, 171. Prior to that, Jackson [18] had demonstrated its presence in humai blood. PEA is ubiquitous in human tissue [ 161. Its amphetamine-like effects are particularly pronounced in animals pretreated with monoamine oxidase inhibitors (MAOI) [19 - 211. Jonsson et al. [22] and Fuxe et al. [ 231 report. ed, respectively, that i.p. administration of PEA resulted in a depletion of norepinephine (NE) and dopamine (DA), so that the excitatory effects of PEA have been attributed in part to the release of catecholamines (CA) in brain. It should be pointed out, however, that Giardina et al. [24] demonstrated by microiontophoretic studies that PEA produced effects opposite to those of CA in some cortical neurons. Furthermore, Feldman and Lebovitz [ 251 demonstrated that although PEA inhibited insulin secretion indirectly through CA release, this action was not terminated by pretreatmen with reserpine, nor was it blocked by phenoxybenzamine, suggesting that either PEA may exert its own direct action in the inhibitory process or that other amines such as DA may contribute to the action. Urinary PEA has been associated, although in no simple 1:l relationship with a number of pathophysiological conditions in man, including abnormall: high excretion in phenylketonurics [ 26, 271 and in schizophrenics [ 281, decreased excretion reported by us in patients suffering from myocardial infarction [17] and in depressed patients [lo, 12, 291. However, the broad
301
peripheral tissue distribution of this adrenergic amine would lead one to suggest that it probably mediates more than CNS functions. The results of these preliminary studies indicate that the withdrawal syndrome may be clearly characterized by (1) 1ow urinary excretion of 2-phenylethylamine and (2) depression. It was interesting to note that while patients showed a significant depression index by the Beck inventory during abstinence, corresponding with progressive decrease in urinary PEA, the levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) reported by us remained, unchanged at 1.03 ? 0.08 pg/mg creatinine (stable); 1.01 + 0.05 pg/mg creatinine (gradual detoxification); and 1.15 * 0.12 pg/mg creatinine (post detoxification) [ 301. Decrease in urinary excretion of MHPG, a compound identified by Axelrod et al. [ 311 and others [32 - 371 as the naturally occurring o-methylated CNS metabolite of norepinephrine, has been associated with endogenous depression in some groups of patients [38 - 401. Furthermore, it was evident that the nurses depression ratings did not seem to be an adequate measure for the type of depression found in these narcotic patients. It should be noted that in a recent study no correlations were obtained between severity of illness as measured by nurses depression ratings and MHPG excretion in patients with diagnosable affective disorders (Jones et al. [40] ). It is, therefore, possible that the self report inventory is indeed a better index of the subjective mood state of the patients, and if this is the case PEA is likely to reflect the biochemical component associated with this peculiar mood alteration in drug-dependent patients. Decreased PEA associated depression may be post amphetamine-type depression [41]. The type of clinical depression observed here clearly characterizes the abstinent state in general in opiate-dependent humans and is identifiable by the Beck’s depression inventory. PEA excretion in patients stabilized on methadone approximately equalled the PEA excretion in healthy normals and was much less after complete withdrawal. Furthermore, the depression indices increased concurrently with decreased PEA excretion. If persistent use of methadone, and for that matter heroin, has not resulted in a permanent physiological lesion in the amine synthesizing systems, then the markedly low excretion of PEA after detoxification may be an index of the original capacity in these patients to synthesize, store and utilize PEA, suggesting that our original proposition that narcotics are consumed in an effort to self-medicate against an inherent affective disorder may be valid and, therefore, deserves further investigation. Results of our preliminary experiments suggest that platelet monoamine oxidase (MAO) in methadonemaintained patients appears to be less than normal indicating that methadone HCl may exhibit MAO-inhibiting properties peripherally as was found to be the case centrally [42]. Thus the excretion of higher levels of PEA in methadone maintained patients should be expected giving more credence to our proposition that abstinent patients may present with the original innate condition of their amine synthesizing pathways prior to narcotic usage. Clinically, these results may have far reaching implications in treatment. In the studies reported here, abstinence was attained with low levels of PEA
302
and depression. Antidepressant drugs increase levels of PEA [ 431 and ameliorate depressive symptomatology. It is common knowledge that post detoxification depression contributes to relapse and re-use of narcotics. Therefore, it is logical for us to further suggest that the studies reported here may provide a biochemical and clinical basis for the use of tricyclic antidepressants in the treatment of post detoxification relapse in some patients. This proposition is particularly interesting in view of the recent findings by Doggett et al. [44] that morphine, the narcotic antagonists cyclazozine and naloxone may each possess some components of clinical antidepressant activity. In fact, a combination of these drugs with methadone at low doses (20 mg’and below) may, indeed, form a rational therapeutic maneuver for the management of some patients during detoxification from opiate drugs.
Acknowledgements This study was supported in part by the Addiction Research and Treatmt Corporation, 22 Chapel Street, Brooklyn, New .York. The amphetamine kits were kindly supplied by Syva Corporation, Palo Alto, California. We wish to thank Joan O’Connell, RN, and the ISPI Hospital staff, Carol Huber, Narendra S. Pate1 and Bertha Wallace, for excellent technical assistance. The preliminary experiments on the levels of MAO in platelets have been the results of collaborative efforts between Dr. G. Pandey of ISPI and the principal author.
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