None of the in vitro hydroxylase inhibitors of this type appears to have been tested in vivo. It seems unlikely that selective inhibition of the hydroxylases could be achieved in view of the wide variety of oxidative enzyme systems that would be affected.
D. Amino Acid Analogs
1. INHIBITION OF TYROSINE HYDROXYLASE
The most widely studied in vivo inhibitor of tyrosine hydroxylase is a-methyl-p-tyrosine ( a- M P T ) . a-Methyl-m-tyrosine (a-MMT) is also an effective inhibitor in vitro but its in vivo actions are complicated because it is rapidly metabolized to the false transmitter agents a-methyl-m-tyramine and metaraminol (m-hydroxynorephedrine). They displace catecholamines from their storage sites (164, 165, 185), and this displacement is believed to play a significant part in the in vivo effects (46, 166, 167, 186). The a-MPT is apparently converted to catechols only to an insignificant extent (44,187,188).
Materials that are themselves amine releasers or, like a-methyl-DOPA and a-MMT, are rapidly metabolized to amine-releasing agents generally have a greater effect on NE levels than on dopamine levels and act more in the periphery than in the brain. Tyrosine hydroxylase inhibitors such as a-MPT, on the other hand, generally have greater and more lasting effects on brain dopamine than on brain NE levels (152, 189) and are usually more effective in brain and heart than in adrenals or spleen (185). Behaviorally, amine releasers such as a-MMT are more
2. AMINO ACID HYDROXYLASE INHIBITORS 75 active in producing hyperactivity in mice treated with an MAO inhibitor (190), as well as in sedative and anticonvulsant action (191, 192), than are true tyrosine hydroxylase inhibitors.
In a large number of studies, a-MPT, or its methyl or ethyl esters, have been shown to reduce the catecholamine content of animal tissues without significantly affecting serotonin levels (193). Activity is limited to the L isomer (44, 194)- After a single dose, the peak effect is at 2-4 hours with recovery in 36 hours. Brain tyrosine hydroxylase drops to a lower percentage of its control value than does NE (146). Some representative figures on the effects of this inhibitor on amines in various animal tissues are given in Table X (157, 193, 195). Indications are that a dose of 1 mmole/kg of a-MPT has little effect in vivo on the phenylalanine hydroxylase activity of rat liver (162).
Large single doses (100 mg/kg) of a-MPT produce renal damage in rats. Equivalent inhibition without damage can be achieved by the use of multiple smaller doses of the amino acid (100) or large single doses of the more soluble methyl (H 44/68) (157, 196) or ethyl ester.
Large variations among animals in the rate of depletion of amines after a-MPT administration have been shown to depend on factors such as age (189), environmental temperature (155, 157, 159, 197, 198), and stimulus. Depletion is slower from nerves that have been transected than from intact nerves (198-200). It is also slower from brains of animals adapted to living under low- rather than high-stimulus condi
tions (44, 201, 202). Depletion can be accelerated by severe stress (203, 204) or by prolonged and intense stimulation with electrodes (205, 206).
The rate of depletion has been reported to be greater in rats dosed with morphine (207) or in mice congregated in groups (208) although
T A B L E X
NOREPINEPHRINE AND D O P A M I N E L E V E L S IN SOME T I S S U E S OF A N I M A L S T R E A T E D WITH a - M P T
Norepinephrine (dopamine) as % of control levels
Dose and time between Nictitating Brown Ref-Animal dose and sacrifice Brain Heart membrane fat erence
Rat 1.3 m m o l e / k g , 3 hours 5 4 ( 3 2 ) 75 157 Cat 0.55 m m o l e / k g , bid for 28 10 38 193
2 days
Hamster 5.2 m m o l e / k g , 4 hours 43 195
76 E. G. MCGEER AND P. L. MCGEER
the latter has been questioned (209). The NE in the supernate of heart and brain is depleted more rapidly than the particle-bound amine (210).
a-Methyl-p-tyrosine has been widely used for behavioral studies in animals and the effects noted are mainly mild sedation, reduction in locomotor activity, and some disruption of rewarding behavior (Table X I ) . Recently it has been reported that a-MPT in monkeys selectively decreases R E M sleep despite a slight increase in total sleeping time
(211). Peripherally, a-MPT inhibits the contractile response of the spleen to sympathetic nerve stimulation (187). The hypothesis that a-MPT-induced behavioral effects are related to depletion of NE (212) and/or dopamine (213) in the brain is supported by a parallel time course of effects (214), by potentiation of behavioral effects by pretreat-ment with reserpine, tetrabenazine, or chlorpromazine (214, 215), and by partial reversal with L - D O P A (44, 216, 217), norepinephrine (212), or amphetamine (216, 218). Administration of L - D O P A alone (219, 220), or with an M A O inhibitor, effectively restores the levels of both dopamine and NE in a-MPT-treated animals (221), while the levels of NE can be selectively restored by administration of 3,4-dihydroxy-phenylserine (213,221).
Oral doses of from 300 to 4000 mg/day of a-MPT have been given to 14 patients with pheochromocytoma and to 6 with essential hyper
tension (222-224). The catecholamine production, as measured by urinary changes, was reduced by 23-70%. Blood pressure was decreased in the pheochromocytoma patients but those with essential hypertension failed to display any significant reduction in blood pressure. Sedation was commonly observed by the end of the first day of treatment but waned somewhat with continued therapy. Upon withdrawal of the a-MPT, a psychic stimulation was observed with diminished sleep requirements for several days. Up to 88% of the a-MPT was recovered in the urine unchanged, and the sum of all catechol metabolites of the drug accounted for less than 0.5% of the administered dose.
Brodie et al. (225, 226) treated seven manic and four depressed pa
tients 2-4 times daily with 250 mg of a-MPT. Five of the seven manic patients showed improvement while two showed some worsening. Three of the four depressed patients showed an increase in depression. As in the patients treated by Engelman (222, 224), & significant diminution in sleep was noted after discontinuance of a-MPT in these mentally ill individuals. Similar doses of a-MPT given to 13 schizophrenic patients for up to 8 weeks (226a) had no demonstrable antipsychotic and no depressive effects.
The methyl ester of 3a-dimethyltyrosine (H 59/64) is another potent
2. AMINO ACID HYDROXYLASE INHIBITORS 77 inhibitor of tyrosine hydroxylase in vitro. Both the behavioral and bio
chemical effects in vivo are less than with a comparable dose of a-MPT.
A dose of 500 mg/kg of H 59/64 (1.1 mmole/kg of the L isomer) reduced rat brain NE to about 35% and dopamine to 50% of controls at 2-4 hours (227). The depletion was equivalent to that obtained with 0.5 mmole/kg of L-a-MPT (192) and was less than that obtained with 250 mg/kg of methyl DL-a-methyltryrosinate (H 44/68) (0.6 mmole/kg of the L isomer) (220).
5-Bromotryptophan is also a powerful in vitro inhibitor of tyrosine hydroxylase that reduces brain NE and tyrosine hydroxylase levels in vivo without affecting brain 5-HT (146). 5-Iodotryptophan is more active in vitro but was not tested in vivo because of limited availability.
The few behavioral studies done with 5-bromotryptophan indicate that it increases the threshold for self-stimulation in rats as does a-MPT (228). The halotryptophans have so far not been studied extensively in vivo and have not been administered to humans.
3-Iodotyrosine, although a more powerful in vitro inhibitor of tyrosine hydroxylase than a-MPT (see Table I X ) , is much less active in vivo.
This is probably because of the rapid dehalogenation in the body (229) to metabolites that are relatively inactive as inhibitors (139). Frequent repetition of high doses of 3-iodotyrosine in guinea pigs (3 X 200 mg/kg) lowered NE levels in the brain stem by 66% without affecting 5-HT levels. Heart NE was lowered only by 40% and splenic NE was un
changed. Behavioral effects resembled those in a-MPT-treated animals in that ptosis, muscle flaccidity, and reduction in motor activity were observed. Similar biochemical and behavioral results were obtained in guinea pigs with two doses of 100 mg/kg of either 2-iodo-a-methyl-DL-tyrosine or 3,5-diiodo-2-iodo-a-methyl-DL-tyrosine (229). 3-Iodotyrosine (200 mg/kg sc or ip) is also reported to lower brain NE or dopamine to about 36% of control in rats (230, 231) but to have less effect on heart NE and little or no effect on adrenal catecholamines (231). Feeding 1.5 gm/kg X day of 3-iodotyrosine to mice had no effect on brain cate
cholamine levels or motor activity (232).
2-Iodo-L-tyrosine and 3,5-diiodo-L-tyrosine are normally produced during the biosynthesis of thyroid hormones. A thyroid disorder is known in which there is a defect in the dehalogenase that normally metabolizes the iodotyrosines (233, 234). It is not known whether these patients have any impairment of tyrosine hydroxylation (229).
In line with the relatively low in vivo activity of 3-iodotyrosine in animals, no pharmacological effects and no inhibition of catecholamine synthesis could be detected in a pheochromocytoma patient given this
78 E. G. MCGEER AND P. L. MCGEER
T A B L E X I
SOME BEHAVIORAL EFFECTS OF p-CHLOROPHENYLALANINE AND CX-METHYL-p-TYROSINE IN ANIMALS" Lever pressing for food Lever pressing for water Hunger U245R, 246R, 243R, 247R) l(250R) 271C, 272R, 273C, 274R, 275Mn, 276C
U292R, 293M, 294M, 295R,
U297R, 298R, 299R, 277C', 288R} 290C, 300R% 252R, 232M, 193G and C, 212R, 265R and M ,
2. A M I N O ACID H Y D R O X Y L A S E I N H I B I T O R S 79
Amphetamine - (308M, 309R) i(310M, 249R, 811R,
309R} 312R b
,191R, 813M, 44R, 192R, 314R, 308M); t, U or - (315R)
p-Chloroamphetamine l(308M) -(315R), (I) (308M)
Mescaline i(316R) l(44&), -(816R)
a - M M T \(283R)
reserpine - (326R) US26R)
b
Tremorine l(327M), -(328) -(327M)
a
Key: f, indicates facilitates, increases, or activates; j , depresses or retards; —no change; ( t ) , slight facilitation; (J,), slight depression. Letter after reference number indicates species used: R, rats;
C, cat; G, guinea pig; H, hamster; M, mouse; Mn, monkey; Hu human; Ra, rabbit. 6
Reversed or partially reserved by L - D O P A .
e
Tolerance develops.
d
Depression of some learned behavior by PCP has been attributed to a "perceptual disorientation"
(290) or a "decrease in emotionality" (250) rather than to a direct effect on learning.
« Reversed by 5-HTP.
* Cats on chronic PCP treatment do not show insomnia but do show abnormal behavior said to resemble, in some respects, that of acutely ill schizophrenics (330).
0 Partially reversed by lithium pretreatment.
h
Did not alter normal latency response but prevented stress-induced increases in seizure latency (329). 1
A few reports (297, 331) suggest that PCP does not cause a true increase in sexual drive but the weight of evidence appears to favor the supposition (281) that PCP stimulates sexual activity but probably has no effect on maximal activity (332, 333); PCP-induced sexual stimulation is dependent on testosterone (298).
i Not correlated with brain 5HT levels.
compound. The same patient had, however, shown effects when treated with a-MPT {212).
a-Methyl-5-hydroxytryptophan (a-Me-5-HTP) has been shown to re
duce NE levels in heart {143, 235-237) and brain {143, 236, 238) without appreciable effects on actual 5-HT levels, although there is an increase
T A B L E X I (Continued)
80 E. G. MCGEER AND P. L. MCGEER
in apparent 5-HT as measured fluorometrically due to the a-Me-5-HT formed in vivo {235, 238, 238a). A dose of 1 mmole/kg a-Me-5-HTP has little or no effect on rat liver phenylalanine hydroxylase activity in vivo {162). There is still controversy over whether the NE depletion is due primarily to tyrosine hydroxylase inhibition by a-Me-5-HTP or to release by its metabolite, a-Me-5-HT {143, 236-238). Both mech
anisms probably play a role, just as they do in the in vivo actions of a-methylphenylalanine. This is another significant inhibitor of tyro
sine hydroxylase in vitro that has been found to cause a greater and more enduring depletion of mouse heart NE than does a-MPT. It is, however, less effective than a-MPT in reducing central catecholamine levels. Metaraminol (m-hydroxynorephedrine) has been found in the hearts, brains, and adrenals of mice, rats, and dogs treated with a-methylphenylalanine. The patterns of biochemical and pharmacological events after a-methylphenylalanine administration are in between those seen after administration of a-MPT (a tyrosine hydroxylase inhibitor) and of a-MMT (primarily an amine releaser) {239).
2. INHIBITION OF TRYPTOPHAN HYDROXYLASE
p-Chlorophenylalanine (PCP, fenclonine) [see Table X I {240-333)]
is the most widely used in vivo inhibitor of tryptophan hydroxylase {281, 334). Administration of PCP depletes brain 5-HT markedly and for a prolonged period of time; 5-HT concentrations are also depressed in the spleen, colon, and blood {334). Brain NE levels are slightly de
pressed {286) and may be so depressed for a period of several days {335). As with other hydroxylase inhibitors, the rate of 5-HT depletion after PCP administration varies with environmental temperature {155).
The extent and duration of either 5-HT or NE depletion varies from one brain area to another {336), and there is one report that the effect of PCP on brain NE is not as dose dependent as the effect on 5-HT (335). Brain tryptophan hydroxylase levels are markedly lowered, while tyrosine hydroxylase levels are only moderately and more transiently affected (147). The enzyme inhibition can be correlated with the 5-HT depletion. Although PCP is a competitive inhibitor of tryptophan hydroxylase in vitro, it causes an irreversible inactivation of the enzyme in vivo (337). Recently, Gal et al. (338), using radioactive PCP, have produced evidence indicating that PCP is incorporated into various pro
teins, presumably in place of phenylalanine moieties. It is suggested that incorporation into tryptophan hydroxylase may be responsible for the prolonged inhibition of hydroxylase activity. The uptake of tritiated
2. AMINO ACID HYDROXYLASE INHIBITORS 8 1
tyrosine or tryptophan into rat brain is not affected by PCP (335).
Since its introduction by Koe and Weissman (151) in 1966, PCP has been widely used for behavioral studies (Table X I ) . There is some con
troversy as to whether the behavioral effects of PCP can all be attributed to depletion of brain 5-HT or whether the more limited depletion of catecholamines (240, 2%6, 286, 339, 340) and/or the severe inhibition of phenylalanine hydroxylase (see Section IV,D,3) play significant roles.
Effects such as insomnia (269, 270, 279) or sexual excitement (281) that can be reversed with 5-HTP presumably involve a serotonergic mechanism.
A dose of 3 0 0 0 mg/day PCP was administered to six healthy vol
unteers, producing profound decreases in blood 5-HT and urinary 5-HIAA. Minor symptoms were noted including dizziness, fatigue, nausea, headache, uneasiness, and constipation. All symptoms disap
peared the day after the drug was stopped (341)- In addition, PCP has been administered to patients with the carcinoid syndrome, bringing about a reduction in 5-HIAA excretion to near normal levels with a concomitant decrease in the gastrointestinal symptoms of the syndrome (281, 342, 342a, 342b). Effects on hot flushes are less dramatic. Similar re
lief of diarrhea with minimal or no effects on the flushing attacks has been reported in carcinoid patients treated with a 5-HT antagonist (343).
The carcinoid patients on PCP showed side effects similar to those re
ported in normals, with additional psychic symptoms ranging from de
pression to hallucinations (342, 342a). In one carcinoid patient under treatment with PCP distinct hypothermia was noted (344)- In another, the expected impairment of phenylalanine hydroxylation with elevation of blood phenylalanine levels was demonstrated (345).
The 6-halotryptophans are another class of powerful in vitro tryptophan hydroxylase inhibitors that have been shown to be effective in vivo in reducing brain 5 - H T as well as tryptophan hydroxylase levels (147). The effect is much more transient than that of PCP and there was no drop in catecholamines at the doses used. Limited behavioral studies suggest that 6-fluorotryptophan, like PCP, increases the threshold for medial forebrain bundle self-stimulation rats (228), but extensive in vivo studies of these compounds have not yet been done.
3. INHIBITION OF PHENYLALANINE HYDROXYLASE
There are strong in vivo interactions between inhibitors of tryptophan hydroxylase and phenylalanine hydroxylase. This has a particular appli
cation in phenylketonuria, which is characterized by an absence of liver
82 E. G. MCGEER AND P. L. MCGEER
phenylalanine hydroxylase, an impairment of serotonin metabolism, and profound damage to normal brain functioning.
p-Chlorophenylalanine is a weak inhibitor of phenylalanine hydroxyl
ase in vitro but a powerful and irreversible inhibitor in vivo {131, 160).
Administration to rats produces a phenylalanine/tyrosine imbalance in plasma and brain (346, 347) as well as changes in brain lipids (348) similar to those observed in phenylketonuria. Rats injected with 316 mg/kg ip of either PCP or a-methyl-PCP (a poor inhibitor of tryp
tophan hydroxylase) were found to have liver phenylalanine hydroxylase activities only about 36% of that in controls (132). A similar dose of p-fluorophenylalanine reduced the activity to 5% (132). Inhibition of phenylalanine hydroxylase may be involved in such behavioral effects of PCP in young rats as learning deficiencies (244) or changes in seizure threshold (349). It has been cited as the mechanism by which PCP (as well as a,/?,/?-trimethyl-DOPA and p-fluorophenylalanine) antag
onize the potent convulsant and lethal effects of m-fluorophenylalanine in rats (160). In contrast, a-MPT slightly potentiates the effects of m-fluorophenylalanine (350). The cataractogenic effects of chronic PCP in rats (244) have also been attributed to its effects on phenylalanine metabolism; no ocular toxicity was found in monkeys (351).
The effects of excess phenylalanine on brain 5-HT have been exten
sively studied because phenylketonurics show lower than normal blood 5-HT levels (352) and infusion of radioactive tryptophan into two phenylketonurics and two normal volunteers showed a 30% reduction in the patients of the conversion to radioactive 5-HIAA in the urine (353). Feeding excess (5-7% diet supplement) of L-phenylalanine to weanling rats or guinea pigs leads to a marked reduction in brain 5-HT levels (354-360), and a lesser reduction is achieved by feeding phenyl-pyruvic acid (4.5%) (354). Rats so treated show no effect on habituation
(361) or on a successive discrimination problem (362) but perform sig
nificantly worse in a maze (357, 362). It has been reported that the decrement in performance does not correlate with the decrease in brain 5-HT levels (355, 362), although the opposite effects of higher brain 5-HT and better maze performance have been obtained by feeding 5%
supplements of L-tryptophan (357). Monkeys fed excess phenylalanine also show behavioral and learning defects as well as a decreased excretion of 5-HIAA (363). Administration of 316 mg/kg ip of L-phenylalanine (160, 364) or repeated injections (365) had no significant effect on liver phenylalanine hydroxylase activity, but rats injected with higher doses
(820 mg/kg) (366) or fed 4% DL-phenylalanine for 8 weeks (364) or 7% phenylalanine for 2-4 weeks (367) showed significantly lowered
2. AMINO ACID HYDROXYLASE INHIBITORS 83 phenylalanine hydroxylase levels. Brain lipids (368) and seizure thresh
old (368a) are changed in newborn rats injected with excess phenyl
alanine just as with PCP.
Although one group (365) has reported lower tryptophan hydroxylase activities in rats after repeated phenylalanine injections, this has not been confirmed and it seems unlikely that the lowering of brain 5-HT is due to tryptophan hydroxylase inhibition. Very similar effects on brain 5-HT levels are obtained by dietary supplements of L-leucine (357, 369, 369a) or L-valine (370), and these compounds are inactive as inhibitors of tryptophan hydroxylase by liver (150) or brain (83) homogenates.
A more probable mechanism for the action of these amino acids appears to be interference with uptake of tryptophan into the brain or with transport of either tryptophan or 5-HTP across brain cell membranes.
Recently (870a, 370b) it has been suggested that a combination of PCP and phenylalanine given to rats produces a syndrome biochemically and behaviorally closer to phenylketonuria than does administration of either amino acid alone.