CHAPTER 2

Amino Acid Hydroxylase Inhibitors

Edith G. McGeer and Patrick L. McGeer

I. Introduction 45 II. Properties of the Enzymes 46 A. Hydroxylase 46 B. Hydroxylase 50 C. Hydroxylase 54 III. In Vitro Inhibitors of the Hydroxylases 56 A. Catechols 57 B. Iron-Complexing Agents 63 C. Compounds Capable of Easy Oxidation/Reduction 63 D. Amino Acid Analogs 65 IV. In Vivo Inhibitors of the Hydroxylases 71 A. Catechols 71 B. Iron-Complexing Agents 73 C. Compounds Capable of Easy Oxidation/Reduction 74 D. Amino Acid Analogs 74 E. Miscellaneous in Vivo Inhibitors 83 V. Indirect Mechanisms of Inhibition 84 A. Direct Feedback (Product) Inhibition 84 B. Substrate Availability 85 C. Hormonal Influences 86 D. Indirect Feedback (Interneuronal) Inhibition 88 VI. Conclusion 89 References 90

I. INTRODUCTION

There are three principal reactions occurring in animals which involve the introduction of a hydroxyl group into the aromatic ring of an amino acid. These reactions are the hydroxylations of tryptophan to 5-hydroxy- tryptophan (5-HTP), of phenylalanine to tyrosine, and of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). There are certain similarities in

45 46 E. G. MCGEER AND P. L. MCGEER these reactions but important differences as well. An understanding of these similarities and differences is needed for an interpretation of the various actions of different types of inhibitors. In each hydroxylation molecular oxygen is required (1) as well as the same, or a highly similar, pteridine cofactor. The general reaction is presumed to be

hydroxylase XH4 + ArH + 02 > ArOH + H20 + XH2 where X is a pteridine and ArH represents the amino acid substrate. The reaction is in each case irreversible. Ferrous ion may be involved in all three hydroxylations, although the evidence in most cases rests mainly on the inhibitory action of some iron-chelating agents. Some nonenzymic oxidation o2+f tryptophan (2) and phenylalanine (3) occurs in the presence of 02, Fe , and a tetrahydropteridine. Each hydroxylase is highly concentrated in a particular, and different, functional area of the body. Phenylalanine hydroxylase is highly specific to the parenchymal cells of the liver. is localized in chromaffin cells of the adrenal medulla, postganglionic nerve cells of the sympathetic nervous system, and dopaminergic and noradrenergic nerve cells of the brain. is found principally in the -producing parenchymal cells of the pineal gland, the serotonergic neurons of the raphe system of the brain, the argentaffin cells of the gut, and mouse mast cells. Melanin formation also involves hydroxylation of tyrosine. The reac­ tion takes place in melanocytes, which are originally derived from the neural crest. The enzyme involved, tyrosinase, is a copper-containing enzyme and is different from the tyrosine hydroxylase of the adrenal medulla or nerve cells. It may carry3 the reaction sequenc3 e further than DOPA (4), although conversion of [ H]tyrosine to [ H]DOPA in mouse melanoma in vivo has been taken as support for the postulate that DOPA is the physiological intermediate in melanin formation (5).

II. PROPERTIES OF THE ENZYMES

A. Tyrosine Hydroxylase

1. ASSAY The most convenient methods of assay for tyrosine hydroxylase are radiometric, although one fluorescent method has been published (6). One widely used technique is to measure the tritiated water released following conversion of 3,5-tritio-L-tyrosine to 5-tritio-DOPA (7, 8). 2. AMINO ACID HYDROXYLASE INHIBITORS 47

One mole of singly tritiated water is formed for each mole of tyrosine that is converted to DOPA, and the water is separated by freeze-drying it into a counting vial or by passage through an ion-exchange column, which absorbs the tritiated amino acids.14 Another metho14 d is to measure the [ C]DOPA formed during incuba­ tion with [ C]L-tyrosine. A DOPA decarboxylase inhibitor such as NSD-1034 (JV-methyl-Af-3-hydroxyphenylhydrazine14 ) is employed to prevent further of DOPA. The [ C]DOPA is isolated on an alumina column, eluted, and counted (4, 9). Both methods are rapid and simple and employ tyrosine concentrations below saturation14. Experi­ ments with brain homogenates incubated with a mixture of [ C] tyrosine and tritiated tyrosine indicate that the two methods give entirely com­ parable results (10). The tritiated tyrosine metho14 d gives higher blanks (and lower test to blank ratios) than the C method, presumably be­ cause of exchange of tritium between tyrosine and water molecules during the incubation and isolation procedures (10a).

2. LOCATION

The most active site of tyrosine hydroxylation in the body is in the adrenal medulla, which has an activity (Vmax) of the order of 1000 nmoles/hour X gm of wet tissue (9). This is about 10 times the activity of the caudate nucleus, the putamen, and the substantia nigra, which have activities at least five times those in any other brain area (11, 12). High activities are found in abnormal chromaffin tissue such as pheochromocytoma and neuroblastoma (13). Considerable activity is also found in tissue having a high concentration of sympathetic nerve endings (14) such as the superior mesenteric artery (15) and the heart. The heart tyrosine hydroxylase activity is greatly reduced in congestive heart failure (16). The high-activity tissue from adrenals, brain, and heart has been used for most inhibitor studies. The tyrosine hydroxylase in the chromaffin cells of the adrenal medulla can be easily solubilized (17-20a). The same is true of the tyrosine hydroxylase of the heart, vas deferens (21), or substantia nigra, but the enzyme in the caudate nucleus and putamen is much more firmly bound to particles (22, 23). Subcellular localization studies of caudate tyrosine hydroxylase have shown it to be highly localized to the nerve ending or synaptosomal fraction (24-27), as would be expected of an enzyme associated with synthesis of a neurotransmitter or neuromodula­ tor. The substantia nigra contains cell bodies of dopaminergic neurons whose axons terminate in the caudate and putamen. Despite the different 48 E. G. MCGEER AND P. L. MCGEER

behavior of the enzyme in these different brain locations, it is neverthe­ less associated with the same neuronal system. Presumably the enzyme is soluble in the soma of the cell where it is being synthesized by ribo- somes but becomes membrane bound when it reaches the synaptosomal areas (28).

3. PROPERTIES

The adrenal enzyme has been purified 400- to 1000-fold (17, 19, 28). The enzyme purified 400- to 500-fold was reported to require a pteridine cofactor plus a thiol compound, molecular oxygen, and ferrous ions for maximal activity (Table I). Recently, using enzyme purified more than 1000-fold2+ , Shiman et al. (19) have found that catalase can substitute for Fe ions and that either acts to protect the enzyme against inactiva- tion by the H202 generated by nonenzymic oxidation of tetrahydrobiop- terin. A synthetic pteridine, 5,6-dimethyltetrahydropteridine (DMPH4), is generally used for in2+ vitro studies. High concentrations of DMPH4 (>1-10 mM) or of Fe (>2.5 mM) are each inhibitory in some prepa­ rations (29), bu2+t the inhibitory effect of excess DMPH4 may be lessened by adding Fe (8). Highly purified adrenal enzyme is not inhibited by excess cofactor (19). The effects of varying concentrations of 02 depend to some extent on the concentration of cofactor present (30). Structural requirements for cofactor activity apparently include a 2-amino group, a 4-hydroxy group, and an unsubstituted nitrogen in the 5 position (8). As with liver phenylalanine hydroxylase, the order of activity of various pteridines tested is tetrahydrofolate < DMPH4 < 6-methyltetrahydropteridine < tetrahydrobiopterin (19, 31). The natural in vivo cofactor in the adrenal medulla has been reported to be tetrahy­ drobiopterin (31, 32). This compound has been isolated from liver and is believed to be the endogenous cofactor for phenylalanine hydroxylase (33), but its origin in the body is still obscure. Exogenous cofactor does not stimulate the particle-bound tyrosine hy­ droxylase in brain homogenates [cf. Table I (34-36), also 9, 22, 37], This may not, however, reflect a lack of need for cofactor. It could indicate that sufficient cofactor is already contained within the particles or that the exogenous cofactor is not taken up by the particles. Cofactor activity showing the chemical characteristics of tetrahydrobiopterin has been measured in brain and kidney as well as in liver (38), which sug­ gests its ubiquitous nature. The partially purified adrenal enzyme does not hydroxylate D-tyrosine, m-tyrosine, , or tryptophan. It does convert phenylalanine to 2. AMINO ACID HYDROXYLASE INHIBITORS 49

TABLE I RELATIVE ACTIVITIES OF ADRENAL AND BRAIN TYROSINE HYDROXYLASE PREPARATIONS IN ASSAY SYSTEMS LACKING VARIOUS COMPONENTS

Tyrosine hydroxylase source

Partially purified enzyme fro m adrenal Crude Crude rat adrenal brain Supernate ParticulatM e homogenatd e homogenatd e System (%Y (%) (%) (7c) e Complete system 100 100 100 100 Minus DMPH4 2.3 0 5 86 Minus DMPH4 and SH 5 109 Minus SH 2+ 38 14.5 107 Minus Fe2+ 22 20 78.5 100 Minus Fe , SH, and DMPH4 109 Minus 02 0.8 5 1 2 Minus enzyme 0 0 0 0 b Data from 3J+. 0 Data from 35. Recent studies (35a) suggest that the trypsin treatment used in the purification of particulate enzyme produces a tyrosine hydroxylase that is only a fragment of the nativd e form. e Data from 36. 2+ Complete system contains DMPH4, 2-mercaptoethanol (SH), Fe , O2 of air, and enzyme. tyrosine, suggesting that the enzyme has the capacity to hydroxylate in the para position if the meta position is not substituted (39, Jfl). The highly purified adrenal enzyme is said to hydroxylate phenylalanine as rapidly as it does tyrosine if tetrahydrobiopterin, rather than DMPH4, is used as cofactor (19). The ability of tyrosine hydroxylase to accept phenylalanine as a substrate is one of the many significant interactions between the principal hydroxylating enzymes which may have some im­ portance in vivo. The liver is obviously the principal site for the normal conversion of phenylalanine to tyrosine for it is the liver phenylalanine hydroxylase that is missing in phenylketonuria (41). The small conversion of phenyl­ alanine to tyrosine that has been observed in phenylketonuria has been attributed to tyrosine hydroxylase action rather than to any residual phenylalanine hydroxylase or to nonenzymic conversion (17). 50 E. G. MCGEER AND P. L. MCGEER

Tyrosine hydroxylase appears to be the rate-limiting step in catechol­ amine synthesis. The Vnmx for tyrosine hydroxylase in catecholamine- synthesizing tissues is generally more than 100-fold lower than that for DOPA decarboxylase or /^-hydroxylase, the other two en­ zymes involved in (NE) synthesis (34)- Some recent studies on DOPA decarboxylase activity in human brain tissue (42-43b) have indicated low levels of activity and suggested that this step may also be important in the regulation of catecholamine synthesis in human brain. It is probable, however, that the low values of DOPA decarboxy­ lase so far measured in human brain tissues do not indicate the true in vivo activity. The lack of effect of inhibitors of DOPA decarboxylase or dopamine /^-hydroxylase {44) on central catecholamine levels is a strong argument for tyrosine hydroxylase being rate limiting for cate­ cholamine synthesis and emphasizes the significance of metabolic inhibi­ tors of this hydroxylase (45, 46). The Km for tyrosine hydroxylas5 e toward substrate has been variously reported 5 as 4-10 X 10" M for the adrenal medulla 6 (28, 30, 34, 36), 2 X 10~ M for guinea pig heart (47), and 5 X 10" M for crude rat brain homogenates (36). Values fo4 r tyrosine in the brain and adrenal medulla are of the order of 10~ M, a concentration that would fully saturate the enzyme. It is not surprising therefore that tyrosine loads do not have an appreciable effect on central catecholamine synthesis (34). Even in starvation, tissue tyrosine levels do not drop appreciably, which suggests that lowering tyrosine levels in the diet would also have little influence on the rate of catecholamine synthesis. The Km towar4 d DMPH4 of the partially purified adrenal enzyme is about 5 X 10" M U).

B. Tryptophan Hydroxylase

1. ASSAY

Attempts to assay tryptophan hydroxylase by release of tritium as in the tyrosine hydroxylase assay were unsuccessful because of a phe­ nomenon known as the "NIH shift" (48). The tritium in 5-tritiotrypto- phan is not released to form wate 14r but instead shifts to the 4 position on the indole ring (49). The C method analogous to that used for tyrosine hydroxylas14 e also present14 s problems. Measurement of the conver­ sion of [ C] tryptophan to [ C]5-hydroxytryptophan (5-HTP) is com­ plicated by difficulties in separating the two amino acids. 2. AMINO ACID HYDROXYLASE INHIBITORS 51

The simplest method for determination o14f tryptophan hydroxylas14 e is to measure the conversion of methylene-[ C] tryptophan to [ C]sero- tonin in the presence of a (MAO) inhibitor to pre­ vent conversion of the (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by 14the large amounts of MAO present in most 5-HT-formin14 g tissues. The [ C]5-HT can be easily separated from the [ C] trypto­ phan on an ion-exchange column (50, 51). This method presumes the presence of sufficient amounts of 5-HTP decarboxylase to convert completely all of the 5-HTP formed to 5-HT. While this is the case for most crude homogenates (51), it cannot be presumed to apply in cases where partially purified enzyme is used, where compounds are added that might inhibit 5-HTP decarboxylase, or where the tissues might lack adequate quantities of the decarboxylase. In such cases, the deficit of decarboxylase can be made up by adding a partially purified L-amino acid decarboxylase (52-54) or a rat kidney supernate which has virtually no tryptophan hydroxylase (52-54) but which has large amounts of an enzyme that can decarboxylate 5-HTP (51). Another metho14 d of tryptophan hydroxylase assay involves the formation of [ C] 5-HTP in an assa14y system containing a decarboxylase inhibitor, its separation from [ C] tryptophan by chromatography, and measure­ ment of its radioactivit 14 y (54-56). A 14third radioactive method measures the amount of C02 formed from [l- C]L-tryptophan and depends not only on the presence of excess decarboxylase but on the greater activity of the decarboxylase toward 5-HTP than toward tryptophan (57). Less sensitive methods involve fluorometric measurement of the 5-HT or 5-HTP formed (58-60).

2. LOCATION The first reports of tryptophan hydroxylation illustrate the difficulties of cross interaction of hydroxylases and nonenzymic oxidation. A super­ natant fraction from rat liver that required a pyridine nucleotide, oxy­ gen, and a relatively high concentration of substrate (61), and a particu­ late fraction from rat or guinea pig intestinal mucosa that required ascorbic acid and cupric ions but not oxygen (62), were reported to convert tryptophan to 5-HT. Further investigation showed that the ac­ tivity in liver supernate was really phenylalanine hydroxylase, for which L-tryptophan is a relatively poor and physiologically unimportant sub­ strate \63, 64). Doubt was cast on the intestinal mucosa system by the demonstration of nonenzymic oxidations in the presence of ascorbic acid and transition metals (65). 52 E. G. MCGEER AND P. L. MCGEER

A specific tryptophan hydroxylase has now been shown in a number of tissues in the following approximate order of activity: mouse mast cell tumor (52, 53, 58, 66), pineal gland (53, 56, 67), carcinoid tumor (53, 55), brain stem (63, 68, 69) and other brain areas (51, 70), and, to a much lesser extent, stomach and duodenum (53, 56). Human pineal has been shown to have activity (71). The combination of a low concen­ tration of the hydroxylase in stomach and duodenum with a relatively high concentration of 5-HT has been hypothesized to mean either an endogenous inhibitor or a low turnover rate. Brain and pineal hy­ droxylase activities, on the other hand are closely parallel to the 5-HT distribution (51, 69, 71a). The weight of evidence indicates that the majority of brain tryptophan hydroxylase is particle bound (51, 70) while that of the pineal gland, carcinoid tumor, or mouse mast cell tumor appears to be either primarily supernatant or else easily solubilized. There is one report that brain tryptophan hydroxylase, like tyrosine hydroxylase, is associated with nerve endings (71a). This is consistent with histochemical evidence for distinct serotonergic, dopaminergic, and noradrenergic neurons in brain with the amines being concentrated in the neuronal axons and nerve endings (72).

3. PROPERTIES

Although there has been considerable controversy with regard to the cofactor requirements of the enzyme, it appears that, in all its forms, it requires oxygen at a higher partial pressure than does tyrosine hy­ droxylase. The supernatant tryptophan hydroxylase, whether prepared from pineal gland, carcinoid cells, brain stem homogenates, or mouse mast cells, requires exogenous DMPH4 together with 2-mercaptoethanol (Table II) (52, 53, 58, 71a, 73-75). The particle-bound enzyme, on the other hand, whether in brain (51, 54, 70, 71a) or in neoplastic mouse mast cells (58), is not activated by exogenous DMPH4. An early report that slices showed much more tryptophan hydroxylase activity than did homogenates was attributed to the presence of an endogenous cofac­ tor that was released on homogenation and, at least in the case of pineal tissue, could be replaced b2+y exogenous DMPH4 (56). A requirement for Fe has been demonstrated only for mast cell en­ zyme (75), but the inhibition of tryptophan hydroxylase from all source2+ s by metal-chelating agents such as «,a'-dipyridyl suggests that Fe is generally involved. Ferrous ions are reported to stimulate the activity of tryptophan hydroxylase from guinea pig brain stem by 20-30% when 2. AMINO ACID HYDROXYLASE INHIBITORS 53

TABLE II TRYPTOPHAN HYDROXYLASE ACTIVITIES IN ASSAY SYSTEMS LACKING VARIOUS COMPONENTS

Enzyme source

Mouse Brain stem b Beef a Carcinoida mast solublae Crude rat System pineal (%) cells (%) cells (%)° (%) brain (%) 0 Complete system 100 100 100 100 100 2+ Minus DMPH4 9 0 6 4 98 Minus Fe 123 100 7.3 125 109 Minus 02 9 0 10 Minus SH 2.3 26 3.6 18 129 2 Minus DMPH4, Fe + and SH 138 a b Data from 78. 0 Data from 51. 2+ Complete system contains DMPH4, Fe , 02, 2-mereaptoethanol (SH), and enzyme.

2+ the Fe concentration is below 10 fiM but to be rather inhibitory at 100 pM (76). The Km toward substrate of tryptopha5 n hydroxylase has been vari­ ously reported as about 1-4 X 10~ M for the enzyme from malignant mouse mast cells (52, 58, 66, 75) and from brain (50, 51). Lovenberg et al. (52) reported a Km toward tryptophan for4 the enzyme from beef pineal and rat brain of the order of 3-5 X 10~ M, but this seems high in view of the other data in the literature5 . The normal tissue content of tryptophan in brain is about 4 X 10~ M and is thus at best barely sufficient to saturate the enzyme. It is thus apparent why the 5-HT content of tissues varies with the tryptophan level in the diet (77-80), why it is increased in brain by intraperitoneal injections of tryptophan (81), and why it is depressed by agents such as L-a-methyltryptophan that decrease brain tryptophan levels. The latter is presumably the result of stimulation of liver tryptophan pyrrolase (82). The substrate/enzyme relation for tryptophan hydroxylase is therefore quite different from that for tyrosine hydroxylase, where the tyrosine levels in tissue are normally saturating (see Section V,B). Tryptophan hydroxylase does not hydroxylate D-tryptophan (54, 75> 83). Phenylalanine was initially reported to be a substrate for trypto- 54 E. G. MCGEER AND P. L. MCGEER phan hydroxylase and this was suggested to have some implications in phenylketonuria (52). Later work with partially purified enzyme from brain stem (54, 58, 68) or carcinoid tumor (84), however, indicated that tryptophan hydroxylase does not hydroxylate either phenylalanine or tyrosine, although both act as competitive inhibitors. The data on enzyme purified from malignant mouse mast cells are conflicting, with one report (58) indicating that phenylalanine is not hydroxylated and another (75) suggesting that phenylalanine is an excellent substrate. Tryptophan hydroxylase, like tyrosine hydroxylase, appears to be rate limiting in the synthesis of serotonin. Under normal conditions, there is more than a 100-fold excess of the L-amino acid decarboxylase (which decarboxylates both DOPA and 5-HTP) in 5-HT-forming tissues, and decarboxylase inhibitors do not have significant effects on 5-HT levels in vivo (46). This again points out the importance of metabolic inhibitors of the hydroxylases which are not only rate limiting but are the point of enzymic distinction between the 5-HT and catecholamine biosynthetic pathways.

C. Phenylalanine Hydroxylase

1. ASSAY Phenylalanine hydroxylase is generally assayed by measuring the amount of tyrosine formed using the fluorometric method of Waalkes and Udenfriend (85) or the colorimetric method of Udenfriend and Cooper (86). Somewhat mor14e sensitive radiometric procedures involve the hydroxylatio 14 n of either 14f C] phenylalanine or p-tritiophenylalanine. In the C method, the [ C] tyrosine formed is separated by paper chromatography and counted (87). During the hydroxylation of p-tritio- phenylalanine, the tritium is not lost to water but instead does an "NIH shift" into the meta position (48, 88), necessitating treatment of the 3-tritiotyrosine formed with an agent such as iV-iodosuccinimide to re­ lease the tritium as water. The tritiated water is separated from the radioactive amino acids and counted, as in the assays for tyrosine hy­ droxylase (89, 90). The assay is most conveniently done using synthetic DMPH4 as cofactor, in the presence of either 2-mercaptoethanol or dithiothreitol, and either ferrous ions or catalase to protect the DMPHt from destruction and to minimize nonenzymic hydroxylations. Fre­ quently, the assay systems also involve the use of a second, nonspecific liver enzyme (dihydropteridine reductase) plus TPNH to maintain the pteridine cofactor in the active, reduced form (91-93). 2. AMINO ACID HYDROXYLASE INHIBITORS 55

2. LOCATION Phenylalanine hydroxylase seems to be specifically and uniquely lo­ cated in the liver and is a supernatant enzyme. The small amount of hydroxylation of phenylalanine that can be demonstrated in brain is probably due to tyrosine hydroxylase (94).

3. PROPERTIES Phenylalanine hydroxylase has been purified manyfold from a super- nant fraction of liver and has recently been obtained in forms that are 85-90% pure (95). The partially purified 2+enzyme requires a reduced pteridine cofactor 2an+ d is stimulated by Fe . Further evidence for a dependency on Fe is the inhibitio2+ n by agents such as a,a'-dipyridyl and the reactivation with Fe of preparations inactivated by dialysis against a,a:'-dipyridyl (96). As with tyrosine hydroxylase, DMPH4 is commonly used as the exogenous cofactor but this is less active than 6-monomethyltetrahydropteridine or tetrahydrobiopterin (31, 32), which has been isolated 5from liver (33). The Km toward DMPH4 is of the order of 4-6 X 10~ M (87, 97). The Km 4 toward substrate has been reported to be o4f the order of 1-3 X 10~ M for rat liver enzyme (98) and 1-9 X 10" M for human enzyme (87, 99), but determination was handicapped for some time by the oft-reported inhibition of the enzyme by excess substrate (86, 91, 98, 100). The maximum velocity in rat liver is of the order of 27 /mioles/hour X gm of tissue (98), which is some 50- to 1000-fold higher than the maximum velocities found for tyrosine and tryptophan hy­ droxylases in adrenal, brain, gut, and other tissues where they exist. The specific phenylalanine hydroxylase appears to develop after birth (98,101, 102), although some have suggested that the lack of hydroxylase activity in newborn liver is due to a deficiency of cofactor rather than a deficiency of enzyme (97). Maximal activity in rats is reached at about 50-60 days of age (103). It is the specific phenylalanine hy­ droxylase that is lacking in phenylketonuria (41), and some have sug­ gested that the specific enzym1e is also qualitatively or quantitatively abnormal in dilute lethal (dyd ) mice (99, 104-106). Further investiga­ tions, however, have indicated no abnormality in phenylalanine hy­ droxylase in such mice if studied at an age (14-16 days) when the effects of starvation or a terminal state are avoided (107). The partially purified enzyme does not hydroxylate D-phenylalanine, D-tryptophan (108), or tyrosine (17, Jfi, 91). It does, however, hy- 56 E. G. MCGEER AND P. L. MCGEER droxylate (17, 40, 91) L-tryptophan, /?-2-thienylalanine, 2-, 3-, or 4-fluorophenylalanine (109), p-chloro- or p-bromo- (but not p-iodo-) (109a) phenylalanine (giving the m-halotyrosine) (110), and p-methyl- phenylalanine (giving m-methyltyrosine and p-hydroxymethylphenyl- alanine) (111). The enzyme is clearly distinct from the nonspecific aryl hydroxylase or hydroxylases (112) in liver microsomes, which (a) detoxify foreign aromatic materials such as aniline or nitrobenzene (96, 118, 114) j (b) convert tyramine to dopamine (115), and (c) hydroxylate melatonin (116) and other indoles (117-119) in the 6 position. This 6-hydroxylation is believed to be the major route of melatonin metabo­ lism (120-123) and is inhibited by chlorpromazine (124). The hydroxyl­ ation of tyramine to dopamine is inhibited by desmethylimipramine or ^-diethylaminoethyl diphenylpropylacetate but not by amethopterin (125). Despite extensive work and the availability of purified enzyme, the full complexities of the phenylalanine hydroxylase system, and particu­ larly the factors involved in keeping the cofactor in the active, reduced form, remain to be elucidated. The tyrosine and tryptophan hydroxylase systems may eventually prove to be equally complex.

III. IN VITRO INHIBITORS OF THE HYDROXYLASES

The physiological importance of the hydroxylases and the availability of easy methods of assay have led to extensive screening of compounds as possible inhibitors of these enzymes. Less extensive work has been done with phenylalanine hydroxylase than with tyrosine or tryptophan hydroxylase but some inhibitors are known. The compounds that have high activity as inhibitors of any of the hydroxylases may be generally classified into four groups.

A. Catechols B. Iron-complexing agents C. Quinones, ketones, and similar compounds capable of easy oxidation/reduction D. Amino acid analogs Other compounds have considerably less activity. The hydroxylases are all relatively insensitive to sulfhydryl reagents (such as mercuric chloride or p-chloromercuribenzoate), cyanide, and iodoacetate (9, 83, 126), al­ though Gal et al. reported some inhibition of "mitochondrial" brain 2. AMINO ACID HYDROXYLASE INHIBITORS 57 tryptophan hydroxylase by either p-chloromercuribenzoat-4 e or cyanide (54) and of liver phenylalanine hydroxylase by 10 M p-chloromercuri- benzoate, BAL, or sodium borohydride (127). Petrack et al. (35) found purified adrenal tyrosine hydroxylase to be markedly inhibited on pre­ incubation with mercuric chloride or p-hydroxymercuribenzoate. One re­ port suggested that folic acid antagonists such as amethopterin or 2-amino-3-hydroxypteridine are good inhibitors of phenylalanine hy­ droxylase (91), but others indicated little or no inhibitor4 y effect on phenylalanine hydroxylase (10-24% inhibition at 10" M) (59, 126), on tryptophan hydroxylase (54, 69, 83), or on tyrosine hydroxylase (9). Since all three hydroxylations are oxidation/reduction reactions ap­ parently involving molecular oxygen, ferrous ions, and the same or a highly similar pteridine cofactor, it may be expected that compounds in classes A, B, and C will be active against all three enzymes. More specific inhibition would be expected in class D.

A. Catechols

A number of o-catechols were shown by Udenfriend et al. (89) to inhibit tyrosine hydroxylase by competition with the pteridine cofactor. The o-catechol function was necessary for activity; neither phenolic derivatives nor 3-methoxy-4-hydroxyphenyl derivatives showed any significant inhibitory action. Both the requirement for an o-catecholic function and the mechanism as competitive toward cofactor have been confirmed by others in various systems (128, 129). The inhibition of par­ ticle-bound hydroxylases by catechols is not affected by adding pteridine cofactor (83), but this may be due to the failure of exogenous cofactor to be taken up by the particles. It has been suggested that some cate­ chols, such as n-propyl gallate or a,/?,/?-trimethyl-DOPA (126), may have a dual effect and inhibit not only by competition with pteridines but by competition with ferrous ions as well. Johnson et al. (128) re­ ported that inhibition of tyrosine hydroxylase by a-n-propyldopacet- amid2+e (H 22/54) was markedly decreased in the absence of exogenous Fe ; the inhibition by various arterenones such as 3,4-dihydroxy-a:-di- methylaminoacetophenone was, however, not appreciably affected. Since many catechols are easily oxidized to quinones, they may also act partly by nonspecific effects on the oxidation/reduction systems and thus be a special subgroup of the type of inhibitors discussed in Section III,C. As might be expected, catechols active against tyrosine hydroxylase TABLE III PERCENT INHIBITION BY REPRESENTATIVE CATECHOL S IN VARIOUS ASSAY SYSTEMS

a-n- Methyl- Concentration (m M) of Propyl- amino- a- dopacet- aceto- Methyl- Dopa­ Sub­ In­ amide catechol DOPA L-DOPA mine Ref­ Enzyme Source strate DMPH 4 hibitor (%) (%) (%) (%) (%) erence Tyrosine Adrenal 0.1 0.5 0.025 66 35 hydroxylase 0.05 1 0.02 50 39 0.05 1 2 52 50 39 0.1 'Pa 0.1 67 30 4 0.1 1.7 0.1 78 55 128 0.05-0.1 1 0.05 70 130 Brain stem 0.02 0 0.1 80 37 0.012 0.1 84 25 68 56 4 58 Whole brain 0.005 0 0.1 79 57 68 69 9

Tryptophan Mast cell 0.125 0.2 0.1 90 60 70 30 75 hydroxylase 0.06 0.5 0.005 23 65 128 0.1 0.2 0.5 90 50 66 Whole brain 0.01 0 0.1 51 27 60 47 83 Brain mito­ chondria 0.0&1 0 0.7 58 54 6 Phenylalanine Liver 10b 0 0.1 100 108 hydroxylase 10 0 0.001 50 50 108 10 3 0.003 68 47 53 126 10 3 0.003 58 33 126 10 3 0.1 47 126 1.5 0 0.01 50 59 1.5 0 0.1 10 10 59 0.5 0 0.5 <50 50 >50 131 b ° T, concentration 5 mM in tetrahydrofolate . With tryptophan as substrate. 2. AMINO ACID HYDROXYLASE INHIBITORS 59 were also generally found to be active against tryptophan hydroxylase and, to the extent that they have been tested, against phenylalanine hydroxylase. The exact percentage of inhibition given by a particular catechol can be expected to vary with the assay conditions used. Some representative data on a few catechols that have been tested by a number of investigators in different systems are shown in Table III {ISO, 1S1). Early reports on the inhibition of the hydroxylation of tryptophan by liver preparations indicated that almost all o-catechols were highly active (108, 132), but these studies, as pointed out in Section II,B, were using a nonphysiological substrate. More extensive explorations using tyrosine hydroxylase from brain or adrenals, tryptophan hydrox- xylase from brain or mast cell tumors, and phenylalanine hydroxylase from liver indicate that the nature of the substitution on the catechol is important to inhibitory activity. Acidic catechols, such as 3,4-dihydroxy- benzoic acid and 3,4-dihydroxyphenylacetic acid, tend to be relatively inactive against all three hydroxylases. The most potent derivatives seem to be those with a neutral side chain such as a-chloro-3,4-dihy- droxyacetophenone, ethyl /?-3,4-dihydroxyphenylpropionate, or a-n-pro- pyldopacetamide (H 22/54). A ketone group next to the ring appears to potentiate activity, perhaps because it adds to the length of the oxida­ tion/reduction resonance system. Further ring substitution, as in 6-hy- droxydopamine or 2-methyl-a-methyl-DOPA, seems to reduce or elimi­ nate activity. In optically active compounds, such as DOPA, the D isomer is much less active than the L isomer. Thus, all but three or four of the most active catechols so far found (Table IV) may be classed as side-chain-substituted derivatives of 3,4-dihydroxyphenylacetamide (dopacetamide), 3,4-dihydroxyphenylethylamine, or 3,4-dihydroxy- phenyl ketone. The compounds in Table IV are listed in the approximate order of their activity against brain tyrosine hydroxylase. It is evident that, in general, this order also holds for adrenal tyrosine hydroxylase and even, to a great extent, for tryptophan and phenylalanine hydroxylases. The latter two enzymes, however, seem to be somewhat more vulnerable than tyrosine hydroxylase to certain of the less substituted derivatives such as catechol itself or a-methyldopacetamide. Dopamine and norepinephrine are included in Table IV because they have been proposed as physiological feedback inhibitors (see Section V,A). Their in vitro effect, however, is not as dramatic as that of some of the other catechols. The inhibitory action of apomorphine toward tyrosine hydroxylase is of some interest since this compound has been suggested as a thera- TABLE IV INHIBITORY ACTIVITY OF VARIOUS CATECHOLS RELATIVE TO ACTIVITY OF METHYLAMINOACETOCATECHOL (I) OR a-PROPYLDOPACETAMIDE (II ) °

Phenyl­ Tryptophan alanine hy­ 6 Tyrosine hydroxylase hydroxylase droxylase Type Brain Adrenal Brain Mast cell Liver

Inhibitor K D A M P IP IP IF IP IP IP P P IP IP IP 11™

Esculetin (6,7-Dihydroxy- coumarin) X 100 101 n-Propyl gallate X 140 Dihydroxyphenyl ethyl ketone X 122 190 Chloromethyl dihydroxyphenyl ketone X 11 6 176 C5 r o Dihydroxyphenyl ketone X 115 173 Epinine (A -methyldopamine) X 102 107 n-Propyldopacetamide 100 100 100 100 100 36 100 100 100 100 iV,iV-Dibenzylarternone X 83 Methylaminoacetocatechol X 100 71 71 100 100 3,4-Dihydroxybenzaldehyde X 96 56 146 L-DOPA 88 81 15 45 115 78 57 a-Pyridylaminoacetocatechol X 78 92 3,4-Dihydroxyphenyl a-methyl- aminoethyl ketone X 77 77 iV-Methylepinephrine X 76 DL-Isopropylnorepinephrine X 76 125 a-n-Butyldopacetamide X 76 a-Isobutyldopacetamide X 70 3,4-Dihydroxybenzophenone X 63 a-Methoxydopacetamide X 90 a-Methyldopacetamide X < 19 80 a-Hydroxydopacetamide X 80 Aminoacetocatechol X 63 42 111 1-Piperidinoacetocatechol X 59 115 1-Pyrrolidinoacetocatechol X 5 9 128 Dopamine X 87 67 57 92 33 10 Dimethylaminoacetocatechol X 51 116 Cobefrin (a-methyldopamine) X 73 106 9 5 Ethyl 3,4-dihydroxyphenyl- propionate X 73 130 Apomorphine X 73 3,4-Dihydroxyacetophenone X 5 0 5-Hydroxyarterenone X 50 a-Ethyldopacetamide X 5 0 90 tt-Isopropyldopacetamide X 48 DL-N-Ethylnorepinephrine X 72 107 L-«-Methyl-DOPA X 72 20 30 54 56 67 10 17 L-Epinephrine X 59 56 75 Isopropylaminoacetocatechol X 5 7 49 93 100 61 L-Norepinephrine X 4 9 30 50 55 72 10 55 a-Methylnorepinephrine X 47 49 Catechol X 45 49 6 158 a The activities of each compound are expressed as a percentage of the activity in the same assay system of either methylamino­ acetocatechol (I) or a-n-Propyldopacetamide (H 22/54) (II); a figure of 50 under a column labeled I would mean that particular compounb d was half as active an inhibitor as methylaminoacetocatechol. Active catechols are classifiable into derivative s of ?1 ?2 HO X (K) (D) HO CH—CH—N (A) Y HO HO andc other (M). k d Data from 9, 83. °h Data from 130. 1Data from 75. e Data from 4- i Data from 35. m Data from 59. Data from 128. Data from 83. Data from 126. f Data from 39. * Data from 66. TABLE V 0 PERCENT INHIBITION BY VARIOUS COMPLEXING AGENTS IN in Vitro ASSAYS OF AROMATIC AMINO ACID HYDROXYLASES

Tryptophan hydroxylase Phenylalanine hydroxylase Tyrosine hydroxylase Brain mito- Liver Brain Adrenal & c c d A Brain chondria Inhibitor 0.1 l 0.1 0.1 0.1' 0.1" 0.1 0.1' 0.01* 0.4'.* 0.1* 0.6™ 1.2*

4-Isopropyltropolone 90 90 8-Hydroxyquinoline 13" 86 49 60 5-Iodo-8-hydroxyquinoline 47 73 Bilirubin 51 <15 o-Phenanthroline 100 57

62 2,9-Dimethy 1-1,10-phenanthroline 56 85 a-a'-Dipyridyl 24" 79 31 39 80 33 Desferrioxiamine 82 2-(4-Thiazolyl)benzimidazole 60 Dibenzo [/,/i]quinoxaline r 52 2,4,6-Tripyridyl-s-triazine 28 Ethyl 3-amino-4fl -pyrrolo- isoxazole-5 (6i/)-carboxylate 50 a-Nitroso-/3—naphthol 28 70 79" Sodium diethyldithiocarbamate 49" 33 <15 <15 50 31 Ethylenediaminetetraacetic acid 42" 26" <15 35 <15 e ° Value above each column indicates millimola r concentration of inhibi­ f k b Data from 8. > Data from 76. tor; values within table indicate percent inhibition by inhibitor. 0 Data from 135. 1 c h Supernatant fraction. Data from 108 Data from 136. m Data from 83. d Data from 126. Data from 4. n Data from 54. Data from 9. * Data from 137. Tryptophan as substrate. 2. AMINO ACID HYDROXYLASE INHIBITORS 63 peutic agent in Parkinson's disease (133). Its usefulness would presum­ ably depend on stimulation of dopaminergic receptors and might be lessened by any inhibition of tyrosine hydroxylase. The inhibitory action of apomorphine is decreased by increasing concentrations of DMPH4 in the incubation medium (134) •

B. Iron-Complexing Agents

As discussed in Section II, stimulation of the hydroxylases by ferrous ions has been observed in onl2+ y a few preparations, and the supposition that these enzymes are Fe dependent rests mainly on the inhibition generally observed with iron-complexing agents such as a,a'-dipyridyl and o-phenanthroline. A list of compounds that have been reported to inhibit substantially one or more of the hydroxylases and whose struc­ tures suggest that they may act by complexing with ferrous ions is given in Table V (135-137). The potency of some of these agents seems well established but that of others such as ethylenediaminetetraacetic acid and sodium diethyldithiocarbamate is controversial, and the weight of evidence suggests little or no activity. The chelating agents as a group do not appear as active as the cate­ chols discussed in Sectio2+n III,A. Partial reversal of the inhibition in vitro on addition of Fe ions to the incubation medium has been shown for o-phenanthroline (137), for ethyl 3-amino-4#-pyrrolo[3,4-c]isox- azole-5(6AT)-carboxylate, which is the most active of a series of pyr- roloisoxazoles tested as inhibitors of adrenal tyrosine hydroxylase (136), for a,a'-dipyridyl (76), and for 4-isopropyltropolone (135).

C. Compounds Capable of Easy Oxidation/Reduction

A number of quinones, ketones, and related compounds (Table VI) (138-140) have been found to inhibit tryptophan, tyrosine, and/or phenylalanine hydroxylase but2 +do not appear to be truly competitive with substrate, cofactor, or Fe ions (29, 138, 141)- It has been specu­ lated that these compounds act by nonspecific interference with the oxi­ dation-reduction processes. Such an interference might be expected in view of the quinoidal nature of the cofactor product7 . The strong inhibi­ tion of tyrosine hydroxylase [Ki = 3.6 X 10" M (29) ] by the quinoida2l+ antibiotic, aquayamycin, could be partially reversed by addition of Fe but was increased [as was that of some simpler quinones (140)] with TABLE VI PERCENT INHIBITION BY VARIOU S COMPOUNDS CAPABLE OF EASY OXIDATION/REDUCTIO0 N IN in Vitro ASSAYS OF AROMATIC AMINO ACID HYDROXYLASES

Phenylalanine Tryptophan hydroxylase Tyrosine hydroxylase hydroxylase Brain Liver c d eBrain Adrenal stem Brain Inhibitor 0.3* 0.08 l > 0.V 1* 0.1* 0.00037* 0.001''* 0.1*-< m Aquayamycin 5 0 78 l,2-Naphthoquinone m 98 50 1,^Naphthoquinone™ 7 0 l,3-Dichloro-l,4-naphthoquinone 55 2-Methyl-l,4-naphthoquinone 61 Menadione (vitamin K) 50 2,5-Dimethoxybenzoquinone 7 5

64 6-Amino-7-chloroquinoline-5,8-quinone 7 9 1,8-Dihy droxy-4,5-dinitroanthraquinone 4 7 Adrenochrome 42 33 Sodium indophenol 5 4 55 2,6-Dichlorobenzoindophenol 80 73 3-Hydroxy-2-naphthaldehyde 8 7 <20 Phenylpyruvic acid 5 5 42 p-Hydroxyphenylpyruvic acid < 5 47 20 <15 2,5-Dihydroxyphenylpyruvic acid 68 <10 p-Chlorophenylpyruvic acid 5 5 Indole-3-pyruvic acid 54 <15 Af,V-Dimethyl-p-phenylenediamine 76 63 A^,iV-Diethyl-p-phenylenediamine <1 5 75 Methylene blue 43 73 Azur eosin (Giemsa stain) 36 68 *S-Methylcysteine 5 6 a d e Value above each column indicates millimola r Data from 132. k*' Data from 29. concentratiob n of inhibitor; values within table With tryptophan as substrate. 1> Data from 76. indicatc e percent inhibition by inhibitor. *h Data from 73. m Supernatant. Data from 99, 138. o Data from 139. Data from 83. Data from 138. Data from 140. Corresponding diols inactive (138). 2. AMINO ACID HYDROXYLASE INHIBITORS 65 increasing cofactor concentrations. These findings suggest that such quinones might inhibit in the same way as do high concentrations of cofactor (29). The inhibition of tryptophan hydroxylase by aquayamycin was almost eliminated by addition of dithiothreitol to the medium, sug­ gesting interference with an oxidation-reduction system (76). Some of these compounds, such as sodium indophenol and 2,6-dichloro- benzoindophenol, have shown approximately equal activity toward tyro­ sine and tryptophan hydroxylases, but others have shown marked differ­ ences in activity toward the two enzymes in the in vitro systems used. This may be largely a matter of pH effects on unstable compounds. Thus, for example, pyruvate derivatives such as indole-3-pyruvic acid and 2,5-dihydroxyphenylpyruvic acid were very active against tyrosine hydroxylase in vitro at pH 6.2 but had only minimal action against tryptophan hydroxylase at pH 7.8 (83). Inhibition of phenylalanine hydroxylase by phenylpyruvic acid and other "abnormal" oxidative me­ tabolites of phenylalanine is not great enough to be of significance in phenylketonuria (141)- Some substituted p-phenylenediamine derivatives are included in this category of inhibitors as well as and azur eosin, each of which has a dialkylamino group in a position para to the nitrogen on the phenothiazine ring system. They may thus be considered analo­ gous to substituted p-phenylenediamines. These compounds should all be capable of easy oxidation to a free-radical semiquinone form. Inhibi­ tion of yeast growth and of certain enzymes such as succinic dehydro­ genase by iV,Af-dimethyl-p-phenylenediamine has previously been at­ tributed to such easy oxidation (11+2). As illustrated by this example, it may be expected that inhibitors of this class would affect a wide variety of oxidative enzyme systems.

D. Amino Acid Analogs

The most important group of inhibitors of the hydroxylases so far discovered is comprised of certain substituted derivatives of phenyl­ alanine, tyrosine, and tryptophan. Table VII (143-147) lists most of the compounds of this class that have been found to show considerable inhibitory activity toward any one of the hydroxylases, while Table VIII (148-151) indicates the percentage of inhibition found for a few aromatic amino acids in various in vitro assay systems. It is evident that the reported results are quite discordant in some cases as are, for example, the data for 3,5-diiodotyrosine. More detailed studies with 3,5- TABLE VII 0 PERCENT INHIBITIO NOF AROMATIC AMINO ACID HYDROXYLASE FOUNS D in Vitro WITH SOME AMINO ACID ANALOGS

Tyrosine hydroxylase Tryptophan Phenylalanine b Adrenalc d Brain e hydroxylase, brain hydroxylase, liver Inhibitor 0.1 0.1 0.2 oT OJL' 1* I* V

Phenylalanine derivatives a-Methyl-DL- 86 46 <50 3-Iodo-a-methyl-DL- 84 52 3-Iodo-DL- 68 84 68 56 3-Bromo-DL- 41 82 70 33 <50 4-Fluoro-a-methyl-DL- 85 4-Amino-a-methyl-DL- 77 3-Bromo-o;-methyl-DL- 76 32

6 4-Fluoro-DL- 75 64 77 88 58 4-Chloro-a-methyl-DL- 71 32 L_y 30 78 64 3- Hy droxy-a-me t hy 1-L- 62 54 41 <15 2-Fluoro-DL- 60 78 0 <50 /S-Methyl-DL- 0 51 31 4-Chloro-DL- 41 50 71 31 2-hydroxy-DL- 30 50 63 3-Hydroxy-DL- 49 69 3-Chloro-a-methyl-DL- 48 16 4-Iodo-DL- 47 78 20 3-Iodo-o-methoxy-DL- 43 90 4-Nitro-DL- <15 80 <50 Tyrosine derivatives 3-Iodo-a-methyl-DL- 94 3-Iodo-L- 90 97 36 a-Methyl-L- 100 <15 3,5-Diiodo-L- <15 81 33 2-Fluoro-<*-methyl-DL 95 2-Chloro-a-methyl-DL- 84 3-Bromo-a-methyl-DL- 73 2a-Dimethyl-DL- 69 <15 <15 3-Amino-L- 65 <15 3-Nitro-L- 64 a-Ethyl-DL- 60 3-Chloro-a-methyl-DL- 60 3-Fluoro-L- 50 ryptophan derivatives a-methyl-5-hydroxy-DL- 80 5-Iodo-DL- 100 42 5-Bromo-DL- 98 45 5-Chloro-2-methyl-DL- 96 23

67 5-Chloro-DL- 90 44 5-Methyl-DL- 75 30 2-Methyl-DL- 68 28 5-Hydroxy-DL- 30 59 <20 L-> 20 58 a-Methyl-DL- 17 52 27 6-Methyl-DL- 45 53 5-Fluoro-DL- 42 50 6-Chloro-DL- <15 75 6-Fluoro-DL- <15 80

° Value above each column indicates millimolar concentration of inhibitor; values within table indicate f Data from 83, 147. b h percent inhibition by inhibitor. 0 Data from 126. c Data from 1^0. l Data from 145. d Data from 143. ' Data from 131. e Data from 144, 145. 1 Unsubstituted Data from 9, 146. Compound. TABLE VIII PERCENT INHIBITION OF AROMATIC AMINO ACID HYDROXYLASES BY SOM E AMINO ACID ANALOGS IN VARIOUS in Vitro ASSAY SYSTEMS

Concentration (mM) of (%) (%) (%)

Sub­ Inhi­ Ref- Enzyme Source strate DMPH4 bitor L- D- 4-F- 4-C1- L- 3-1- l- d- 5-F- 5-HO- =-Me- erence

Tyrosine Adrenal 0.1 1 0.01 85 72 40 hydroxylase 0.1 0.5 0.025 83 67 85 0.2 1 0.1 20 US 0.1 0 0.1 30 75 41 90 <15 HO 0.1 2 0.05 67 HO 0.1 0 0.05 94 HO 0.1 b 0.1 10 0 61 60 4 0.01 5 0.1 100* 5-15 148 b 0.05 0.02 50 89 0.05 2 0.2 54< 33 30 H4,14$ Brain 0.005 0 0.1 78 64 50 97 52 9.H b 0.012 0.1 78 7 50 4 0.008 0 0.1 80 80 65 23 00 0.02 0 0.1 60 87 Heart 0.01 4 0.1 HS Tryptophan Brain 0.01 0 0.1 64 77 69 27 36 <10 33 <20 83, H7 hydroxylase 0.009 0 0.08 56 54 Mitochondria 0.009 0 0.04 Activate 54 Mast cell 0.125 0.2 1 30 35 75 0.125 0.2 0.1 55 75 Phenylalanine Liver 0.3 0.07 0.5 50« 149 hydroxylase 10 3 0.1 0 126 0.43 0.09 5 30« 149 16* 0 0.1 22 47 2 108 0.12 1 0.01 0 0 40 10* 3 0.1 14 67 14 126 10* 3 1 19 126 10 3 1 88 126 20* 0 0.1 80 39 132 20* 1 0.1 8 1 182 1 1 1 58 31 145 16* 0 0.2 40 0 32 150 16* 0 0.1 39 151 0 3.2 0 c 0.1 0 151 b Noncompetitive with substrate. Competitive with substrate. Concentration 5mAf in tetrahydrofolate. * With tryptophan as substrate. 2. AMINO ACID HYDROXYLASE INHIBITORS 69

TABLE IX APPROXIMATE Ki FOR SOME AMINO ACID DERIVATIVES AS INHIBITORS OF TYROSINE AND/OR TRYPTOPHAN HYDROXYLASE*

Tryptophan Tyrosine hydroxylase hydroxylase 6 c d Inhibitor Adrenal Brain Brain 7 3-Iodo-a-methyl-DL-tyrosine 1.8 X 10~7 6 3-Iodo-L-tyrosine 3.9 X 10" 1.9 X 10"6 5-Iodo-DL-tryptophan 2.5 X 10"6 5-Bromo-DL-tryptophan 6 4.2 X 10" 3,5-Diiodo-L-tyrosine 9.3 X 10"5 a-Methyl-L-tyrosine 1.7 X 10" 4 L-Tryptophan 1.5 X 10"3 5-Fluoro-DL-tryptophan 3.6 X lO- 1.5 X 10"<{ p-Chloro-DL-phenylalanine 1.6-2 X 10- 6-Fluoro-DL-tryptophan 9-12 X 10"'

°6 Approximate Ki expressed as molar value. c Data from 39. d Data from 146. Data from 147.

diiodotyrosine suggest that it is as potent an in vitro inhibitor of tyrosine hydroxylase as a-methyl-p-tyrosine (Table IX). The extensive screening of amino acid analogs as inhibitors of tyrosine and tryptophan hydroxylases allows some conclusions as to the relation­ ship between structure and activity (9, 39, 83, lift, 144-147). The posi­ tion and nature of the substituents appear to be critical for activity and for selectivity toward each of the hydroxylases. Electrophilic substitution in the para position of phenylalanine or in the 6 position of tryptophan by a nitro group or halogen seems to increase the activity toward tryptophan hydroxylase while decreasing the activity toward tyrosine hydroxylase. Substitution of a halogen in the meta position of phenylalanine or tyrosine or in the 5 position of tryptophan causes a strong enhancement of inhibitory action toward tyrosine hydroxylase without much effect on the activity toward trypto­ phan hydroxylase. Thus, 3-iodotyrosine and 5-iodotryptophan are effec­ tive tyrosine hydroxylase inhibitors in vitro but are relatively weak against tryptophan hydroxylase. p-Chlorophenylalanine and 6-fluoro- tryptophan, on the other hand, are potent inhibitors of tryptophan hy­ droxylase but have little action on tyrosine hydroxylase. In both series 70 E. G. MCGEER AND P. L. MCGEER of inhibitors, the nature of the halogen appears to be much more critical for tyrosine hydroxylase inhibition than for tryptophan hydroxylase in­ hibition. Introduction of a methyl group on the a position of the side chain seems to decrease inhibitory potency toward tryptophan hydrox­ ylase with possible enhancement of inhibitory potency toward tyrosine hydroxylase. Among the phenylalanine and tyrosine derivatives, esters appear to be approximately equal to the parent amino acid as inhibitors of either hydroxylase. However, other alterations of the side chain, such as N substitution, hydroxyl substitution as in phenylserine, decarboxyla­ tion, rearrangement as in /?-aminophenylpropionic acid, or lengthening or shortening as in 3-iodophenylglycine or 4-(3-iodophenyl)-2-methyl- 2-aminobutanoic acid, appear to destroy inhibitory activity. No amino acid inhibitor of phenylalanine hydroxylase has yet been found with an in vitro activity comparable to those of the most potent amino acid inhibitors of tyrosine and tryptophan hydroxylases. Halo- genated phenylalanines are the only type that have shown promise in the limited screening so far done. The weight of evidence suggests that 2-halogenated phenylalanines have little or no action against phenyl­ alanine hydroxylase {131, H5), although there is one report (126) of 2-fluorophenylalanine showing promise. The distinction in activity be­ tween 3- and 4-substituted derivatives is not as clear-cut as for inhibition of the other hydroxylases, and the nature of the halogen appears critical. If the substitution is in the 3 position, the order of activity is I > Br > CI > F, but in the 4 position the reverse order holds. Thus, the most active inhibitors in these series are 3-iodophenylalanine and 4-fluorophenylalanine (145). Introduction of an a-methyl group into the halogenated phenylalanines does not enhance inhibition of phenylalanine hydroxylase (14$)- This is one of a number of examples in which struc­ tural requirements for phenylalanine hydroxylase inhibition are closer to those for tryptophan than for tyrosine hydroxylase inhibition. As has been found for tyrosine and tryptophan hydroxylases, reduction or lengthening of the alanine side chain destroys inhibitory potency. Some amino acids have shown stimulatory effects on the hydroxylases. Phenylalanine, for example, is said to enhance hydroxylation of trypto­ phan by brain (54) and liver (149) at low concentrations and to inhibit it at high concentrations. The inhibition of phenylalanine hydroxylase by excess substrate has already been mentioned. Thyroxine has been reported to stimulate tyrosine hydroxylase from adrenals (148) and brain (23) in vitro but the in vivo effects may be more complex (see Section V,C). Competition with substrate has been demonstrated to be the mech- 2. AMINO ACID HYDROXYLASE INHIBITORS 71 anism of action for the in vitro inhibition of tyrosine hydroxylase by phenylalanine (144) , 3-iodophenylalanine (144, H5), L-3-iodotyrosine (148) j 3,5-diiodo-L-tyrosine (148), 5-halotryptophans (146), and a-methyl-p-tyrosine (4, 148) despite one report (140) that the last- named compound is competitive with cofactor and not with substrate. It has also been demonstrated for tryptophan hydroxylase inhibition by p-chlorophenylalanine and 6-fluorotryptophan (147). a-Methyl- 5-hydroxytryptophan, on the other hand, has been reported to be com­ petitive toward pteridine cofactor (148) for tyrosine hydroxylase inhibi­ tion as have p-halogenated phenylalanines for phenylalanine hydroxylase (182).

IV. IN VIVO INHIBITORS OF THE HYDROXYLASES

Hydroxylase inhibition in vivo presents more complex challenges than inhibition in vitro. A compound active in vitro may be rapidly metabol­ ized to an inactive derivative or, conversely, to a more active derivative. Its effect on the rate of hydroxylation in vivo may be on the control mechanisms that normally regulate the synthesis rather than on. the hydroxylation enzyme per se. The compounds most easily classified are those that have been demonstrated to inhibit the hydroxylases in vitro.

A. Catechols

Most of the catechols have only minimal effects in vivo. Very large doses of a variety of catechol derivatives such as 3,4-dihydroxy-6- methyl-o-methylaminoacetophenone (6-methylarterenone) (128, 180), ethyl 3,4-dihydroxyphenylpropionate, or 3,4-dihydroxybutyrophenone (88) have been used in rats without significant changes in brain 5-HT, brain tryptophan hydroxylase, or brain tyrosine hydroxylase, a-n- Propyldopacetamide (H 22/54) has been reported to reduce mouse or rat brain 5-HT (152-156), NE, and dopamine levels (155, 157) after single IP doses of 300-500 mg/kg. The extent of depletion was dependent on the environmental temperature (155). Pineal 5-HT was particularly affected (158). Ross and Haljasmaa (156) also found brain 5-HT de­ creases after the administration of a variety of related catechols includ­ ing 2,3-dihydroxy isomers of compounds such as H 22/54. Both H 22/54 and a,/?,/?-trimethyl-DOPA have been reported to decrease levels of NE 72 E. G. MCGEER AND P. L. MCGEER

in rat heart, spleen, liver, and adrenals with the extent of depletion in each organ being affected by the environmental temperature {157, 159). Others (83, 128) have used similar doses of H 22/54 in mice, rats, and guinea pigs without finding significant effects on brain 5-HT levels. There were questionable effects on NE concentrations. a,/?,/?-Trimethyl- DOPA does not affect endogenous brain levels of 5-HT or NE (126). Replenishment of 5-HT by 5-HTP in rats treated with H 22/54 has been taken as evidence that the drug is acting in vivo on tryptophan hydroxylation (154). Assays in vitro on the livers of rats sacrificed after injection of H 22/54 or a,/?,/Mrimethyl-DOPA in vivo show sig­ nificant decreases in the ability to hydroxylate phenylalanine (160, 161) or tryptophan (126, 132, i62), although the inhibition can be largely overcome by addition of pteridine cofactor to the in vitro assay system (132). Similar results have been obtained with a variety of 2,3- and 3,4-dopacetamides related to H 22/54 (156). Rats treated with large doses of H 22/54 show a slight decrease in locomotor activity but no change in estrous behavior such as obtained with more potent inhibitors of tryptophan hydroxylase (153). Rats treated with apomorphine (25 mg/kg sc) sho14w a 50% decrease in the synthesis in telencephalon an14d brain stem of [ C] catecholamines from intraventricularly injected [ C]tyrosine (134)- The interactions of apomorphine with the catecholinergic system (163) are complex, how­ ever, and direct inhibition of tyrosine hydroxylase probably plays a very minor role in its physiological actions. Apomorphine is thought to have a more prominent action on dopaminergic receptors. The catechol, «-methyl-DOPA, has been shown to lower slightly brain 5-HT (156) and dopamine levels and, more profoundly, brain and heart NE levels (46, 164). This effect is, however, primarily due to displace­ ment of normal amines by the a-methyldopamine and a-methyl-NE formed in vivo (46, 164-167) rather than to any inhibitory effect on either the hydroxylases or the decarboxylase (168-170). Equivalent degrees of decarboxylase inhibition can be reached in vivo by compounds such as «-methyl-2,3-dihydroxyphenylalanine with little or no effect on tissue amine levels (46). Gal et al. (171) showed that administration of a-methyl-DOP4 A in amounts calculated to produce a concentration of 1-5 X 10" M in brain did not impair the ability of brain tissue to hydroxylate tryptophan in vivo. Another catechol, 6-hydroxydopamine, has been found to deplete catecholamines from heart and sympathetic nervous system (172-173a) on peripheral administration and from brain on intraventricular admin­ istration, but this is also due, not to any inhibitory effect on tyrosine 2. AMINO ACID HYDROXYLASE INHIBITORS 73 hydroxylase, but to an immediate displacement effect (166) coupled with a destructive action on the terminal binding sites (174) and neurons themselves (173, 175-178e). The damage to adrenergic nerve terminals by 6-hydroxydopamine is at least partially reversible in the sympathetic nervous system (179). In summary, the catechols have not yet yielded a drug of great practi­ cal utility as a hydroxylase inhibitor in vivo. It may be expected that any active catechol will interfere to some extent with any enzyme that requires a pteridine cofactor and possibly with other oxidative systems; H 22/54 (158), related dopacetamides (180), and 3',4'-dihydroxy-2- methylpropiophenone (181) have, for example, been shown to inhibit catechol O-methyltransferase (COMT) in vivo.

B. Iron-Complexing Agents

The chelating agents so far identified as inhibitors do not generally appear as active in vitro as the catechols, but the few experiments that have been done suggest that they may be more active in vivo. Thus, Taylor et al. (137) found that, when «,a'-dipyridyl was administered intraperitoneal^ to rats, there was a dose-related decrease in adrenal medulla tyrosine hydroxylase activity as measured in vitro. In animals given 100 mg/kg, the adrenal tyrosine hydroxylase activity was 54% of control at 3 hours, 40% at 6 hours, and then gradually rose to normal levels by about 12 hours. The depletion of NE in adrenals, brain, and heart was more marked (down to 20-25%) and prolonged (>24 hours) than would be expected from the extent and duration of tyrosine hy­ droxylase inhibition observed. It is probable that a,a:'-dipyridyl was also acting on dopamine ^-hydroxylase, which is known to be affected by a variety of copper-chelating agents, including a,a'-dipyridyl and o-phenanthroline (182). A depletion of dopamine in animals treated with a,a'-dipyridyl was suggested on the basis of the development of a mild tremor and ptosis (137); the relationship between development of tremor in rats and the dopamine content of the brain, however, is not clear enough to conclude that development of tremor indicates dopamine depletion. Intraperitoneal injection of 100 mg/kg of a,a'-dipyridyl into rats was found to decrease the phenylalanine hydroxylase activity in liver, measured in vitro, to about 40% of control after 1 hour (160). Another chelating agent, 4-isopropyltropolone, in a dose of14 100 mg/kg ip has been shown to lower the conversion of injected [ C]tyrosine 74 E. G. MCGEER AND P. L. MCGEER 14 to [ C] catecholamines in vivo in brain, heart, and adrenals by about 60-80% (135). have also been shown to inhibit dopamine /^-hydroxylase (182, 183) and COMT (180, 184) by chelation with the metallic proteins of these enzymes, so again a multiple mechanism may be operating in the in vivo effects. The chelating agents, even more than the catechols, may be expected, if active at all, to affect a variety of enzyme systems in vivo, including all three aromatic amino acid hydroxylases. They would appear therefore to be of limited usefulness in specific and selective inhibition of a single hydroxylase.

C. Compounds Capable of Easy Oxidation/Reduction

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-MPT). 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

TABLE X NOREPINEPHRINE AND DOPAMINE LEVELS IN SOME TISSUES OF ANIMALS TREATED WITH a-MPT

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 mmole/kg, 3 hours 54(32) 75 157 Cat 0.55 mmole/kg, bid for 28 10 38 193 2 days Hamster 5.2 mmole/kg, 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 XI). Recently it has been reported that a-MPT in monkeys selectively decreases REM 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-DOPA (44, 216, 217), norepinephrine (212), or amphetamine (216, 218). Administration of L-DOPA alone (219, 220), or with an MAO 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 IX), 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-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

TABLE XI SOME BEHAVIORAL EFFECTS OF p-CHLOROPHENYLALANINE AND CX-METHYL-p-TYROSINE IN ANIMALS"

p-Chlorophenylalanine a-Methyl-p-tyrosine (or its methyl (or its methyl ester H 69/17) ester H 44/68)

Self-stimulation l(228R, 240R and C), i(228R, 218R), (j) (24IR) — (241R) Brightness discrimination learning U242R) Maze learning -(242R, 248R), |(^R) b C Active avoidance U245R,d 246R, 243R, 247R) l(248R, 216R, 249R ) Conditional avoidance l(250R) , ](245R) \(250aR and C, 220R, 251R, 252Ry 253G} 254G), -(192R) Sensitivity to pain UUSR, d 255R) c Lever pressing for food l(256R) i(257R) C h Lever pressing for water (I) (257R) , i(258R} 217R ) Hunger i(259R) Body temperature - (260R) Habituation to auditory stimulation (i) (261R) c C Locomotor activity (i) (153R, 243R, e i(257R , 264R , 248R, T (245R, 263R )} 252R, 232M, 193G and - (262M) C, 212R, 265R and M, 266R) Rotarod performance l(248R, b 251R, 252R) Tremor and catatonia ](267M) Sedation or sleep i(268R and C, 269C, 270C, \(283R} 193G and C, 271C, 272R, C273C, 274R, 220R), -(192R, 258R) 275Mn, 276C , 277C% 278M, e279R, C280Hu, 28mue , 282C >0 "Conflict" l(284R , 285R) e Aggression \(286M} 287R) ](288R , 289R, 290C, Interspecies aggression 291R°) U292R, 293M, 294M, 295R, (|) (293M, 294M-), Seizure susceptibility U297R, 298R, 299R, 277C', Mounting d 288R} 290C, 300R% 291Re )* Estrous behavior ](277 , 801R, 153R, l(303R) 288R), -(302R) Sexual development of females l(304R) 2. AMINO ACID HYDROXYLASE INHIBITORS 79

TABLE XI (Continued)

p-Chlorophenylalanine a-Methyl-p-tyrosine (or its methyl (or its methyl ester H 69/17) ester H 44/68)

Self-admin, of EtOH l(305R, 306R, S07R') (i)(305R) Interaction with be­ havioral effects of b Amphetamine - (308M, 309R) i(310M, 249R, 811R, 309R} 312R ,191R, 813M, 44R, 192R, 314R, 308M); t, U or - (315R) p-Chloroamphetamine l(308M) -(315R), (I) (308M) Mescaline i(316R) l(44&), -(816R) a-MMT \(283R) Morphine abstinence symptoms -(317M), i(318M) U319R, 320R) e b b Analgesia - (320aM) 1(321M} 320R) General l(322R} 320R ), -(322aR, \(320R , 322aR , 313M), 320aM) - (44R, 823M) LSD-25 l(316R), |WR) i or -(325Rs), -(824R) i(255M) (i) (255M) Meperidine b Chlorpromazine or reserpine - (326R) US26R) Tremorine l(327M), -(328) -(327M) a

Key: f, indicates facilitates, increases, or activates; j, depresses or retards; —no change; (t), sligh6 t facilitation; (J,), slight depression. Letter after reference number indicates species used: R, rats; C, ecat; G, guinea pig; H, hamster; M, mouse; Mn, monkey; Hu human; Ra, rabbit. d Reversed or partially reserved by L-DOPA. Tolerance develops. 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 resembleh , in some respects, that of acutely ill schizophrenics (330). 0 Partially reversed by lithium pretreatment. 1 Did not alter normal latency response but prevented stress-induced increases in seizure latency (329). 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 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, ) [see Table XI {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 81 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 XI). 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 3000 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-HT 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.

E. Miscellaneous in Vivo Inhibitors

Amethopterin, which is reported to be an inhibitor of phenylalanine hydroxylase in vitro, is also active in vivo as evidenced by phenylalanine tolerance tests on patients under treatment for choriocarcinoma or leukemia with this folic acid antagonist (371). p-Chloroamphetamine and p-chloro-iV-methylamphetamine lower both 5-HT and 5-HIAA levels in rat brain (372), suggesting that the mech­ anism may be inhibition of 5-HT synthesis and not release, since releas­ ing agents generally cause an elevation in 5-HIAA levels. These amphetamines are not inhibitors of 5-HTP decarboxylase (373) so the supposition was that they might inhibit the hydroxylation step. The de­ pletion resembles that caused by PCP in that it varies from one brain area to another but is more selective in that NE levels are normal 24-96 hours after a single dose of either of these chloroamphetamines (336). Like PCP, p-chloroamphetamine suppresses sleep in cats (269) and de­ creases intraspecies aggression in rats (374). Unlike PCP, however, p-chloroamphetamine increases the convulsive threshold in both rats and mice, although no effect on brain 5-HT levels was noted in the mice (372). It seems to have much more stimulant effect on locomotor activity in mice than does PCP (308), and this has been attributed to am­ phetaminelike interactions with brain catecholamines rather than to any 84 E. G. MCGEER AND P. L. MCGEER

effect on brain 5-HT (375). Unlike PCP, the p-chloroamphetamines have no effect on liver phenalalanine hydroxylase (376). The mechanism by which these p-chloroamphetamines deplete brain 5-HT is still unclear, but they are not active in vitro as tryptophan hydroxylase inhibitors (376), and the demonstration that they inhibit the conversion of 5-HT to 5-HIAA in rat brain (373, 377) suggests that they act by an unusual mechanism of 5-HT release without metabolism to 5-HIAA (376, 378). Th14e evidence that p-chloroamphetamine decreases the amoun14 t of [ C]5-HT formed in brain from intraventricularly injected [ C]tryp­ tophan (379) could be explained as interference with uptake. Other com­ pounds such as "fenfluoramine" [ (m-trifluoromethylphenyl)-2-ethyl- aminopropane], a clinical anorexogenic agent, seem to deplete brain 5-HT selectively by a similar release mechanism (380) and to cause, if anything, an increased rather than a decreased turnover of 5-HT (381).

V. INDIRECT MECHANISMS OF INHIBITION

There are several possibilities for the inhibition (or stimulation) of metabolic hydroxylations other than by direct action on the hy­ droxylases. The limited information presently available may be grouped into four broad categories: (a) direct feedback (product) inhibition, (b) substrate availability, (c) hormonal influences, and (d) indirect (interneuronal) feedback. Exploitation of these possibilities must await a fuller understanding of the natural rate-controlling mechanisms.

A. Direct Feedback (Product) Inhibition

The fact that catecholamines are themselves inhibitors of tyrosine hydroxylase has led to the suggestion that product inhibition may play a crucial role in the rapid regulation of catecholamine synthesis 382-384). Product inhibition of dopamine /^-hydroxylase has also been suggested as part of the regulatory mechanism for norepinephrine synthe­ sis (385, 386). It has been argued tha-t4 the normal cofactor concentra­ tion in tissues is of the order of <10 M (38) so that norepinephrine would be more effective in 3vivo than in in vitro assays where DMPH4 is often used at about 10~ M. This, however, does not allow for the probable compartmentalization of the cofactor in vivo and the possible 2. AMINO ACID HYDROXYLASE INHIBITORS 85 attainment of high local concentrations at sites of tyrosine hydroxylase action. The decrease in brain tyrosine hydroxylase activity in animals treated with MAO inhibitors has been attributed to an increase in product inhibi­ tion {382, 387) although interneuronal mechanisms may play a role. Likewise, the increase of tyrosine hydroxylase activity on nerve stimula­ tion in vivo {74, 388) or by stresses such as temperature or electric shock {389) has been attributed to a decrease in feedback inhibition by nerve-induce+ d release of norepinephrine {382). Other factors such as K ion stimulation {390), hormonal interaction or transsynaptic in­ duction of enzyme synthesis could also be important. Decreased feedback inhibition has been cited as responsible for the apparent increase in brain tyrosine hydroxylase in rats treated with diethyldithiocarbamate or CS2, agents that inhibit dopamine /^-oxidase {391). 2Th+ e changes in tyrosine hydroxylase activity in brain slices when Ca ions {392) or ouabain {393) are added to the medium have been attributed to changes in feedback inhibition du2+e to release of catecholamines rather than to any direct effect of the Ca or ouabain. Tyrosine hydroxylase isolated from a human pheochromacytoma was found to be less sensitive to inhibition by NE than was enzyme from bovine adrenal medulla, suggesting either a species difference or an adaptation of the enzyme due to the high concentrations of NE in pheochromocytoma tissue {394). Serotonin and 5-HTP are not inhibitors of tryptophan hydroxylase (69, 83), nor is tyrosine an inhibitor of phenylalanine hydroxylase (108, 126y 151). Hence, direct product inhibition is not likely to be an im­ portant mechanism in the control of these hydroxylation reactions (387, 395), although high levels of 5-HT (396) during development may permanently repress development of the hydroxylase that produces it.

B. Substrate Availability

Tissue concentrations of phenylalanine and tyrosine are such as to saturate their hydroxylating enzymes. Tryptophan in brain is normally present at a concentration close to or below the Km of tryptophan hy­ droxylase. Substrate concentration therefore probably plays a role in the control of tryptophan but not phenylalanine or tyrosine hydroxyla­ tion. As already mentioned (Section II,B,3), brain 5-HT levels are de­ creased by a tryptophan-deficient diet although tryptophan deficiency per se has no effect on the amount of hydroxylating enzymes in brain 86 E. G. MCGEER AND P. L. MCGEER

or liver (397). A parallelism between 5-HT synthesis and altered brain tryptophan levels following a number of drugs or environmental treat­ ments [for example, amphetamine (398), reserpine, LiC03, cyclic AMP, PCP, or a 4-40°C environment] (399) is further supporting evidence. Agents that induce liver tryptophan pyrrolase might be expected to lower brain tryptophan levels and thus diminish 5-HT synthesis. This mechT­ anism could explain the decreased synthesis of brain 5-HT in rats follow - ing injection of hydrocortisone (400, 401) or the synthetic glucocortico- steroid, betamethasone (402). The effect is potential because some of the products of pyrrolase action such as kynurenine and 3-hydroxy- kynurenine apparently inhibit uptake of tryptophan into the brain (403). An inverse relation between tryptophan pyrrolase and brain serotonin levels does not, however, hold for all drugs or stress treatments (404) • The p02 in the tissue may play a regulatory role in these hydroxyla- tions under some conditions, particularly in -4tryptophan hydroxylation since this has a Km for 02 of mor5 e than 10 M while that of tyrosine hydroxylase is about 7.4 X 10" M (395). Information presently being collected in a number of laboratories suggests increased turnover of both brain NE and 5-HT under high p02 (202, 395, 405, 406) although the amine levels may be depleted (407-409) or unchanged (410), possibly because of potent stimulation of MAO (395, 406). Anoxia lowers brain NE levels (411, 41%)- Hypobaric oxygen, on the other hand, increases brain dopamine (413) as well as peripheral tissue serotonin (414)- Again, decreased MAO activity or some other factor could be involved. An adaptive increase in tryptophan pyrrolase (prevented by actinomycin D) (415) and in phenylalanine hydroxylase (using tryptophan as a substrate) may occur after prolonged exposure to hypobaric conditions We).

C. Hormonal Influences

Various hormones have been suggested to play roles in the regulation of hydroxylation reactions but the situation is at present unclear. Rats treated chronically with hydrocortisone do not show the low brain 5-HT levels seen after single injections (401), suggesting that there may be an induction of tryptophan hydroxylase to compensate for the initially lowered 5-HT levels attributed to lowered brain tryptophan levels. Azmitia et al. (417-419) have suggested that adrenocorticosteroids regu­ late tryptophan hydroxylase biosynthesis and thus control the turnover of brain 5-HT. Hydrocortisone has also been shown to influence NE 2. AMINO ACID HYDROXYLASE INHIBITORS 87 uptake into brain slices (420) and may thereby affect tyrosine hy­ droxylase activity by a modification of product inhibition. The considerable evidence indicating an intimate interrelation between the thyroid gland and the sympathetic nervous system (421) suggests that thyroid hormones may play a role in controlling tyrosine hydroxyl­ ase activity. There is, however, little evidence for any direct effect of thyroid hormones on catecholamine production or metabolism (422). Ten-day treatment of rats with thyroxine leads to a diminished synthesis of catecholamines, presumably by some indirect mechanism (423) since the only in vitro effect of thyroxine on tyrosine hydroxylase is a slight stimulation (23). Ovariectomy is said to increase brain NE synthesis in rats and to decrease the sensitivity of such synthesis to «-MPT (424, 425). Hy- pophysectomized animals, on the other hand, have been reported to show a decrease in adrenal tyrosine hydroxylase (426). Adrenocorticotropic hormone increases tyrosine hydroxylase activity in such animals. The interrelation of thyroid hormones with serotonin synthesis is also not clear (427), but hypothyroid rats are said to show phenylalanine hydroxylation in brain (428) and, since a true phenylalanine hydroxylase does not occur in brain, this may indicate an increase in tryptophan hydroxylase. Increased serotonin synthesis is also indicated in insulin- treated rats but it is not known whether this is due directly to insulin, to low blood glucose levels, or to some secondary effects of hypoglycemia (429). Despite one conflicting report (317), it appears that 5-HT turnover and tryptophan hydroxylase activity are increased in morphine-tolerant (318, 430-434) and methadone-treated (435) animals. Brain and adrenal tyrosine hydroxylase activities also appear to be increased in morphine- tolerant animals (433, 436-438). The stimulant effects of morphine on the hydroxylases are indirect (439) and hormonal influences are suspected but are not proven (434)- The mechanism may well involve some type of interneuronal stimulation since it is prevented by cyclo- heximide (318), which blocks protein synthesis. Increased turnover of both catecholamines (204, 440-442) and serotonin (443) under various stressful conditions such as forced tem­ perature adaption, electroshock, restraint, or X irradiation may also in­ volve hormonal mechanisms although feedback inhibition or induction are equally possible. The effects of stresses, such as cold, on tyrosine hydroxylase activity are generally most evident in adrenal tissue and may not occur in dopaminergic neurones in the brain (204, 441, 444)- There is conflicting evidence as to the effect of various stresses on brain 88 E. G. MCGEER AND P. L. MCGEER

amine levels (402) and on enzymes. Whole body radiation, for example, has been reported to increase brain tyrosine hydroxylase and liver tryptophan pyrrolase (445) but to decrease the hydroxylation of tryp­ tophan by liver (446). Both a-MPT and PCP were shown not to affect various pituitary responses of rats (447); PCP does not bring about adrenal cortical ac­ tivation (448).

D. Indirect Feedback (Interneuronal) Inhibition

A variety of studies indicate that the amount of tyrosine hydroxylase synthesized in a neuron, and thus ultimately the rate of catecholamine synthesis, is affected, presumably by an interneuronal mechanism, by the amount of catecholinergic activity on the receptors of adjacent neurons (449, 467). This mechanism of regulation depends on induction or repression of enzyme synthesis. It is relatively slow and requires several days to become manifest. Chronic treatment with presumed blockers of catecholamine receptor sites such as chlorpromazine (129, 450-458), thioproperazine (459, 459a), phenoxybenzamine (460-463), or benzoctamine (464) increases the amount of tyrosine hydroxylase in adrenal, brain, and/or sympathetic ganglia. So does treatment with reserpine ( 462-468), which depletes the amines. Experiments on chicks (469) suggest that the effect of reserpine on brain tyrosine hydroxylase may be permanent, or long lasting, if the reserpine is administered during development. These effects of chlor­ promazine (457, 470) and reserpine (466) have been said to be highly dose dependent and, in the brain, may not be evident in NE neurons (453). Chronic treatment with reserpine also leads to increases in brain tryptophan hydroxylase, presumably by an analogous mechanism (397, 467). These agents have no effect on tyrosine or tryptophan hydroxylase in vitro (9, 83, 471). Their mechanism of action in vivo probably in­ volves induction of enzyme biosynthesis. The reserpine response is blocked by the protein synthesis inhibitors actinomycin D or cyclo- heximide (465). Catecholamine excretion data on depressed patients under treatment with imipramine have been interpreted as indicating the converse effect, i.e., feedback (interneuronal) repression of synthesis (472, 473), but imipramine has multiple effects on catecholamine transport and metabol­ ism that make interpretation uncertain (474)- Recently (475) chronic 2. AMINO ACID HYDROXYLASE INHIBITORS 89

treatment of rats with 1000 mg/kg of L-DOPA daily for 5-7 days was shown to cause a progessive reduction of adrenal tyrosine hydroxylase. Brain tyrosine hydroxylase levels were unchanged. The decrease of up to 50% in adrenal tyrosine hydroxylase was attributed to feedback re­ pression of synthesis of the enzyme due to the high levels of NE in DOPA-treated animals. Similar feedback (interneuronal) control mechanisms have been hy­ pothesized to explain the effects on tyrosine hydroxylase of 6-hydroxy- dopamine (467, 468, 476) or amphetamine (468) in the adrenals (increasing), of amphetamine in the caudate (477) (reducing), and of in the brain (increasing) (478). Recently it has been suggested (467) that cyclic AMP may play an important role in interneuronal control mechanisms. It has been reported that LSD-25 reduces both the synthesis and turnover of brain serotonin (479) but, like the hallucinogenic muscinol, it increases brain 5-HT levels (480). The mechanism of action is unclear but may involve interneuronal mechanisms. Brain tryptophan hydroxyl­ ase activity is not inhibited by LSD in vitro (83). Interneuronal feed­ back has also been suggested as a factor in the inhibition of the conver­ sion of tryptophan to serotonin in the brains of cocaine-treated mice (398) and in the increased rate of serotonin synthesis in rats treated with lithium (481) or morphine (see Section V,C).

VI. CONCLUSION

The development of relatively specific metabolic inhibitors of tyrosine, tryptophan, and phenylalanine hydroxylases has added a powerful tool to the armamentarium of scientists attempting to identify the role of the central and peripheral amines in various physiological functions. Unfortunately, interpretation of the results obtained to date is difficult because the powerful inhibitors of phenylalanine hydroxylase so far dis­ covered are likewise inhibitors of other enzymes, generally tryptophan hydroxylase. The full clinical potentiality of these hydroxylase inhibitors also remains to be explored. The limited studies to date suggest that tyrosine hydroxylase inhibitors should find clinical usefulness in the treatment of various chromaffin tissue tumors such as pheochromo- cytomas and other adenomas of this type. Further exploration in essen­ tial hypertension may be warranted, although preliminary results are discouraging and a primary defect in catecholamine metabolism in this 90 E. G. MCGEER AND P. L. MCGEER condition has never been proven. The report that a-MPT reduced excre­ tion of DOPA and retarded tumor growth in hamsters with melantotic melanoma (482) suggests another condition in which tyrosine hydroxyl­ ase inhibitors might be worth further testing, although the initial clinical trial in a patient with metastatic malignant melanoma was unsuccessful (483). It is also possible that tyrosine hydroxylase inhibitors may be useful in such extrapyramidal disorders as chorea which might benefit from decreased dopaminergic tone. Further exploration of the clinical usefulness of tryptophan hydroxyl­ ase inhibitors will also undoubtedly be done, with possibilities, suggested by animal studies, lying not only in the field of carcinoid tumors but in sexual problems and drug addiction. The activity of the 5-HT depletor, p-chloro-iV-methylamphetamine, as an antidepressant (378, 484-486) suggests possible use of tryptophan hydroxylase inhibitors, alone or with DOPA, in some forms of depression, while the beneficial effects obtained with a serotonin antagonist in the dumping syndrome (487) suggests another area for clinical trial. Trial of either type of hydroxylase inhibitor in various mental disorders, which are now treated with amine-depleting and -blocking agents, in different types of sleep disorder or in sexual problems may be worthwhile. One difficulty with PCP for such exploration is that it is such a powerful inhibitor in vivo of phenylalanine hydroxylase; other agents such as the 6-halotryp- tophans should probably be tested for their in vivo effects on phenyl­ alanine hydroxylase and, if inactive, might prove safer for the exploration of the clinical usefulness of tryptophan hydroxylase inhibitors. The search for new and more potent inhibitors of these hydroxylases will undoubtedly be continued. It would appear unlikely, however, in view of the wide screening for direct inhibitors of tryptophan and tyrosine hydroxylases, that agents much more potent than those presently known will be found. This probability focuses more attention on the indirect mechanisms of inhibition discussed in Section V, and these will undoubtedly grow in importance as more information is gathered. REFERENCES

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