99mTc-DTPA-amino acids conjugate as specific SPECT pharmaceuticals for tumor imaging.

(99m)Tc-Diethylene triamine pentaacetic acid-bis (amide) conjugates have been synthesized and evaluated as a potential radiopharmaceutical for tumor imaging. The compounds were synthesized by the condensation reaction of DTPA bis(anhydride) with different l-amino acids (methyl tryptophan, and 5-hydroxy tryptophan) and were characterized on the basis of IR, NMR, and Mass spectroscopy. (99m)Tc-labeled compounds were found stable for about 24 h under physiological conditions with more than 95% radiolabeling yield. Blood kinetic studies of all these complexes showed a bi-exponential pattern as well as quick wash out from the blood circulation. The biological t(1/2)(F) and t(1/2)(S) were found to be 20 +/- 0.001 min for DTPA-(Me-Trp)(2) and 18 +/- 0.001 min for DTPA-(5HT)(2) and t(1/2) (slow) 5 h 45 min +/- 0.001, 5 h 30 +/- 0.001 min for DTPA-(Me-Trp)(2), and DTPA-(5HT)(2), respectively. Imaging and biodistribution studies were performed in mice bearing Ehrlich ascites tumor (EAT) tumors in right thigh. Radioconjugate derived from l-5-hydroxytryptophan exhibited remarkable localization at tumor site; whereas radiotracer derived from l-methyl tryptophan shows relatively less accumulation at the tumor site. Tumor-to-muscles ratios were 5.07 +/- 0.001, and 4.2 +/- 0.001 at 1 and 4 h for (99m)Tc-DTPA-(Me trp)(2) and 4.97 +/- 0.001 and 5.8 +/- 0.001 at 1 and 4 h after postinjection for (99m)Tc-DTPA-(5HT)(2), respectively. The preliminary results with these amino acid based ligands are encouraging to carrying out further in vivo experiments for targeted tumor imaging.

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry.

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.

An enzymatic assay for metabolites of perfluoro-tagged 5-hydroxytryptophan.

l-5-hydroxytryptophan (5-HTP) with two types of multiple (19)F-atom tags bonded at various positions onto the indole ring (positions 4, 6, or 7) was exposed to aromatic l-amino acid decarboxylase (AADC) in lysates of Escherichia coli JM109 which had been transformed with the plasmid pKKAADCII. Resulting samples were analyzed with HPLC. In the first study, which investigated a straight-chain seven-atom tag, a novel peak, putatively perfluoro-tagged serotonin, was detected. A second study demonstrated that 5-HTP was converted to 5-HT in transformed E.coli lysate but not in untransformed lysate. A third study, investigating a tag with nine fluorine atoms all in the same nuclear environment, identified the isomer serving as the best substrate for AADC. This novel molecule had the tag bonded at the 6 position on the indole ring. Isomers that fit into the active site of AADC are likely to follow the biosynthetic path for serotonin in vivo and are potentially useful in (19)F magnetic resonance spectroscopy studies. The enzymatic assay described here provides an efficient and cost-effective tool for screening new compounds.

Tissue immunoassay for 19F-tagged 5-hydroxytryptophan.

A new tool for magnetic resonance, L-6-heptafluorobutyryl-5-hydroxytryptophan, was synthesized and investigated using an antibody to perfluoroalkyl moieties developed previously. To be useful as an imaging agent, the compound must cross the blood brain barrier and then be concentrated in vesicles in serotonergic neurons in order to accumulate in sufficient quantity for in vivo detection to be possible. The novel imaging compound was administered in ova to domestic chicks (Gallus domestics) to investigate the bioavailability and uptake dynamics of the compound in this model organism. Typical immunoassay methods were ineffective, so a new technique was developed which binds amines and amino acids to the walls of acid-functionalized cuvettes. The first study established the presence of higher quantities of the tags in neural and liver tissue than in heart tissue. A second study investigated regional differences, with the midbrain containing more tagged compounds than the frontal lobe sample, and the frontal lobe sample containing more than the occipital or cerebellum samples. These studies demonstrate that the compound follows the pathway of endogenous serotonin. A third study investigated uptake dynamics of the novel compound. Maximum concentration of the tagged molecule in the brain was achieved three days after injecting Incubation Day 14 eggs, suggesting that it bioaccumulates in vivo. This new immunoassay technique used to detect the novel compound in tissue samples demonstrated good repeatability.

Double-blind crossover study with levorotatory form of hydroxytryptophan in patients with degenerative cerebellar diseases.

OBJECTIVE: To determine whether treatment with the levorotatory form of hydroxytryptophan (L-5-hydroxytryptophan), a controversial experimental drug, can improve the conditions of patients with ataxia. DESIGN: A double-blind crossover study with the levorotatory form of hydroxytryptophan was performed in 39 patients with degenerative cerebellar diseases. SETTING: Patients were selected from an ongoing prospective follow-up study at two university hospitals. PATIENTS: We studied 19 patients with Friedreich's ataxia, 13 with cerebellar atrophy, and seven with olivoponto-cerebellar atrophy. INTERVENTION: The levorotatory form of hydroxytryptophan was given orally in a dose of 1000 mg/d. Each treatment phase, with the levorotatory form of hydroxytryptophan or the placebo, lasted 10 months, after which the treatment of patients was crossed over to the other phase. MAIN OUTCOME MEASURES: Ataxia was documented and quantified by using a clinical score, posturography, and measurement of grip force and the rapid-syllable repetition rate. RESULT: The levorotatory form of hydroxytryptophan had no significant effect on cerebellar symptoms. CONCLUSION: Long-term treatment with a high dose of the levorotatory form of hydroxytryptophan does not improve the conditions of patients with ataxia.

Enantiospecific syntheses of alpha-(fluoromethyl)tryptophan analogues: interactions with tryptophan hydroxylase and aromatic L-amino acid decarboxylase.

alpha-Fluoromethyl amino acids are enzyme-activated irreversible inhibitors of amino acid decarboxylases. Aromatic L-amino acid decarboxylase (AADC) is the enzyme responsible for the final step in the biosynthesis of both dopamine and serotonin via decarboxylation of L-dopa and 5-hydroxy-L-tryptophan, respectively. Our goal is to utilize antagonists of the serotonin-producing enzymes (tryptophan hydroxylase and AADC) as the basis for a chemotherapeutic approach to the treatment of carcinoid tumors, a rare tumor type characterized by the overproduction of serotonin. We report here an enantiospecific synthesis of alpha(S)-(fluoromethyl)tryptophan [(S)-11a] and alpha(S)-(fluoromethyl)-5-hydroxytryptophan [(S)-11b], as well as the (R)-enantiomers, based upon recent methodology involving the face-selective alkylation of cyclic tryptophan tautomers. Our synthetic route provided both enantiomers of 11a and 11b with greater than 97% enantiomeric purity based upon evaluation of the NMR spectra of their Mosher's acid derivatives. (S)-11a was evaluated as a substrate for P815 tryptophan hydroxylase and determined to have an apparent Km of 4.31 +/- 1.07 mM, essentially half the value previously reported for the racemic mixture of 11a with rat brain stem tryptophan hydroxylase. (R)-11a was not a substrate for P815 tryptophan hydroxylase. (S)-11b was evaluated as an enzyme-activated irreversible inhibitor of murine liver AADC and determined to have a KI of 24.3 +/- 3.01 microM and a k2 of 2.26 +/- 0.44 min-1. (R)-11b was not an inhibitor of murine liver AADC.

Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization.

Muscle cells were dispersed separately from circular and longitudinal muscle layers of guinea pig intestine, and 5-hydroxytryptamine (5-HT) receptors were characterized in naive cells and in cells in which one receptor type was preserved by selective receptor protection. In naive cells from both regions, 5-HT caused contraction and stimulated increases in cytosolic free calcium concentration ([Ca2+]i) (3-fold; p < 0.01) and cAMP levels (40-60%; p < 0.01) that were inhibited, respectively, by the 5-HT2 antagonist ketanserin and the 5-HT1p antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP). In circular muscle cells, where agonist-induced increase in [Ca2+]i is mediated by Ca2+ release from inositol (1,4,5)trisphosphate-sensitive stores, 5-HT caused an increase in inositol (1,4,5)trisphosphate levels that was inhibited by ketanserin. In cells maximally contracted with a non-5-HT agonist (cholecystokinin octapeptide), 5-HT caused relaxation when the contractile effect mediated by 5-HT2 receptors was blocked with ketanserin; relaxation and the concomitant increase in cAMP were inhibited by 5-HTP-DP. The singular contributions of the Ca2+ and cAMP signaling pathways were identified in cells where only one receptor type was preserved. In cells with only 5-HT2 receptors, 5-HT caused contraction and an increase in [Ca2+]i but not in cAMP levels; contraction and the increase in [Ca2+]i were inhibited by ketanserin. Conversely, in cells with only 5-HT1p receptors, 5-HT caused relaxation and an increase in cAMP levels but not in [Ca2+]i; relaxation and the increase in cAMP levels were inhibited by 5-HTP-DP. The two signaling pathways were functionally linked, converging to regulate the level of [Ca2+]i. Thus, the increase in [Ca2+]i was augmented 1) when cAMP production was inhibited by 5-HTP-DP in naive cells or 2) when cAMP production was suppressed in cells where 5-HT1p receptors were inactivated and only 5-HT2 receptors were preserved. The results imply that the increase in cAMP levels mediated by 5-HT1p receptors acted to attenuate the increase in [Ca2+]i mediated by 5-HT2 receptors. We conclude that the response to 5-HT in muscle cells is a compound effect involving activation of two receptor types coupled to distinct signaling pathways that converge on [Ca2+]i as the determinant of mechanical activity.

The genetics of conidiophore pigmentation in Aspergillus nidulans.

The grey-brown pigmentation of Aspergillus nidulans conidiophores depends on the functions of two 'ivory' loci. ivoB codes for a developmental specific phenol oxidase, and mutants accumulate its substrate N-acetyl-6-hydroxytryptophan. ivoA mutants are unable to make this substrate. ygA mutants are also poorly pigmented, and extracts require copper salts to activate both the phenol oxidase and conidial laccase. ivoA and ivoB mutants partially suppress the spore colour phenotype of ygA mutants. Comparisons of morphology, phenol oxidase and substrate accumulation in morphological mutants at the brlA locus suggest that the brlA protein regulates ivoA, ivoB and morphogenetic loci independently. The medA locus, which also affects morphology and pigmentation, may code for a modifier of brlA function. abaA mutants which are blocked at a later stage of development than brlA or medA mutants have low phenol oxidase levels, implying that by this stage of development the activity of the ivoB locus is declining.

Localization of serotonin receptors in the rat thalamus by electrophysiology and the action of 5-HTP-DP-hex.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological properties of alpha-mono- and alpha-difluoromethyl derivatives of tryptophan and 5-hydroxytryptophan.

The syntheses of alpha-mono- and alpha-difluoromethyl derivatives of tryptophan and 5-hydroxytryptophan are described. In an attempt to selectively regulate serotonin synthesis, alpha-(mono- and difluoromethyl)tryptophan were tested in vivo as precursors (or prodrugs) of their 5-hydroxy analogues. Although alpha-(mono- and difluoromethyl)-5-hydroxytryptophans are potent irreversible inhibitors of aromatic amino acid decarboxylase (equipotent to alpha-difluoromethyl-Dopa), only alpha-(monofluoromethyl)tryptophan affects the level of serotonin in vivo (small decrease), alpha-(difluoromethyl)tryptophan being a very poor substrate of the activating (or helper) enzyme, tryptophan hydroxylase.

[Isolation of 5-benzyl hydroxytryptophan enantiomers by using microbial aminoacylase]. / Poluchenie énantiomerov 5-benziloksitriptofana s pomoshch'iu mikrobnoi aminoatsilazy.

The process of asymmetric deacylation of an acetyl derivative of 5-benzyl hydroxytryptophan racemate in the presence of native microbial aminoacylase was studied. The effect of pH, temperature, concentration of the enzyme and substrate were investigated. Conditions for preparation and isolation of individual amino acids were defined by optimization of N-Ac-DU-5-BOT hydrolysis.

Influence of a serotonin receptor antagonist, 5-HTP-DP-hex, on spinal and thalamic nociceptive neurons in rats.

The antinociceptive properties of a new synthetic dipeptide (N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, or 5-HTP-DP-hex) were studied in rats by an electrophysiological method. After an i.p. injection of alpha-chloralose and urethane, the animals were prepared for stereotaxic approach to the nucleus ventralis posterolateralis of the thalamus. With tungsten microelectrodes, individual nociceptive neurons in the nucleus were identified by the sequence of spikes emitted in response to single-pulse stimulation of the sciatic nerve. In addition to the usual short-latency spikes, a nociceptive neuron fired late spikes at regular intervals within 500 ms following each stimulus. When the spikes were accumulated in poststimulus time histograms, the short-latency spikes compiled an intensity-related (I) peak. The late spikes formed modality-related (M) peaks with spacing characteristic of nociception. Intracarotid infusion of 5-HTP-DP-hex (1 mg/kg) elevated the delayed portion of the I peak and the first M peak. This effect was followed in 25 min by suppression of all M peaks. The control record could be reinstated at any time by 5-hydroxytryptophan (3.5 mg/kg), or by natural recovery in 2.5 h. Responses evoked from a thalamic nociceptive neuron by single-pulse stimulation of the spinothalamic tract were modified by 5-HTP-DP-hex in a similar manner, except that no elevation of the activity peaks was observed. As shown previously, elevation of the delayed I peak and M1 indicated an increased input of A-delta and C fibers, respectively. The increased input lowers the response threshold and may represent hyperalgesia. Suppression of the M peaks may result from altered function of the positive feedback loop in the nociceptive system at the thalamic level, and may represent analgesia. Naloxone, methysergide, as well as ketanserin had no significant effect on the response histograms. These findings suggested that 5-HTP-DP-hex, a known serotonin receptor antagonist, targeted its action on very specific receptors, and thus interfered with particular synaptic activity within the spinal cord and on the thalamic level.

Analgesic properties of a systemically-administered synthetic dipeptide of 5-hydroxytryptophan.

Synthetic peptides of 5-hydroxytryptophan (5-HTP), including N-acetyl-5-HTP-5-HTP amide (5-HTP-ACETYL-DP), specifically inhibit the binding of serotonin to serotonin binding protein. 5-HTP-ACETYL-DP also produces a long-lasting, opiate-sensitive analgesia following central, but not systemic administration. The present study evaluated an apolar derivative of 5-HTP dipeptide, N-hexanoyl-5-HTP-5-HTP amide (5-HTP-HEX-DP), for its analgesic properties in rats following systemic administration. 5-HTP-HEX-DP (5-50 mg/kg) significantly increased jump thresholds in a dose-dependent manner with peak analgesia occurring at 2.5 hr after injection, and lasting up to 5 hr. In the tail-flick assay, 5-HTP-HEX-DP (20 mg/kg) produced a significant antinociceptive effect at 1 hr post-injection using both high and low intensity levels of radiant heat. While 5-HTP-HEX-DP and morphine each elicited analgesia following acute administration, chronic (14 days) incremental dosing with 5-HTP-HEX-DP or morphine resulted in persistent analgesia in 5-HTP-HEX-DP-treated animals, and a loss of analgesia in morphine-treated rats. Thus, significant tolerance to morphine, but not 5-HTP-HEX-DP analgesia developed using this protocol. Hence, 5-HTP-HEX-DP is a systemically-active analgesic which fails to develop tolerance when administered daily over 14 days.

[Treatment of leprosy by human metabolites]. / Traitement de la lèpre par des métabolites humaines.

We are interested for other human metabolites than desoxyfructo-serotonin (DFS), showing antileprosy activity. This is the case of desoxyfructo-5-hydroxytryptophan and of some liposoluble derivatives of DFS. The time of resorption and penetration into M. leprae infected tissue, is very different for these metabolites. For this reason the simultaneous application of these compounds may represent some advantage in the treatment of multibacillar form of leprosy. The use of DFS together with the antileprosy diet "NAL" have the supplementary advantage to stabilize the DFS level in the serum during the treatment.

The enteric neural receptor for 5-hydroxytryptamine.

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.

Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus.

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP-DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5-hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

[Effect of 6-hydroxydopamine and 5,6-Dihydroxytryptophan on the luliberin and luteinizing hormone levels in female rats under constant illumination]. / Vliianie 6-oksidofamina i 5,6-oksitriptofana na uroven' liuliberina i liuteiniziruiushchego gormona u samok krys pri postoiannom osveshchenii.

Administration of 5,6-oxytryptophan to female rats' 3rd cerebral ventricle, as opposed to 6-oxydopamine, prevents the decrease in the luteinizing hormone contents in the blood occurring under constant illumination. Administration of 6-oxydopamine against the background of desmethylimipramine caused a significant increase of the luliberine contents in the anterior hypothalamus while preserving its normal level in the medio-basal portion.

[Problems in clinical testing of a psychotropic substance examplified by L-5-HTP-ester (author's transl)]. / Probleme bei der klinischen Prüfung einer psychotropen Substanz am Beispiel eines L-5-HTP-esters

An i.v. injectable form of the serotonin-precursor L-5-hydroxytyptophan (Ro 3-5940) was investigated for its acute psychotropic effect. The difficulties are presented which had to be overcome as it was not known which effects could be expected. It was shown that for the intensive psychotropic effect found, it was not possible to use a long and detailed self-rating scale. The problem of informed consent is discussed.