Biopterin cofactor biosynthesis: independent regulation of GTP cyclohydrolase in adrenal medulla and cortex.

Guanosine triphosphate cyclohydrolase, the enzyme that is apparently rate-limiting in biopterin biosynthesis, is increased in adrenal cortex and medulla of rats treated with insulin or reserpine. Denervation and hypophysectomy block the increase in medullary and cortical enzyme activity, respectively, whereas cycloheximide presents the increase in both tissues. These results provide evidence for induction and regulation of guanosine triphosphate cyclohydrolase.

Dihydrofolate reductase in primary brain tumors, cell cultures of central nervous system origin, and normal brain during fetal and neonatal growth.

Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors in rats. Specific activities of DHFR in cell cultures derived from human and rat primary intracranial gliomas and sarcomas were up to 6 times those found in adult rat liver. The presence of DHFR in neoplasms of central nervous system origin is relevant to the development of folate antagonists which, unlike methotrexate, can readily cross the blood-brain barrier. In normal developing rat brain, DHFR specific activity was high in embryos at 19 days of gestation and declined thereafter, until at 20 days after birth the activity was very low. The methotrexate titration assay was used to measure enzyme levels in the brains of fetal and newborn rats, and good correlation with the spectrophotometric assay was observed. The pattern was different in liver, showing maximum activity 11 days after birth and retaining high activity in adult liver. Both the cofactor requirement and the sensitivity to methotrexate indicate that the enzyme in the brain is DHFR.

Biochemical and chemotherapeutic studies on 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW 301U), a novel lipid-soluble inhibitor of dihydrofolate reductase.

The lipophilic diaminopyridopyrimidine BW 301U (2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine) is as active as methotrexate as an inhibitor of dihydrofolate reductase and mammalian cell growth. This compound was selected from among related pyridopyrimidines and other lipid-soluble diaminoheterocyclic compounds as having the most favorable combination of properties as a potent inhibitor of dihydrofolate reductase with minimal effects on histamine metabolism. In contrast to methotrexate, entry of BW 301U into cells is rapid and is not temperature dependent, indicating passage across cell membranes by diffusion. There is no competition between BW 301U and leucovorin (folinic acid) for uptake into Sarcoma 180 cells in culture. When BW 301U is added to culture medium, deoxyuridine incorporation ceases within the first few min, and this inhibition persists when cells are transferred to drug-free medium. Both leucovorin and thymidine are required to protect cells in culture from the cytotoxicity of BW 301U. The effect on thymidine biosynthesis appears to be indirect since BW 301U is inactive as an inhibitor of thymidylate synthetase. Hypoxanthine and thymidine restore growth by only 50% in cultures containing BW 301U, and complete restoration of growth requires the further addition of adenosine and either uridine or cytidine to the medium. In vivo, BW 301U is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors.

Hormonal regulation of guanosine triphosphate cyclohydrolase activity and biopterin levels in the rat adrenal cortex.

The regulation of GTP-cyclohydrolase (GTP-CH) activity and tetrahydrobiopterin (BH4) levels in the adrenal cortex were studied in intact and hypophysectomized rats. Treatment with a single dose of reserpine (5 mg/kg) or insulin-induced hypoglycemia (4 h) elevated adrenocortical BH4 3-fold by 10 h; BH4 levels remained elevated after 24 h and returned to control levels by 48-72 h. GTP-CH was significantly increased 24 h after hypoglycemic shock, and the increased enzyme activity preceded the changes in BH4 levels after reserpine treatment. Two and a half hours of stress by immobilization also increased GTP-CH activity and BH4 levels in the adrenal cortex. The activities of sepiapterin reductase and dihydrofolate reductase, putative enzymes in the biosynthetic pathway from GTP to BH4, were not increased by reserpine. Both reserpine and insulin increased the apparent maximum velocity for GTP, with no increase in the affinity of the enzyme for its substrate, further suggesting that the experimental treatments induce the synthesis of GTP-CH. Hypophysectomy completely blocked the reserpine-dependent increase in both cortical GTP-CH activity and BH4 content. The administration of purified porcine ACTH to intact and hypophysectomized rats elevated adrenocortical GTP-CH activity and cofactor levels. Synthetic ACTH-(1-24) also enhanced the enzyme activity and BH4 levels in the adrenal cortex. Thus, pituitary control of adrenal cortical GTP-CH synthesis and biopterin levels appears to be mediated through the secretion of ACTH. The changes in enzyme activity and cofactor levels after activation of the hypothalamo-hypophyseal axis or ACTH administration suggest that BH4, a cofactor for certain monooxygenases, has some function, as yet unknown, in the adaptive changes of the adrenal cortex in response to stress.

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate.

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.

Synthesis and antitumor activity of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine.

The synthesis of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW301U, 7) by a route that has general applicability to the preparation of many 6-(substituted benzyl)-5-methylpyrido[2,3-d]pyrimidines is described. The key intermediate, 2,4-diamino-7,8-dihydro-6-(2,5-dimethoxybenzyl)-5-methyl-7-oxopyrido[2,3-d]pyrimidine (4), is converted to the 7-chloro compound 5 by treatment with a 1:1 complex of N,N-dimethylformamide--thionyl chloride, and 5 is hydrogenolyzed with palladium on charcoal in the presence of potassium hydroxide to yield 7. BW301U is a potent lipid-soluble inhibitor of mammalian dihydrofolate reductase and has significant activity against the Walker 256 carcinosarcoma in rats.

Synthetic analogues of tetrahydrobiopterin with cofactor activity for aromatic amino acid hydroxylases.

Tetrahydrobiopterin (THB) analogues with 6-alkoxymethyl substituents, 3a-j, where the substituents were straight- and branched-chain alkyl ranging from methyl to octyl, have been synthesized by the Taylor method from pyrazine ortho amino nitriles by guanidine cyclization, hydrolysis in aqueous NaOH, and catalytic hydrogenation over Pt in trifluoroacetic acid (TFA). The best of these compounds, 3b, is an excellent cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (V = 154% of THB), and tryptophan hydroxylase, does not destablize the binding of substrate (Kmtyr = 23 microM), and is recycled by dihydropteridine reductase (V = 419% of THB). The compounds are being evaluated as cofactor replacements in biopterin-deficiency diseases.

Inhibitors of histamine metabolism in vitro and in vivo. Correlations with antitrypanosomal activity.

The effects of antimalarial and antitrypanosomal drugs on the activity of histamine N-methyl transferase and diamine oxidase in vitro, as well as diamine oxidation and histamine levels in vivo, were examined. Diamidine antitrypanosomal drugs which interfere with polyamine metabolism were found to be potent inhibitors both in vitro and in vivo. Antrycide ( quinapyramine ) and isometamidium were the best inhibitors of both enzymes. Ki values for histamine N-methyl transferase were 3 X 10(-8) M for both compounds, and the inhibition was competitive for histamine. Antrycide and isometamidium were both non-competitive inhibitors of diamine oxidase, having Ki values of 6 X 10(-9) M and 3 X 10(-9) M respectively. Isometamidium elevated histamine levels in rat kidney 2-fold and produced a long-term inhibition of putrescine oxidation in vivo. Among the compounds examined, only known active antitrypanosomal agents inhibited both histamine N-methyl transferase and diamine oxidase in vitro as well as putrescine oxidation in vivo. These observations suggest that the enzymes acting on histamine and putrescine as substrates can be used to select compounds which interfere with polyamine metabolism and that persistence of such compounds in vivo, as indicated by inhibition of putrescine oxidation, correlates with favorable chemotherapeutic properties as antitrypanosomal agents.

Endocrine-dependent regulation of tetrahydrobiopterin levels and guanosine triphosphate cyclohydrolase activity.

The role of endocrine organs in the regulation of tetrahydrobiopterin (BH4) levels and guanosine triphosphate cyclohydrolase (GTP-CH) activity was studied in the spleen, bone marrow and brain of rats and mice. Following hypophysectomy, BH4 levels and GTP-CH activity were significantly decreased in both spleen and bone marrow. Fourteen days after hypophysectomy GTP-CH activity and BH4 levels were approximately 25% of control levels in both tissues. In contrast, BH4 levels and GTP-CH activity in brain were not significantly different from control values. The decrease in GTP-CH activity and BH4 levels in spleen and marrow could not be reversed by high doses of ACTH or by a pituitary extract. Removal of the thyroid gland resulted in significant decreases in BH4 levels and GTP-CH activity in spleen; marrow and brain levels were not affected. BH4 levels in spleens of thyroidectomized rats returned to control values following treatment with either triiodothyronine or thyroxine. Adrenalectomy and castration had no effect on biopterin metabolism in bone marrow, spleen or brain. Tissue levels of BH4 and GTP-CH were also studied in mutant mouse strains having mutations in either pituitary or thyroid functions in order to examine further the role of these tissues in the regulation of the biosynthesis of this cofactor. The results of this study indicate that factors secreted from the pituitary are important in the regulation of BH4 levels and GTP-CH activity in spleen and bone marrow and that the thyroid gland also plays a role in regulation in the spleen. Levels of BH4 and GTP-CH in the brain, if regulated, appear to be independent of the endocrine tissues studied.

Antitumor phthalanilides active in acute and chronic Trypanosoma brucei brucei murine infections.

A series of phthalanilides and related compounds were tested on a short-term, fulminating, mouse infection of Trypanosoma brucei brucei (EATRO 110 isolate). The most effective compound was [4,4'-bis (4-methylimidazolin-2-yl)-terephthalanilide] which had a cure rate of 75% at 0.1 mg/kg body weight and 100% at 0.5 mg/kg when administered as three single daily intraperitoneal injections starting 24 hours post-infection. Several related phthalanilides and similarly substituted ureas showed definite but lower activity. In tests with a chronic neurotropic T. b. brucei isolate (TREU 667), cure rates greater than 90% were obtained with 10 or 25 mg/kg [4,4'-bis(4-methylimidazolin-2-yl)-terephthalanilide]. Cured animals survived for at least 200 days after infection with no evidence of recrudescence of parasitemia or of toxicity; blood or brain homogenates of cured animals were non-infective to immunosuppressed animals. These studies indicate that this series of compounds, previously studied as antitumor agents, should be re-examined as potential trypanocides.

Stimulation of retinal dopamine biosynthesis in vivo by exogenous tetrahydrobiopterin: relationship to tyrosine hydroxylase activation.

Intravitreal injection of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), increases 3,4-dihydroxyphenylalanine (DOPA) accumulation in retinas of dark-adapted rats, as does exposure to light. In contrast, BH4 had no significant effect on DOPA accumulation in retinas of light-exposed rats. The levels of endogenous retinal BH4 and the uptake of injected BH4 into the retinal tissue were not affected by light exposure. These data indicate that TH is not saturated with endogenous BH4 in the retinas of dark-adapted rats. In addition, the observations support the interpretation that the decrease in apparent Km of TH for the cofactor in response to light exposure is of sufficient magnitude to allow near saturation of TH by endogenous BH4 and, thus, is causally related to the increase of dopamine biosynthesis in response to short-term photic stimulation.

A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of 3H2O and adsorption of [3H]-tyrosine by charcoal.

A rapid, simple and sensitive assay has been developed for tyrosine-3-monooxygenase, the enzyme catalyzing the rate-limiting step in catecholamine biosynthesis. The assay is based upon the release of 3H2O from 3H-[3,5]-L-tyrosine with adsorption of the isotopic substrate (and its metabolites) by an aqueous slurry of activated charcoal. This method routinely yields low blank values and is simpler than the procedure requiring the use of cation exchange columns to separate the isotopic substrate from the 3H2O formed during the hydroxylation reaction.

Biopterin cofactor biosynthesis: GTP cyclohydrolase, neopterin and biopterin in tissues and body fluids of mammalian species.

Levels of GTP cyclohydrolase, neopterin and biopterin were determined in tissues and body fluids of humans, monkey, dog and mouse. Highest levels of GTP cyclohydrolase and biopterin were found in pineal, liver, spleen, bone marrow, whole adrenal gland and small intestine. High levels of biopterin were found in the urine of all species examined. High levels of neopterin were found only in the urine of humans and monkeys, very low levels could be detected in dog, while none could be detected in mouse, rat, guinea pig or hamster urine.

Specific TLC tissue residue determination of sulfadiazine following fluorescamine derivatization.

A spectrodensitometric method for the direct determination of sulfadiazine at the tissue residue level (0.1 ppm) is based upon the measurement of the fluorescence of a sulfadiazine-fluorescamine derivative formed directly on a TLC plate by dipping it into a fluorescamine solution. The linear dynamic range for the assay is about 150 from 200 to 0.2 ng, the lower limit of sensitivity. Recoveries from various spiked tissues including milk, eggs, liver, kidneys, muscle, skin, and fat varied with the tissue type but were reproducible. The assay technique has also been used for the assay of sulfamethoxazole and has been explored for use in specifically assaying sulfonamide mixtures.

Urinary biopterin and neopterin excretion and pituitary-adrenal activity in psychiatric patients.

The urinary excretion of biopterin and neopterin, pterin compounds related to tetrahydrobiopterin, the cofactor for the initial steps in monoamine synthesis, was serially measured in a heterogeneous group of psychiatric patients and compared to excretion in control subjects, to state of illness, and to the results of the dexamethasone suppression test. Patients with major depression had increased excretion of biopterin compared to normal subjects. There was no relationship between biopterin or neopterin excretion and postdexamethasone cortisol levels. Pterin excretion did not significantly change with improvement in mood or with conversion from nonsuppressor to suppressor status. The meaning of increased urinary biopterin is presently unclear, although its relation to hormonal state and sympathoadrenal tone deserves further study.

Urinary excretion of biopterin and neopterin in psychiatric disorders.

Levels of urinary neopterin and biopterin were determined in patients having a diagnosis of schizophrenia, unipolar depression, or bipolar depression. Both neopterin and biopterin levels were significantly higher in the urine of patients with unipolar depression than in the urine of the control group. Subclassification of patients into primary and secondary depression demonstrated a significant elevation of urinary biopterin in both groups, whereas urinary neopterin was elevated only in those patients with primary depression. In patients with bipolar depression, neopterin excretion was elevated, but biopterin excretion did not differ from controls. No significant differences were found in schizophrenic patients.