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The metabolism of the 14 C-niflumic acid † specific activity 16 mc/mM has been studied in the rat, after intraperitoneal administration. With doses of 50 mg/kg, the blood level is of 75 μg/ml at the 1st hr, and of 6 μg/ml at the 24th hr. The product is nearly all bound to the plasma proteins. The liver retains, after 1 hr, 4 per cent of the injected activity and the stomach 0.5 per cent only traces (0.08 per cent) have been detected in the brain. The technique of autoradiography in the mouse, after administration of the product per os and i.p. confirms these results. About 5 per cent of 14 CO 2 is found with respect to the injected activity, after 12 hr. The radioactivity eliminated in the urine is about 2 per cent of the injected activity after 1 hr, reaches 30 per cent at the 12th hr, is practically completed after 24 hr. The radioactivity found in the faeces is generally of the order of 0.01 per cent. Five catabolites were found by paper chromatography in the urine. Niflumic acid is not eliminated unchanged, and the greatest quantity appears to be conjugated glucuronide. One of the catabolites, after paper Chromatographie study in various systems, corresponds to 2-aminonicotinic acid. Possible metabolic pathways of the Niflumic acid discussed.

The metabolism and excretion of orally administered or injected [14C]chlorphentermine has been studied in man, rhesus monkey, marmoset, rabbit, guinea-pig and rat. These species excreted 55–95% of the administered radioactivity in the urine over 5 days. Two metabolites were characterised by thin-layer and paper chromatography, gas-liquid chromatography and g.c.-m.s. and these were N-hydroxychlorphentermine and 1-(4'-chlorophenyl)-2-methyl-2-nitropropane. There are marked species differences in the excretion of N-oxidation products which were found in the urine of human volunteers. rhesus monkeys, rabbits and guinea-pigs, but not in the urine of marmosets or rats. The rat, rabbit and marmoset also excreted an unidentified unstable acid-labile precursor of chlorphentermine. The results are discussed in relation to the toxicity of the drug and to the metabolism of amphetamines in general.

It was confirmed that a biologically active substance was released when pretreated bovine colostrum (colostrokininogen) was incubated with human salivary kallikrein. It was also demonstrated that colostrokininogen belongs to the immunolactoglobulin of colostric proteins. Crude colostrokinin was purified by means of adsorption on carboxymethylcellulose, Sephadex gel filtration (G-25, G-10 and G-15) and CM-Sephadex column chromatography. The active fraction obtained after CM-Sephadex column chromatography is homogeneous in thin-layer chromatography (TLC) and paper electrophoresis. The peptide spot and biological activity migrated similarly in paper electrophoresis. The vasodilator activity of 1 mg colostrokinin in dogs corresponds to that of 258 μg bradykinin. The pharmacological activities of colostrokinin qualitatively agreed with those of kinins, such as bradykinin. Vasodilation in dogs, stimulation of smooth muscle of rats, and increase in capillary permeability in guinea pigs were caused by colostrokinin. Finally, it was indicated that colostrokinin might aid in the transport of high molecular weight substances across the neonatal gut of ungulates.

After intravenous administration of N 6 ( Δ 2 -isopentenyl)adenosine-8- 14 C (IPA-8- 14 C) to human subjects, several metabolites were isolated along with a small quantity of unchanged IPA. The ultraviolet spectra and paper chromatography of purified metabolites led to the identification of 6- N -(3-methyl-3-hydroxybutylamino)purine, hypoxanthine, adenine and some N 6 -alkylated adenines and N -alkylated xanthines. A large quantity of the radioactivity (50 per cent) from IPA-8- 14 C was excreted in the form of non-ultraviolet absorbing compounds, suggesting saturation or cleavage or both of the purine portion of IPA. Orally administered IPA produced comparable urine levels and gave a very similar metabolic picture. These results indicate that IPA is rapidly metabolized in the body, but not to uric acid, as is found for common purine nucleosides. Incubation of N 6 -( Δ 2 isopentenyl)adenine with xanthine oxidase resulted in rapid oxidation, presumably, to 2,8-dihydroxy- N 6 ( Δ 2 -isopentenyl)adenine. On the basis of these findings in vivo and in vitro , a catabolic pathway for IPA is suggested.

Six subjects were injected i.v. within 1 min with 4.8 μc (34 μg) of DL-adrenaline-2- 14 C. The urine was collected via an indwelling catheter at 2 min and 5 min after injection and thereafter at 10-min intervals for 1 hr, and continued for 6 and 24 hr. Urinary adrenaline and its metabolic products were separated and identified by means of column fractionation and paper chromatography. The radioactivity of each metabolite was measured by liquid scintillation. The basic metabolites, including adrenaline, are metadrenaline and an unknown. The acidic metabolites are: 3-methoxy-4-hydroxy-phenylglycol; 3,4-dihydroxyphenylglycol; sulfate conjugate of metadrenaline; glucuronic acid conjugate of metadrenaline; 3-methoxy-4-hydroxymandelic acid; 3,4-dihydroxymandelic acid; 3-methoxy-4-hydroxyphenylacetic acid; 3,4-dihydroxyphenyl-acetic acid; 3-methoxy-4-hydroxybenzoic acid; 3-methoxy-4-hydroxyphenylglycol sulfate; 3,4-dihydroxyphenylglycol sulfate and several unknowns. No radioactivity was recovered during the 0 to 2-min post injection period. However, the highest percentage of recovered adrenaline and 3,4-dihydroxymandelic acid appeared in the 2 to 5-min postinjection period. The greatest amount and the highest percentage of radioactivity recovered as metadrenaline appeared 5 to 10 min after injection. In terms of total radioactivity the amounts of adrenaline, metadrenaline, and 3,4-dihydroxymandelic acid so rapidly decline that by 30 min after injection these compounds each represent less than 3.5 per cent. Concomitant with this decline there is a rapid increase in the radioactivity of 3-methoxy-4-hydroxymandelic acid such that by 20 min after injection it represents more than 50 per cent of the total radioactivity found in the urine; after 40 min this metabolite begins to decrease. There is a gradual increase in the metadrenaline sulfate; in the 6 to 24 hr period it represents 53.0 ± 1.6 per cent of the total radioactivity. The importance of understanding the early formation of these adrenaline metabolites is emphasized, and the probable mechanisms involved in the metabolism and inactivation of circulating adrenaline are discussed.

The effects of digitonin and digitoxin on the phospholipid metabolism of mammalian tissue culture cells have been studied. Digitonin stimulates the incorporation of inorganic orthophosphate- 32 P into the total phospholipid. The phospholipid was then separated on silicic acid-impregnated glass paper chromatography, and the specific activity of each phosphatide was calculated. It was found that digitonin caused a linear increase in the specific activity of phosphatidyl inositol and diphosphatidyl glycerol at the time periods studied. The increase in the specific activities of phosphatidyl choline and phosphatidyl ethanolamine was delayed but to the same extent (more than twofold) after 120-min incubation. The specific activities of phosphatidyl serine and sphingomyelin were low and not significantly affected. The increase was similar in specific activity values of phospholipids of digitonintreated HeLa cells as that of the controls in the nuclear, mitochondrial, and microsomal fractions. Digitonin failed to stimulate the incorporation of palmitic-l- 14 C acid into the phospholipids of HeLa cells. A nonsteroid surface-active agent (Triton X-100) did not stimulate the incorporation of 32 P, into the phospholipids of HeLa cells, but other steroids, desoxycholate, and estradiol did stimulate such incorporation of 32 P i . Digitoxin had little effect on the phospholipid labeling of HeLa cells. However, digitoxin stimulated the incorporation of 32 P, into phosphatidyl inositol and sphingomyelin fraction of human heart culture cells. The specific interaction of various steroids with the target-cell membranes and its relation to phospholipid metabolism is discussed.

Various biological and chemical properties of colostrokinin were studied. Vasodilatation in dogs, stimulation of smooth muscles of rats and increasing capillary permeability in guinea pigs were observed as the pharmacological actions. Among these, the change in capillary permeability is the strongest when compared to the activity of bradykinin. Colostrokinin activity is destroyed by chymotrypsin, but only slightly by trypsin. It is more stable in acid than in alkaline solution. This substance is dialyzable, and is soluble in water, in 67% ethanol and in 75% acetone, but not in absolute acetone. The isoelectric point is close to pH 7.4. Phenylalanine and serine were detected as the N -terminal and the C-terminal amino acids respectively. The molecular weight, on the basis of amino acid analysis, was calculated to be about 2000. Colostrokinin absorbs light close to λ = 260 m μ .

Four subjects were infused with 0.063 μg/kg/min of dl-adrenaline-2-14C (10 μc) for 8 hr. Urine was collected at 2-hr intervals for 12 hr and then at the end of 18 and 24 hr. The urinary metabolites were separated and quantitated by combination column and filter paper chromatography. The radioactivity recovered in the urine reached a constant level 4 hr after the beginning of the infusion and continued at a constant level for 4 hr. The primary metabolites of adrenaline, i.e. metradrenaline and 3,4-dihydroxymandelic acid (DOMA), were recovered in constant amounts after 2 hr, whereas the secondary metabolites, 3-methoxy-4-hydroxymandelic acid (MOMA) and metadrenaline sulfate, were not recovered in constant amounts until after 4 hr. During this period of constant urinary radioactivity, the infusion rate was 1.25 μc/hr, DOMA was recovered in the urine at 0.018 μc/hr, free metadrenaline at 0.031 μc/hr, metradrenaline sulfate at 0.30 μc/hr and MOMA at 0.24 μc/hr. By extrapolating these results in terms of the normal endogenous rate of adrenaline secretion (860 μc/24 hr), it can be calculated from Table 1 that on the order of 100–300 μg/hr of MOMA normally occurs in the urine as the result of adrenaline metabolism. Since the normal urinary output of MOMA is 3–5 mg/24 hr, this then means that most of the MOMA is derived from endogenous noradrenaline.

Nicotine-1′-oxide, prepared by the oxidation of (−)-nicotine with hydrogen peroxide, has been resolved into two optically active stereoisomers by fractional precipitation with ammonium reineckate. (−)-Nicotine is enzymically oxidized into both isomers of nicotine-1′-oxide in vitro. These enzyme systems are stereospecific since guinea-pig and rabbit liver form similar amounts of each stereoisomer white BALB/c mouse and hamster liver and guinea-pig lung synthesize chiefly laevo-rotatory nicotine-1′-oxide. Both stereoisomers of nicotine-1′-oxide were identified in the urine of cigarette smokers.

Brain levels of Δ 1 -tetrahydrocannabinol (Δ 1 -THC) and its major metabolite (7-hydroxy-Δ 1 -THC) following intravenous injection of tritium-labelled Δ 1 -THC into mice were measured and correlated with inhibition of spontaneous motor activity. Metabolism of Δ 1 -THC to 7-hydroxy-Δ 1 -THC was fast and both substances rapidly penetrated the C.N.S. Peak brain levels of both compounds occurred within 20 min of injection and the correlation between pharmacological response and brain concentration of either compound was equally good. Twenty min after injection more than 50 per cent of the radioactivity in blood was irreversibly bound, whereas only 12 per cent of the activity in brain was unextractable. SKF 525A (25 mg/kg i.p.) produced only a slight increase in the brain level of Δ 1 -THC but the level of 7-hydroxy-Δ 1 -THC was increased nearly three-fold, presumably due to the inhibition of subsequent metabolic reactions. Piperonyl butoxide (100 mg/kg i.p.) produced an increase in the brain level of Δ 1 -THC but no change in the level of 7-hydroxy-Δ 1 -THC. Higher doses of either inhibitor produced behavioural changes in the absence of Δ 1 -THC. It is concluded that 7-hydroxy-Δ 1 -THC is centrally active, but that it has not yet been demonstrated that the effects of injected Δ 1 -THC are due solely to its primary metabolite.

Pronethalol is metabolised by two main pathways, side chain oxidation, and ring hydroxylation and conjugation. Five metabolites have been identified by comparison with synthetic substances. Four of these, 2-amino-1-(2-naphthyl)ethanol, 2-naphthylglycollic acid, 2-naphthylglyoxylic acid, and 2-naphthoic acid are formed by degradation of the isopropylaminoethanol side chain. The fifth, the 7-hydroxy-analogue of pronethalol, is partly present in the free form, but the major amount is present as a glucuronide in which the glucuronic acid residue is attached to the phenolic hydroxyl group. In several species of animals, and in humans, the differences in the major metabolites are of a quantitative rather than a qualitative nature. We have been unable to determine the nature of the proximate carcinogen responsible for producing thymic tumours in mice, but it is unlikely to be one of the major metabolites. The 7-hydroxy-analogue of pronethalol is about five times more potent than pronethalol as a β-adrenergic blocking agent and is probably responsible for some, but not all, the pharmacological activity of pronethalol.

Increased urinary Porter-Silber values were found after administration of benzyl N -benzyl carbethoxyhydroxamate (W-398) to man. Investigation of this phenomenon showed that it was not due to steroidal compounds or to the known metabolites of W-398, hippuric and benzoic acids, but to an additional metabolite, α-(benzyloxy-amino)benzyl glucuronide. The structure of this excretory product was established by the isolation and identification of its β-glucuronidase hydrolytic product, O -benzyl benzaldoxime. O -benzyl benzaldoxime accounted for about one-third of the excreted radioactivity in urine from patients receiving N -benzyl-α- 14 C-labeled W-398.

The distribution and metabolic fate of [3H]harmaline-HCl were studied in rats. Thirty min after subcutaneous injection, high radioactivity was found in the small intestine, liver, adrenals, kidneys and lungs. A rapid turnover and elimination was evident after the first hour, as most of the tissues, except the liver, kidneys and intestines, had decreased nearly 50 per cent in levels of radioactivity. About 40 per cent of the harmaline was bound to human serum or rat serum proteins in vitro. The blood levels, however, were low at all times in vivo. The peak concentration in the brain occurred at 1 hr postinjection. The major route of excretion of harmaline and its metabolites was through the kidneys; a total of 62 per cent of the injected dose was excreted in the urine during 96 hr as compared to only 11·5 per cent in the feces over the same period. The major fate of harmaline in rats was demethylation to form harmalol, which was predominantly excreted as the glucuronide conjugate. Six to 10 per cent of the radioactivity was identified as the sulfate conjugate of harmol, which was formed by the dehydrogenation of harmalol. During the first 8 hr, unchanged harmaline in the urine amounted to about 25 per cent; however, this decreased to only 7 per cent during the 8–24 hr period.

After oral administration about 45 per cent of 2′,3′,5′-tri- O -acetyl-6-azauridine (TA-6-azauridine) is eliminated in the urine of rats, as well as in man, in the form of its deacetylation products. In the urine of rats, TA-6-azauridine is excreted almost exclusively in the form of free 6-azauridine whereas in man a substantial amount of mono- O -acetyl-azauridine (MA-6-azauridine) also was found. Furthermore, about 35 per cent of the dose was found in the form of MA-6-azauridine in the faeces of rats collected during 48 hr after the administration. Neither TA-6-azauridine nor its deacetylation products are excreted in the bile of rats. 6-Azauracil was not detected as a deribosylation product of 6-azauridine neither in the urine or faeces nor in the bile of rats under study.

Extracts of some 24-hr samples of urine from schizophrenic patients accelerated the caeruloplasmin-catalysed oxidation of noradrenaline but inhibited the oxidation of 5-hydroxytryptamine; these effects were not observed with extracts prepared in an identical manner from the urine of pre-operative or healthy controls. The oxidation of noradrenaline by caeruloplasmin was inhibited by extracts of urine from some members of each group of people and the oxidation of noradrenaline by cupric ions was catalysed by some of these urine extracts.

Pretreatment with either pyrogallol, tropolone or 8-hydroxyquinoline enhanced markedly the mean stereotyped behavior scores after apomorphine treatment in the rat. Experiments in vitro , using rat liver or brain catechol- O -methyltransferase (COMT) preparations and 14 C-methyl- S -adenosyl- l -methionine, demonstrated that apomorphine was methylated by this enzyme system. The apparent K m values for dopamine and apomorphine were 2.6 × 10 −4 M and 1.4 × 10 −3 M, respectively, for liver COMT. Pyrogallol and tropolone inhibited the methylation of apomorphine in vitro competitively when the apomorphine concentration was varied. These results suggest that methylation by COMT may represent an important metabolic pathway for the deactivation of apomorphine in vivo .

The excretion of radioactive metabolites in urine, feces, and expired air after the oral administration of 5 or 25 mg/kg of [ 3 H, 14 C]caffeine to male CD-1 mice was investigated. Of the total radioactivity, 64–90 per cent was recovered in the urine. The major identifiable urinary metabolites were 1,7-dimethylxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethyluric acid and 1-methyluric acid. Of the administered' caffeine, 3–6 per cent was recovered from the urine. Excertion of identifiable metabolites occured rapidly and was 80 per cent complete in 8 hr following the 25 mg/kg dose. No consistent difference in urinary excretion of radioactivity was seen when mice fed caffeine since weaning were compared to those not previously exposed to caffeine, although the fecal excretory route was enhanced in the former.

The metabolic disposition of allopurinol [4-hydroxypyrazolo(3,4-d)-pyrimidine) was determined in mice, dogs, and human subjects. The drug is a substrate for, as well as an inhibitor of, xanthine oxidase and is converted in all species to the corresponding xanthine analog, alloxanthine, which is its major metabolite. Neither is bound to human plasma proteins, and both are distributed more or less equally in total body water in the mouse. Both analogs are cleared rapidly by the mouse and dog kidney. In the human subject allopurinol is cleared rapidly, but alloxanthine resembles uric acid in having a slow clearance responsive to probenecid. The accumulation of alloxanthine during prolonged therapy with allopurinol may contribute significantly to the therapeutic effects of the drug in the control of hyperuricemias.

The properties of a methionine-activating enzyme in human erythrocytes and those of other species are described. Several compounds were examined for their capacity to inhibit the enzyme. Electrophoretic mobility of the enzyme from human and animal tissues was investigated by starch block electrophoresis. Possible clinical applications of this new assay are discussed.

Administration of4(5)-(3,3-dimethyl-1-triazeno)imidazole-5(4)-carboxamide (DIC), a structural analogue of 4(5)-aminoimidazole-5(4)-carboxamide (AIC), i.V. or P.O., to patients with cancer was followed by increased urinary AIC excretion. After a single i.v. administration of 4.5 mg/kg of DIC, AIC excretion in the 24-hr period after DIC administration was 20 per cent of the dose of DIC given. When the same dose was given p.o., about 16.5 per cent of the dose of DIC was excreted as AIC within 24 hr. One patient given DIC p.o. at a dose of 1.5 mg/kg every 6 hr for four consecutive doses excreted about 13.6 per cent of each dose as AIC in each 6-hr period. Two patients were given DIC-2- 14 C p.o. Ion-exchange chromatography of plasma samples resulted in the detection of a peak of radioactivity in the column eluate fraction containing AIC. After ion-exchange chromatography of urine samples, 20.9 and 8.7 per cent, respectively, of the radioactivity appeared in the AIC fractions. 14 C-AIC was isolated from urine and characterized by paper-chromatographic behavior, ultraviolet fluorescence and color reaction after diazotization and coupling identical to that of authentic AIC, and was recrystallized to constant specific activity. The specific activity of the urinary 14 C-AIC from one patent was 78 per cent of that of the DIC-2- 14 C given. These data suggest that in man most of the increased urinary AIC excretion observed after DIC administration is the direct result of metabolism of DIC. The mechanism(s) of action of DIC as an antitumor agent in man is unknown.

Six isomers of angiotensin II, differing from one another only with respect to changes made in the aspartic acid situated at the amino end, were incubated with rat serum, human plasma or rat kidney homogenate. The rate at which the isomers were inactivated was studied by measuring pressor effects in nephrectomized rats. In parallel tests, fragments were demonstrated by means of paper chromatography. Compounds with an α-L-Asp 1 configuration were inactivated much more rapidly in serum or plasma than α-D-, β-L-, β-D- or desamino-angiotensin II. When the substances were incubated with kidney homogenate, however, the differences were less marked. Chromatographic studies revealed that, when the α-L-compounds were incubated, not only peptide fragments occurred as a result of aminopeptidasic degradation, but also small quantities (in free form) of all the amino acids contained in the molecule. The α-D-, β-L-, β-D- and desamino-compounds were found to be protected against aminopeptidasic degradation, whereas endopeptidasic degradation proceeded in the same way as in the case of the α-L-compounds. Here, in addition to free amino acids, the isomeric tetrapeptides Asp-Arg-Val-Tyr were also demonstrable. Whereas in the serum of nephrectomized rats approximately 90 per cent of the total degradation process was due to the activity of aminopeptidases and only 10 per cent to that of endopeptidases, the corresponding activity ratio for the enzymes in rat kidney homogenate or in human plasma was approximately 60 per cent: 40 per cent.

Several proteases from human kidney tissue were partially purified by ammonium sulfate precipitation followed by Sephadex G-75 gel filtration chromatography. The incubations of column fractions with 14C-tetradecapeptide renin substrate indicated four major renin activity peaks between the molecular weights of 25,000 and 80,000. The highest specific activity was found in Fraction B (mol. wt 39,500). Significant renin-like activity was also found in Fraction A (mol. wt 58,000) and Fraction C (mol. wt 33,500). None of the fractions contained angiotensinase activity. Disc-gel electrophoresis indicated that Fractions B and C were heterogeneous. The conversion of 14C-tetradecapeptide renin substrate to angiotensin I by major fractions was differentially inhibited by low concentrations of pepstatin. Both the rate of substrate conversion and the sensitivity to pepstatin were influenced by pH. Generally, the proteases were more sensitive to pepstatin at physiological pH. Regardless of pH, all of the kidney proteases were completely inhibited by 10−3 M pepstatin. The conversion of human substrate by human plasma proteases was found to be even more sensitive to pepstatin. The concentration of 10−5 M pepstatin essentially eliminated plasma renin activity. These results indicate that pepstatin can serve as an efficient inhibitor of both human kidney and plasma renin proteases and as such will be useful in the study of the kinetics of these systems in both research and clinical situations.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α1-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10−7 was present in number equivalent to concentration of α1-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α1-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α1-acid glycoprotein is the main propranolol binding protein in human serum.

Diaryl o -( α -hydroxy)tolyl phosphates are cyclized with liberation of an aryl group to yield aryl saligenin cyclic phosphates, which usually are potent antiesterase agents. This cyclization is catalyzed by various plasmata, but not by liver homogenate. The cyclizing activity of plasma is associated with the albumin component when this fraction is prepared either by salting-out with ammonium sulfate or by using Cohn's method and ethanol (fraction V). The activity of the cyclizing enzyme could not be separated from albumin either by Sephadex G-100 or by DEAE-cellulose column chromatography. p -Nitrophenyl acetate, which is also hydrolyzed by the albumin fraction, inhibits the cyclization of diaryl o -( α -hydroxy)tolyl phosphates.

Salicyl-coenzyme A (salicyl-CoA) has been prepared by organic synthesis, and its properties compared to those of benzoyl-CoA. Salicyl-CoA reacts with glycine acyl transferase prepared from both beef and human liver to form salicyluric acid. For both enzymes the rate with salicyl-CoA is approximately 50 per cent of the rate with benzoyl-CoA. In addition, there is a small non-enzymatic acylation of glycine by salicyl-CoA.

Several modifications of 1-β- d -arabinosylcytosine (ara-C) are described with particular regard to prolonging its presence in the tissues and lowering its toxicity. Precursors containing an isourea-ether or amidine group and a phosphate group were synthesized and studied in vitro. The most promising compound satisfying these criteria is O2:2'-anhydro-1-β- d -arabinosylcytosine 3'-phosphate (anhydro-ara-CMP). We have followed the disappearance of the anhydro-ara-C chromphore spectrophotometrically in phosphate, bicarbonate, citrate, lactate and Tris-HCl buffers of various concentration and pH. We found that the ultraviolet change was due to two concomitant firstorder reactions. The main process was a general-base catalyzed hydrolysis leading to the formation of 1-β- d -arabinosylcytosine 3'-phosphate (ara-CMP), but the formation of 2',3'-cyclic CMP was also observed to some extent. The rate constants and the arabino to ribo ratio depended on the ionic strength and the pH. In the course of studies of 14C-labeled compound in human blood in vitro, the presence of ara-C and its favorable rate of formation could be observed. It was concluded that the administration of a single dose of anhydro-ara-CMP will produce an effect similar to continuous infusion of decreasing amounts of ara-C.

N-(purin-6-ylcarbamoyl- L -threonine riboside-8-14C (Ado-CO-thr-8-14C) and N-(purin-6-ylcarbamoyl- l -[U-14C]threonine riboside (Ado-CO-thr-14C) were synthesized by using adenosine-8-14C and threonine [U-14C], respectively, as labeled starting materials. The Ado-CO-thr-8-14C and Ado-CO-thr-14C were given intravenously and orally to rat and to man. Of the 2·4 × 106 dis/min administered intravenously to rats, 73 per cent was excreted in urine. More than 79 per cent of this excreted activity was in unchanged Ado-CO-thr, 2·2 per cent in the free base, N-(purin-d-ylcarbamoyl)- l -threonine (Ade-CO-thr), and less than 2 per cent in adenosine. Radioactivity in adenine, inosine, hypoxanthine and uric acid was less than 0·1 per cent. When the labeled Ado-CO-thr was administered orally, less than 5 per cent of the radioactivity was excreted in urine. Similar results were obtained in man. These data revealed that Ado-CO-thr is quite a stable modified nucleoside in vivo. Intravenously administered, Ado-CO-thr was not incorporated into rat t-RNA. In vitro, it did not serve as a substrate for adenosine deaminase, xanthine oxidase and adenosine kinase. Incubation of Ado-CO-thr with urease, acylase, protease, peptidases and similar other enzymes did not lead to any structural change in the molecule. It is suggested that naturally occurring urinary Ado-CO-thr originates from t-RNA and is excreted quantitatively in urine.

The experiments were made to determine whether our previously reported finding of decreased 5-hydroxytryptamine binding by blood platelets from children with Down's syndrome (mongolism) involved changes in platelet monoamine oxidase Platelets from normal and mongol children were incubated with 5-hydroxytryptamine. The efflux of the 5-hydroxytryptamine metabolites 5-hydroxyindolylacetic acid and 5-hydroxytryptophol was measured during a 2-hr incubation. There were highly significant reductions in the efflux of 5-hydroxytryptamine and 5-hydroxyindolylacetic acid from mongol platelets. When 5-hydroxytryptamine loaded platelets were incubated with reserpine, 5-hydroxytryptamine release was greatly augmented in both groups, but there was no increased efflux of 5-hydroxyindolylacetic acid. We conclude that mongol platelets form abnormally low amounts of 5-hydroxyindolylacetic acid in vitro. However, the decreased 5-hydroxytryptamine binding reported before cannot be related to any defects of platelet monoamine oxidase until the enzyme has been characterized, since there is the possibility that the enzyme in mongoloid cells is not homogeneous but abnormally heterogeneous.

Cytosine arabinoside, labeled with tritium, was administered to five patients with far-advanced neoplasms. The analog was rapidly deaminated to uracil arabinoside; the latter accounted for between 86 and 96 per cent of the radioactivity excreted in the urine. Incorporation of tritium-labeled cytidine into DNA by suspensions of human leukemic leukocytes was inhibited in the presence of cytosine arabinoside. Although administration of cytosine arabinoside at therapeutic levels depressed the ability of leukemic leukocytes to incorporate cytidine into DNA in vitro , there was no correlation between the degree and duration of this effect and the clinical response to the drug. Tritium-labeled cytosine arabinoside entered human leukemic leukocytes very rapidly when incubated with the cells in vitro ; there was a small but significant incorporation of the analog into both DNA and RNA.

Glycan is an essential molecule that controls and drives life in a precise direction. The paucity of research in glycobiology may impede the significance of its role in the pandemic guidelines. The SARS-CoV-2 spike protein is heavily glycosylated, with 22 putative N-glycosylation sites and 17 potential O-glycosylation sites discovered thus far. It is the anchor point to the host cell ACE2 receptor, TMPRSS2, and many other host proteins that can be recognized by their immune system; hence, glycosylation is considered the primary target of vaccine development. Therefore, it is essential to know how this surface glycan plays a role in viral entry, infection, transmission, antigen, antibody responses, and disease progression. Although the vaccines are developed and applied against COVID-19, the proficiency of the immunizations is not accomplished with the current mutant variations. The role of glycosylation in SARS-CoV-2 and its receptor ACE2 with respect to other putative cell glycan receptors and the significance of glycan in host cell immunity in COVID-19 are discussed in this paper. Hence, the molecular signature of the glycan in the coronavirus infection can be incorporated into the mainstream therapeutic process.

It was thought that originally long non-coding RNAs (lncRNAs) were a kind of RNAs without any encoding function. Recently, a variety of studies have shown that lncRNAs play important roles in many life activities. The abnormal expression of lncRNAs in tumor microenvironment (TME) usually promotes the proliferation, migration, and drug resistance of tumor cells through direct or indirect effects, which also usually predicts the poor prognosis. The regulation of lncRNAs expression in TME could significantly inhibit tumor progress. However, the interaction between lncRNAs and TME has not been fully defined at present. Therefore, this paper provided the systemic summary of their interaction and natural products and chemicals targeting lncRNAs in cancer treatment. Currently, the strategies of cancer treatment still have their limits. Understanding the relationship between TME and lncRNAs can help us to realize breakthrough strategy for tumor treatment.

Currently, the only available effective treatment option for obesity and its comorbidities is weight loss surgery (WLS). Long-term maintenance of weight loss after surgery cannot be explained by caloric restriction or malabsorption alone and has been attributed to unexplained changes in eating behavior. Whether these behavioral changes are related to altered taste or reward functions, or both, are subject to debate. In contrast to reduced food cravings and food addiction following WLS, recent clinical studies have revealed that bariatric surgery patients are prone to an increased risk for substance use disorder (SUD), especially alcohol use disorder (AUD). The substitution of drugs for previously stimulating foods, and the emergence of SUD after WLS, supported by preclinical studies, strongly suggest that manipulation of gut-brain signals may bring about changes in the reward system. This paper reviews current clinical and basic science research and discusses potential underlying mechanisms of reward-related behaviors. Specifically, it explores relevant neural and hormonal changes that present post WLS and their effects on dopaminergic reward pathway and highlights targets for potential pharmacological interventions. Special emphasis is given to recent work suggesting that different types of WLS procedures such as Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) have differential effects on alcohol consumption in humans and rats. These differential effects may hold the key not only to understanding increased substance use following WLS but may also help elucidate the contribution of gut-brain signals to regulation of reward, in general.

Purpose The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. Methods WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. Results Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. Conclusions Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.

Parathyroid hormone (PTH) is an 84-amino-acid peptide hormone that is secreted by the parathyroid gland. It has different administration modes in bone tissue through which it promotes bone formation (intermittent administration) and bone resorption (continuous administration) and has great potential for application in sbone defect repair. PTH regulates bone metabolism by binding to PTH1R. PTH plays an osteogenic role by acting directly on mesenchymal stem cells, cells with an osteoblastic lineage, osteocytes, and T cells. It also participates as an osteoclast by indirectly acting on osteoclast precursor cells and osteoclasts and directly acting on T cells. In these cells, PTH activates the Wnt signaling, cAMP/PKA, cAMP/PKC, and RANKL/RANK/OPG pathways and other signaling pathways. Although PTH(1-34), also known as teriparatide, has been used clinically, it still has some disadvantages. Developing improved PTH-related peptides is a potential solution to teriparatide's shortcomings. The action mechanism of these PTH-related peptides is not exactly the same as that of PTH. Thus, the mechanisms of PTH and PTH-related peptides in bone metabolism were reviewed in this paper.

Epithelial mesenchymal transition (EMT) is a biological process that plays a critical role in cancer progression. Blocking the process of EMT can be an essential strategy in the development of anticancer drugs. In this paper, we briefly introduce recent research trends and issues of EMT, focusing on the relationship of EMT with the entire cycle of inflammation (from initiation to resolution of inflammation). Recent findings on the relationship between EMT and immune evasion are discussed. We will also explore the importance of controlling inflammation by anti-inflammatory compounds and specialized pro-resolving lipids to inhibit EMT process in cancer.

Chromosomal translocations - leading to the expression of fusion genes - are well-studied genetic abberrations associated with the development of leukemias. Most of them represent altered transcription factors that affect transcription or epigenetics, while others - like BCR-ABL - are enhancing signaling. BCR-ABL has become the prototype for rational drug design, and drugs like Imatinib and subsequently improved drugs have a great impact on cancer treatments. By contrast, MLL-translocations in acute leukemia patients are hard to treat, display a high relapse rate and the overall survival rate is still very poor. Therefore, new treatment modalities are urgently needed. Based on the molecular insights of the most frequent MLL rearrangements, BET-, DOT1L-, SET- and MEN1/LEDGF-inhibitors have been developed and first clinical studies were initiated. Not all results of these studies have are yet available, however, a first paper reports a failure in the DOT1L-inhibitor study although it was the most promising drug based on literature data. One possible explanation is that all of the above mentioned drugs also target the cognate wildtype proteins. Here, we want to strengthen the fact that efforts should be made to develop drugs or strategies to selectively inhibit only the fusion proteins. Some examples will be given that follow exactly this guideline, and proof-of-concept experiments have already demonstrated their feasibility and effectiveness. Some of the mentioned approaches were using drugs that are already on the market, indicating that there are existing opportunities for the future which should be implemented in future therapy strategies.

General anaesthesia is used widely in surgery and during interventional medical procedures, but little is known about the exact neural mechanisms for how unconsciousness arises from administering an anaesthetic drug. Computational modelling of brain dynamics has already provided valuable insights into the neural circuitry involved in generating this state. Current theories for the origin of electroencephalographic (EEG) features in brain activity under GABAergic anaesthetic drugs have been proposed through modelling results. While much attention has been paid to describing alpha and delta oscillations, burst suppression, paradoxical excitation and the possibility of hysteresis during transitions to and from unconscious state, these models have focused only on the role of the thalamocortical system. Recent empirical findings suggest that anaesthetic drugs may act directly on the neural circuitry regulating sleep and wake states and circadian rhythms in the hypothalamus. Coupled with the common behavioural features found in physiological sleep and general anaesthesia, this evidence serves as a foundation for the 'shared circuits hypothesis' which proposes that anaesthetic-induced unconsciousness arises predominantly through modulation of the hypothalamic sleep-wake switch. This paper reviews the key findings from computational models describing brain states during the administration of anaesthetic drugs, with a focus on those enhancing GABAergic inhibition given their widespread use in practice and that almost all models of anaesthesia have focused on these drugs. We draw physiological and behavioural links between brain states during sleep and anaesthesia, and aim to highlight the importance of computational modelling in advancing our understanding of anaesthesia by considering sleep and circadian mechanisms in generating unconsciousness in future work.

Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDA-MB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

Prof. Geoffrey Burnstock originated the concept of purinergic signaling. He demonstrated the interactions and biological roles of ionotropic P2X and metabotropic P2Y receptors. This review paper traces the historical origins of many currently used antagonists and agonists for P2 receptors, as well as adenosine receptors, in early attempts to identify ligands for these receptors – prior to the use of chemical libraries for screening. Rather than presenting a general review of current purinergic ligands, we focus on common chemical scaffolds (privileged scaffolds) that can be adapted for multiple receptor targets. By carefully analyzing the structure activity relationships, one can direct the selectivity of these scaffolds toward different receptor subtypes. For example, the weak and non-selective P2 antagonist reactive blue 2 (RB-2) was derivatized using combinatorial synthetic approaches, leading to the identification of selective P2Y(2), P2Y(4), P2Y(12) or P2X2 receptor antagonists. A P2X4 antagonist NC-2600 is in a clinical trial, and A(3) adenosine agonists, show promise for chronic pain. P2X7 antagonists have been in clinical trials for depression (JNJ-54175446), inflammatory bowel disease (IBD), Crohn’s disease, rheumatoid arthritis, inflammatory pain and chronic obstructive pulmonary disease (COPD). P2X3 antagonists are in clinical trials for chronic cough, and an antagonist named after Burnstock, gefapixant, is expected to be the first P2X3 antagonist filed for approval. We are seeing that the vision of Prof. Burnstock to use purinergic signaling modulators, most recently at P2XRs, for treating disease is coming to fruition.

Geoffrey Burnstock, the founder of the field of purinergic signaling research passed away in Melbourne, Australia on June 3rd, 2020, at the age of 91. With his death, the world of biomedical research lost one of its most passionate, creative and unconventional thought leaders. He was an inspiration to the many researchers he interacted with for more than 50 years and a frequent irritation to those in the administrative establishment. Geoff never considered himself a pharmacologist having being trained as a zoologist and becoming an autonomic neurophysiologist based on his evolving interests in systems and disease-related research. By the end of his life he had: published some 1550 papers; been cited more than 125,000 times; had an h-index of 156 and had supervised over 100 Ph.D. students. His indelible legacy, based on a holistic, data-based, multidisciplinary, unconventional “outside the box” approach to research was reflected in two of the seminal findings in late 20th century biomedical research: the purinergic neurotransmitter hypothesis and the concept of co-neurotransmission, both of which were initially received by his peers with considerable skepticism that at times verged on disdain. Nonetheless, while raising hackles and threatening the status quo, Geoff persevered and prevailed, becoming a mentor for several generations of biomedical researchers. In this review we provide a joint perspective on Geoff Burnstock’s legacy in research.

Many malignant tumors use endogenous nitric oxide (NO) to promote survival, growth, and metastatic migration. This NO, which is typically generated by inducible nitric oxide synthase (iNOS), can also antagonize various anti-cancer therapies and its source is most often assumed to be constitutive or pre-existing iNOS. In this paper, we provide evidence (i) that many different cancer cells exhibit resistance to oxidative killing by photodynamic therapy (PDT), and (ii) that cells surviving the challenge grow, migrate and invade more aggressively, as do non-targeted bystander cells. Accompanying these effects are activation or upregulation of pro-survival/progression effector proteins such as NF-κB, Akt, and Survivin. Observed in the author's laboratory, these responses were not attributed to basal iNOS/NO in most cases, but rather to NO from enzyme that was strongly upregulated by photodynamic stress. Each of these effects and how they can be mitigated by inhibitors of iNOS activity or transcription, or by NO scavengers will be discussed. When approved for clinical use, such pharmacologic agents could improve PDT efficacy as well as reduce potentially negative side-effects of this therapy.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6β2-containing (α6β2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6β2* agonist exhibiting functional selectivity toward other nAChRs, including α4β2, α3β4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6β2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6β2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

Chondrocytes and their mesenchymal cell progenitors secrete a variety of bioactive molecules, including adenine nucleotides and nucleosides, but these molecules are not usually highlighted in review papers about the secretome of these cells. Ageing and inflammatory insults compromise chondrocytes ability to keep ATP/adenosine synthesis, release and turnover. Cartilage homeostasis depends on extracellular adenosine levels, which acting via four P1 purinoceptor subtypes modulates the release of pro-inflammatory mediators, including NO, PGE2 and several cytokines. Native articular cartilage is challenged by synovial fluid flow during normal joint motion transiently increasing ATP release and adenosine formation in the joint microenvironment. Excessive joint motion and shockwave trauma are deleterious to cartilage homeostasis due to HIF-1α overexpression, resulting in disproportionate ecto-5'-nucleotidase/CD73 production, adenosine accumulation and superfluous A2B receptors activation. Scarcity of data however exists on the putative interplay between coexistent high affinity (A2A and A3) and low affinity (A2B) adenosine receptors activation affecting stem cells fate towards preferential chondrogenic or osteogenic lineages in the human cartilage. Hints gathered in this commentary result mainly from studies using human immortalized cell lines and animal (e.g. rodent, equine, bovine) tissue samples. The available data point towards adenosine A2A and A3 receptors having cartilage protective roles, while excessive adenosine accumulation may be detrimental via low affinity A2B receptors activation, with little reference to the putative role of the adenosine forming enzyme ecto-5'-nucleotidase/CD73. Thus, emphasizing the multiple pathways responsible for controlling adenosine signalling in cartilage will certainly impact on the search for novel therapeutic targets for highly disabling articular disorders.