Antiviral effect of formycin and formycin B.
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The riddle of formycin A insulinotropic action.
Formycin A augments insulin release evoked by glucose (5.6 mm or more), this effect not being rapidly reversible. The mechanism responsible for the insulinotropic action of formycin A was investigated in isolated pancreatic islets. It could not be ascribed to facilitation of glucose metabolism. On the contrary, formycin A inhibited glucose oxidation, lowered ATP content, and impaired glucose-stimulated protein biosynthesis. The insulinotropic action of formycin A was apparently attributable to its conversion to formycin A 5'-triphosphate, both this process and the secretory response to formycin A being abolished by the inhibitor of adenosine kinase 5-iodotubercidin. In agreement with the latter view, adenosine receptor antagonists such as 8-cyclopentyl-1, 3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine failed to suppress and, instead, augmented the insulinotropic action of formycin A. Unexpectedly, however, formycin A failed to decrease 86Rb efflux, this coinciding with a low efficiency of formycin A 5'-triphosphate to inhibit KATP-channel activity in excised membranes and with the fact that formycin A increased gliben-clamide-stimulated insulin release. The secretory response to formycin A represented a Ca2+-dependent process suppressed in the absence of extracellular Ca2+ or presence of verapamil and associated with an increased net uptake of 45Ca. Nevertheless, the view that formycin A exerts any major effect upon intracellular Ca2+ redistribution, protein kinase C activity, or cyclic AMP net production also met with objections such as the minor secretory effect of formycin A in islets exposed to a high concentration of K+ in the presence of a diazoxide analog, the resistance of formycin A insulinotropic action to bisindolylmaleimide, the poor increase of cyclic AMP content in formycin A-stimulated islets, and the pronounced enhancement by forskolin or theophylline of insulin release from islets exposed to formycin A. It is concluded, therefore, that the mechanism of action of formycin A in the pancreatic beta-cell remains to be elucidated.
Adenosine↗
The effect of formycin B on mRNA translation and uptake of purine precursors in Leishmania mexicana.
Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.
Adenine↗
Biochemical genetic analysis of formycin B action in Leishmania donovani.
Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.
Allopurinol↗
Antileishmanial activity of human red blood cells containing formycin A.
Formycin B is the most active antileishmanial agent in vitro because it is metabolized by the parasites to formycin A phosphates. The in vivo use of formycin B may be limited by its toxicity to humans due to the slight similar metabolism of the drug in human cells. The obligatory intramacrophage localization of Leishmania in man suggests that encapsulation of drugs within macrophage-directed carriers, such as human red blood cells (RBCs), might enhance the therapeutic-toxic ratio. Since uncharged formycin B would be poorly metabolized by the RBC and would diffuse from the carrier, RBCs were incubated with formycin A so that approximately 90% of the formycin A was taken up by the cells, phosphorylated by RBC enzymes to charged formycin A triphosphate, and retained in the cells. In vitro, 81% of Leishmania-infected macrophages phagocytized IgG-coated RBCs containing this active form of formycin B, and multiplication of organisms within macrophages could be suppressed by approximately 80%. The 50% effective dose of the formycin A-RBC formulation was 0.02 microM, whereas the 50% effective dose of unencapsulated drug was 0.84 microM. This report of in vitro activity of human RBCs containing formycin A and coated with IgG indicates that this formulation should be tested for antileishmanial activity in vivo.
Animals↗
Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.
The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.
Adenosine Diphosphate↗
Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells.
In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.
Adenosine↗
Formycin B elimination from the cerebrospinal fluid of the rat.
The goal of this study was to determine whether specific transport systems are involved in nucleoside elimination from the cerebrospinal fluid (CSF). First, in vitro studies were carried out in isolated choroid plexus tissue slices from rat to ascertain the mechanisms of transport of formycin B, a model nucleoside analogue. 3H-Formycin B accumulated against a concentration gradient in the presence of an Na+ gradient in the isolated ATP-depleted choroid plexus tissue slices. This accumulation was reduced by high concentrations of unlabeled formycin B. Nitrobenzylthioinosine (NBMPR), an equilibrative nucleoside transport inhibitor, inhibited the uptake of formycin B in the absence of an Na+ gradient. These data suggest that both equilibrative and secondary active Na(+)-nucleoside transport systems are present in rat choroid plexus. In vivo, formycin B, together with inulin as a bulk flow marker, was injected into the lateral ventricle of the anesthetized rat with the aid of a stereotaxic device, and CSF was sampled from the cisterna magna at various times after injection. Twelve rats were randomized and divided into a low- and a high-dose group. The CSF clearance (CLCSF) of formycin B was significantly higher than the CLCSF of inulin in both animal groups (P < 0.01), indicating that formycin B is cleared from CSF by a pathway(s) in addition to bulk flow. Formycin B CLCSF was significantly lower in the high-dose group than in the low-dose group (P < 0.05), suggesting a saturable CSF elimination. The CLCSF of formycin B was also significantly reduced in animals treated with NBMPR (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Biochemical pharmacology and toxicology of formycin alone and in combination with 2'-deoxycoformycin (pentostatin).
The toxicology and pharmacology of formycin both as a single agent and combined with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF) were examined in outbred Swiss mice heterozygous for the nude gene (nu/+). The LD10 for formycin alone given on a daily x 5 schedule was 21 mg/kg. When the animals were pretreated with 1 mg/kg of dCF 1 hour prior to each dose of formycin, toxicity was approximately doubled, ie, LD10 was reduced to 10 mg/kg. Death was associated with hepatic toxicity in both treatment regimens; suppression of leukocyte counts was mild except at doses greater than the LD10. Formycin nucleotides were detected by high-performance liquid chromatography in the livers of mice treated with formycin either alone or combined with dCF. When isolated rat hepatocytes were incubated for 2 hours with either formycin or dCF plus formycin, analog nucleotides accumulated in the cells. Cellular ATP decreased to below the limits of detection, whereas a large peak corresponding to formycin-5'-triphosphate was present. This replacement of cellular ATP by formycin-5'-triphosphate may help explain the hepatic toxicity observed.
Adenosine Deaminase Inhibitors↗
Use of formycin B as a general substrate for measuring facilitated nucleoside transport in mammalian cells.
Formycin B, a C-nucleoside analog of inosine, is not catabolized by human erythrocytes and mouse P388 leukemia cells and is only very inefficiently phosphorylated in these cells. This relative inertness allows the measurement of its transport into and out of the cells uncomplicated by metabolic conversions. We have measured the zero-trans and equilibrium exchange flux of formycin B in these cells by rapid kinetic techniques. The Michaelis-Menten constants and maximum velocities for formycin B transport in both types of cell were similar to those previously reported for uridine and thymidine. Nevertheless, the differential mobility of the substrate-loaded and empty carrier of human erythrocytes was less for formycin B than uridine as substrate. Formycin B influx was inhibited by other nucleosides in accordance with their affinities for the carrier, but unaffected by purines. The inhibition of formycin B influx by nitrobenzylthioinosine and dipyridamole was also identical to that observed with uridine as substrate (IC50 = 10 and 30 nM, respectively). Formycin B accumulated in both types of cell to 30-40% higher concentrations than were present in the medium. This concentrative accumulation was not due to active transport, metabolism or partitioning into membrane lipids. It seems to reflect binding of formycin B to intracellular components, but does not interfere significantly with measurements of its transport.
Animals↗
Metabolism and mechanism of action of formycin B in Leishmania.
Formycin B is a potent inhibitor of growth of the promastigote forms of Leishmania tropica, L. mexicana, L. braziliensis, and L. donovani. The metabolic products formed in these organisms are formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates, with formycin A 5'-triphosphate predominating. In addition, formycin A is extensively incorporated into RNA. From the metabolic profile, we conclude that formycin B is first converted to the 5'-monophosphate by the nucleoside phosphotransferase found in Leishmania and is subsequently converted to cytotoxic adenosine nucleotide analogs of formycin A that become incorporated into RNA. The metabolism and mechanism of formycin B appears to be qualitatively similar to that of allopurinol riboside, but quantitative differences and species selectivity suggest that these agents may have a different spectrum of activity as potential anti-leishmanial agents.
Antibiotics, Antineoplastic↗
Metabolism of formycin B by Leishmania amastigotes in vitro. Comparative metabolism in infected and uninfected human macrophages.
Formycin B is metabolized by cutaneous Leishmania amastigotes within cultured human macrophages to give formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates. Formycin A is also incorporated into RNA. The activity of formycin B against amastigotes was correlated with the levels of formycin A metabolites formed in the parasites. Uninfected macrophages also convert formycin B into the same products, but the levels are markedly lower than those seen in infected macrophages. The results suggest that a sufficient therapeutic index exists to warrant consideration of formycin B as an anti-leishmanial drug in humans.
Animals↗
Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells.
At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na+-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [3H]formycin B. Uptake of [3H]formycin B into these cells was inhibited by replacement of Na+ in the buffer with choline, or by blocking Na+/K+ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]formycin B, cells were loaded with [3H]formycin B (10 microM) then washed and resuspended in buffer. Replacement of Na+ in the buffer with choline enhanced [3H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na+ concentrations of 30 mM or less. Resuspending loaded cells into Na+ buffer containing 2 mM ouabain or 10 microM monensin, a Na+ ionophore, significantly enhanced [3H]formycin B release during 20 min by 39% or 29%, respectively. Release of [3H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na+-dependent nucleoside transporters. Release was also inhibited significantly by 100 microM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na+-independent nucleoside transporters. In the absence of Na+, the permeants adenosine and uridine enhanced [3H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.
Adenosine Triphosphate↗
Interaction of 2',2'-difluorodeoxycytidine (gemcitabine) and formycin B with the Na+-dependent and -independent nucleoside transporters of Ehrlich ascites tumor cells.
The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2',2'-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 approximately 400 microM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.
Animals↗
Insulinotropic action of formycin A.
The adenosine analogue formycin A is phosphorylated to its triphosphate ester in a sequence of reactions catalyzed by adenosine kinase and adenylate kinase. Formycin A triphosphate is an ATP analogue that is currently used to probe for ATP binding sites. Considering the key role ascribed to ATP in the coupling of metabolic to cationic events in the process of glucose-stimulated insulin release, we investigated whether formycin A displays insulinotropic action in rat pancreatic islets. Formycin A (10 microM to 1.0 mM) caused a concentration-related increase of insulin release evoked by 8.3 mM D-glucose and prevented the fall in insulin output otherwise observed over two successive incubations of 90 min each. Formycin A (1.0 mM) also augmented insulin secretion at low (5.6 mM) and high (16.7 mM) concentrations of D-glucose. At the low hexose concentration, the secretory response to formycin A was comparable to that evoked by either glibenclamide or glipizide. At higher concentrations of D-glucose, however, formycin A was more potent than the hypoglycemic sulfonylureas in enhancing insulin output. These findings support the role of ATP in glucose-stimulated insulin release and, therefore, suggest that ATP mimetics represent a new class of insulinotropic agents that have potential utility in the treatment of non-insulin-dependent diabetes mellitus.
Animals↗
Dynamics of the cationic, bioelectrical and secretory responses to formycin A in pancreatic islet cells.
The dynamics of the cationic, bioelectrical and secretory responses to formycin A were monitored in pancreatic islet cells in order to assess whether this adenosine analogue, which is known to be converted to formycin A 5'-triphosphate in isolated islets, triggers the same sequence of ionic events as that otherwise involved in the process of nutrient-stimulated insulin release and currently attributed to an increase in adenosine 5'-triphosphate (ATP) generation rate. Unexpectedly, formycin A first increased 86Rb outflow, decreased 45Ca outflow and inhibited insulin release from prelabelled islets perifused at physiological or higher concentrations of D-glucose. This early inhibitory effect of formycin A upon insulin release coincided, in perforated patch whole-cell recordings, with an initial transient increase of ATP-sensitive K+ channel activity. A positive secretory response to formycin A, still not associated with any decrease in K+ conductance, was only observed either immediately after formycin A administration to islets already exposed to glibenclamide or during prolonged exposure to the adenosine analogue. This coincided with an increase of cytosolic Ca2+ concentration in intact B-cells and a greater increase of membrane capacitance in response to depolarization in B-cells examined in the perforated patch whole-cell configuration. The latter stimulation of exocytotic activity could not be attributed, however, to any increase in peak or integrated Ca2+ current. Thus, the mode of action of formycin A, or its 5'-triphosphate ester, in islet cells obviously differs from that currently ascribed to endogenous ATP in the process of nutrient-stimulated insulin release.
Adenosine Triphosphate↗
Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture.
The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2'-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10(-6) M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10(-5) M of each analog. In the presence of 10(-6) M 2'-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxy-cytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [3H]formycin and [3H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2'-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [3H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.
Adenosine↗
Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.
The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.
Adenosine Triphosphatases↗