Development and validation of LC/MS/MS method for Triciribine and its monophosphate metabolite in plasma and RBC: Application to mice pharmacokinetic studies.

Triciribine (TCN) is a tricyclic nucleoside analog of adenosine and an inhibitor of Akt kinase. Triciribine 5'-monophosphate (TCNP) is a water-soluble analog of Triciribine and has progressed to Phase I and II clinical trials in oncology. TCNP is also an endogenous anabolite of TCN similar to other nucleoside phosphates. Clinical development of TCNP has been hampered by high pharmacokinetic variability due to complex interplay of TCN-TCNP conversion and reconversion in plasma, erythrocytes (RBC) and peripheral organs. TCN has been demonstrated to be an efficacious agent in mice models of acute lung injury at low doses (0.5 mg/kg/day) although its pharmacokinetic-pharmacodynamic (PK/PD) relationship remained unclear. We have developed and validated a sensitive, specific and robust LC/MS/MS assay for quantitation of TCN and TCNP in plasma and RBC. Using a simple protein precipitation method, quantitation of these analytes was accomplished with recoveries exceeding 85% and with a run time of 4 min. This assay was used to determine the pharmacokinetic parameters of TCN and TCNP in mice after single dose intravenous administration at 1, 3 and 10 mg/kg. TCNP accumulates in RBC, has low clearance and a half-life of 18 to 23 h. Unlike other nucleoside phosphates, TCNP was found to be relatively stable in mice plasma serving as a secondary depot. TCN levels were low and with high clearance relative to hepatic blood flow. A combination of sustained levels of TCNP in RBC and plasma serves as a depot for TCN to elicit robust therapeutic activity in acute lung injury mice models.

Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR.

5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-ß Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1).

AIM: Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin. METHODS: Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells. RESULTS: AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes. CONCLUSIONS: We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.

Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Phase I clinical, pharmacokinetic, and pharmacodynamic study of the Akt-inhibitor triciribine phosphate monohydrate in patients with advanced hematologic malignancies.

Akt, a serine/threonine protein kinase, is constitutively phosphorylated and hyperactivated in multiple cancers, including acute myeloid leukemia. High levels are linked to poor survival and inferior responses to chemotherapy, making Akt inhibition an attractive therapeutic target. In this phase I/II study of TCN-PM, a small-molecule Akt inhibitor, TCN-PM therapy was well tolerated in patients with advanced hematological malignancies, and reduced levels of phosphorylation of Akt and its substrate Bad were shown, consistent with inhibition of this survival pathway and induction of cell death. Further investigation of TCN-PM alone or in combination in patients with high Akt levels is warranted.

A HPLC method for simultaneous determination of 5-aminoimidazole-4-carboxamide riboside and its active metabolite 5-aminoimidazole-4-carboxamide ribotide in tumor-bearing nude mice plasma and its application to pharmacokinetics study.

A HPLC method with on-line solid phase extraction (SPE) and column switching was developed for simultaneous determination of 5-aminoimidazole-4-carboxamide riboside (AICA riboside) and its active metabolite 5-aminoimidazole-4-carboxamide ribotide (AICA ribotide) in nude mice plasma. Plasma sample was deproteinized by adding a half volume of 10% trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA. 50 µl aqueous fraction was injected onto a WAX-1 SPE column, and AICA ribotide was trapped on the SPE column, while AICA riboside was eluted from the SPE column. The chromatographic separation of AICA riboside was achieved on CG16 column, and separation of AICA ribotide was performed on HILIC-10 and WAX-1 column. The columns temperature was maintained at 40 °C, and the optimal detection wavelength was 268 nm for both AICA riboside and AICA ribotide. The total analytical run time was 40 min. The proposed method was linear over the range of 0.1-500 µg/ml for AICA riboside and 0.03-50 µg/ml for AICA ribotide. The lower limit of quantification (LLOQ) was 100 and 30 ng/ml for AICA riboside and AICA ribotide, respectively. The sensitivity, accuracy and precision of this method were within acceptable limits during validation period. The method was successfully applied to investigate the pharmacokinetics characteristics of AICA riboside and its active metabolite AICA ribotide in nude mice bearing MCF-7 cell xenografts.

Intravenous AICAR during hyperinsulinemia induces systemic hemodynamic changes but has no local metabolic effect.

AMPK activation may stimulate glucose uptake in skeletal muscle, but the results in humans have so far been inconclusive. The authors investigated whether infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide-riboside (AICAR), increased whole-body glucose infusion rate (GIR) and forearm skeletal muscle glucose uptake (FGU) during hyperin-sulinemia in vivo in healthy humans. Ten participants (paired data: n = 8) underwent 2 euglycemic hyperinsulinemic clamps (60 mU·m(-2)·min(-1), 120 minutes) with concomitant AICAR (67 mg·kg(-1)) or placebo (saline) administration over the last 60 minutes. The authors also measured forearm blood flow (FBF; plethysmography), heart rate, blood pressure, and AICAR and AICA-ribotide (ZMP) concentrations in plasma and erythrocytes. FGU and GIR (T = 95-120 min) did not differ between insulin + AICAR and insulin + placebo. Compared with insulin + placebo, insulin + AICAR raised heart rate more profoundly (T = 60-120 minutes: from 58 ± 3 to 70 ± 3 vs 60 ± 4 to 63 ± 4 bpm for placebo; P < .05 between treatments) and lowered blood pressure significantly. AICAR plasma concentrations increased significantly during AICAR infusion; AICAR was rapidly taken up by erythrocytes and phosphorylated to ZMP. In conclusion, AICAR does not seem to have a direct effect on systemic or local glucose uptake in humans. AICAR increases heart rate and decreases blood pressure, most likely by systemic vasodilation.

Phase I pharmacokinetic and pharmacodynamic study of triciribine phosphate monohydrate, a small-molecule inhibitor of AKT phosphorylation, in adult subjects with solid tumors containing activated AKT.

PURPOSE: Triciribine phosphate is a potent, small-molecule inhibitor of activation of all three isoforms of AKT in vitro. AKT is an intracellular protein that, when activated, leads to cellular division; it is dysregulated in a large number of malignancies, and constitutively activating AKT mutations are present in a minority of cancers. PATIENTS AND METHODS: In this phase I study triciribine phosphate monohydrate (TCN-PM) was administered to subjects whose tumors displayed evidence of increased AKT phosphorylation (p-AKT) as measured by immunohistochemical analysis (IHC). TCN-PM was administered over 30 min on days 1, 8 and 15 of a 28-day cycle. Tumor biopsy specimens, collected before treatment and on day +15, were assessed for p-AKT by IHC and western blot analyses. RESULTS: Nineteen subjects were enrolled; 13 received at least one cycle of therapy, and a total of 34 complete cycles were delivered. One subject was treated at the 45 mg/m(2) dose before the study was closed due to its primary objective having been met. No dose-limiting toxic effects were observed. Modest decreases in tumor p-AKT following therapy with TCN-PM were observed at the 35 mg/m(2) and 45 mg/m(2) dose levels, although definitive conclusions were limited by the small sample size. CONCLUSIONS: These preliminary data suggest that treatment with TCN-PM inhibits tumor p-AKT at doses that were tolerable. Although single agent activity was not observed in this enriched population, further combination studies of TCN-PM with other signal transduction pathway inhibitors in solid tumors is warranted.

Cross-linked polymeric nanogel formulations of 5'-triphosphates of nucleoside analogues: role of the cellular membrane in drug release.

Activation of cytotoxic nucleoside analogues in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogues may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5'-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, the 5'-triphosphate of an antiviral nucleoside analogue, 3'-azido-2',3'-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analogue 5'-triphosphate using a one-pot procedure. The corresponding 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating IC50 values 130-200 times lower than those values for AZT alone. The exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic P85. Cellular trafficking of nanogel particles, contrasted by polyethylenimine-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was observed following interactions of drug-loaded nanogels with cellular membranes. A drug release mechanism triggered by interaction of the drug-loaded nanogels with phospholipid bilayer is proposed. The results illustrate therapeutic potential of the phosphorylated nucleoside analogues formulated in nanosized cross-linked polymeric carriers for cancer chemotherapy.

Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS.

Amino acid phosphoramidates of nucleosides have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside monophosphate prodrugs. To access their ability to deliver 3'-azido-3'-deoxythymidine (AZT) 5'-monophosphate to cells, the decomposition pathway of an 18O-labeled AZT amino acid phosphoramidate was investigated by capillary reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC-ESI(-)-MS/MS). 18O-labeled L-AZT tryptophan phosphoramidate methyl ester ([18O]2) was synthesized with an 18O/16O relative ratio of 1.22 +/- 0.18. For CEM cells, a human T-lymphoblast leukemia cell line, incubated with [18O]2, values of 1.55 +/- 0.37, 0.34, and 0.13 were found for the 18O/16O relative ratio of intracellular AZT-MP for time intervals of 0.5, 4, and 20 h, respectively. The decrease in the level of labeled AZT-MP in CEM cells corresponded to a rapid increase in the amount of intracellular AZT presumably by dephosphorylation of AZT-MP. In contrast, for peripheral blood mononuclear cells (PBMCs), the 18O/16O relative ratio values of intracellular AZT-MP were 1.43, 1.06, and 0.61 for time intervals of 0.5, 4, and 20 h, respectively. Intracellular AZT in PBMCs was nearly undetectable for each time interval. Taken together, these results are consistent with the detection of direct P-N bond cleavage by CEM cells and PBMCs. However, AZT phosphoramidates are able to more effectively deliver AZT-MP to PBMCs than to CEM cells. Differential expression of 5'-nucleotidase in CEM cells relative to PBMCs is likely the reason for this discrepancy. Although applied to a phosphoramidate pronucleotide, the judicious use of 18O labeling and LC-MS is a general approach that could be applied to the investigation of the intracellular fate of other pronucleotides.

The pK(a) of the internucleotidic 2'-hydroxyl group in diribonucleoside (3'-->5') monophosphates.

Ionization of the internucleotidic 2'-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariat-RNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pK(a) of 2'-OH in mono- and diribonucleoside (3'-->5') monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pK(a) values for internucleotidic 2'-OH of eight different diribonucleoside (3'-->5') monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pK(a) is 12.31 (+/-0.02) for ApG and 12.41 (+/-0.04) for ApA, 12.73 (+/-0.04) for GpG and 12.71 (+/-0.08) for GpA, 12.77 (+/-0.03) for CpG and 12.88 (+/-0.02) for CpA, and 12.76 (+/-0.03) for UpG and 12.70 (+/-0.03) for UpA. By comparing the pK(a)s of the respective 2'-OH of monomeric nucleoside 3'-ethyl phosphates with that of internucleotidic 2'-OH in corresponding diribonucleoside (3'-->5') monophosphates, it has been confirmed that the aglycons have no significant effect on the pK(a) values of their 2'-OH under our measurement condition, except for the internucleotidic 2'-OH of 9-adeninyl nucleotide at the 5'-end (ApA and ApG), which is more acidic by 0.3-0.4 pK(a) units.

AMP-activated protein kinase: possible target for treatment of type 2 diabetes.

A considerable amount of data have accumulated showing that contraction of muscle has an acute insulin-like effect, triggering the uptake of glucose. Chronic muscle contraction, as seen in endurance training has effects on insulin sensitivity, enhancing the effect of insulin on glucose uptake. Endurance training results in an increase in levels of GLUT4 in the muscle. This increase in GLUT4 is thought to be responsible in part for the enhancement of insulin sensitivity. Recent experiments have demonstrated that acute and chronic effects of muscle contraction on glucose uptake and the increase in GLUT4 may be due to activation of a protein kinase, AMP-activated protein kinase (AMPK). This kinase is activated by the increase in 5'-AMP and the decline in creatine phosphate that occur during muscle contraction. Phosphorylated AMPK then presumably phosphorylates undefined target proteins, which in turn increase glucose uptake and transcription of the GLUT4 gene. Experiments have demonstrated that this kinase, normally activated during exercise, can be activated artificially in muscle by injecting non-exercising rats with 5-aminoimidazole-4-carboxamide-riboside (AICAR), an adenosine analog. AICAR is taken up into muscle and phosphorylated to form an analog of 5'-AMP. Acute (stimulation of glucose uptake into muscle) and chronic (increase in GLUT4) effects of exercise can be reproduced by injection of this drug. These observations open the door to the possibility of treatment of patients with type 2 diabetes with AMPK activators.

Phosphorylation of triciribine is necessary for activity against HIV type 1.

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.

Temperature dependence of the release of ATP hydrolysis products from the 10S conformation of smooth muscle myosin.

The transition of smooth muscle myosin to the folded 10S monomeric conformation dramatically inhibits the release of the ATP hydrolysis products, ADP and Pi. In this work, we examined the influence of temperature on the time course of product release from the 10S conformer of chicken gizzard smooth muscle myosin. Release was monitored by single turnover assays, using either [gamma-32P]ATP or the fluorescent ATP analog, formycin triphosphate (FTP). For all temperatures over the range 15-35 degrees C, single exponential kinetics described the observed product release from 10S myosin. A 10 degrees C increase in temperature resulted in a fourfold increase in the rate constant for the observed product release. Using single turnover analysis, we found a similar temperature dependence for the apparent rate constants for product release from the extended 6S monomeric conformation of myosin. However, at any given temperature, the rate constant for 6S myosin was approximately 1.5 orders of magnitude greater than that for the 10S. These results are consistent with a kinetic scheme in which 10S myosin must undergo transition to the 6S conformation prior to product release.