Pharmacokinetics of IDX184, a liver-targeted oral prodrug of 2'-methylguanosine-5'-monophosphate, in the monkey and formulation optimization for human exposure.

IDX184 is a phosphoramidate prodrug of 2'-methylguanosine-5'-monophosphate, developed to treat patients infected with hepatitis C virus. A mass balance study of radiolabeled IDX184 and pharmacokinetic studies of IDX184 in portal vein-cannulated monkeys revealed relatively low IDX184 absorption but higher exposure of IDX184 in the portal vein than in the systemic circulation, indicating >90 % of the absorbed dose was subject to hepatic extraction. Systemic exposures to the main metabolite, 2'-methylguanosine (2'-MeG), were used as a surrogate for liver levels of the pharmacologically active entity 2'-MeG triphosphate, and accordingly, systemic levels of 2'-MeG in the monkey were used to optimize formulations for further clinical development of IDX184. Capsule formulations of IDX184 delivered acceptable levels of 2'-MeG in humans; however, the encapsulation process introduced low levels of the genotoxic impurity ethylene sulfide (ES), which necessitated formulation optimization. Animal pharmacokinetic data guided the development of a tablet with trace levels of ES and pharmacokinetic performance equal to that of the clinical capsule in the monkey. Under fed conditions in humans, the new tablet formulation showed similar exposure to the capsule used in prior clinical trials.

Challenges and solutions in the bioanalysis of BMS-986094 and its metabolites including a highly polar, active nucleoside triphosphate in plasma and tissues using LC-MS/MS.

BMS-986094, a nucleotide polymerase inhibitor of the hepatitis C virus, was withdrawn from clinical trials because of a serious safety issue. To investigate a potential association between drug/metabolite exposure and toxicity in evaluations conducted after the termination of the BMS-986094 development program, it was essential to determine the levels of BMS-986094 and its major metabolites INX-08032, INX-08144 and INX-09054 in circulation and the active nucleoside triphosphate INX-09114 in target and non-target tissues. However, there were many challenges in the bioanalysis of these compounds. The chromatography challenge for the extremely polar nucleoside triphosphate was solved by applying mixed-mode chromatography which combined anion exchange and reversed-phase interactions. The LC conditions provided adequate retention and good peak shape of the analyte and showed good robustness. A strategy using simultaneous extraction but separate LC analysis of the prodrug BMS-986094 and its major circulating metabolites was used to overcome a carryover issue of the hydrophobic prodrug while still achieving good chromatography of the polar metabolites. In addition, the nucleotide analytes were not stable in the presence of endogenous enzymes. Low pH and low temperature were required for blood collection and plasma sample processing. However, the use of phosphatase inhibitor and immediate homogenization and extraction were critical for the quantitative analysis of the active triphosphate, INX-09114, in tissue samples. To alleviate the bioanalytical complexity caused by multiple analytes, different matrices, and various species, a fit-for-purpose approach to assay validation was implemented based on the needs of drug safety assessment in non-clinical (GLP or non-GLP) studies. The assay for INX-08032 was fully validated in plasma of toxicology species. The lower limit of quantification was 1.00ng/mL and the linear curve range was 1.00-500.00ng/mL using a weighted (1/x(2)) linear regression model. Intra-assay and inter-assay precision (CV, %) ranged from 2.3% to 5.5% and accuracy within ±2.2% from nominal. INX-08032 was found to be stable in acidified mouse plasma for at least 24h in wet ice bath, 125 days at -70°C and following at least three freeze-thaw cycles. No endogenous components in plasma were found to interfere with the measurement. The extraction recovery was between 90% and 95%. The assays for BMS-986094, INX-08144, INX-09054 and INX-09114 were qualified with wider acceptance criteria for accuracy and precision. Analyte stability was also evaluated to guide sample collection, storage, and processing. These assays were successfully applied to an investigative toxicokinetic and tissue metabolite profiling study described in the article.

Overcoming stability challenges in the quantification of tissue nucleotides: determination of 2'-C-methylguanosine triphosphate concentration in mouse liver.

A conventional, rapid and high throughput method for tissue extraction and accurate and selective LC-MS/MS quantification of 2'-C-methylguanosine triphosphate (2'-MeGTP) in mouse liver was developed and qualified. Trichloroacetic acid (TCA) was used as the tissue homogenization reagent that overcomes instability challenges of liver tissue nucleotide triphosphates due to instant ischemic degradation to mono- and diphosphate nucleotides. Degradation of 2'-MeGTP was also minimized by harvesting livers using in situ clamp-freezing or snap-freezing techniques. The assay also included a sample clean-up procedure using weak anion exchange solid phase extraction followed by ion exchange chromatography and tandem mass spectrometry detection. The linear assay range was from 50 to 10000 pmol/mL concentration in liver homogenate (250-50000 pmol/g in liver tissue). The method was qualified over three intraday batches for accuracy, precision, selectivity and specificity. The assay was successfully applied to pharmacokinetic studies of 2'-MeGTP in liver tissue samples after single oral doses of IDX184, a nucleotide prodrug inhibitor of the viral polymerase for the treatment of hepatitis C, to mice. The study results suggested that the clamp-freezing liver collection method was marginally more effective in preventing 2'-MeGTP degradation during liver tissue collection compared to the snap-freezing method.

Protección contra el daño miocárdico por isquemia-reperfusión en la práctica clínica / Protection Against Myocardial Ischemia-reperfusion Injury in Clinical Practice

Pese a recibir lo más tempranamente posible la terapia de reperfusión, un amplio número de los pacientes que sufren infarto agudo de miocardio con elevación del segmento ST tienen infartos que comprometen su supervivencia y su calidad de vida. Parte de la muerte celular secundaria a una oclusión coronaria transitoria ocurre durante la reperfusión, por mal manejo del calcio en el sistema retículo sarcoplasmático-mitocondria, activación de calpaínas, estrés oxidativo y fallo mitocondrial, favorecidos por la rápida normalización del pH intracelular. Varios ensayos clínicos han demostrado que se puede limitar el tamaño del infarto mediante estrategias no farmacológicas —como el poscondicionamiento isquémico y el condicionamiento isquémico remoto— o farmacológicas —como la estimulación de la síntesis de guanosina monofosfato cíclico, la insulina, los agonistas del péptido glucagonoide tipo 1, los bloqueadores beta o la ciclosporina. Diversos ensayos clínicos han dado resultados negativos, en la mayoría de los casos por falta de datos preclínicos consistentes o un diseño equivocado, en particular, administración tardía. Son necesarios, pues, ensayos clínicos grandes con variables clínicas primarias y terapias combinadas y que consideren edad, sexo y comorbilidades, para que la protección contra el daño por reperfusión se convierta en un tratamiento estándar para los pacientes con infarto agudo de miocardio con elevación del segmento ST (AU)

Even when reperfusion therapy is applied as early as possible, survival and quality of life are compromised in a considerable number of patients with ST-segment elevation acute myocardial infarction. Some cell death following transient coronary occlusion occurs during reperfusion, due to poor handling of calcium in the sarcoplasmic reticulum-mitochondria system, calpain activation, oxidative stress, and mitochondrial failure, all promoted by rapid normalization of intracellular pH. Various clinical trials have shown that infarct size can be limited by nonpharmacological strategies—such as ischemic postconditioning and remote ischemic conditioning—or by drugs—such as cyclosporine, insulin, glucagon-like peptide-1 agonists, beta-blockers, or stimulation of cyclic guanosine monophosphate synthesis. However, some clinical studies have yielded negative results, largely due to a lack of consistent preclinical data or a poor design, especially delayed administration. Large-scale clinical trials are therefore necessary, particularly those with primary clinical variables and combined therapies that consider age, sex, and comorbidities, to convert protection against reperfusion injury into a standard treatment for patients with ST-segment elevation acute myocardial infarction (AU)

IDX184 in combination with pegylated interferon-α2a and ribavirin for 2 weeks in treatment-naive patients with chronic hepatitis C.

BACKGROUND: IDX184 is a liver-targeted nucleotide prodrug that selectively inhibits HCV NS5B polymerase. METHODS: This randomized, double-blind, placebo-controlled, ascending-dose study investigated the antiviral activity, safety and pharmacokinetics of IDX184 plus pegylated interferon-α2a and ribavirin (P/R) in treatment-naive patients with genotype-1 HCV. A total of 81 patients with baseline HCV RNA≥5 log10 IU/ml, alanine aminotransferase ≤3× upper limit of normal and compensated liver disease were dosed. Sequential cohorts of 20 patients, randomized 16:4 (active:placebo), received IDX184 for 14 days at rising daily doses of 50, 100, 150 or 200 mg in combination with P/R for 14 days. RESULTS: At the end of triple dosing, HCV RNA changes from baseline (mean ±sd log10) and proportion of patients achieving undetectable viral load (<15 IU/ml) based on the efficacy-evaluable population were -2.7 ±1.3 (13%), -4.0 ±1.7 (50%), -4.2 ±1.9 (50%), -4.1 ±1.2 (40%), -4.3 ±1.5 (29%) and -3.7 ±1.2 (25%) for the 50 mg once daily, 50 mg twice daily, 100 mg once daily, 150 mg once daily, 100 mg twice daily and 200 mg once daily IDX184 doses, respectively. P/R alone resulted in a reduction of -1.5 ±1.3 log10 with only 6% of patients with undetectable viral load. Patients with genotypes-1a or -1b responded similarly. No viral breakthrough or resistance associated with IDX184 was observed. Anti-HCV activity of IDX184 correlated with plasma exposure of its nucleoside metabolite 2'-methylguanosine. Most adverse events were mild or moderate in severity and were consistent with those associated with P/R. The most common adverse events were fatigue and headache. CONCLUSIONS: IDX184 in combination with P/R for 14 days was well tolerated and demonstrated greater antiviral activity with more patients achieving undetectable viral load than P/R.

Short-term monotherapy with IDX184, a liver-targeted nucleotide polymerase inhibitor, in patients with chronic hepatitis C virus infection.

IDX184 is a liver-targeted prodrug of 2'-methylguanosine (2'-MeG) monophosphate. This study investigated the safety, tolerability, antiviral activity, and pharmacokinetics of IDX184 as a single agent in treatment-naïve patients with genotype-1 chronic hepatitis C virus (HCV) infection. Forty-one patients with baseline HCV RNA ≥ 5 log(10) IU/ml, alanine aminotransferase (ALT) ≤ 2.5× the upper limit of normal, and compensated liver disease were dosed. Sequential cohorts of 10 patients, randomized 8:2 (active:placebo), received 25, 50, 75, and 100 mg of IDX184 once daily for 3 days, with a 14-day follow-up. There were no safety-related treatment discontinuations or serious adverse events. The adverse events and laboratory abnormalities observed for IDX184- and placebo-treated patients were similar. At the end of the 3-day treatment period, changes from baseline in HCV RNA levels (means ± standard deviations) were -0.5 ± 0.6, -0.7 ± 0.2, -0.6 ± 0.3, and -0.7 ± 0.5 log(10) for the 25-, 50-, 75-, and 100-mg doses, respectively, while viral load remained unchanged for the pooled placebo patients (-0.05 ± 0.3 log(10)). Patients with genotype-1a and patients with genotype-1b responded similarly. Serum ALT levels decreased, especially at daily doses ≥ 75 mg. During the posttreatment period, plasma viremia and serum aminotransferase levels returned to near pretreatment levels. No resistance mutations associated with IDX184 were detected. Plasma exposure of IDX184 and its nucleoside metabolite 2'-MeG was dose related and low. Changes in plasma viral load correlated with plasma exposure of 2'-MeG. In conclusion, the results from this proof-of-concept study show that small doses of the liver-targeted prodrug IDX184 were able to deliver significant antiviral activity and support further clinical evaluation of the drug candidate.

Safety and pharmacokinetics of IDX184, a liver-targeted nucleotide polymerase inhibitor of hepatitis C virus, in healthy subjects.

IDX184 is a nucleotide prodrug designed to enhance formation in the liver of the active triphosphate of 2'-methylguanosine (2'-MeG), a potent and specific polymerase inhibitor of the hepatitis C virus (HCV). In the present study, single ascending oral doses of 5, 10, 25, 50, 75, and 100 mg IDX184 were administered sequentially to cohorts of 8 healthy subjects, randomized 6:2, active/placebo. Plasma and urine pharmacokinetic sampling was performed over a period of 120 h after dosing. Upon absorption, IDX184 rapidly disappeared from plasma, with a mean half-life (t(1/2)) of approximately 1 h, while plasma concentrations of 2'-MeG gradually increased. Consistent with a liver-targeting approach, plasma exposure of IDX184 and 2'-MeG was low and was also dose related: the mean maximum concentrations ranged from 1.1 to 17 ng/ml for IDX184 and 1.7 to 19 ng/ml for 2'-MeG, and the respective mean total area under the curve ranged from 1.2 to 22.7 and 17.3 to 334 ng·h/ml. Mean 2'-MeG plasma concentrations 24 h after dosing were 0.6 to 3 ng/ml for the 25- to 100-mg doses. Mean 2'-MeG t(1/2) values ranged from 18 to 43 h for doses of 25 mg and above. Mean cumulative urine excretion was 0.2% and 12 to 20% of administered doses for the unchanged IDX184 and 2'-MeG, respectively. IDX184 was safe and well tolerated; no serious adverse events (SAEs), dose-dependent adverse events (AEs), or dose-limiting toxicities were observed. The incidence of AEs and laboratory abnormalities was low and was similar among subjects receiving IDX184 or a placebo. All AEs were mild to moderate and resolved at the end of study. The favorable safety and pharmacokinetic profiles support further clinical evaluation of IDX184 in HCV-infected patients.

Self-organization of guanosine 5'-monophosphate on mica.

Self-assembled aggregates of guanosine 5'-monophosphate (GMP) on the surface of muscovite mica were investigated by atomic force microscopy (AFM). Aqueous solutions of sodium, potassium and ammonium GMP salts were studied. For solution concentrations c < 0.005 wt% only small islands of deposited material are present on the surface. For c approximately 0.02 wt%, in addition to the islands and patches, also linear aggregates called G-wires are formed. The wire-like aggregates are on average 1.9 nm high and can be several micrometers long. They exhibit a profound directional growth along the six main crystallographic axes of the basal plane of mica. For c > 0.1 wt% flat terraces with the height of 2.5 nm appear. They are formed of G-wires lying with their long axis parallel to the substrate and stacking in a hexagonal arrangement. The morphology of the adsorbates is independent of the type of salt used to prepare the initial solution. This signifies that intrinsic potassium ions from the substrate play much more important role in the GMP adsorption than cations from the solution.

[Effect of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels].

OBJECTIVE: To observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels. METHODS: Using whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution. RESULTS: (1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists. CONCLUSIONS: Dual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria.

In previous work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE, Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course and mechanisms of guanine nucleotide transport are characterized. Two distinct mechanisms of transport were found to be capable of moving guanine nucleotides across the inner membrane. The first carrier was saturable, displayed temperature dependence, preferred GDP to GTP, and did not transport GMP or IMP. When incubated in the absence of exogenous ATP, this carrier had a V(max) of 946 +/- 53 pmol. mg(-1). min(-1) with a K(m) of 2.9 +/- 0.3 mM for GDP. However, transport of GTP and GDP on this carrier was completely inhibited by physiological concentrations of ATP, suggesting that this carrier was not involved with guanine nucleotide transport in vivo. Because transport on this carrier was also inhibited by atractyloside, this carrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive to atractyloside, displayed temperature dependence, and was capable of transporting GMP, GDP, and GTP at approximately equal rates but did not transport IMP, guanine, or guanosine. GTP transport via this mechanism was slow, with a V(max) of 48.7 +/- 1.4 pmol. mg(-1). min(-1) and a K(m) = 4.4 +/- 0.4 mM. However, because the requirement for guanine nucleotide transport is low in nondividing tissues such as the heart, this transport process is nevertheless sufficient to account for the matrix uptake of guanine nucleotides and may represent the physiological mechanism of transport.