A multicenter phase 1/2 study investigating the safety, pharmacokinetics, pharmacodynamics and efficacy of a small molecule antimetabolite, RX-3117, plus nab-paclitaxel in pancreatic adenocarcinoma.

Background RX-3117 is an oral small molecule antimetabolite, cyclopentyl pyrimidyl nucleoside that is activated by cancer cells over-expressing uridine cytidine kinase 2 (UCK2). Single agent RX-3117 demonstrated efficacy in a phase I trial in patients with metastatic (met) pancreatic adenocarcinoma (PC). RX-3117 plus nab-paclitaxel (nab-Pac) was evaluated as a first line treatment in met-PC cancer. Methods This was a multicenter open label phase I/II 2-stage study investigating the combination of RX3117 plus nab-Pac in the frontline treatment of patients with met-PC. The phase I portion comprised a dose de-escalation design with primary objectives of determining the safety, tolerability and recommended phase 2 dose (RP2D) of RX-3117 (orally 700, 600, or 500 mg/day for 5 consecutive days with 2 days off/week) plus nab-Pac (intravenous (IV) 125 mg/m2 once weekly) for 3 weeks with 1 week off per a 4-week cycle. The primary objective was to determine the antitumor efficacy. Results 46 patients were enrolled (22 male/24 female; median age 67; 91% Caucasian). The RP2D of RX-3117 plus nab-Pac was 700 mg/day. No dose-limiting toxicities were observed (DLTs). The overall response rate (ORR) was 23.1% and disease control rate (DCR) 74.4%. RX-3117 pharmacokinetics (PK) results were similar to previously reported monotherapy phase 1 trial. All patients experienced a treatment emergent adverse event (TEAE) with the most common diarrhea, nausea, and fatigue.10.9% of patients experienced a serious adverse event (SAE) related to the combination. Conclusion RX-3117 plus nab-Pac in newly diagnosed met-PC patients demonstrated tolerability, safety, and early treatment efficacy.

Computational Analysis of Molnupiravir.

In this work, we report in-depth computational studies of three plausible tautomeric forms, generated through the migration of two acidic protons of the N4-hydroxylcytosine fragment, of molnupiravir, which is emerging as an efficient drug to treat COVID-19. The DFT calculations were performed to verify the structure of these tautomers, as well as their electronic and optical properties. Molecular docking was applied to examine the influence of the structures of the keto-oxime, keto-hydroxylamine and hydroxyl-oxime tautomers on a series of the SARS-CoV-2 proteins. These tautomers exhibited the best affinity behavior (-9.90, -7.90, and -9.30 kcal/mol, respectively) towards RdRp-RTR and Nonstructural protein 3 (nsp3_range 207-379-MES).

Nucleoside analogue 2'-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin.

Hepatitis E virus (HEV) infection has emerged as a global health issue, but no approved medication is available. The nucleoside analogue 2'-C-methylcytidine (2CMC), a viral polymerase inhibitor, has been shown to inhibit infection with a variety of viruses, including hepatitis C virus (HCV). Here, we report that 2CMC significantly inhibits the replication of HEV in a subgenomic replication model and in a system using a full-length infectious virus. Importantly, long-term treatment with 2CMC did not result in a loss of antiviral potency, indicating a high barrier to drug resistance development. However, the combination of 2CMC with ribavirin, an off-label treatment for HEV, exerts antagonistic effects. Our results indicate that 2CMC serves as a potential antiviral drug against HEV infection.

Phase I dose-escalating study of TAS-106 in combination with carboplatin in patients with solid tumors.

BACKGROUND: TAS-106 was designed to inhibit RNA synthesis by blocking RNA polymerases I, II, and III. METHODS: This was a single-center, open-label, phase I study to identify the maximum tolerated dose (MTD), pharmacokinetics, and biologic effects of the combination of TAS-106 and carboplatin, following a standard 3 + 3 design. This phase I trial was comprised of a regimen of a 60-min IV infusion of carboplatin on day 1 of each 21-day cycle followed by a 24-h infusion of TAS-106, also on day 1 of each cycle. RESULTS: 39 patients were treated (21 male, 18 female, median age 62 years, range 21-80 years). Median number of prior therapies was 4. Maximum Tolerated Dose (MTD) was 3 mg/m(2) TAS-106 with AU 4 carboplatin. Dose-limiting toxicities were neutropenia and thrombocytopenia, with and without growth factor support. While no patients achieved a complete or partial response, four patients had stable disease lasting ≥4 months, including one patient each with ovarian, non-small cell lung, basal cell and colorectal cancer. CONCLUSIONS: In summary, the combination of TAS-106 and carboplatin was well-tolerated, and further studies in non-small cell lung and ovarian cancer are warranted to assess the efficacy of this drug combination.

Phase I and pharmacokinetic study of 3'-C-ethynylcytidine (TAS-106), an inhibitor of RNA polymerase I, II and III,in patients with advanced solid malignancies.

BACKGROUND: TAS-106 is a novel nucleoside analog that inhibits RNA polymerases I, II and II and has demonstrated robust antitumor activity in a wide range of models of human cancer in preclinical studies. This study was performed to principally evaluate the feasibility of administering TAS-106 as a bolus intravenous (IV) infusion every 3 weeks. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with escalating doses of TAS-106 as a single bolus IV infusion every 3 weeks. Plasma and urine sampling were performed during the first course to characterize the pharmacokinetic profile of TAS-106 and assess pharmacodynamic relationships. RESULTS: Thirty patients were treated with 66 courses of TAS-106 at eight dose levels ranging from 0.67-9.46 mg/m(2). A cumulative sensory peripheral neuropathy was the principal dose-limiting toxicity (DLT) of TAS-106 at the 6.31 mg/m(2) dose level, which was determined to be the maximum tolerated dose (MTD). Other mild-moderate drug-related toxicities include asthenia, anorexia, nausea, vomiting, myelosuppression, and dermatologic effects. Major objective antitumor responses were not observed. The pharmacokinetics of TAS-106 were dose-proportional. The terminal elimination half-life (t(1/2)) averaged 11.3 ± 3.3 h. Approximately 71% of TAS-106 was excreted in the urine as unchanged drug. Pharmacodynamic relationships were observed between neuropathy and: C(5min;) AUC(0-inf;) and dermatologic toxicity. CONCLUSIONS: The recommended phase II dose of TAS-106 is 4.21 mg/m(2). However, due to a cumulative drug-related peripheral sensory neuropathy that proved to be dose-limiting, further evaluation of this bolus every 21 day infusion schedule will not be pursued and instead, an alternate dosing schedule of TAS-106 administered as a continuous 24-hour infusion will be explored to decrease C(max) in efforts to minimize peripheral neuropathy and maximize antitumor activity.

Robust antiviral activity of R1626, a novel nucleoside analog: a randomized, placebo-controlled study in patients with chronic hepatitis C.

UNLABELLED: The nucleoside analog R1479 is a potent and highly selective inhibitor of nonstructural protein 5B-directed hepatitis C virus (HCV) replication in vitro. R1626, a tri-isobutyl ester prodrug of R1479, was developed to increase bioavailability and improve antiviral activity. A multicenter, observer-blinded, randomized, placebo-controlled, multiple ascending dose, phase 1b study was designed to evaluate the safety, pharmacokinetics, and antiviral activity and to potentially identify the maximum tolerated dose of R1626 in patients with chronic hepatitis C. Forty-seven treatment-naïve patients infected with HCV genotype 1 were treated with R1626 orally at doses of 500 mg, 1500 mg, 3000 mg, or 4500 mg or placebo twice daily for 14 days with 14 days of follow-up. Safety, tolerability, pharmacokinetics, and antiviral activity were assessed. Doses up to and including 3000 mg twice daily were well tolerated after 14 days of treatment. There was an increase in frequency of adverse events at the highest dose (4500 mg). Reversible mild to moderate hematological changes were observed with increasing doses. R1626 was efficiently converted to R1479, with dose-proportional pharmacokinetics observed over the entire dose range. The pharmacokinetics of R1479 were linear over the dose range evaluated. Dose-dependent and time-dependent reductions in HCV RNA were observed. Mean decreases (median; range) in viral load after 14 days of treatment with doses of 500, 1500, 3000, and 4500 mg were 0.32 (0.22; 0.01-0.71), 1.2 (0.8; 0.49-2.46), 2.6 (2.7; 1.27-3.93) and 3.7 (4.1; 2.15-4.39) log(10), respectively. No resistance to R1479 was observed after 14 days of treatment with R1626. CONCLUSION: These data support further studies of R1626 in combination with peginterferon alfa-2a and ribavirin for the treatment of patients with chronic HCV infection.

RX-3117 (fluorocyclopentenyl cytosine): a novel specific antimetabolite for selective cancer treatment.

INTRODUCTION: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.

Clinical evaluation of the lightening effect of cytidine on hyperpigmented skin.

BACKGROUND: Melanocytes, which reside in the basal layer of the epidermis, produce the pigment melanin in cytoplasmic organelles known as melanosomes. Melanosomes are transferred to keratinocytes which provide the color in our skin. Recently, Diwakar et  al reported the crucial roles of protein glycosylation in both melanogenesis and melanosome transfer to keratinocytes, and each was inhibited by the nucleotide cytidine. OBJECTIVE: The main objective of this study was to determine the clinical effects of topical application of cytidine to the hyperpigmented regions of the face in a group of human volunteers. METHODS: A randomized, vehicle-controlled study was conducted for 12 weeks on healthy Korean female subjects. Cytidine was formulated into the lotion at concentrations of 2%, 3%, and 4% (w/w) and compared to the vehicle control formulation. The clinical outcomes were evaluated by performing visual assessment grading, measuring melanin index, skin brightness, and skin color parameters. In vitro skin penetration studies were conducted using Franz cell chambers for the 2% cytidine test formulation. RESULTS: The test group showed significant improvements in the visual assessment scores, melanin index, skin brightness, and skin color compared to the control group. Although significant dose-dependent improvements were seen in the clinical study, the in vitro Franz cell studies indicated that the clinical efficacy and potency of cytidine might be further enhanced by formulating a better topical delivery system, which will be the goal of our future studies. CONCLUSIONS: This randomized, double-blind, 12-week clinical study successfully demonstrated the efficacy of cytidine on skin depigmentation in a dose-dependent manner.

Physicochemical properties of the nucleoside prodrug R1626 leading to high oral bioavailability.

The nucleoside analog R1479 is a potent and highly selective inhibitor of NS5b-directed hepatitis C virus (HCV) RNA polymerase in vitro. Because of its limited permeability, lipophilic prodrugs of R1479 were screened. Selection of the prodrug involved optimization of solubility, permeability, and stability parameters. R1626 has dissociation constant, intrinsic solubility, log partition coefficient (n-octanol water), and Caco-2 permeability of 3.62, 0.19 mg/mL, 2.45, and 14.95 x 10(-6) cm/s, respectively. The hydrolysis of the prodrug is significantly faster in the Caco-2 experiments than in hydrolytic experiments, suggesting that the hydrolysis is catalyzed by enzymes in the cellular membrane. Using GastroPlus, the physical properties of R1626 successfully predict the dose dependence of the pharmacokinetics in humans previously studied. The program predicts that if the particle size of R1626 is less than 25 microm, it will be well absorbed. Prodrugs with a solubility of greater than 100 microg/mL and permeability in the Caco-2 assay greater than 3 x 10(-6) cm/s are expected to achieve a high fraction absorbed.

A mass balance and disposition study of the DNA methyltransferase inhibitor zebularine (NSC 309132) and three of its metabolites in mice.

PURPOSE: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer. EXPERIMENTAL DESIGN: Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine. RESULTS: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine. CONCLUSIONS: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.

Pharmacokinetics and toxicity of the novel oral demethylating agent zebularine in laboratory and tumor bearing dogs.

The purpose of this study was to determine the plasma pharmacokinetics (PK) and toxicity of zebularine, an oral cytidine analog with demethylating activity, in dogs. Plasma zebularine concentrations were determined by HPLC-MS/MS following an oral zebularine dose of 8 or 4 mg kg-1 . Plasma zebularine clearance was constant. Mean maximum concentration (Cmax ) was 23 ± 4.8 and 8.6 ± 1.4 µM following 8 and 4 mg kg-1 , respectively. Mean half-life was 5.7 ± 0.84 and 7.1 ± 2.1 following 8 and 4 mg kg-1 , respectively. A single 8 mg kg-1 dose was well tolerated. Daily 4 mg kg-1 treatment in three laboratory dogs resulted in grade 4 neutropenia (n = 3), grade 1 anorexia (n = 2) and grade 1 or 2 dermatologic changes (n = 2). All adverse events resolved with supportive care. A 4 mg kg-1 dose every 21 days was well tolerated. A follow-up dose escalation study is in progress with a lower starting dose.

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

PURPOSE: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. EXPERIMENTAL DESIGN: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. RESULTS: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)). CONCLUSIONS: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.

Pharmacokinetics and metabolism of cyclopentenyl cytosine in nonhuman primates.

The plasma and cerebrospinal fluid pharmacokinetics of cyclopentenyl cytosine (CPE-C) were studied following i.v. bolus and continuous i.v. infusion in male rhesus monkeys. Following an i.v. bolus dose of 100 mg/m2 plasma elimination of CPE-C was biexponential with a mean t1/2 alpha of 8.4 min, a mean t1/2 beta of 36 min, and a total clearance (CLTB) of 662 ml/min/m2, which is 5- to 10-fold higher than clearance rates in rodents and dogs. Less than 20% of the total dose of CPE-C was excreted unchanged in the urine. The remainder was excreted as the inactive deamination product cyclopentenyl uridine (CPE-U). The ratio of the areas under the plasma concentration versus time curves of CPE-U to CPE-C was 7.0 +/- 2.4 following i.v. bolus CPE-C. The cerebrospinal fluid:plasma ratios of CPE-C and CPE-U were 0.08 and 0.30, respectively. Continuous i.v. infusion of CPE-C was compared to continuous infusion of 1-beta-D-arabinofuranosylcytosine in two monkeys. Steady state plasma concentrations, normalized to a dose of 12.5 mg/m2/h of CPE-C and an equimolar dose of 1-beta-D-arabinofuranosylcytosine, were 2.1 and 0.53 microM, respectively. The steady state concentrations of their corresponding uridine metabolites (CPE-U and 1-beta-D-arabinofuranosyluridine) were 8.2 and 15.5 microM. The rapid elimination of CPE-C by deamination in the primate resulted in a much higher CLTB and considerably lower total drug exposure than in rodents and dogs that clear CPE-C at a much lower rate by renal excretion. These significant interspecies differences in the disposition of CPE-C should be considered in the selection of a starting dose and schedule for human trials and suggest that a pharmacologically directed dose escalation scheme should be used in the planned phase I studies.

In situ cyclopentenyl cytosine infusion for the treatment of experimental brain tumors.

Cyclopentenylcytosine (CPEC; NSC 375575) is a pyrimidine nucleoside analogue that has potent antitumor effects when tested in vitro and also when tested in experimental tumors outside the central nervous system. CPEC exerts its antiproliferative effect through inhibition of CTP synthetase and consequent depletion of CTP and dCTP pools required for cell replication. Due to its poor penetration of the bloodbrain barrier, CPEC has failed to demonstrate therapeutic efficacy in experimental brain tumors after systemic administration. We therefore examined the in vivo activation, distribution, and antitumor effect of CPEC after long-term regional infusion of the drug directly into experimental brain tumors in rats. HPLC analysis of CPEC incubated with homogenized human brain and brain tumor tissue showed minimal degradation of the drug over 24 h. Analysis of rat cerebral 9L gliosarcoma infused with tritium-labeled CPEC demonstrated intratumoral accumulation of the active metabolite CPEC-triphosphate and concomitant depletion of CTP to a much greater extent in tumor tissue than in the adjacent brain. Tumor tissue UTP also decreased, but no significant effects on other ribonucleoside triphosphates were detected. Only trace amounts (< 1%) of CPEC and its metabolites reached peripheral sites, including the liver and kidneys, after intratumoral infusion. Rats treated with continuous intratumoral infusion of CPEC for 4 weeks using s.c. implanted osmotic pumps survived significantly longer than control rats receiving intratumoral saline or i.p. CPEC (P < 0.0001). Long-term intratumoral infusion of CPEC was not associated with any detectable toxicity. Our results support the feasibility of using intratumoral administration of CPEC as a regional therapy for malignant brain tumors.

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells.

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.

Phase I clinical trial of continuous infusion cyclopentenyl cytosine.

Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5'-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i.v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i.v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 microM to 3.1 microM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146 +/- 38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2:1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6% +/- 3.1% at 22 h (mean +/- SE, n = 16), and remained inhibited by 67.6% +/- 7.7% (n = 10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose-dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24-48 h after completion of the CPE-C infusion during their first (n = 2), third (n = 1) and sixth cycles (n = 1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2 per h). Hypotension was not seen in patients receiving < or = 2.5 mg/m2 per h CPE-C.(ABSTRACT TRUNCATED AT 400 WORDS)

[Molecular mechanism of N4-aminocytidine mutagenesis].

N4-Aminocytidine is strongly mutagenic towards E. coli, S. typhimurium, B. subtilis and coliphages phi X174 and M13mp2. It also causes mutations in mammalian cell lines and somatic cell mutations in D. melanogaster. The sequence analysis of deoxyribonucleic acid (DNA) from mutated phages revealed that N4-aminocytidine induces both adenine-thymine (AT) to guanine-cytosine (GC) and GC to AT transitions. No transversions are detectable. When E. coli and the mammalian cells were cultured in the presence of [3H]-N4-aminocytidine, [3H]-N4-aminodeoxycytidine was found in their DNA. It is likely that N4-aminocytidine is metabolized within the cells into N4-aminodeoxy-cytidine 5'-triphosphate (dCamTP), which is then incorporated into DNA, thereby causing base-pair transitions. To prove this hypothesis, we studied the incorporation of dCamTP into polynucleotides in the in vitro DNA synthesis catalyzed by E. coli DNA polymerase I large fragment (Klenow enzyme) and DNA polymerase alpha from a mouse cell line. Both polymerases catalyze incorporation of dCamTP into DNA efficiently in place of dCTP opposite guanine, and less efficiently, but to a significant extent, in place of dTTP opposite adenine. These observations prove the erroneous nature of dCamTP as a substrate for DNA synthesis. DNA containing N4-aminocytosine was prepared by the incorporation of dCamTP into single-stranded phage DNA annealed to complementary oligonucleotides. The DNA was transfected to E. coli cells. The analysis of progeny phages indicates that N4-aminocytosine residue in DNA causes A to G or G to A mutation in the position opposite to the site where N4-aminocytosine should be incorporated.

Quantification of 2'-deoxy-2'-ß-fluoro-4'-azidocytidine in rat and dog plasma using liquid chromatography-quadrupole time-of-flight and liquid chromatography-triple quadrupole mass spectrometry: Application to bioavailability and pharmacokinetic studies.

2'-Deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) is a novel pyrimidine analog that inhibits not only the replication of the hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV but also the replication of lamivudine-resistant HBV, 4'-azidocytidine or 2'-ß-methylcytidine-resistant HCV, and nucleoside reverse-transcriptase inhibitor-resistant HIV variants. The present study was undertaken to evaluate the absolute oral bioavailability of FNC in rats and the pharmacokinetic properties of FNC after intragastric administration of single and multiple doses in rats and dogs. A sensitive high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) method and a reliable high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC/QqQ MS/MS) method were established for the determination of FNC in the rat and dog plasmas, respectively. The sample preparation involved a protein-precipitation method with methanol after the addition of lamivudine as an internal standard. FNC was analyzed by LC using a YMC-Pack Pro C18 column (150mm×4.6mm, 3µm) with methanol (containing 0.3% formic acid): 10mM ammonium acetate (containing 0.3% formic acid, pH 2.8) (35:65, v/v) as the mobile phase. Both mass spectrometers were equipped with an electrospray ionization interface in the positive-ion mode. The linear range was from 2.00 to 2000.00ngmL(-1) in rat plasma and 0.50 to 400.00ngmL(-1) in dog plasma. The intraday and interday precision were less than 10.55%, and the accuracy was in the range of -5.86 to 5.13%. The mean recoveries were greater than 82.70% and 82.97% for FNC and IS, respectively. The HPLC/Q-TOF MS and HPLC/QqQ MS/MS methods were both successfully applied in the pharmacokinetic studies of FNC in rats and dogs.

Effect of salt intake on bioavailability of mizoribine in healthy Japanese males.

Bioavailability of mizoribine in subjects with the concentrative nucleoside transporter 1 (CNT1, SLC28A1) 565-A/A allele is significantly lower than that in subjects with the SLC28A1 565-G/G allele. The aims of the present study were to investigate the cellular uptake of mizoribine in CNT1- and CNT2-expressing Madin-Darby canine kidney type II (MDCKII) cells, and to evaluate the effect of salt intake on bioavailability of mizoribine in healthy Japanese volunteers with SLC28A1 565-A/A and -G/A alleles. Eight healthy males participated in the present study, and took 150 mg mizoribine concomitantly with/without 300 mg salt. Bioavailability of mizoribine was estimated by total cumulative urinary excretion of the drug. Mizoribine was taken up Na(+)-dependently into not only CNT1-expressing but also CNT2-expressing MDCKII cells, indicating that mizoribine is a substrate for both CNT1 and CNT2. Mean bioavailability of mizoribine taken with salt (83.8%) was significantly higher than that taken without salt (73.0%). These findings suggest that the salt intake is expected to improve the bioavailability of mizoribine in patients with insufficient intestinal absorption.

A phase I study to determine the safety and pharmacokinetics of intravenous administration of TAS-106 once per week for three consecutive weeks every 28 days in patients with solid tumors.

BACKGROUND: The nucleoside 3'-c-ethynylcytidine (TAS-106) was designed to inhibit RNA synthesis which occurs throughout the cell cycle except for the M phase. TAS-106 is incorporated into cells, is rapidly phosphorylated to a monophosphate form, and is preferentially distributed into malignant cells. Preclinical studies showed that TAS-106 has a wide antitumor spectrum against human cancer xenografts. This phase I study was conducted in order to determine the recommended phase II dose of TAS-106 administered once per week for three consecutive weeks, every 28 days in patients with solid tumors. PATIENTS AND METHODS: Patients were enrolled in cohorts of three, starting at 0.22 mg/m(2)/dose. Patients received at least two doses in order to be evaluable in each dose cohort. Dose escalation was stopped if two or more patients experienced dose limiting toxicity at any dose level. RESULTS: In 20 evaluable patients, TAS-106 was given at the following dose levels (mg/m(2)/dose): 0.22 (3 pts), 0.33 (3 pts), 0.66 (3 pts), 0.99 (1 pt), 1.32 (3 pts), 2.64 (3 pts) and 3.96 (1 pt). Three additional patients were evaluated at 2.64 mg/m(2)/dose for further characterization of toxicity and safety. A total of 16 patients completed courses 1 and 2. All 21 patients enrolled experienced at least one adverse event. The AE attributed to the study drug was grade 2 peripheral neuropathy characterized by peripheral sensory neuropathy, numbness, tremor, pain, and hyperesthesia involving the fingers, hands, toes, and feet. CONCLUSION: Due to neurotoxicity the MTD was the 2.64 mg/m(2)/dose for the study schedule. No suggested phase II dose was determined. However, at the 1.32 mg/m(2)/dose level, no patients experienced DLTs during course 1 or 2. This could be further studied to determine its viability as a potential phase II dosage.