Development and Validation of a Simple and Reliable HPLC-UV Method for Determining Gemcitabine Levels: Application in Pharmacokinetic Analysis.

Background and Objectives: Gemcitabine has been used to treat various solid cancers, including, since 1997, metastatic pancreatic cancer. Here, we developed an HPLC-UV method to determine serum gemcitabine levels and use it in pharmacokinetic studies. Materials and Methods: The analysis was performed after a single protein precipitation step on a reversed-phase column, isocratically eluted with sodium phosphate buffer and methanol. For the pharmacokinetic study, NOD/SCID mice received a single dose of gemcitabine at 100 mg/kg by either subcutaneous (SC) or intraperitoneal (IP) administration. Blood samples were collected at 5, 15, and 30 min and 1, 2, 4, and 6 h after the administration of gemcitabine for further analysis. Results: The duration of the analysis was ~12.5 min. The calibration curve was linear (r2 = 0.999) over the range of 1-400 µM. The mean recovery of GEM was 96.53% and the limit of detection was 0.166 µΜ. T1/2, Tmax, Cmax, AUC0-t, and clearance were 64.49 min, 5.00 min, 264.88 µmol/L, 9351.95 µmol/L*min, and 0.0103(mg)/(µmol/L)/min, respectively, for the SC administration. The corresponding values for the IP administration were 59.34 min, 5.00 min, 300.73 µmol/L, 8981.35 µmol/L*min and 0.0108(mg)/(µmol/L)/min (not statistically different from the SC administration). Conclusions: A simple, valid, sensitive, and inexpensive method for the measurement of gemcitabine in serum has been developed. This method may be useful for monitoring gemcitabine levels in cancer patients as part of therapeutic drug monitoring.

A Liposomal Gemcitabine, FF-10832, Improves Plasma Stability, Tumor Targeting, and Antitumor Efficacy of Gemcitabine in Pancreatic Cancer Xenograft Models.

PURPOSE: The clinical application of gemcitabine (GEM) is limited by its pharmacokinetic properties. The aim of this study was to characterize the stability in circulating plasma, tumor targeting, and payload release of liposome-encapsulated GEM, FF-10832. METHODS: Antitumor activity was assessed in xenograft mouse models of human pancreatic cancer. The pharmacokinetics of GEM and its active metabolite dFdCTP were also evaluated. RESULTS: In mice with Capan-1 tumors, the dose-normalized areas under the curve (AUCs) after FF-10832 administration in plasma and tumor were 672 and 1047 times higher, respectively, than after using unencapsulated GEM. The tumor-to-bone marrow AUC ratio of dFdCTP was approximately eight times higher after FF-10832 administration than after GEM administration. These results indicated that liposomal encapsulation produced long-term stability in circulating plasma and tumor-selective targeting of GEM. In mice with Capan-1, SUIT-2, and BxPC-3 tumors, FF-10832 had better antitumor activity and tolerability than GEM. Internalization of FF-10832 in tumor-associated macrophages (TAMs) was revealed by flow cytometry and confocal laser scanning microscopy, and GEM was efficiently released from isolated macrophages of mice treated with FF-10832. These results suggest that TAMs are one of the potential reservoirs of GEM in tumors. CONCLUSION: This study found that FF-10832 had favorable pharmacokinetic properties. The liposomal formulation was more effective and tolerable than unencapsulated GEM in mouse xenograft tumor models. Hence, FF-10832 is a promising candidate for the treatment of pancreatic cancer.

Combination of a novel microtubule inhibitor MBRI-001 and gemcitabine synergistically induces cell apoptosis by increasing DNA damage in pancreatic cancer cell lines.

Pancreatic cancer (PC) is a highly malignant cancer with poor prognosis. Although gemcitabine (GEM; 2',2'-difluoro-deoxycytidine) has been used as the first-line chemotherapeutic agent in PC treatment for decades, its limited efficacy remains a significant clinical issue, which may be resolved by GEM combination therapy. In this study, we aimed to investigate the anti-tumor effects of MBRI-001 in combination with GEM in BxPC-3 and MIA PaCa-2 human PC cell lines. In vitro and in vivo results indicate that MBRI-001 showed synergistic activity with GEM. GEM induced apoptosis by increasing DNA damage (phosphorylated core histone protein H2AX (γ-H2AX)), MBRI-001 activated mitochondrial-apoptotic pathway (cleaved poly-ADP ribose polymerase (PARP)). Thus, the combination of the two intensified both apoptosis and DNA damage and showed significantly superior anti-tumor activity compared to each agent alone. The adoption of combination of MBRI-001 with GEM may be beneficial as they act synergistically and thus, can be a potential therapeutic choice for improving the prognosis of PC patients in the future.

Novel Long-Acting Drug Combination Nanoparticles Composed of Gemcitabine and Paclitaxel Enhance Localization of Both Drugs in Metastatic Breast Cancer Nodules.

PURPOSE: To develop drug-combination nanoparticles (DcNPs) composed of hydrophilic gemcitabine (G) and hydrophobic paclitaxel (T) and deliver both drugs to metastatic cancer cells. METHODS: GT DcNPs were evaluated based on particle size and drug association efficiency (AE%). The effect of DcNP on GT plasma time-course and tissue distribution was characterized in mice and a pharmacokinetic model was developed. A GT distribution study into cancer nodules (derived from 4 T1 cells) was performed. RESULTS: An optimized GT DcNP composition (d = 59.2 nm ±9.2 nm) was found to be suitable for IV formulation. Plasma exposure of G and T were enhanced 61-fold and 3.8-fold when given in DcNP form compared to the conventional formulation, respectively. Mechanism based pharmacokinetic modeling and simulation show that both G and T remain highly associated to DcNPs in vivo (G: 98%, T:75%). GT DcNPs have minimal distribution to healthy organs with selective distribution and retention in tumor burdened tissue. Tumor bearing lungs had a 5-fold higher tissue-to-plasma ratio of gemcitabine in GT DcNPs compared to healthy lungs. CONCLUSIONS: DcNPs can deliver hydrophilic G and hydrophobic T together to cancer nodules and produce long acting exposure, likely due to stable GT association to DcNPs in vivo.

Encapsulation of gemcitabine in RGD-modified nanoliposomes improves breast cancer inhibitory activity.

In this study, RGD coated GEM liposomes were prepared by the emulsification-solvent evaporation method. The in vitro and in vivo characterizations were done to evaluate the feasibility of application. The mean particle size of the prepared liposomes was found to be 165.6 ± 15.7 nm. The entrapment efficiency and drug loading of the formulation were 82.4% ± 7.2% and 10.1% ± 1.4%, respectively. The liposomes were negatively charged with a zeta potential of -25.8 mV. The surface morphology of RGD-GEM liposomes was spherical and smooth. After three months of storage at different conditions, lyophilized liposomes appeared to be stable since they showed no collapse or contraction. The Weibull model was the most appropriate kinetic model for RGD-GEM liposomes, showing that the release of GEM from the liposomes was in the manners of both dissolution and diffusion. In vivo, the additive cytotoxicity of RGD-GEM-LPs in our study was caused by the presence of RGD which is more effective in the treatment of breast cancer devoid of toxicity to normal cells. Liposomes could also significantly extend the role of GEM in vivo and showed higher bioavailability than solution.

Respiratory syncytial virus-A dynamics and the effects of lumicitabine, a nucleoside viral replication inhibitor, in experimentally infected humans.

Background: Respiratory syncytial virus (RSV) causes high morbidity, with mortality rates approaching or exceeding that of influenza in adult and infant patient populations, respectively. Lumicitabine (ALS-008176 or JNJ-64041575) is an oral nucleoside analogue prodrug in clinical development to treat RSV infections. This prodrug converts to plasma-circulating ALS-8112, and then to the 5'-active nucleoside triphosphate (NTP) form within host cells. We conducted an RSV-A challenge study in healthy adults to evaluate lumicitabine's activity during an active RSV infection. Objectives: To develop a semi-mechanistic mathematical model describing RSV kinetics, and the pharmacokinetics (PK) and pharmacodynamics (PD) of lumicitabine during treatment. Methods: Nasopharyngeal viral load and concentrations of ALS-8112 and ALS-8144 (uridine metabolite) were measured frequently over the study duration. Population viral kinetic and PK/PD models were developed using NONMEM. The RSV life-cycle was described using a target-cell-limited model that included a physiological delay. Results: The estimated clearances of ALS-8112 and ALS-8144 were 54.2 and 115 L/h/70 kg, respectively. A semi-physiological model was linked to predict ALS-8112 conversion to active intracellular NTP. Extensive and rapid RSV reduction occurred after lumicitabine treatment (EC50 = 1.79 µM), with >99% viral inhibition at 2 h after loading dose. Simulated NTP exposures and time to EC50 attainment suggested that rapid therapeutic effects and reduced dosing frequency are achievable in adult and paediatric patients. Conclusions: The semi-mechanistic model characterizes RSV kinetics and the antiviral effectiveness of lumicitabine in an adult challenge population. This model is applicable to guide dose selection in adult and paediatric patients.

Measurement of urinary arsenic profiles and DNA hypomethylation in a case-control study of urothelial carcinoma.

Environmental exposure to arsenic may be involved in the disturbance of DNA hypomethylation. The aim of this study is the first to explore the effect of interactions of urinary total arsenic levels, arsenic methylation capacity, 8-hydroxy-2'-deoxyguanosine (8-OHdG), plasma folate, and global 5-methyl-2'-deoxycytidine (5-MedC) levels on the risk of urothelial carcinoma (UC). A hospital-based case-control study was constructed. The research involved the histological recruitment and pathological verification of 178 UC patients and 356 age-/sex-matched controls without prior history of cancer. Arsenic species were determined by high-performance liquid chromatography (HPLC)-hydride generation and atomic absorption. 5-MedC levels were detected by HPLC and triple-quadrupole mass spectrometry (MS). 8-OHdG was processed by an online solid-phase extraction LC-MS/MS. Plasma folate levels were measured using the chemiluminescent technology. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by multiple logistic regression analysis. Results indicate that the high levels of total urinary arsenic, inorganic arsenic percentage, and 8-OHdG and the low levels of DMA % and plasma folate were independent factors of UC. In addition, global 5-MedC levels in the first quartile versus fifth quartile significantly increased the twofold OR of UC after potential factors were adjusted (95% CI:1.10-4.03). The interaction of 5-MedC level and high total arsenic level, insufficient arsenic capacity, high 8-OHdG, and low folate levels was insignificant. Results of stepwise logistic regression analysis indicate that high total urinary arsenic levels (Q3 versus Q1), low plasma folate level, and low global 5-MedC (Q4 versus Q5) significantly increased the ORs of UC. The above results suggest that high total arsenic, low plasma folate, and 5-MedC levels affect the ORs of UC independently.

Tenofovir-based preexposure prophylaxis for HIV infection among African women.

BACKGROUND: Reproductive-age women need effective interventions to prevent the acquisition of human immunodeficiency virus type 1 (HIV-1) infection. METHODS: We conducted a randomized, placebo-controlled trial to assess daily treatment with oral tenofovir disoproxil fumarate (TDF), oral tenofovir-emtricitabine (TDF-FTC), or 1% tenofovir (TFV) vaginal gel as preexposure prophylaxis against HIV-1 infection in women in South Africa, Uganda, and Zimbabwe. HIV-1 testing was performed monthly, and plasma TFV levels were assessed quarterly. RESULTS: Of 12,320 women who were screened, 5029 were enrolled in the study. The rate of retention in the study was 91% during 5509 person-years of follow-up. A total of 312 HIV-1 infections occurred; the incidence of HIV-1 infection was 5.7 per 100 person-years. In the modified intention-to-treat analysis, the effectiveness was -49.0% with TDF (hazard ratio for infection, 1.49; 95% confidence interval [CI], 0.97 to 2.29), -4.4% with TDF-FTC (hazard ratio, 1.04; 95% CI, 0.73 to 1.49), and 14.5% with TFV gel (hazard ratio, 0.85; 95% CI, 0.61 to 1.21). In a random sample, TFV was detected in 30%, 29%, and 25% of available plasma samples from participants randomly assigned to receive TDF, TDF-FTC, and TFV gel, respectively. Independent predictors of TFV detection included being married, being older than 25 years of age, and being multiparous. Detection of TFV in plasma was negatively associated with characteristics predictive of HIV-1 acquisition. Elevations of serum creatinine levels were seen more frequently among participants randomly assigned to receive oral TDF-FTC than among those assigned to receive oral placebo (1.3% vs. 0.2%, P=0.004). We observed no significant differences in the frequencies of other adverse events. CONCLUSIONS: None of the drug regimens we evaluated reduced the rates of HIV-1 acquisition in an intention-to-treat analysis. Adherence to study drugs was low. (Funded by the National Institutes of Health; VOICE ClinicalTrials.gov number, NCT00705679.).

Intracellular pharmacokinetics of gemcitabine, its deaminated metabolite 2',2'-difluorodeoxyuridine and their nucleotides.

AIMS: Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) is a prodrug that has to be phosphorylated within the tumour cell to become active. Intracellularly formed gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) are considered responsible for the antineoplastic effects of gemcitabine. However, a major part of gemcitabine is converted into 2',2'-difluoro-2'-deoxyuridine (dFdU) by deamination. In the cell, dFdU can also be phosphorylated to its monophosphate (dFdUMP), diphosphate (dFdUDP) and triphosphate (dFdUTP). In vitro data suggest that these dFdU nucleotides might also contribute to the antitumour effects, although little is known about their intracellular pharmacokinetics (PK). Therefore, the objective of the present study was to gain insight into the intracellular PK of all dFdC and dFdU nucleotides formed during gemcitabine treatment. METHODS: Peripheral blood mononuclear cell (PBMC) samples were collected from 38 patients receiving gemcitabine, at multiple time points after infusion. Gemcitabine, dFdU and their nucleotides were quantified in PBMCs. In addition, gemcitabine and dFdU plasma concentrations were monitored. The individual PK parameters in plasma and in PBMCs were determined. RESULTS: Both in plasma and in PBMCs, dFdU was present in higher concentrations than gemcitabine [mean intracellular area under the concentration-time curve from time zero to 24 h (AUC0-24 h ) 1650 vs. 95 µM*h]. However, the dFdUMP, dFdUDP and dFdUTP concentrations in PBMCs were much lower than the dFdCDP and dFdCTP concentrations. The mean AUC0-24 h for dFdUTP was 312 µM*h vs. 2640 µM*h for dFdCTP. CONCLUSIONS: The study provides the first complete picture of all nucleotides that are formed intracellularly during gemcitabine treatment. Low intracellular dFdU nucleotide concentrations were found, which calls into question the relevance of these nucleotides for the cytotoxic effects of gemcitabine.

Improving Plasma Stability and Bioavailability In Vivo of Gemcitabine Via Nanoparticles of mPEG-PLG-GEM Complexed with Calcium Phosphate.

PURPOSE: Despite being widely used for the treatment of several solid tumors, Gemcitabine (GEM) exhibits several suboptimal pharmacokinetic properties. Therefore, the design of nanoparticle delivery systems is a promising strategy to enhance GEM pharmacokinetic properties. METHODS: In this work, the polymeric material methoxy poly(ethylene glycol)-block-poly(L-glutamic acid)-graft-gemcitabine (mPEG-b-PLG-g-GEM) was synthesized through the covalent conjugation of GEM with the carboxylic group of methoxy poly(ethylene glycol)-block-poly (L-glutamic acid) (mPEG-b-PLG) (mPEG113, Mn = 5000). mPEG-PLG-GEM/CaP nanoparticles were prepared through the simple mixing of calcium and phosphate/mPEG-PLG-GEM solutions. mPEG-PLG-GEM was embedded in the calcium phophate (CaP) backbone via electrostatic interactions. RESULTS: After incubation in plasma at 37°C for 24 h, gemcitabine was degraded by 24.6% for the mPEG-PLG-GEM, 14.7% for the mPEG-PLG-GEM/CaP nanoparticles, and 90% for the free gemcitabine solution. It was observed that mPEG-PLG-GEM and mPEG-PLG-GEM/CaP improved the area-under-curve (AUC) values by 5.26-fold and 6.33-fold compared to free drug, respectively. CONCLUSION: The amide bond linked gemcitabine polymers was able to protect GEM from cytidine deaminase degradation in vivo, and the skeleton formed by the calcium phosphate enhanced the stability and prolonged the half-life of GEM. Importantly, mPEG-PLG-GEM/CaP nanoparticles elevated the GEM plasma concentration in an animal model.

Rapid Homogeneous Immunoassay to Quantify Gemcitabine in Plasma for Therapeutic Drug Monitoring.

BACKGROUND: Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. METHODS: A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2',2'-difluorodeoxyuridine. RESULTS: Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 µ/mL (1.5-125.5 µM). Correlation with validated LC-MS/MS methods was R = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2',2'-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-8°C). CONCLUSIONS: The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

Evaluating the association of multiple single nucleotide polymorphisms with response to gemcitabine and platinum combination chemotherapy in urothelial carcinoma of the bladder
.

OBJECTIVE: To examine germline single nucleotide polymorphisms (SNPs) as markers of response to gemcitabine platinum (GP) combination chemotherapy in urothelial carcinoma (UC). METHODS: Saliva or blood was prospectively collected from 216 patients treated with GP for UC of the bladder between 1991 and 2011. Based on reported associations with gemcitabine and cisplatin response or putative mechanisms of gemcitabine or cisplatin/carboplatin activity, we selected SNPs of interest and were able to genotype 59 SNPs (using the SequenomMass ARRAYiPLEX platform) in 261 patients randomly split 2/3 into a training set (n = 174) and 1/3 into a test set (n = 87). Logistic regression was used to test the association between response to GP and SNPs. RESULTS: The median age at diagnosis was 64 years (range: 28 - 85) for the discovery set and 67 years (range: 30 - 84) for the validation set. Males composed 76% and 69%, and white non-Hispanics composed 88% and 91% of the training and test validation sets, respectively. Three SNPs on GALNTL4 (rs7937567, rs12278731, and rs9988868) and one intergenic SNP (rs1321391) were significantly associated with response to GP in the training set and were used to build a SNP score. However, when assessed in the test set, the SNP score was not significantly associated with response. CONCLUSION: Multiple SNPs selected from previous studies failed to predict response to GP in this cohort. Larger studies capable of accounting for population-based allele frequency heterogeneity may be required for replication of genetic alterations important to pharmacogenomics.
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Validated UHPLC-MS/MS assay for quantitative determination of etoposide, gemcitabine, vinorelbine and their metabolites in patients with lung cancer.

A fully valid UHPLC-MS/MS method was developed for the determination of etoposide, gemcitabine, vinorelbine and their metabolites (etoposide catechol, 2',2'-difluorodeoxyuridine and 4-O-deacetylvinorelbine) in human plasma. The multiple reaction monitoring mode was performed with an electrospray ionization interface operating in both the positive and negative ion modes per compound. The method required only 100 µL plasma with a one-step simple de-proteinization procedure, and a short run time of 7.5 min per sample. A Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) provided chromatographic separation of analytes using a binary mobile phase gradient (A, 0.1% formic acid in acetonitrile, v/v; B, 0.1% formic acid in water, v/v). Linear coefficients of correlation were >0.995 for all analytes. The relative deviation of this method was <10% for intra- and inter-day assays and the accuracy ranged between 86.35% and 113.44%. The mean extraction recovery and matrix effect of all the analytes were 62.07-105.46% and 93.67-105.87%, respectively. This method was successfully applied to clinical samples from patients with lung cancer.

Alterations in Pharmacokinetics of Gemcitabine and Erlotinib by Concurrent Administration of Hyangsayukgunja-Tang, a Gastroprotective Herbal Medicine.

Gemcitabine and erlotinib are the chemotherapeutic agents used in the treatment of various cancers and their combination is being accepted as a first-line treatment of advanced pancreatic cancer. Hyangsayukgunja-tang (HYT) is a traditional oriental medicine used in various digestive disorders and potentially helpful to treat gastrointestinal adverse effects related to chemotherapy. The present study was aimed to evaluate the effect of HYT on the pharmacokinetics of gemcitabine and erlotinib given simultaneously in rats. Rats were pretreated with HYT at an oral dose of 1200 mg/kg/day once daily for a single day or 14 consecutive days. Immediately after pretreatment with HYT, gemcitabine and erlotinib were administered by intravenous injection (10 mg/kg) and oral administration (20 mg/kg), respectively. The effects of HYT on pharmacokinetics of the two drugs were estimated by non-compartmental analysis and pharmacokinetic modeling. The pharmacokinetics of gemcitabine and erlotinib were not altered by single dose HYT pretreatment. However, the plasma levels of OSI-420 and OSI-413, active metabolites of erlotinib, were significantly decreased in the multiple dose HYT pretreatment group. The pharmacokinetic model estimated increased systemic clearances of OSI-420 and OSI-413 by multiple doses of HYT. These data suggest that HYT may affect the elimination of OSI-420 and OSI-413.

Preclinical studies of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in pediatric brain tumors.

Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.

Fixed-dose-rate administration of gemcitabine in cancer-bearing cats: A pilot study.

Gemcitabine is an antimetabolite chemotherapy agent with schedule-dependent metabolism and efficacy. The purpose of this study was to identify the fixed-dose-rate (FDR) of gemcitabine administration in cancer-bearing cats that achieved a target plasma concentration (TPC) of 10 to 20 µM. Fifteen client-owned cats received gemcitabine infusions administered at various FDR for 1 to 6 hours. Plasma gemcitabine and dFdU (2',2'-difluorodeoxyuridine), the major gemcitabine metabolite, were quantitated by high performance liquid chromatography. Cats treated with an FDR less than 2.5 mg/m2 per minute failed to achieve TPC, whereas cats treated with an FDR of 10 mg/m2 per minute quickly exceeded the target range. An FDR of 5 mg/m2 per minute provided the longest duration of exposure without exceeding the upper limit of the TPC. Plasma dFdU concentration mirrored plasma gemcitabine concentrations. These data suggest that in order to maintain TPC of gemcitabine in cats the FDR lies between 2.5 and 5 mg/m2 per minute. A Phase II study to evaluate efficacy and toxicity of this approach is underway.

Administration de gemcitabine à vitesse et à dose fixes chez des chats atteints du cancer : une étude pilote. La gemcitabine est un agent de chimiothérapie antimétabolite ayant un métabolisme et une efficacité qui dépendent du plan thérapeutique. Cette étude visait à identifier la vitesse et la dose fixes (VDF) d'administration de la gemcitabine chez des chats atteints du cancer qui avaient atteints une concentration plasmatique cible (CPC) de 10 à 20 µM. Quinze chats appartenant à des clients ont reçu des infusions de gemcitabine administrées à diverses VDF pendant 1 à 6 heures. La gemcitabine et la dFdU (2',2'-difluorodeoxyuridine) dans le plasma, le métabolite majeur de la gemcitabine, ont été quantifiés par chromatographie liquide à haute performance. Les chats traités à l'aide de VDF de moins de 2,5 mg/m2 par minute n'ont pas réussi à atteindre la CPC, tandis que les chats traités à l'aide de VDF de 10 mg/m2 par minute ont rapidement dépassé la zone cible. Des VDF de 5 mg/m2 par minute ont fourni la durée d'exposition la plus longue sans dépasser la limite supérieure de la CPC. La concentration de dFdU dans le plasma a reflété les concentrations de gemcitabine dans le plasma. Ces données suggèrent qu'fin de maintenir la CPC de la gemcitabine chez les chats, les VDF doivent se situer entre 2,5 et 5 mg/m2 par minute. Une étude de phase II pour évaluer l'efficacité et la toxicité de cette approche est actuellement en cours.(Traduit par Isabelle Vallières).

Enhanced pH-Responsiveness, Cellular Trafficking, Cytotoxicity and Long-circulation of PEGylated Liposomes with Post-insertion Technique Using Gemcitabine as a Model Drug.

PURPOSE: The in vitro and in vivo properties of PEGylated pH-sensitive liposomes (PSL) prepared by pre- and post-insertion techniques were investigated. METHODS: A pre-insertion or post-insertion technique was used for PSL PEGylation. For the first time, confocal laser scanning microscopy coupled with a modified calcein self-quench assay was applied to evaluate the endosome escape capability. PSL cellular uptake was evaluated using macrophages and the cytotoxicity using a gemcitabine (model drug)-resistant MIA PaCa-2 cells. The pharmacokinetics of PSL encapsulated gemcitabine was investigated in rats. RESULTS: PEGylation reduced the pH-sensitivity in a concentration-dependent manner (0.5-5% mol). Both PEGylation methods reduced the uptake of PSL by macrophages by over 60%. Cytotoxicity was ranked in the order: post-inserted PSL ≥ pre-inserted PSL > non-PSL > gemcitabine solution, consistent with the confocal microscopic observation and pH-sensitivity. Both pre and post-inserted PSL resulted in significant reductions (p < 0.05) in plasma clearance (58.6 and 38.4 ml/h/kg), increases in the area-under-the-concentration-time curve (56.9 and 87.1 µM · h) and half-life (6.1 and 6.2 h) compared to gemcitabine solution (152.9 ml/h/kg, 22.2 µM · h and 1.4 h). CONCLUSION: PEGylation by post-insertion offers advantages over pre-insertion to obtain PSL with enhanced pH-sensitivity, more effective intra-cytoplasmic delivery, and a superior pharmacokinetics.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

SLC28A3 genotype and gemcitabine rate of infusion affect dFdCTP metabolite disposition in patients with solid tumours.

BACKGROUND: Gemcitabine is used for the treatment of several solid tumours and exhibits high inter-individual pharmacokinetic variability. In this study, we explore possible predictive covariates on drug and metabolite disposition. METHODS: Forty patients were enrolled. Gemcitabine and dFdU concentrations in the plasma and dFdCTP concentrations in peripheral blood mononuclear cell were measured to 72 h post infusion, and pharmacokinetic parameters were estimated by nonlinear mixed-effects modelling. Patient-specific covariates were tested in model development. RESULTS: The pharmacokinetics of gemcitabine was best described by a two-compartment model with body surface area, age and NT5C2 genotype as significant covariates. The pharmacokinetics of dFdU and dFdCTP were adequately described by three-compartment models. Creatinine clearance and cytidine deaminase genotype were significant covariates for dFdU pharmacokinetics. Rate of infusion of <25 mg m(-2) min(-1) and the presence of homozygous major allele for SLC28A3 (CC genotype) were each associated with an almost two-fold increase in the formation clearance of dFdCTP. CONCLUSION: Prolonged dFdCTP systemic exposures (≥72 h) were commonly observed. Infusion rate <25 mg m(-2) min(-1) and carriers for SLC28A3 variant were each associated with about two-fold higher dFdCTP formation clearance. The impacts of these covariates on treatment-related toxicity in more selected patient populations (that is, first-line treatment, single disease state and so on) are not yet clear.

Comparison of the absolute level of epigenetic marks 5-methylcytosine, 5-hydroxymethylcytosine, and 5-hydroxymethyluracil between human leukocytes and sperm.

5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 104 deoxynucleosides, and 5.209 versus 0.492 per 106 deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (>10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome.