In vivo evaluation of pharmacokinetic drug-drug interactions between fluorinated pyrimidine anticancer drugs, 5-fluorouracil and capecitabin, and an anticoagulant, warfarin.

Warfarin is a common anticoagulant and has demonstrated interactions with several drugs. Among them, as a serious adverse event, a case of death due to the enhanced warfarin action owing to its combined use with a fluoropyrimidine anticancer drug has been reported, but the detailed mechanism has not been elucidated.Some reports have advocated that fluorinated pyrimidine anticancer drugs reduce cytochrome P450 2C9 expression, leading to the enhanced pharmacological effects of warfarin.The purpose of this study was to clarify the mechanisms of drug-drug interactions between warfarin and 5-fluorouracil (5-FU) and capecitabine in vivo using rats. Rats were administered warfarin in combination with 5-FU (15 mg/kg/d) or capecitabine (15 mg/kg/d) for 7 d. Prothrombin time (PT) and activated partial thromboplastin time were significantly prolonged in the warfarin plus 5-FU or capecitabine groups compared with those in the warfarin alone group. No significant difference was observed in the area under the plasma concentration-time curve of the warfarin alone group compared with the warfarin with 5-FU or capecitabine groups.These data suggest that the enhancement of warfarin efficacy caused by the combination of 5-FU or capecitabine was due to a pharmacological interaction rather than a pharmacokinetic interaction.

Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that tends to be resistant to chemotherapy. Multidrug resistance proteins (MRPs) contribute to the chemoresistance of these tumors. The objectives of the study were to document MRP expression profiles in two representative human intrahepatic and extrahepatic CCA cells lines (HuCCT1 and KMBC, respectively) and gemcitabine-induced cytotoxicity prior to and following MRP knockdown. METHODS: Multidrug resistance protein mRNA and protein expression were documented by real-time reverse transcription-polymerase chain reaction and western blots, respectively. MRP knockdown was achieved with lentivirus small hairpin RNA constructs. RESULTS: Prior to gemcitabine exposure, MRP1, MRP2, MRP4, MRP5, and MRP6 mRNA were expressed in HuCCT1 cells and MRP1, MRP3, MRP4, and MRP5 in KMBC cells. Following gemcitabine exposure, MRP5 and MRP6 expressions were significantly upregulated in HuCCT1 cells and MRP5 in KMBC cells. In HuCCT1 cells, although MRP5 knockdown had no effect, MRP6 knockdown significantly increased gemcitabine-induced cytotoxicity. In KMBC cells, MRP5 knockdown significantly increased gemcitabine cytotoxicity. CONCLUSIONS: Inhibition of MRP6 expression in intra-hepatic and MRP5 in extra-hepatic should be explored as potential treatments for CCA in humans.

The DNA repair helicase RECQ1 has a checkpoint-dependent role in mediating DNA damage responses induced by gemcitabine.

The response of cancer cells to therapeutic drugs that cause DNA damage depends on genes playing a role in DNA repair. RecQ-like helicase 1 (RECQ1), a DNA repair helicase, is critical for genome stability, and loss-of-function mutations in the RECQ1 gene are associated with increased susceptibility to breast cancer. In this study, using a CRISPR/Cas9-edited cell-based model, we show that the genetic or functional loss of RECQ1 sensitizes MDA-MB-231 breast cancer cells to gemcitabine, a nucleoside analog used in chemotherapy for triple-negative breast cancer. RECQ1 loss led to defective ATR Ser/Thr kinase (ATR)/checkpoint kinase 1 (ChK1) activation and greater DNA damage accumulation in response to gemcitabine treatment. Dual deficiency of MUS81 structure-specific endonuclease subunit (MUS81) and RECQ1 increased gemcitabine-induced, replication-associated DNA double-stranded breaks. Consistent with defective checkpoint activation, a ChK1 inhibitor further sensitized RECQ1-deficient cells to gemcitabine and increased cell death. Our results reveal an important role for RECQ1 in controlling cell cycle checkpoint activation in response to gemcitabine-induced replication stress.

Gemcitabine induced cytotoxicity, DNA damage and hepatic injury in laboratory mice.

The present study was conducted to demonstrate cytotoxicity, apoptosis and hepatic damage induced by gemcitabine in laboratory mice. Animals were treated with a single dose of gemcitabine (415 mg/kg body wt), equivalent to a human therapeutic dose, and sacrificed after 1, 2 and 3 weeks. A significant decrease in mean body weight and absolute liver weight was registered. The levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased as a result of this induced stress. Various structural changes were observed in the liver tissue of treated mice, as evident in the histological sections. Specifically, gemcitabine exposure was able to induce apoptosis in liver cells, and the incidence of TUNEL positive liver cells was increased compared to the control group. DNA fragmentation appeared on agarose gel and flow cytometry analysis confirmed the induction of apoptosis. These findings in gemcitabine-treated animal tissues suggest that inhibition or disruption of cells' DNA synthesis may be the mechanism by which this drug induces toxicity in the animal body.

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity.

Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2'-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino-imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that >60% of neutral KP1212 is present in the enol-imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH <7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.

Gemcitabine: Selective cytotoxicity, induction of inflammation and effects on urothelial function.

Intravesical gemcitabine has recently been introduced for the treatment of superficial bladder cancer and has a favourable efficacy and toxicity profile in comparison to mitomycin c (MMC), the most commonly used chemotherapeutic agent. The aim of this study was to assess the cytotoxic potency of gemcitabine in comparison to MMC in urothelial cell lines derived from non-malignant (UROtsa) and malignant (RT4 and T24) tissues to assess selectivity. Cells were treated with gemcitabine or mitomycin C at concentrations up to the clinical doses for 1 or 2h respectively (clinical duration). Treatment combined with hyperthermia was also examined. Cell viability, ROS formation, urothelial function (ATP, acetylcholine and PGE2 release) and secretion of inflammatory cytokines were assessed. Gemcitabine displayed a high cytotoxic selectivity for the two malignant cell lines (RT4, T24) compared to the non-malignant urothelial cells (UROtsa, proliferative and non-proliferative). In contrast, the cytotoxic effects of MMC were non-selective with equivalent potency in each of the cell lines. The cytotoxic effect of gemcitabine in the malignant cell lines was associated with an elevation in free radical formation and was significantly decreased in the presence of an equilibrative nucleoside transporter inhibitor. Transient changes in urothelial ATP and PGE2 release were observed, with significant increase in release of interleukin-6, interleukin-8 and interleukin-1ß from urothelial cells treated with gemcitabine. The selectivity of gemcitabine for malignant urothelial cells may account for the less frequent adverse urological effects with comparison to other commonly used chemotherapeutic agents.

Preclinical Evaluation of the Short-Term Toxicity of 4-(N)-Docosahexaenoyl 2´, 2´- Difluorodeoxycytidine (DHA-dFdC).

PURPOSE: This study was designed to test the short-term toxicity of DHA-dFdC in a mouse model and its efficacy in a mouse model of leukemia at or below its repeat-dose maximum tolerated dose (RD-MTD). METHOD: A repeat-dose dose-ranging toxicity study was designed to determine the tolerability of DHA-dFdC when administered to DBA/2 mice by intravenous (i.v.) injection on a repeat-dose schedule (i.e. injections on days 0, 3, 7, 10, and 13). In order to determine the effect of a lethal dose of DHA-dFdC, mice were injected i.v. with three doses of DHA-dFdC at 100 mg/kg on days 0, 3, and 5 (i.e. a lethal-RD). The body weight of mice was recorded two or three times a week. At the end of the study, major organs (i.e. heart, liver, spleen, kidneys, lung, and pancreas) of mice that received the lethal-RD or RD-MTD were weighed, and blood samples were collected for analyses. Finally, DHA-dFdC was i.v. injected into DBA/2 mice with syngeneic L1210 mouse leukemia cells to evaluate its efficacy at or below RD-MTD. RESULTS: The RD-MTD of DHA-dFdC is 50 mg/kg. At 100 mg/kg, a lethal-RD, DHA-dFdC decreases the weights of mouse spleen and liver and significantly affected certain blood parameters (i.e. white blood cells, lymphocytes, eosinophils, and neutrophil segmented). At or below its RD-MTD, DHA-dFdC significantly prolonged the survival of L1210 leukemia-bearing mice. CONCLUSION: DHA-dFdC has dose-dependent toxicity, affecting mainly spleen at a lethal-RD. At or below its RD-MTD, DHA-dFdC is effective against leukemia in a mouse model.

Response to and toxicity of gemcitabine for recurrent ovarian cancer according to number of previous chemotherapy regimens.

AIM: Gemcitabine is used not only as a second-line, but also as a third-line or higher regimen for taxane/platinum-resistant recurrent ovarian cancer. The purpose of this study was to clarify the response to and toxicity of gemcitabine for recurrent ovarian cancer according to the number of previous chemotherapy regimens. METHODS: The subjects were patients with taxane/platinum-resistant recurrent ovarian cancer on gemcitabine treatment at the present hospital between June 2007 and September 2013. We retrospectively reviewed the medical records. Response and adverse events were assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. and the Common Terminology Criteria for Adverse Events v4.0, respectively. RESULTS: The subjects consisted of 65 patients. The median number of previous chemotherapy regimens was 3 (range, 1-7). Overall response rate was 4.6%, and disease control rate (DCR) was 40.0%. DCR versus one, two, three, and ≥four previous chemotherapy regimens was 83.3%, 45.0%, 36.4%, and 23.5%, respectively. Grade 3/4 neutropenia, anemia, and thrombocytopenia occurred in 52.3%, 9.2%, and 9.2% of patients, respectively. Prevalence of grade 3/4 neutropenia according to one, two, three, and ≥four previous chemotherapy regimens was 66.7%, 55.0%, 54.5%, and 41.2%, respectively. Prevalence of anemia, thrombocytopenia, and almost all the non-hematological toxicities also did not increase with an increase in the number of previous chemotherapy regimens. CONCLUSIONS: Although DCR decreased as the number of previous chemotherapy regimens increased, the toxicities did not increase. Gemcitabine may be relatively safe in heavily pretreated ovarian cancer patients.

cRGD grafted siRNA nano-constructs for chemosensitization of gemcitabine hydrochloride in lung cancer treatment.

PURPOSE: The aim of present investigation was to effectively deliver ribonucleotide reductase subunit 1 (RRM1) targeted siRNA and assess chemo-sensitization of lung cancer cells against Gemcitabine hydrochloride. It was hypothesised that effective and selective delivery of RRM1 siRNA will help in the treatment of lung cancer chemotherapy using Gemcitabine hydrochloride by reducing drug dose and thereby, reduces dose related toxicity of Gemcitabine hydrochloride. METHODS: In this investigation, cRGD grafted siRNA nano-constructs were developed for efficient and targeted intracellular delivery of siRNA. Developed formulations were characterized for gel retardation assay, particle size, zeta potential, cryo transmission electron microscopy, serum stability, in vitro cytotoxicity, qualitative and quantitative cell uptake, gene expression, and chemo-sensitization. RESULTS: Complete complexation of siRNA with cRGD grafted nano-constructs was found at N/P ratio of 2.0. Naked siRNA was found to degrade within 6 h in presence of 50% serum while nano-constructs protected the complexed siRNA even after 24 h. RRM1 level significantly reduced when siRNA was delivered in nano-construct form as compared to naked siRNA. Pre-exposure of RRM1 siRNA decreased the IC50 value of Gemcitabine hydrochloride 5 folds in A-549 cells compared to Gemcitabine hydrochloride alone. CONCLUSION: These results suggest the application of present siRNA delivery strategy to potentiate the chemotherapeutic effect by means of chemosensitization which may be utilized for effective and thorough remission of lung cancer.

A comparison of cardiomyocyte cytotoxic mechanisms for 5-fluorouracil and its pro-drug capecitabine.

1. 5-Fluorouracil (5-FU) and its prodrug capecitabine are key chemotherapeutic agents in the treatment of many gastrointestinal tract adenocarcinomas. In addition to their beneficial antitumor effects, they also possess undesired cardiac toxicity. In the present study, we investigated the cytotoxic mechanisms of 5-FU and capecitabine in freshly isolated rat cardiomyocytes. 2. 5-FU and capecitabine cytotoxicities were associated with reactive oxygen species (ROS) formation, lipid peroxidation and rapid glutathione depletion. Increased intracellular ROS could target mitochondria, and our findings confirmed that the cardiomyocytes mitochondrial membrane potential (ΔΨm) was rapidly decreased by 5-FU and capecitabine. Mitochondrial dysfunction subsequently initiates downstream events that trigger caspase-3 activation, and our results showed that 5-FU and capecitabine activated caspase-3 which leads to apoptosis or necrosis. However, 5-FU acted much more powerful than capecitabine at inducing several cytotoxicity markers in heart cardiomyocytes. In addition, 5-FU but not capecitabine caused lysosomal membrane leakiness when it was incubated with cardiomyocytes. All cytotoxicity markers were prevented by antioxidants, ROS scavengers, mitochondrial permeability transition (MPT) pore sealing agents and lysosomotropic agents. 3. Our findings showed that the cytotoxic action of 5-FU and capecitabine on cardiomyocytes are mediated by oxidative stress and subsequent mitochondrial dysfunction which causes caspase-3 activation and cell death.

Discovery of genetic biomarkers contributing to variation in drug response of cytidine analogues using human lymphoblastoid cell lines.

BACKGROUND: Two cytidine analogues, gemcitabine and cytosine arabinoside (AraC), are widely used in the treatment of a variety of cancers with a large individual variation in response. To identify potential genetic biomarkers associated with response to these two drugs, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data, including 1.3 million SNPs and 54,000 basal expression probesets to perform genome-wide association studies (GWAS) with gemcitabine and AraC IC50 values. RESULTS: We identified 11 and 27 SNP loci significantly associated with gemcitabine and AraC IC50 values, respectively. Eleven candidate genes were functionally validated using siRNA knockdown approach in multiple cancer cell lines. We also characterized the potential mechanisms of genes by determining their influence on the activity of 10 cancer-related signaling pathways using reporter gene assays. Most SNPs regulated gene expression in a trans manner, except 7 SNPs in the PIGB gene that were significantly associated with both the expression of PIGB and gemcitabine cytotoxicity. CONCLUSION: These results suggest that genetic variation might contribute to drug response via either cis- or trans- regulation of gene expression. GWAS analysis followed by functional pharmacogenomics studies might help identify novel biomarkers contributing to variation in response to these two drugs and enhance our understanding of underlying mechanisms of drug action.

Concomitant gemcitabine therapy negatively affects DC vaccine-induced CD8(+) T-cell and B-cell responses but improves clinical efficacy in a murine pancreatic carcinoma model.

BACKGROUND: Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model. MATERIALS AND METHODS: Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8(+) T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively. RESULTS: Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8(+) T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8(+) T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination. CONCLUSIONS: Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.

The adenosine transporter, ENT1, in cardiomyocytes is sensitive to inhibition by ethanol in a kinase-dependent manner: implications for ethanol-dependent cardioprotection and nucleoside analog drug cytotoxicity.

The adenosine transporter 1 (ENT1) transports nucleosides, such as adenosine, and cytotoxic nucleoside analog drugs. ENT1 is well established to play a role in adenosinergic signaling in the cardiovascular system by modulating adenosine levels. Moderate ethanol consumption is cardioprotective and underlying mechanisms of action are not clear although adenosinergic signaling has been implicated. Here, we show that ethanol (5-200 mM) significantly reduces ENT1-dependent [(3)H] 2-chloroadenosine uptake (by up to 27 %) in the cardiomyocyte cell line, HL-1. Inhibition or absence of ENT1 is known to be cardioprotective, suggesting that the interaction of ethanol with ENT1 may promote adenosinergic cardioprotective pathways in the cardiovasculature.Ethanol sensitivity of adenosine uptake is altered by pharmacological activation of PKA and PKC. Primary cardiomyocytes from PKCε-null mice have significantly greater sensitivity to inhibition (by approximately 37 %) of adenosine uptake by ethanol than controls. These data suggest that the presence of ethanol may compromise ENT1-dependent nucleoside analog drug cytotoxicity, and indeed, ethanol (5 mM) reduces the cytotoxic effects of gemcitabine (2 nM), an anti-cancer drug, in the human cancer cell line, HTB2. Thus, the pharmacological inhibition of ENT1 by ethanol may contribute to ethanol-dependent cardioprotection but compromise gemcitabine cytotoxicity.

Synthesis, reactivity and biological activity of N(4)-boronated derivatives of 2'-deoxycytidine.

By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp(3) hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented.

A pilot study: dose adaptation of capecitabine using mobile phone toxicity monitoring - supporting patients in their homes.

PURPOSE: Real-time symptom monitoring using a mobile phone is potentially advantageous for patients receiving oral chemotherapy. We therefore conducted a pilot study of patient dose adaptation using mobile phone monitoring of specific symptoms to investigate relative dose intensity of capecitabine, level of toxicity and perceived supportive care. METHODS: Patients with breast or colorectal cancer receiving capecitabine completed a symptom, temperature and dose diary twice a day using a mobile phone application. This information was encrypted and automatically transmitted in real time to a secure server, with moderate levels of toxicity automatically prompting self-care symptom management messages on the screen of the patient's mobile phone or in severe cases, a call from a specialist nurse to advise on care according to an agreed protocol. RESULTS: Patients (n = 26) completed the mobile phone diary on 92.6 % of occasions. Twelve patients had a maximum toxicity grade of 3 (46.2 %). The average dose intensity for all patients as a percentage of standard dose was 90 %. In eight patients, the dose of capecitabine was reduced, and in eight patients, the dose of capecitabine was increased. Patients and healthcare professionals involved felt reassured by the novel monitoring system, in particular, during out of hours. CONCLUSION: It is possible to optimise the individual dose of oral chemotherapy safely including dose increase and to manage chemotherapy side effects effectively using real-time mobile phone monitoring of toxicity parameters entered by the patient.

Chidamide, a novel histone deacetylase inhibitor, synergistically enhances gemcitabine cytotoxicity in pancreatic cancer cells.

Pancreatic cancer is a lethal human malignancy with an extremely poor prognosis and urgently requires new therapies. Histone deacetylase inhibitors (HDACIs) represent a new class of anticancer agents and have shown promising antitumor activities in preclinical models of pancreatic cancer. In this study, we sought to determine the antitumor effects of a novel HDACI, chidamide (CS055), in pancreatic cancer cells alone or in combination with gemcitabine. Treatments of BxPC-3 or PANC-1 pancreatic cancer cell lines with chidamide resulted in dose- and time-dependent growth arrest, accompanied by induction of p21 expression. When combined in a sequential schedule, chidamide synergistically enhanced gemcitabine-induced cell growth arrest and apoptosis, accompanied by cooperative downregulation of Mcl-1 and loss of mitochondrial membrane potential (ΔΨm). Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression. Our results suggest that chidamide has a therapeutic potential for treating pancreatic cancer, especially in combination with gemcitabine.

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium.

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO-sGC-cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients.

Evaluation of the effect of gemcitabine on cochlea by otoacoustic emission in experimental animal model.

OBJECTIVES: In this study, we aimed to search whether gemcitabine - a commonly used antimetabolite type antineoplastic agent-has ototoxic effect. STUDY DESIGN AND SETTING: An experimental animal research. SUBJECTS AND METHODS: We evaluated the effect of gemcitabine on hearing through its possible effect on cochlea by using otoacoustic emission method on experimental rat model. For this purpose 16 healthy adult male Wistar albino rats were used and these were divided into 4 groups. The rats in the 1st, 2nd, 3rd and the 4th groups were given 40-160-240 and 320 mg/kg of intraperitoneal gemcitabine respectively. Distortion product otoacoustic emission measurements on both ears of each rat were performed before and 1 week after administration of gemcitabine. The mean of signal to noise ratio of emission values obtained from each rat before and after the drug administration were calculated. RESULTS: Rats in the 1st group had no statistically significant difference at the emission rates before and after gemcitabine administration. A statistically significant decrease was observed in the emission rates at emission values of only 5 kHz in the 2nd group, at 4-6 kHz in the 3rd group, at 4-5-6-8 kHz in the 4th group. CONCLUSIONS: Gemcitabine causes a decrease in otoacoustic emission values on experimental rat models at high doses and especially at high frequencies. Larger clinical and laboratory studies are needed to determine whether this decrease in emission rates are permanent and whether it has any permanent effect on hearing.  

Evaluation of single and combination therapies with tenofovir disoproxil fumarate and emtricitabine in vitro and in a robust mouse model supporting high levels of hepatitis B virus replication.

Next-generation therapies for chronic hepatitis B virus (HBV) infection will involve combinations of established and/or experimental drugs. The current study investigated the in vitro and in vivo efficacy of tenofovir disoproxil fumarate (TDF) and/or emtricitabine [(-)-FTC] alone and in combination therapy for HBV infection utilizing the HepAD38 system (human hepatoblastoma cells transfected with HBV). Cellular pharmacology studies demonstrated increased levels of (-)-FTC triphosphate with coincubation of increasing concentrations of TDF, while (-)-FTC had no effect on intracellular tenofovir (TFV) diphosphate levels. Quantification of extracellular HBV by real-time PCR from hepatocytes demonstrated the anti-HBV activity with TDF, (-)-FTC, and their combination. Combination of (-)-FTC with TDF or TFV (ratio, 1:1) had a weighted average combination index of 0.7 for both combination sets, indicating synergistic antiviral effects. No cytotoxic effects were observed with any regimens. Using an in vivo murine model which develops robust HBV viremia in nude mice subcutaneously injected with HepAD38 cells, TDF (33 to 300 mg/kg of body weight/day) suppressed virus replication for up to 10 days posttreatment. At 300 mg/kg/day, (-)-FTC strongly suppressed virus titers to up to 14 days posttreatment. Combination therapy (33 mg/kg/day each drug) sustained suppression of virus titer/ml serum (<1 log(10) unit from pretreatment levels) at 14 days posttreatment, while single-drug treatments yielded virus titers 1.5 to 2 log units above the initial virus titers. There was no difference in mean alanine aminotransferase values or mean wet tumor weights for any of the groups, suggesting a lack of drug toxicity. TDF-(-)-FTC combination therapy provides more effective HBV suppression than therapy with each drug alone.

Cytidine deaminase single-nucleotide polymorphism is predictive of toxicity from gemcitabine in patients with pancreatic cancer: RTOG 9704.

The aim of this study is to validate the prognostic and predictive value of the non-synonymous cytidine deaminase (CDA) Lys²7Gln polymorphism for hematological toxicity and survival using a randomized phase III adjuvant trial (Radiation Therapy Oncology Group (RTOG) 9704) in pancreatic cancer in which one treatment arm received gemcitabine. CDA is involved in gemcitabine inactivation, and there is conflicting data on the role of the non-synonymous CDA Lys²7Gln polymorphism in predicting toxicity and survival in cancer patients treated with gemcitabine. RTOG 9704 randomized 538 patients after pancreatic resection to receive radiotherapy with either 5-fluorouracil (5-FU) or gemcitabine. CDA Lys²7Gln polymorphism genotype was analyzed. We tested an association between CDA single-nucleotide polymorphism genotype and the survival outcome by the Cox proportional hazard model adjusting for other covariates, as well as toxicity by the logistic regression model. There is statistically significant more severe hematological toxicity in patients treated with gemcitabine with either the homozygote wild-type genotype (Lys/Lys) alone (odds ratio (OR)=0.06, P=0.01), or in combination with the heterozygote (Lys/Gln; OR=0.14, P=0.03) when compared with homozygote variant genotype (Gln/Gln) when adjusted for other covariates. This was not seen in the non-gemcitabine treated arm. There are no genotype differences with respect to survival outcome. In conclusion, in this prospective randomized adjuvant study of patients with pancreatic cancer, the CDA Lys²7Gln polymorphism is validated as a predictive marker of gemcitabine hematological toxicity, but not with treatment response or survival.