Cellular pharmacology of 5-fluorouracil in a human colon adenocarcinoma cell line selected for thymidine kinase deficiency

A human colon adenocarcinoma cell line (GC3TK-) was selected for thymidine kinase (TK) deficiency from cloned parental cells (GC3C1) by exposure to 5-bromodeoxyuridine (BrdUrd). The cellular pharmacology of 5-fluorouracil (FUra) and the influence of physiological concentrations of thymidine (dThd; 0.1 to 1 microM) on FUra cytotoxicity during brief exposure in both cell lines were examined. The uptake of FUra during a 1-hr drug exposure, its metabolism to ribo- and deoxyribonucleotides, incorporation into RNA, and inhibition of thymidylate synthase were similar in GC3C1 and GC3TK- cells as were the IC50 values for FUra (26 and 23 microM respectively). TK deficiency did not reduce the intracellular concentrations of FdUMP generated from FUra. In GC3C1, at FUra concentrations up to 100 microM, cytotoxicity was prevented by co-administration of dThd (0.1 to 20 microM). The relationship between cell survival and thymidylate synthase inhibition was close under these conditions. At higher drug concentrations, less dThd protection was observed, and none was detected in GC3TK- cells. Thus, the metabolism of FUra did not appear to be altered substantially in GC3C1 cells selected for TK deficiency. Also in these cells, at concentrations of FUra less than 100 microM, FUra cytotoxicity appeared to be mediated via the inhibition of thymidylate synthase.     

Skin delivery of 5-fluorouracil from ultradeformable and standard liposomes in-vitro

The potential use of ultradeformable and standard liposomes as skin drug delivery systems was investigated in-vitro. An improved experimental design gave a good measure for skin deposition of drug. This avoided the contamination that can occur due to incomplete washing of the donor before direct determination of the amount of drug in the skin. The design used aqueous ethanolic receptor which is believed to diffuse into skin, disrupting deposited liposomes (if any) and thus releasing both bound and free drug. The receptor fluid was refined by testing different concentrations of ethanol. The applied dose was also optimized. Using the improved design and the optimum dose, an ultradeformable formulation was compared with four traditional liposomes for skin delivery of 5-fluorouracil (5-FU). The best receptor was 50% aqueous ethanol and the optimum dose was 20 microL. The ultradeformable formulation was superior to standard liposomes in the skin delivery of 5-FU. Of the traditional liposomes, the non-rigid preparation was the best. However, stabilization of the liposome membrane with cholesterol abolished the benefit of this non-rigid preparation. It was concluded that ultradeformable vesicles are promising agents for skin delivery of drugs.     

Enzyme regulatory site-directed drugs: study of the interactions of 5'-amino-2', 5'-dideoxythymidine (5'-AdThd) and thymidine triphosphate with thymidine kinase and the relationship to the stimulation of thymidine uptake by 5'-AdThd in 647V cells

5'-Amino-2',5'-dideoxythymidine (5'-AdThd) is a nontoxic thymidine (dThd) analogue capable of antagonizing the feedback inhibition exerted by thymidine triphosphate (dTTP) on thymidine kinase (EC 2.7.1.21). In intact cells, this results in stimulation of thymidine uptake by 5'-AdThd. We have studied the interaction between 5'-AdThd and thymidine kinase purified from 647V cells. We found that 5'-AdThd inhibited competitively thymidine kinase activity (Ki of 0.5 microM) in the absence of dTTP whereas dTTP inhibited thymidine kinase activity in a noncompetitive manner. However, in the presence of dTTP, 5'-AdThd was able to stimulate enzyme activity in a mode that suggests competition with dTTP for the regulatory site. Altered interactions were observed at high substrate (dThd) concentrations, with dThd showing competitive kinetics with dTTP. In intact cells, we evaluated the hypothesis that antagonism of feedback inhibition could account for stimulation of dThd uptake by 5'-AdThd. If inhibition of thymidine kinase activity by dTTP is critical, then depletion of cellular dTTP by methotrexate should reduce the ability of 5'-AdThd to stimulate dThd uptake. Indeed, this was the case. If the dTTP pools were repleted by the addition of higher concentrations of dThd, the ability of 5'-AdThd to stimulate dThd uptake was restored. Furthermore, effects of 5'-AdThd on nucleoside phosphorylase or cytoplasmic 5'-nucleotidase activity (dTMP breakdown) could not account for the stimulation of dThd uptake in 647V cells. In summary, our results indicate that 5'-AdThd interacts with thymidine kinase at the dTTP-binding site, resulting in stimulation of enzyme activity and stimulation of dThd uptake in intact cells.     

Pharmacokinetics of 5-fluorouracil: inter-relationship with biochemical kinetics in monitoring therapy

The major difficulty in using pharmacokinetic concepts for monitoring individual therapy with 5-fluorouracil is based on the mostly intracellular location of the active nucleotide metabolites of 5-fluorouracil with no clear correlation of plasma levels of the drug. In addition, the basic biochemical mechanism of 'thymine-less cell death' following inhibition of de novo thymidylate synthesis by 2'-deoxy-5-flurouridine 5'-monophosphate (FdUMP) is poorly understood, and only some of the biochemical determinants of therapeutic response to 5-fluorouracil are known. Individualised therapy with 5-fluorouracil requires an intergrated approach which should include methods of pharmacokinetics and biochemical kinetics. 5-Fluorouracil stands as an example for most of the pyrimidine and purine metabolites to which similar consideration apply in monitoring of cancer chemotherapy.     

Inhibition by interferon of biochemical transformation induced by cloned herpesvirus thymidine kinase genes

To learn whether interferon could prevent the biochemical transformations induced by cloned herpesvirus thymidine kinase (TK) genes, LM(TK-) mouse fibroblast cultures were pretreated for 24 h with 2.4-40 international units (I.U.)/ml mouse alpha + beta interferon, and subsequently transformed to the TK+ phenotype with recombinant plasmids containing the herpes simplex virus type 1 (HSV-1) TK gene (pAGO and pMH110) and the marmoset herpesvirus (MarHV) TK gene (pMAR035). Mouse alpha + beta interferon inhibited transformation and the inhibition was interferon dose-dependent. Transformation was also inhibited when LM(TK-) cells were pretreated for 2-5 h with 40 I.U./ml interferon. Maximal inhibitions of TK+ colony formation were observed following a 9-20 h pretreatment period with interferon. In contrast, 40 I.U./ml interferon treatment for 20 h did not reduce the rate or extent of LM(TK-) cell growth. Experiments in which cultures were first treated with plasmid pAGO and only afterwards treated with interferon also showed that, as the interferon concentration used, interferon did not inhibit the outgrowth of transformated colonies. Enzyme assays showed that pretreatment with interferon inhibited the induction of TK activity in cells that had been transfected with pAGO DNA.     

Bis(pivaloyloxymethyl) thymidine 5'-phosphate is a cell membrane-permeable precursor of thymidine 5'-phosphate in thymidine kinase deficient CCRF CEM cells

Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM(2)-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM(2)-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk-). After 72 h incubation at 37 degrees C, TdR showed significant antiproliferative activity (IC(50)=27 microM) against CEM cells but was weakly effective (IC(50)=730 microM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5'-monophosphate (POM(2)-dTMP) was equally inhibitory (IC(50)=5 microM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-(3)H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk- with 5 microM POM(2)-[methyl-(3)H]dTMP. The incorporation of [methyl-(3)H]TdR into HClO(4)-insoluble cell residue by CEM tk- was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk- incorporated radioactivity from POM(2)-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM(2)-dTMP can penetrate into cells and serve as a source of dTMP.     

Biochemical modulation of 5-fluorouacil through dihydropyrimidine dehydrogenase inhibition: a Southwest Oncology Group phase II trial of eniluracil and 5-fluorouracil in advanced resistant colorectal cancer

Purpose. To investigate thehypothesis that a systemic agent designedto inhibit dihydropyrimidine dehydrogenase(DPD), the first enzyme in thefluoropyrimidine degradative pathway, couldimprove the effective amount of5-fluorouracil (5-FU) delivered to a tumorresulting in enhanced response. Patients and methods. Eligibility includedcytologically or pathologically verifieddiagnosis of colorectal cancer thatrecurred during or within 12 months ofcompletion of adjuvant therapy,representing patients generally consideredresistant to fluorinated pyrimidinetherapy. Stratification was into twocohorts: recurrence while receivingadjuvant therapy, and relapse within 12months of completing adjuvant therapy.Treatment consisted of 28 days of oraltherapy every five weeks with eniluraciland 5-FU administered in a 10:1 ratio. Thedaily dose of eniluracil was 10 mg/m²with 5-FU 1 mg/m², divided into twodoses. Results. Twenty-five patientsare evaluable for response: 9 relapsedduring therapy and 16 relapsed within oneyear of adjuvant therapy. In the firstgroup, there was one partial response (9%;95% CI 0–41%); in the second cohort therewas one confirmed complete response (5%;95% CI 0–23%) and one unconfirmed partialresponse, for an overall response rate of10%. Conclusions. This regimen lackssignificant activity in this targetpopulation. Pre-treatment intratumoral DPDexpression was not assessed, therefore themechanism of fluorinated pyrimidineresistance cannot be specificallyattributed to elevated DPD levels.Attempting restoration of chemotherapysensitivity through blockade of enzymes orsignal transduction molecules responsiblefor resistance is rational, provided thattumor target expression is the basis fortrial entry.     

Loss of murine tumor thymidine kinase activity in vivo following 5-fluorouracil (FUra) treatment by incorporation of FUra into RNA.

The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage.     

杂氮硅三环与5-氟尿嘧啶结合物的合成

杂氮硅三环具有免疫增强和抗肿瘤作用，依据药物设计的拼合原理设计了杂氮硅三环与5-氟尿嘧啶结合的协同前药，以期寻找高效低毒的抗肿瘤化合物。合成了2个未见报道的杂氮硅三环与5-氟尿嘧啶的结合物。其结构经质谱、核磁共振氢谱及元素分析确证。     

甲硝唑大鼠肠吸收实验研究

目的考察甲硝唑在大鼠不同肠段的吸收动力学特征、吸收部位及吸收机制,为其结肠定位缓控释制剂的设计提供理论依据。方法采用大鼠在体灌流肠吸收实验,考察了甲硝唑的吸收部位和吸收动力学特征。结果甲硝唑在大鼠肠道内无特定吸收部位,各肠段吸收速率常数按十二指肠、空肠、结肠、回肠顺序依次上升,吸收速率常数分别为0.078、0.136、0.155、0.168/h。在2～20μg/ml的浓度范围内,甲硝唑的吸收量与浓度有良好的线性关系。结论甲硝唑在大鼠全肠段均有吸收,吸收符合一级动力学特征,吸收机制为被动扩散,适于制备结肠定位缓控释制剂。     

Colonic drug delivery of 5-fluorouracil: an in vitro evaluation

Compression coating has been found to be useful for colonic drug delivery. The aim of the present investigation was to design a formulation with a considerably reduced coat weight and gum concentration for colonic delivery of 5-fluorouracil for the treatment of colorectal cancer. Rapidly disintegrating core tablets containing 50 mg of 5-fluorouracil were prepared and compression coating with 175 mg of granules containing a mixture of xanthan gum (XG) and guar gum (GG) in varying proportions was done. With this coat weight, a highly retarded drug release was observed. After 24h of dissolution the mean percent drug release from the compression coated XG:GG 20:20, 20:10 and 10:20 tablets were found to be around 18+/-1.23%, 20+/-1.54% and 30+/-1.77%, respectively. So, the coat weight was further reduced to 150 mg. It was observed that reduction of coat weight did not affect the initial drug release rate in simulated upper gastrointestinal tract (GIT) conditions. At the end of 24h of dissolution the amount of drug released increased to 25+/-1.22%, 36.6+/-1.89% and 42.6+/-2.22%, respectively in XG:GG 20:20, 20:10 and 10:20 tablets. Studies of XG:GG (10:20) tablets in presence of colonic contents showed an increased cumulative percent drug release of 67.2+/-5.23% in presence of 2% cecal content and 80.34+/-3.89% in presence of 4% cecal content after 19 h of incubation.     

Leucovorin, 5-fluorouracil,and gemcitabine: A phase I study

Gemcitabine is a chemotherapy agent with efficacy in the treatment of lung, pancreas, bladder and breast cancer. It inhibits DNA synthesis by interfering with cytidine triphosphate production and also inhibits the activity of ribonucleotide reductase. Gemcitabine may potentiate fluorouracil's inhibition of thymidylate synthase. This inhibition would be expected to be sequence dependent, occurring only if gemcitabine were administered following fluorouracil (5FU).The combination of leucovorin, 5-FU, and gemcitabine was assessed in this phase I trial. Eligibility requirements included refractory solid tumor malignancy; adequate hematologic, renal and hepatic reserve; no prior therapy with the combination of leucovorin and 5FU, or with gemcitabine; ECOG performance status 0–2, and signed informed consent.Eleven men and nine women were eligible. The median age was 52.5 years and the median performance status was 1. All but three patients had prior chemotherapy. The starting doses were leucovorin 20 mg/m², 5FU 255 mg/m² and gemcitabine 600 mg/m². 5FU and gemcitabine were escalated in tandem to 340 mg/m² and 800 mg/m² and thereafter to 425 mg/m² and 1000 mg/m², respectively. Gemcitabine administration always followed that of 5FU by 30 minutes. The median number of cycles was 2 (range 1–32). Two patients at the starting dose had disease progression within the first cycle with one death on day 28. One patient with cholangiocarcinoma had a partial response and remained on study for 40 months. There were no other responses.The maximum tolerated dose is leucovorin 20 mg/m², 5FU 340 mg/m², and gemcitabine 800 mg/m². The impact of drug sequence remains undetermined.     

Induction of thymidine kinase 1 after 5-fluorouracil as a mechanism for 3′-deoxy-3′-[F]fluorothymidine flare

Imaging the pharmacodynamics of anti-cancer drugs may allow early assessment of anti-cancer effects. Increases in 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) uptake early after thymidylate synthase inhibition (TS) inhibition, the so-called flare response, is considered to be largely due to an increase in binding sites for type-1 equilibrative nucleoside transporter. We investigated the induction of thymidine kinase 1 (TK1) after 5-fluorouracil (5-FU) treatment as one of mechanisms for [¹⁸F]FLT flare. Exposure of nine cancer cell lines to 5-FU for 24 h induced a 2.5- to 3.5-fold increase in [¹⁸F]FLT uptake, significantly higher than the 1.5-fold increase observed 2 h after treatment. The increase of [¹⁸F]FLT uptake 24 h after 5-FU exposure accompanied TK1 induction in most cell lines. In representative cell lines (A431 and HT29), 5-FU time-dependently increased [¹⁸F]FLT uptake, kinase activity and the levels of protein and mRNA for TK1, sequential cyclin E and A induction, and G₁-S phase transition. Cycloheximide treatment and knockdown of TK1 completely inhibited 5-FU-induced [¹⁸F]FLT flare. On the other hand, HCT8 cells showed a biphasic [¹⁸F]FLT flare with lacked TK1 induction in response to the dosage of 5-FU. Cycloheximide did not inhibit 5-FU-induced [¹⁸F]FLT flare in this cells. In vivo dynamic [¹⁸F]FLT-PET and ex vivo analysis in HT29 tumor-bearing mice showed significantly increased [¹⁸F]FLT flux and TK1 activity of tumor tissue 24 h after 5-FU administration (P < 0.05). Conclusively, 5-FU induced TK1 and TK1-mediated high [¹⁸F]FLT flare in most of cell lines. [¹⁸F]FLT-PET may be used to assess pharmacodynamics of TS inhibitor by a mechanism involving TK1 induction.     

Regional and systemic serum concentrations of 5-fluorouracil after portal and intravenous infusion: an experimental study in dogs

The serum and urinary concentrations of 5-FU after continuous portal and jugular infusion have been followed by means of a highly sensitive microbiological assay method. Our data indicate that more than 90% of 5-FU was eliminated in the liver after continuous portal infusion of 0.625 mg x kg-1 x hr-1, corresponding to a dose of 15 mg x kg-1 x 24 hr-1. Negligible amounts of intact 5-FU were excreted into the bile, and the urinary excretion was only a few percent of the amount infused. The arterial concentration was on average tenfold higher during the continuous jugular infusion than after the continuous portal infusion, indicating that the route of administration has a pronounced effect on the disposition of 5-FU. Twenty-three percent of the jugular dose reached the liver; 77% was degraded by extrahepatic metabolism. Of these, degradation in the prehepatic splanchnic area accounted for 15%.     

Double-blind randomised placebo-controlled phase III study of an E. coli extract plus 5-fluorouracil versus 5-fluorouracil in patients with advanced colorectal cancer

The primary aim of this study was to evaluate the toxicity (mucositis, diarrhea and leucopenia) of a therapy with 5-fluorouracil (CAS 51-21-8; 5-FU) plus an E. coli extract (LC-Extract, Laves coli extract, Colibiogen inject, cell-free soluble fraction from lysed E. coli, Laves strain) in comparison with 5-FU plus placebo. Secondary endpoints included general toxicity, response rate according to WHO, survival time and quality of life. 164 patients with advanced colorectal cancer were enrolled in this randomised, placebo-controlled, double-blind, multicenter phase III study. The treatment consisted of 0.167 ml/kg/d LC-Extract or placebo followed by 500-750 mg/m2/d 5-FU on five consecutive days, repeated every three weeks for up to six treatment cycles. 158 (77 verum, 81 placebo) patients were evaluable for toxicity, 144 (72 verum, 72 placebo) evaluable for response. The therapy with LC-Extract was well tolerated. Adverse events that occurred during the study were mainly judged as 5-FU- or tumor-related. Toxicity from treatment with 600 mg/m2/d 5-FU in both treatment groups was very low. After treatment with 750 mg/m2/d 5-FU patients in the placebo-group experienced a higher CTC toxicity than in the LC-Extract groups. Remission rate and survival time showed a slight trend in favour of LC-Extract. These results suggest a positive benefit-risk ratio of the additional application of LC-Extract to 5-FU in the treatment of advanced colorectal cancer especially for administration of high doses of 5-FU.     

Functional effects of imipramine on the rabbit urinary bladder: an in-vitro study

Imipramine is a tricyclic antidepressant that has been demonstrated to be useful in the treatment of certain voiding dysfunctions. Imipramine has a variety of pharmacological effects including direct antimuscarinic activity, inhibition of catecholamine reuptake, direct muscle relaxant, and calcium antagonism. Using the in-vitro whole bladder model we have studied the effect of imipramine on the rate and magnitude of both intravesical pressure generation and bladder emptying in response to field stimulation. The results can be summarized as follows: at concentrations as low as 1 mumol/l imipramine causes a significant inhibition of volume expulsion without significantly affecting pressure generation. Imipramine produced a dose-dependent inhibition of both pressure development and percent volume emptying; however, it was substantially more potent in inhibiting the ability of the bladder to empty than to generate pressure.     

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

Traditional Chinese herbal medicine(TCM) has been shown to enhance the efficacy of standard anticancer agents. However, there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM. OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models. METHODS Cell growth assays, cell cycle analysis,immunoblot analysis and q RT-PCR were performed to investigate the mechanism(s) of action of the formulason human CRC cells. In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with5-FU. RESULTS We identified Huangqin Gegen Tang(HQGGT) which suppressed the in vivo growth of human CRC HT-29 xenografts. HQGGT significantly inhibited the growth of CRC cell lines. HQGGT enhanced the cytotoxicity of5-FU against human 5-FU-resistant cells(H630 R1) and mouse colon cancer cells(MC38). This synergy was the result of suppression of thymidylate synthase expression by HQGGT. HQGGT significantly enhanced the antitumor effect of5-FU in mice bearing MC38 xenografts. Ongoing studies have identified Huangqin as the herb responsible for TS inhibition. CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrimidine chemotherapy for CRC treatment.