Rate Of Intracellular Accumulation Research Articles

A phase II study of biweekly gemcitabine at fixed dose rate infusion plus S1 as first-line chemotherapy in patients with locally advanced or metastatic pancreatic cancer.

357 Background: The combination regimen of gemcitabine plus S1 (GS) as first-line chemotherapy has shown good efficacy in advanced pancreatic cancer. However, it is inconvenient to deliver gemcitabine on days 1 and 8 within one treatment cycle. In addition, previous studies have demonstrated that the gemcitabine administration by fixed dose rate infusion of 10 mg/m2/min should maximize the rate of intracellular accumulation of gemcitabine triphosphate and might improve clinical efficacy. The aim of this study was to evaluate the efficacy and safety of biweekly gemcitabine at fixed dose rate infusion combined with S1 (FGS) in chemonaive patients with locally advanced or metastatic pancreatic cancer. Methods: Forty-three eligible patients with locally advanced or metastatic pancreatic cancer received gemcitabine at a fixed dose of 10 mg/m2/min intravenously over a 2-h period on day 1, together with oral S1 40mg/m2twice daily on days 1 to 10, every 2 weeks. Tumor assessment was performed for every 3 cycles of chemotherapy. Results: All patients were evaluable for toxicity and 42 patients for efficacy. The overall response rate was 35.7% with 1 complete response, 14 partial responses, 17 stable diseases, and 10 progressions. Median time to progression was 5.9 months and median overall survival was 9.6 months. The main grade 3/4 toxicities included neutropenia (30.2%), thrombocytopenia (6.9%), anemia (2.3%), nausea (4.6%), diarrhea (2.3%). Conclusions: The biweekly FGS regimen is active, well tolerated and conveniently delivered as first-line chemotherapy for patients with locally advanced or metastatic pancreatic cancer.

Monitoring the kinetics of CellTrace™ calcein red-orange AM intracellular accumulation with spatial intensity distribution analysis

BackgroundRoutine black box approaches quantify fluorescence intensity to profile the uptake of fluorophores, providing limited insight into microscopic events. Spatial intensity distribution analysis has previously been reported to quantify oligomerisation and number of particles from selected regions and profile intracellular distributions of labelled moieties. MethodsIn this study, the concentration and time-dependent behaviour of CellTrace™ calcein red-orange (AM) intracellular accumulation was examined in colorectal adenocarcinoma cell line and bovine aortic endothelial cells. Monolayers were subjected to fluorescence correlation spectroscopy, fluorescence intensity and SpIDA measurements to determine differences in the rate and extent of intracellular accumulation. ResultsIntracellular accumulation data derived from Spatial intensity distribution analysis were found to correlate with that of fluorescence correlation spectroscopy and fluorescence intensity profiles. The extent of intracellular accumulation was found to be time and concentration-dependent in both cell lines examined, with no significant differences in the rate of intracellular accumulation. ConclusionsSpatial intensity distribution analysis applied at ‘proof of concept’ level is a rapid and user-friendly tool that can be applied to the quantification of intracellular concentration and kinetics of fluorophore uptake. General significanceConfocal imaging as a routinely implemented tool for profiling fluorescently-labelled species is often under-exploited for yielding quantitative parameters.

Doxorubicin release is not a prerequisite for the in vitro cytotoxicity of HPMA-based pharmaceuticals: In vitro effect of extra drug-free GlyPheLeuGly sequences

A systematic study was designed to elucidate differences in cytostatic activity in vitro between HPMA-based doxorubicin conjugates synthesized using different polymerization techniques and differing in peptidyl side chain. A polymer–drug conjugate containing doxorubicin (DOX) bound to HPMA copolymer backbone through the enzymaticaly non-cleavable sequence GlyGly shows low but significant cytotoxicity in vitro in seven cancer cell lines of mouse (EL4, 38C13, 3T3, BCL1) and human (SW620, Raji, Jurkat) origin. The low cytotoxicity can be considerably increased by the presence of additional drug-free GlyPheLeuGly side chains. P1 conjugate, i.e. non-targeted HPMA copolymer bearing doxorubicin bound via a biodegradable GlyPheLeuGly sequence, synthesized by direct copolymerization of HPMA with monomeric doxorubicin and thus without additional drug-free GlyPheLeuGly sequences is less effective compared to PK1 synthesized by polymer analogous reaction and thus containing extra drug-free GlyPheLeuGly sequences. Significant activity-enhancing effect was not seen with other amino acid/oligopeptide sequences (e.g., Gly or GlyGly). The activity-enhancing effect of GlyPheLeuGly sequences is more obvious in the conjugate containing doxorubicin bound to HPMA through GlyGly sequence. Derivatization of the terminal carboxyl group of the extra GlyPheLeuGly side chains (amide, N-substituted amide, free carboxyl) does not significantly influence the cytotoxicity of the conjugates. The presence of the GlyPheLeuGly sequence in the conjugate structure increases its rate of intracellular accumulation. Normal cells (Balb/c splenocytes) accumulate less polymer–doxorubicin conjugate compared to cancer cells (T cell lymphoma EL4, B cell lymphoma Raji and T cell leukemia JURKAT).

Questions About Gemcitabine Dose Rate: Answered or Unanswered?

Even before 2 ,2 -difluorodeoxycytidine (dFdC) was given the name gemcitabine, laboratory studies were investigating the biochemical feature of its phosphorylation to demonstrate that the rate of accumulation of its triphosphate (dFdCTP, gemcitabine triphosphate) was saturated by 10 to 20 mol/L of exogenous gemcitabine. The first investigation that established the saturability for gemcitabine triphosphate accumulation was done in a cell line. This was extended to primary cells in vitro and in peripheralblood mononuclear cells (PBMCs) during therapy. In the clinic, when gemcitabine was administered at different doses during a standard 30-minute infusion, the rate of intracellular accumulation of gemcitabine triphosphate was highest when plasma gemcitabine was about 20 mol/L. Pharmacokinetic studies during therapy further established that such gemcitabine levels (20 mol/L) were achieved in plasma with a dose rate of 10 mg/m/ min, hence coining the term fixed dose rate (FDR) of gemcitabine. At this dose rate, a continuous increase in gemcitabine triphosphate concentration was observed for up to 18 hours. For the FDR delivery, generally 1,000 or 1,500 mg/m is infused during 100 or 150 minutes, respectively. In contrast, a phase I dose escalation trial established that the maximum tolerated dose of gemcitabine was 2,200 mg/m/wk for 3 weeks when administered as a 30-minute infusion. This half-hour administration has been the most used schedule and has been termed the standard (STD) dose schedule of gemcitabine. Saturation kinetics that led to a defined dose rate have been observed for cytarabine, a prototype of nucleoside analogs. The reason that phosphorylated moieties of cytarabine (cytarabine triphosphate) and gemcitabine (gemcitabine diphosphate, gemcitabine triphosphate) are the active and cytotoxic metabolites of these nucleoside analogs further underscored the importance of the dose rate of infusion during clinical trials. Although several clinical trials have been performed with gemcitabine, either with STD doses or with FDR schedules, only a few were designed to ask two specific questions: First, is there a greater accumulation of gemcitabine triphosphate with a FDR of gemcitabine; and, second, does this result in a better clinical outcome (Table 1).

3544 POSTER Phase I study of gemcitabine as a fixed dose rate infusion and S-1 combination therapy in gemcitabine-refractory pancreatic cancer patients.

Purpose There is no standard regimen for gemcitabine (Gem)-refractory pancreatic cancer (PC) patients. In a previous phase II trial, S-1 was found to exhibit marginal efficacy. Gem administration by fixed dose rate infusion of 10 mg/m2/min (FDR-Gem) should maximize the rate of intracellular accumulation of gemcitabine triphosphate and might improve clinical efficacy. We conducted the phase I/II of FDR-Gem and S-1 (FGS) in patients with Gem-refractory PC.

Intracellular Accumulation and Cytotoxicity of Doxorubicin with Different Pharmaceutical Formulations in Human Cancer Cell Lines

The structure of both carrier and anticancer drug affects the intracellular fate of a transported drug. The study investigated in vitro intracellular accumulation and cytotoxic activity of doxorubicin-loaded solid lipid nanoparticles (SLN), doxorubicin in pegylated liposomes (Caelyx) and free doxorubicin. Intracellular doxorubicin levels and cytotoxic activity were determined by high performance liquid chromatography with fluorescence detection, and by the trypan blue dye exclusion assay, respectively. Doxorubicin-loaded SLN inhibited cell growth more strongly than either free or liposomal doxorubicin, in human colorectal adenocarcinoma, HT-29, retinoblastoma Y79, and glioblastoma U373 cell lines. The IC50 values for doxorubicin-loaded SLN were significantly lower after 24 h exposure than those for free doxorubicin in all cell lines; after 48 h exposure they were lower than those for liposomal doxorubicin in HT-29 and Y79 cells. The enhanced cytotoxic activity of doxorubicin-loaded SLN was associated with increased drug incorporation in cells: intracellular doxorubicin levels were significantly enhanced after exposure to drug-loaded SLN versus either free or liposomal drug. Rate of intracellular accumulation and cytotoxic activity also differed among different cell lines; in particular, cells of epithelial origin were found to be more sensitive to doxorubicin-loaded SLN. In conclusion, the greater sensitivity of HT-29, Y79, and U373 cells to doxorubicin-loaded SLN than to the other drug formulations may be due to the capability of the delivery system to enhance drug action, through a marked uptake and accumulation of SLN within the cell.

Continuous but not intermittent administration of growth hormone to hypophysectomized rats increases apolipoprotein-E secretion from cultured hepatocytes.

Hypophysectomy of female rats has been shown to decrease the serum levels of apolipoprotein E (apoE). Continuous but not intermittent administration of GH to hypophysectomized (HX) rats increases these levels to those of normal rats, indicating that the sexually dimorphic secretion of GH is important in the regulation of apoE metabolism. In this study, these effects of GH were further investigated by studying the biosynthesis and secretion of apoE from isolated hepatocytes. Hepatocytes were isolated from HX rats as well as from HX rats that had received hormonal treatment with T4 and cortisol (C) or T4 and C together with GH given either as two daily sc injections (GH x 2) or as a continuous infusion (GHc). Hypophysectomy decreased by 47% the amount of apoE present in the culture medium after a 4-h incubation. Treatment of HX rats with T4 and C alone or in combination with GH x 2 did not influence the amount apoE present in the medium, whereas treatment with T4, C, and GHc increased the amount of apoE to that of normal controls. The different levels of apoE in the medium was not due to differences in the disappearance of apoE, indicating that it was caused by changes in the rate of apoE secretion. Consistent with this, hypophysectomy decreased the rate of intracellular accumulation of apoE measured by incubation of the cells with [35S]methionine for 0, 8, and 20 min. Treatment with T4, C, and GHc increased the rate of accumulation, but T4, C, and GH x 2 had no effect. The differences in the initial rate of intracellular accumulation of apoE were not due to variations in apoE messenger RNA pools or to differences in the degradation of apoE at a step early in the secretory pathway. These results indicate that the differences in the initial rate of accumulation of apoE results from differences in the translational rate. The major amount of apoE that was secreted to the medium appeared in the high-density lipoprotein fraction, whereas small amounts were present in the very-low-density lipoprotein fraction (VLDL). Hypophysectomy decreased the amount of newly secreted apoE in the VLDL fraction. Only therapy with T4, C, and GHc could restore the normal distribution of apoE in the VLDL fraction. In conclusion, the results indicate that the secretory pattern of GH is involved in the regulation of the apoE secretion by influencing the rate of translation.

Mass and in situ Molar Activity of Tyrosine Hydroxylase in the Median Eminence

The effects of thyroidectomy and thyroid hormone replacement on the mass and in situ molar activity of tyrosine hydroxylase (TH) in the median eminence (ME) and superior cervical ganglia (SCG) of male rats were investigated. The tissue specificity of these effects were evaluated by comparing the ME with the superior cervical ganglion (SCG). All animals were thyroparathyroidectomized (Tx) or sham Tx. Tx rats were treated daily for 3 weeks with 0.15 M NaCl (solvent vehicle) or L-thyroxine (T4). Two doses of T4, 10 and 100 micrograms/day/kg BW, were used. Sham Tx rats were treated with 0.15 M NaCl. All animals were studied on the day following the last treatment. The mass of TH was determined using an immunoblot assay, and the in situ activity of TH was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine (DOPA) after administration of an inhibitor of DOPA decarboxylase activity. In the ME, thyro-parathyroidectomy resulted in a 40% increase in the mass and a 100% increase in the in situ molar activity of TH over that of sham Tx rats. Compared to Tx animals given 0.15 M NaCl, Tx rats treated with a low dose of T4 (10 micrograms/day/kg BW) had a reduced quantity of TH in the ME, but the molar activity of the enzyme was increased. Treatment of Tx rats with a high dose of T4 (100 micrograms/day/kg BW) restored TH mass but not the in situ activity of TH in the ME to the level seen in sham Tx rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin does not mediate activation of glucose transport by muscle contraction

The purpose of this study was to evaluate the report that bradykinin is the "muscle activity hypoglycemia factor" responsible for the activation of glucose transport that occurs in response to muscle contractile activity. Stimulation of rat epitrochlearis muscles to contract resulted in approximately a fourfold increase in the rate of intracellular accumulation of the nonmetabolizable glucose analog 3-O-methylglucose. Incubation of the muscles with high concentrations of aprotinin (Trasylol), a polypeptide inhibitor of kallikrein which blocks formation of kinins, did not inhibit the activation of sugar transport by contractile activity. Furthermore incubation of muscles with bradykinin did not have a stimulatory effect on the uptake of 3-methylglucose either at a physiological concentration or at high concentrations. These results provide no support for the claims that aprotinin prevents the activation of sugar transport in muscle by contractile activity or that bradykinin is the muscle activity hypoglycemia factor.

EFFECTS OF INSULIN ON THE INTACT LEVATOR ANI MUSCLE OF THE RAT

ABSTRACT The effects of insulin on the rate of intracellular accumulation and incorporation into the protein of some normal amino acids (glycine-3H, leucine-14C and valine-14C) in the intact levator ani muscle of the rat were studied in vitro. The effect of puromycin on the accumulation of these amino acids as well as on the accumulation of the model amino acid α-aminoisobutyric acid-14C (AIB-14C) was examined. In addition the effect of insulin on the incorporation of adenine-14C into the muscle RNA as well as on the effect of actinomycin D added to the incubation medium were also investigated. The insulin stimulated AIB-14C transport is described by the formulation of Michaelis-Menten and approximate values of Km and Vmax are calculated for this transport in the levator ani muscle. Insulin was found to stimulate the intracellular accumulation of glycine-3H and AIB-14C, while a significant decrease was found in the accumulation of leucine-14C and valine-14C. Insulin also stimulated the incorporation of glycine-3H and leucine-14C into the muscle protein. When puromycin was added to the medium, no radioactivity was found in the protein fraction and insulin then stimulated the intracellular accumulation of glycine-3H, leucine-14C and AIB-14C. A slight but significant stimulation with insulin was found on the incorporation of adenine-14C into the RNA-fraction of the levator ani muscle. The addition of actinomycin D was found to inhibit this effect of insulin, but did not change the insulin stimulation of the AIB-14C accumulation. The results are discussed in relation to our present knowledge of the different in vitro effects of insulin.