Phase I and pharmacokinetic (PK) study of MAG-CPT (PNU 166148): a polymeric derivative of camptothecin (CPT)

Polymeric cytotoxic conjugates are being developed with the aim of preferential delivery of the anticancer agent to tumour. MAG-CPT comprises the topoisomerase I inhibitor camptothecin linked to a water-soluble polymeric backbone methacryloylglycynamide ( average molecular weight 18 kDa, 10% CPT by weight). It was administered as a 30-min infusion once every 4 weeks to patients with advanced solid malignancies. The objectives of our study were to determine the maximum tolerated dose, dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document responses to this treatment. The starting dose was 30 mgm(-2) (dose expressed as mg equivalent camptothecin). In total, 23 patients received 47 courses at six dose levels, with a maximum dose of 240 mgm(-2). Dose-limiting toxicities were myelosuppression, neutropaenic sepsis, and diarrhoea. One patient died after cycle 1 MAG-CPT at the maximum dose. The maximum tolerated dose and dose recommended for further clinical study was 200 mgm(-2). The half-lives of both MAG-CPT and released CPT were prolonged (46 days) and measurable levels of MAG-CPT were retrieved from plasma and urine 4 weeks after treatment. However, subsequent pharmacodynamic studies of this agent have led to its withdrawal from clinical development

A phase I study with MAG-camptothecin intravenously administered weekly for 3 weeks in a 4-week cycle in adult patients with solid tumours

In MAG-camptothecin (MAG-CPT), the topoisomerase inhibitor camptothecin is linked to a water-soluble polymer. Preclinical experiments showed enhanced antitumour efficacy and limited toxicity compared to camptothecin alone. Prior phase I trials guided the regimen used in this study. The objectives were to determine the maximum tolerated dose, dose-limiting toxicities, safety profile, and pharmacokinetics of weekly MAG-CPT. Patients with solid tumours received MAG-CPT intravenously administered weekly for 3 weeks in 4-week cycles. At the starting dose level ( 80 mg m(-2) week(-1)), no dose-limiting toxicities occurred during the first cycle (n = 3). Subsequently, three patients were enrolled at the second dose level ( 120 mg m(-2) week(-1)). Two of three patients at the 80 mg m(-2) week(-1) cohort developed haemorrhagic cystitis ( grade 1/3 dysuria and grade 2/3 haematuria) during the second and third cycles. Next, the 80 mg m(-2) week(-1) cohort was enlarged to a total of six patients. One other patient at this dose level experienced grade 1 haematuria. At 120 mg m(-2) week(-1), grade 1 bladder toxicity occurred in two of three patients. Dose escalation was stopped at 120 mg m(-2) week(-1). Cumulative bladder toxicity was dose-limiting toxicity at 80 mg m(-2) week(-1). Pharmacokinetics revealed highly variable urinary camptothecin excretion, associated with bladder toxicity. Due to cumulative bladder toxicity, weekly MAG-CPT is not a suitable regimen for treatment of patients with solid tumours

A phase I and pharmacokinetic study of MAG-CPT, a water-soluble polymer conjugate of camptothecin

Polymeric drug conjugates are a new and experimental class of drug delivery systems with pharmacokinetic promises. The antineoplastic drug camptothecin was linked to a water-soluble polymeric backbone (MAG-CPT) and administrated as a 30 min infusion over 3 consecutive days every 4 weeks to patients with malignant solid tumours. The objectives of our study were to determine the maximal tolerated dose, the dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document anti-tumour activity. The starting dose was 17 mg m−2 day−1. Sixteen patients received 39 courses at seven dose levels. Maximal tolerated dose was at 68 mg m−2 day−1 and dose-limiting toxicities consisted of cumulative bladder toxicity. MAG-CPT and free camptothecin were accumulated during days 1–3 and considerable amounts of MAG-CPT could still be retrieved in plasma and urine after 4–5 weeks. The half-lives of bound and free camptothecin were equal indicating that the kinetics of free camptothecin were release rate dependent. In summary, the pharmacokinetics of camptothecin were dramatically changed, showing controlled prolonged exposure of camptothecin. Haematological toxicity was relatively mild, but serious bladder toxicity was encountered which is typical for camptothecin and was found dose limiting

Clathrin and LRP-1-Independent Constitutive Endocytosis and Recycling of uPAR

Background: The urokinase receptor (uPAR/CD87) is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA) and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. Methodology/Principal Findings: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. Conclusions/Significance: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism couple

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

A theoretical study of the structure of big endothelin

Protein structure prediction and molecular dynamics simulations have been applied for elaborating the three dimensional structure of Big Endothelin (bigET). BigET is 38-amino acid peptide which is converted, by proteolytic cleavage, into Endothelin, the most potent and long lasting endothelium derived contracting factor identified up to date. The intervention of a specific, yet unknown, protease has been evoked. The determination of bigET tertiary structure will contribute to elucidate its proteolytic conversion. In-vitro experimental data on proteolytic fragmentation of bigET in the presence of known proteases and protein homogenates from endothelial cells have been considered for testing the proposed structure

A THEORETICAL-STUDY OF THE STRUCTURE OF BIG ENDOTHELIN

Protein structure prediction and molecular dynamics simulations have been applied for elaborating the three dimensional structure of Big Endothelin (bigET). BigET is 38-amino acid peptide which is converted, by proteolytic cleavage, into Endothelin, the most potent and long lasting endothelium derived contracting factor identified up to date. The intervention of a specific, yet unknown, protease has been evoked. The determination of bigET tertiary structure will contribute to elucidate its proteolytic conversion. In-vitro experimental data on proteolytic fragmentation of bigET in the presence of known proteases and protein homogenates from endothelial cells have been considered for testing the proposed structure

Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin.

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex