Hyaluronan-Irinotecan improves progression-free survival in 5-fluorouracil refractory patients with metastatic colorectal cancer: a randomized phase II trial.

PURPOSE: The objective of this study was to conduct a randomised phase II study in second-line metastatic colorectal cancer with the purpose of confirming preliminary clinical data indicating that the formulation of irinotecan with the drug carrier, hyaluronan (HA) reduced toxicity of the drug. METHODS: Irinotecan-naïve patients were randomized to receive either irinotecan (350 mg/m(2)) or HA-Irinotecan (HA 1,000 mg/m(2) and irinotecan at 350 mg/m(2)) every 3 weeks for a maximum of eight cycles. RESULTS: Seventy-six patients (41 HA-Irinotecan and 35 irinotecan-alone) were enrolled. There was no significant difference in any individual, or overall, grade 3 or 4 toxicity. There was a trend for increased diarrhea in the HA-Irinotecan-treated patients (20 versus 9%; P = 21), potentially explained by a disproportionate number of baseline toxicity-associated risk factors in this treatment group. The median number of cycles completed was six for HA-Irinotecan patients and two for irinotecan-alone patients (P = 0.005). When compared to the control arm, HA-Irinotecan patients had a significantly longer median progression-free survival of 5.2 versus 2.4 months (P = 0.017) and time to treatment failure (4 vs. 1.8 months; P = 0.007). Median overall survival was 10.1 months for HA-Irinotecan compared to 8.0 months for irinotecan patients (P = 0.196). CONCLUSION: Further studies are required to define the safety of the formulation of irinotecan with HA. While this study was not adequately powered to demonstrate survival differences, these phase II data indicated HA-Irinotecan to be a promising therapy demonstrating improved efficacy compared to irinotecan-alone.

Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney.

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

The analysis and characterisation of immuno-unreactive urinary albumin in healthy volunteers.

OBJECTIVES: To compare the analysis of different forms of intact albumin in urine from healthy volunteers. To determine contamination by common non-albumin proteins on HPLC analysis of urinary albumin and of purified immuno-unreactive albumin. DESIGN AND METHODS: Overnight urine samples collected from healthy volunteers were analysed for total albumin (immunoreactive plus immuno-unreactive) by HPLC and densitometry following native PAGE separation and for immunoreactive albumin by RIA. The contamination by non-albumin proteins of the HPLC analysis of urinary albumin and of immuno-unreactive albumin preparations was determined by ELISA. Immuno-unreactive albumin was tested for Co2+-binding capacity. RESULTS AND CONCLUSIONS: HPLC analysis of healthy urine generates higher ACR values than immunological methods due to the presence of immuno-unreactive albumin. Immuno-unreactive albumin cannot be accounted for by the non-albumin urinary proteins tested. Isolated immuno-unreactive albumin is not recognised by antibodies to common urinary proteins or by an array of anti-albumin antibodies and behaves like serum albumin in terms of HPLC elution, native PAGE migration, and cobalt ion binding.

Reversible angiotensin II-mediated albuminuria in rat kidneys is dynamically associated with cytoskeletal organization.

Angiotensin II (ANG II) is intimately involved in normal renal function, and is estimated to exist at a normal physiological range of 6-10 nM within the renal tubules. The potential role that intrarenal ANG II may play in renal disease was assessed by perfusing isolated rat kidneys with or without excess intratubular levels of ANG II, which may mimic changes in the intrarenal RAS under pathological conditions. The effects of increased systemic ANG II were also determined by infusing rats with ANG II by osmotic pump. In isolated perfused kidneys, ANG II significantly and specifically increased the fractional clearance of albumin to clinical levels, as determined by using radiolabelled albumin. This effect was reversible, as removing ANG II from the perfusate caused the albumin fractional clearance to decrease to pre-ANG II exposure levels. The increase in fractional clearance of albumin was not correlated with renal hemodynamic changes, nor glomerular permeability alterations as measured by the fractional clearance of 36 A Ficoll and immunoglobulin G. Immunochemical analysis using anti-alpha-tubulin antibody of perfused kidney sections revealed that ANG II caused a marked disruption of tubular epithelial cytoskeletal components, through disassembly and reorganization of alpha-tubulin. This disruption was reversible. In vivo, osmotic pump delivery of ANG II at less potent dosage caused a proteinuria (Biuret) and an albuminuria (radioimmunoassay) in rats, from as early as 2 days after pump implantation. These results demonstrate that ANG II may reversibly induce clinical levels of albuminuria. These data point to an important role for renal tubules and the intratubular lumen concentrations of ANG II in the renal processing of albumin.

Albumin fragments in normal rat urine are derived from rapidly degraded filtered albumin.

Filtered albumin is excreted as a heterogeneous population of albumin-derived molecules resulting from degradation during renal passage. In order to understand the dynamics of this degradation process, albumin clearance was studied over a short-term (minutes) and a long-term (7 days) by both radioactivity and radioimmunoassay. The radiolabelled material in the urine was also analysed extensively by using size exclusion chromatography, size selective filtration and high performance liquid chromatography. These studies demonstrate that during renal passage, albumin degradation to fragments in the size range of 500-10,000 occurs in a matter of minutes. The fragments are not detected by using radioimmunoassay. Steady state excretion rates or fractional clearance of radiolabelled albumin occur over a similar time period. Both rates of degradation and approach to steady-state clearance, while rapid, were considerably slower than the transit time for molecules in the Bowman's capsule and early tubular lumen. The results are consistent with an extremely rapid lysosomal uptake of filtered albumin, and degradation and regurgitation of the albumin-derived peptide fragments into the tubular lumen prior to excretion.

Urinary clearance of albumin is critically determined by its tertiary structure.

The excretion of serum albumin in the urine is considered the net result of renal glomerular filtration and tubular uptake. During routine experiments, we observed that a batch of tritium-labeled albumin yielded anomalous results, being excreted in the urine of isolated perfused kidneys at 10 times the rate of normal tritiated albumin. This anomalous albumin, when simultaneously studied with normal carbon 14-labeled albumin, exhibited 10 times greater excretion than normal [(14)C]albumin. Anomalous albumin could not be reversed to normal albumin by means of conditioning with blood. In vivo clearances of anomalous albumin could not be quantitated because anomalous albumin is degraded during circulation. Anomalous albumin appeared to have the same molecular size (as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary electrophoresis, and gel chromatography) and isoelectric-point profile (2-dimensional electrophresis) as normal albumin. Normal albumin could be transformed to anomalous albumin with alkali/heat treatment. Reverse-phase high-pressure liquid chromatography analysis of fragments from tryptic digests of anomalous albumin, alkali/heat-treated albumin, and normal albumin suggest that anomalous albumin and alkali/heat-treated albumin have altered tertiary structure, possibly as a result of denaturation and disulfide exchange. These studies show that the tertiary structure of albumin, beyond simple size and charge, is a critical determinant for albumin processing by the kidney and suggest that a specific albumin-recognition event by the kidneys is critical to normal renal handling of albumin.