Phase I clinical and pharmacokinetic study of titanocene dichloride in adults with advanced solid tumors.

This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the dose-limiting toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4. Forty patients with refractory solid malignancies were treated with a total of 78 courses. Using a modified Fibonacci scheme, 15 mg/m2 initial doses of titanocene dichloride were increased in cohorts of three patients up to level 11 (560 mg/m2) if DLT was not observed. The maximum tolerated dose was 315 mg/m2, and nephrotoxicity was DLT. Two minor responses (bladder carcinoma and non-small cell lung cancer) were observed. The pharmacokinetics of plasma Ti were assessed in 14 treatment courses by atomic absorption spectroscopy. The ratio for the area under the curve(0-infinity) in plasma and whole blood was 1.2. The following pharmacokinetic parameters were determined for plasma, as calculated in a two-compartment model: biological half-life t1/2beta in plasma was 22.8+/-11.2 h (xh +/- pseudo-SD), peak plasma concentration cmax approximately 30 microg/ml at a dose of 420 mg/m2, distribution volume Vss= 5.34+/-2.1 L (xa +/- SD), and a total clearance CItotal = 2.58+/-1.23 ml/min (xa +/- SD). There was a linear correlation between the area under the curve(0-infinity) of Ti in plasma and the titanocene dichloride dose administered with a correlation coefficient r2 of 0.8856. Plasma protein binding of Ti was in the 70-80% range. Between 3% and 16% of the total amount of Ti administered were renally excreted during the first 36 h. The recommended dose for Phase II evaluation is 240 mg/m2 given every 3 weeks with i.v. hydration to reduce renal toxicity.

Proteomic prediction of disease outcome in cancer : clinical framework and current status.

Better than gene sequencing or quantitative amplification, proteomics tools allow the study of tumor phenotype. Indeed, most current prognostic tests in cancer (carcinoembryonary antigen [CEA], prostate-specific antigen [PSA], CA 19-1, CA 125, alpha-fetoprotein [AFP], etc.) are based on the detection and quantification of single proteins in body fluids. However, a common characteristic of these tests is their relatively low predictive value, so that they are usually complemented with other procedures such as biopsy and/or endoscopy. Recently, improved analytical and bioinformatics tools have driven the attention on pattern recognition approaches rather then single-marker tests for prognostic forecasting. It is expected that predicting metastasization on the basis of tumoral protein patterns will soon be a reality. However, currently available technologies either limit the number of proteins that can be analyzed simultaneously or they are expensive, difficult, and time-consuming. Moreover, the tools adapted for expression proteomics might not be the same as those for prognostic studies that require investigation of protein function over time. We believe that clinical proteomics research designed within a precise clinical and pathology framework should be strongly supported, since many prognostic factors are determined not by the tumor itself, but by the patient, the treatment and the environment.

Early urinary markers of target nephron segments as studied in cadmium toxicity.

A number of chemicals may adversely affect one or more of the anatomical structures of the kidney, such as the glomerulus, the tubular apparatus, the medullary, or interstitial cells. To recognize subclinical renal dysfunction, a battery of new, non-invasive tests was applied in comparison to established ones. The study on cadmium exposed subjects, performed within the framework of a collaborative European research project, exemplifies the concept of target selectivity within a nephron. One hundred seventy-two subjects were classified according to urinary cadmium excretion as controls (< 1.5 micrograms/g creatinine), or subjects with moderate or high cadmium body burden (1.5 to 5 micrograms/g creatinine, > 5 micrograms/g creatinine). Twenty-six urinary analytes (such as serum derived proteins, tubular enzymes, eicosanoids) and four plasma markers, related to the function or integrity of specific nephron segments, were investigated in a cross-sectional study. The group with the moderate cadmium body burden showed alterations of proximal tubular integrity, that is, increased excretion of tubular brush-border antigens. The group with higher cadmium body burden revealed an involvement of the whole nephron. The most prominent quantitative changes were found for the glomerular markers high molecular weight proteins, and thromboxane B2 and for the proximal tubular markers retinol binding protein, alpha 1-microglobulin, N-acetyl-beta-D-glucosaminidase, and the intestinal alkaline phosphatase. A diagnostic approach to screen for nephrotoxicity due to environmental hazards like cadmium should include proximal tubular markers (alpha 1-microglobulin and tubular enzymes, that is, intestinal alkaline phosphatase) but the measurement of glomerular markers is also advisable.

The role of proteomics in the diagnosis and outcome prediction in colorectal cancer.

Colorectal cancer is the second most frequent cancer in Western countries. Exogenous factors play a major role in the aetiology of sporadic colorectal cancer representing about 90% of all cases, hereditary cancers accounting for about 10% of patients. Thus, in the large majority of cases, cell dysfunction in CRC results from multiple rather than single, gene interactions. Numerous cellular events and environmental influences modify gene expression or post-translational protein modifications. Changes like glycosylation of proteins and lipids which are a common feature in colorectal cancer and influence cancer cell behaviour, cannot be directly detected by genetic studies. Better than genomics studies, functional proteomics studies allow the investigation of environmental factors over time, allowing the monitoring of metabolic responses to various stimuli. However, proteomics studies also have several drawbacks: a) current tools only allow narrow-range analyses, b) identification of proteins of interest remains cumbersome, c) protein studies address multiple compounds of high complexity, d) large amount of proteins are necessary to allow analysis, e) protein research require specific tools, e.g. tagged antibodies, that first have to be developed. Some protein tests are already in application for CRC: a classical prognostic test in colorectal cancer is based on the detection and quantification of a single protein (CEA) in body fluids. Recently, a screening assay based on APC protein truncation test has also been proposed. However, studies linking large protein expression patterns with clinical outcome in colorectal cancer are still in their infancy. To be able to predict occurrence of disease, and treatment outcome, more studies on genotype-phenotype correlations are needed both in sporadic and in hereditary colorectal cancer.

Comparative study of the acute nephrotoxicity from standard dose cisplatin +/- ifosfamide and high-dose chemotherapy with carboplatin and ifosfamide.

The nephrotoxic effects of different platinum compounds based combination chemotherapies were compared. Chemotherapy consisted of either cisplatin fractionated over 5 days (5 x 20 mg/m2) or given as a single-day infusion (1 x 50 mg/m2) plus ifosfamide (4 g/m2) or high-dose chemotherapy was applied including carboplatin (3 x 500 mg/m2) and ifosfamide (3 x 4 g/m2) fractionated over three consecutive days. Conventional parameters such as serum creatinine and glomerular filtration rate (GFR), as well as urinary protein excretion of N-acetyl-beta-D-glucosaminidase (NAG)) and alpha 1-micro-globulin were assessed in 52 patients. Fractionation over 5 days without adding other nephrotoxic agents, i.e. ifosfamide, prevented decreases in GFR following cisplatin, whereas the combination of conventional dose cisplatin and ifosfamide, given as a single-day infusion, and high-dose carboplatin/ifosfamide yielded a pronounced fall of GFR. All groups showed increases in the urinary excretion levels of serum derived proteins and NAG, but with significant differences; about 2 to 3-fold for 5-days cisplatin, 3 to 5-fold for single-day cisplatin/ifosfamide, and 20 to 35-fold for high-dose chemotherapy. Thus, conventional approaches can reduce but not prevent the nephrotoxicity of cisplatin-based chemotherapy. In particular, high-dose chemotherapy regimens including carboplatin and ifosfamide are associated with comparable or even higher nephrotoxicity to single-day cisplatin/ifosfamide. In the light of the long-term consequences of persistent renal damage prevention of nephrotoxicity should be further improved.

Altered levels of urinary prostanoids in lead-exposed workers.

This paper describes the alteration of the urinary excretion of prostanoids in workers occupationally exposed to lead. For this purpose, the following groups were studied: Group 1 (n = 62): controls; Group 2 (n = 29): risk group; and Group 3 (n = 69): exposed group. Urine samples were collected for prostanoid analysis and lead blood levels were analyzed. Our results demonstrate that urinary excretion of prostanoids is already altered even at levels of lead in blood = 200 micrograms/l. Owing to their sensitivity, urinary prostanoids could be useful markers of early renal changes associated with lead exposure. However, underlying mechanisms should be elucidated and the health significance of such changes should be assessed before any conclusion is drawn.

Isolated glomeruli of the Atlantic hagfish Myxine glutinosa as an alternative in vitro model to study glomerular protein metabolism in pharmaco-toxicology of anticancer drugs.

This study was designed to validate an alternative in vitro system with isolated glomeruli of the Atlantic hagfish Myxine glutinosa as a model to study alterations in glomerular protein metabolisms in pharmaco-toxicology of anticancer drugs. A morphometric characterization of the glomeruli of Myxine glutinosa reveals a calculated glomerular volume of 180 nl/glomerulus. The glomerular extracellular volume, measured as inulin space, is 38.5 nl/glomerulus. Total glomerular protein content of Myxine glutinosa amounts to 3.56 micrograms/glomerulus and total DNA content to 0.44 microgram/glomerulus. Metabolic properties, estimated as glomerular protein synthesis, are comparable with mammalian glomeruli. The glomeruli of Myxine glutinosa are viable in a tissue culture for up to 12 hr. The incorporation rate of radiolabeled amino acids into glomerular, acid-precipitable proteins is almost identical to that of rats (e.g. Myxine glutinosa 1091 +/- 98 DPM/micrograms DNA vs. rat 1340 +/- 84 DPM/micrograms DNA after 4 hr incubation). To evaluate how nephrotoxic substances affect glomerular metabolism in this model, the anticancer drug Adriamycin (ADR) was used to experimentally induce a glomerular lesion. ADR caused an increase in glomerular protein synthesis in isolated glomeruli of Myxine glutinosa, which is in accordance with data found in rats. Cisplatin, in contrast, known to mainly interfere with tubular integrity, had no effect on glomerular protein synthesis, confirming the specificity of the model. The isolated glomeruli of Myxine glutinosa are suggested as a valid alternative multicellular in vitro system for studying alterations in glomerular metabolism under pharmaco-toxicological conditions and for the evaluation of specific target-cell toxicity of selected nephrotoxins.

Risk assessment of nephrotoxicity of cadmium.

Cadmium as an environmental or occupational toxin has been well studied. Exposure can fairly easily be detected by analysis of cadmium in biological material. Different routes of cadmium uptake, such as via airborne particles, cigarette smoke or contaminated food, have been identified. Urinary concentrations associated with renal effects vary, depending on urinary marker of effect, and are as low as 2 micrograms cadmium/g creatinine. Different segments of the nephron are affected by cadmium. Urinary enzymes and serum derived proteins, mainly low-molecular weight proteins, as well as eicosanoids or glycosaminoglycans are among the tubular or glomerular markers of effect. Predisposing factors, such as smoking, dietary habits and low body iron stores have been described. It is concluded that even for low level cadmium exposure effects can be detected with sensitive diagnostic tools.

Changed excretion of urinary proteins and enzymes by chronic exposure to lead.

Fifteen various serum and urine parameters were evaluated as indicators of renal alterations induced by lead in 82 male workers of a battery plant chronically exposed to lead (median of blood lead concentration: 2.03 mumol/l). The control group comprised 44 non-exposed healthy volunteers (0.34 mumol/l). High-molecular-mass proteins (transferrin, immunoglobulin G (IgG), (albumin)) were determined in urine as markers of glomerular integrity; low-molecular-weight proteins and parenchymal enzymes (alpha 1-microglobulin, beta 2-microglobulin, retinol-binding protein, lysozyme, ribonuclease, N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), alkaline phosphatase (AP), gamma-glutamyltransferase (GGT)) as indicators of changes in the proximal tubule; Tamm-Horsfall glycoprotein and kallikrein as markers of the distal tubule. There was a positive correlation between tubular indicators and blood lead concentration as well as the erythrocyte protoporphyrin (EPP). About 30% of the lead-exposed workers showed an increased excretion of alpha 1-microglobulin, NAG, ribonuclease, and/or Tamm-Horsfall protein, whereas the glomerular indicators remained unchanged. The combined determination of NAG and alpha 1-microglobulin in urine could be helpful in the early detection of lead-induced changes in the nephron.

Evaluation of late nephrotoxicity in long-term survivors of Hodgkin's disease.

Chemotherapy (Ctx) and/or radiotherapy (Rtx) are effective in the treatment of Hodgkin's disease (HD) but potentially involve late toxicities, including nephrotoxic side effects. Therefore a follow-up study has been performed to screen patients for late signs of an impaired tubular or glomerular function and to correlate data of renal function with type of therapy and cumulative doses of cytotoxic agents applied. 81 patients in complete remission for at least 2 years and a median follow-up of 96 (39-304) months, and 53 controls were examined. Clinical routine parameters such as creatinine and electrolytes were determined. A differentiation of proteinuria into the albumin, high molecular weight (HMW) and low molecular weight (LMW) fractions made it possible to assess glomerular and tubular function based on the LMW/HMW ratio. The structural protein fibronectin served as an additional, sensitive marker of glomerular integrity. Routine parameters of kidney function did not show any signs of late nephrotoxicity. However, patients treated for HD had a higher ratio of LMW/HMW in comparison to the group of healthy volunteers (p < 0.01), indicating subclinical tubular renal damage. When the cutoff for tubular damage was defined as LMW/HMW > 1.5, 50% of the patients treated with combined modality, and 42 and 37% of the patients with Ctx or Rtx alone had subclinical tubular alterations, respectively. A tendency towards a higher prevalence of subclinical tubular changes was observed in patients with higher cumulative doses of methotrexate or ifosfamide and in patients with combined Ctx and Rtx with radiation fields involving the renal area. Changes in glomerular function were not observed. It is concluded that treatment of HD is not associated with clinically apparent long-term impairment of renal function but can lead to subclinical alterations. Further clinical implications of these subclinical tubular alterations cannot be assessed at present. A differentiation of proteinuria does not have to be performed routinely but might be useful in the follow-up of selected patients with an increased risk.

Cathepsin B and L in isolated proximal tubular segments during acute and chronic proteinuria.

Acute and chronic proteinuria were studied in rats, using lysosomal cathepsin B and L as marker enzymes for tubular protein degradation. The activity of cathepsin B and L has been determined in microdissected segments S1, S2 and S3 of the proximal tubule by an ultramicroassay. Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as a substrate. In normoproteinuric Sprague-Dawley rats, induction of acute unselective glomerular proteinuria with Adriamycin (5 mg/kg body weight) revealed a moderate activity increase of cathepsin B and L in the S2 segment, reaching 12.6 +/- 5.6 versus 8.6 +/- 4.2 pmol.mm-1.min-1 in controls. In contrast, Munich Wistar Frömter (MWF) rats, that are characterized by a genetically determined, chronically elevated glomerular protein excretion, showed a very high activity of cathepsin selectively in S2 of 25.0 +/- 12.1 pmol.mm-1.min-1. Acute proteinuria induced by Adriamycin in chronic proteinuric MWF rats could increase cathepsin activity in the S3 segment only, showing 12.0 +/- 8.3 versus 6.8 +/- 4.0 pmol.mm-1.min-1 in MWF control rats. In conclusion, chronically increased protein filtration changes the functional reserve capacity of the proximal tubule. While acutely induced glomerular proteinuria in normoproteinuric rats stimulates lysosomal proteolytic activity mainly in S2 segment, chronic proteinuric MWF rats may display already a maximally stimulated cathepsin activity in this segment probably due to long-term increased tubular protein load. In case of acute elevation of chronic proteinuria, the consecutive S3 segment shows increased lysosomal function for protein conservation.

Cisplatin nephrotoxicity and protection by silibinin.

BACKGROUND: The anticancer drug cisplatin is know to have toxic side-effects on different segments of the nephron. The flavonoid silibinin has previously been shown to be protective in models of hepatotoxicity. The aim of the present study was to evaluate, whether silibinin can also ameliorate alterations in renal glomerular and tubular function and tubular morphology induced by cisplatin. METHODS: In a rat model renal damage was induced by a single injection of cisplatin (5 mg/kg body weight). The protective effects of silibinin were studied in rats that received the flavonoid (200 mg/kg body weight, i.v.) 1 h prior to the administration of cisplatin. Kidney function was monitored by analysing urinary markers of glomerular and tubular function over a period of 11 days. Animals of a second group, with identical treatment, were sacrificed 4 days after drug application for an evaluation of tubular morphology at the light-microscopical level. RESULTS: Administration of cisplatin caused a decline in kidney function within a day following treatment. Symptoms observed were for example decreases in creatinine clearance and increases in proteinuria, in the urinary activity of the proximal tubular enzymes alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase and in renal magnesium wasting. The effects of cisplatin on creatinine clearance and proteinuria were totally prevented by a pretreatment of the animals with silibinin. Impairment of proximal tubular function was ameliorated, that is enzymuria and magnesium wasting was less pronounced. Silibinin alone had no effect on kidney function. Treatment with silibinin distinctly diminished morphological alterations observed in the S3-segment of the proximal tubule 4 days after cisplatin administration. CONCLUSION: The effects of cisplatin on glomerular and proximal tubular function as well as proximal tubular morphology could totally or partly be ameliorated by silibinin. It is concluded the silibinin can act as a nephroprotectant and it is suggested that it could have beneficial effects on the kidney in clinical settings.

Filtration characteristics of the single isolated perfused glomerulus of Myxine glutinosa.

Using an in vitro microperfusion technique, the filtration characteristics of single isolated glomeruli of the Atlantic hagfish (Myxine glutinosa) were investigated. The suitability of the method as a model to study glomerular function was evaluated. Experiments with a protein-free perfusate led to a filtration coefficient Kf (0.189 +/- 0.181 nl x s-1 x mm Hg-1) that was in the same order of magnitude as determined in vitro for other vertebrates. Morphometric analysis on serial thin sections (1 microns) revealed a glomerular capillary surface (A) of 1.84 +/- 0.72 mm2. A linear relationship between glomerular diameter and A allowed the calculation of the hydraulic conductivity Lp (0.618 +/- 0.384 microliter x s-1 x mmHg-1 x cm-2). The data indicate that the isolated perfused glomerulus of M. glutinosa has filtration characteristics similar to higher vertebrates. The glomeruli of M. glutinosa, which in contrast to glomeruli of other animals are easy to dissect, are a suitable model for the study of glomerular filtration and permeability characteristics of vertebrates. An example of a possible application of the model is a study into the effects of adrenaline on glomerular filtration characteristics. Adrenaline led to increases in ultrafiltration pressure (PUF), single nephron glomerular filtration rate (SNGFR) and filtration fraction (FF); Kf remained unaffected.

Urinary proteins and enzymes as early indicators of renal dysfunction in chronic exposure to cadmium.

We tested the diagnostic sensitivity of various urinary analytes for detecting cadmium-induced nephropathy at an early stage. We investigated 73 healthy persons (control group 1) and individuals exposed to cadmium, either environmentally (n = 36, risk group 2) or occupationally (n = 62, exposed group 3). All data were related to limits of the central 95% reference intervals of the control group. The serum creatinine and ribonuclease values, indicators of the glomerular filtration rate, were not different in the three groups. In the exposed persons (group 3), proximal tubular indicators (low-M(r) proteins lysozyme, ribonuclease, retinol-binding protein, and alpha 1-microglobulin) were more often increased than the glomerular indices (higher-M(r) proteins transferrin, IgG, and albumin). Both the low-M(r) proteins and tubular enzymes were differently altered in their excretion rates. Alanine aminopeptidase, alkaline phosphatase, and N-acetyl-beta-D-glucosaminidase increased even in the risk group 2. alpha 1-Microglobulin was increased in the exposed persons whose cadmium excretion was < 5 mumol/mol creatinine. The combined determination of alpha 1-microglobulin and N-acetyl-beta-D-glucosaminidase exceeded the corresponding upper reference limits in 30% of group 2 and 39% of group 3. We recommend screening for these two analytes to detect cadmium-induced renal dysfunction at an early stage.

A randomized trial comparing the nephrotoxicity of cisplatin/ifosfamide-based combination chemotherapy with or without amifostine in patients with solid tumors.

This study evaluates the degree of kidney damage during cisplatin/ifosfamide-based combination chemotherapy and its possible prevention by amifostine. Thirty-one patients with solid tumors stratified according to pretreatment were randomized to receive VIP- or TIP-chemotherapy with or without amifostine (910 mg/m2) given as a short infusion prior to cisplatin. Chemotherapy consisted of cisplatin (50 mg/m2), ifosfamide (4 g/m2) and either etoposide (500 mg/m2) (= VIP) or paclitaxel (175 mg/m2) (= TIP) repeated at 3 weekly intervals. For all patients the glomerular filtration rate (GFR) measured by creatinine-clearance, serum creatinine, electrolytes and differential urinary protein/enzyme excretion were determined prior to, during and after each cycle. A total of 62 cycles of chemotherapy were evaluable. In the amifostine-group GFR was fully maintained after application of two cycles of chemotherapy, whereas in the control group a > 30%-reduction of median GFR (108 to 80 ml/min) was observed (p < 0.001). Patients receiving amifostine had a lower degree of high molecular weight proteins excretion indicating less glomerular damage. In both groups significant increases of tubular marker profiles peaking at day 3 after chemotherapy were observed with a nearly complete reversibility of these changes prior to the next chemotherapy cycle. The number of patients with low magnesium serum levels during treatment was 17% after amifostine application versus 69% in control patients. The results seem to indicate that treatment with amifostine can preserve GFR after application of two cisplatin/ifosfamide-based chemotherapy cycles. This may be advantageous if repetitive cycles of chemotherapy or subsequent administration of high dose chemotherapy is planned.

The use of reduced doses of amifostine to ameliorate nephrotoxicity of cisplatin/ifosfamide-based chemotherapy in patients with solid tumors.

This study evaluates the degree of kidney damage during cisplatin/ifosfamide-based combination chemotherapy and its possible prevention by amifostine. Thirty-one patients with solid tumors stratified according to pretreatment were randomized to receive cisplatin/ifosfamide-based chemotherapy with or without amifostine (1000 mg absolute) given as a short infusion prior to cisplatin. Chemotherapy consisted of cisplatin (50 mg/m2), ifosfamide (4 g/m2) and either etoposide (500 mg/m2) (VIP regimen) or paclitaxel (175 mg/m2) (TIP regimen) repeated at 3 weekly intervals. For all patients the glomerular filtration rate (GFR) measured by creatinine clearance, serum creatinine, electrolytes and differential urinary protein excretion were determined prior to, during and after each treatment cycle. A total of 62 cycles of chemotherapy were evaluable. In the amifostine arm the GFR was almost completely maintained after application of two cycles of chemotherapy (121 to 108 ml/min), whereas in the control group a 30% reduction of the GFR (105 to 80 ml/min) was observed. In both groups marked increases of glomerular and tubular marker profiles peaking at day 3 after chemotherapy were found with a nearly complete reversibility of these changes prior to the next chemotherapy cycle. Patients receiving amifostine had a lower degree of hypomagnesemia, as well as a lower urinary excretion of N-acetyl-glucosaminidase and albumin, indicating less tubular damage compared to the control patients. Treatment with 1000 mg amifostine resulted in an almost complete preservation of GFR. This corresponded to a slightly reduced excretion of tubular marker proteins and a lower incidence of hypomagnesemia during chemotherapy in amifostine patients compared to controls. This dose of amifostine may be sufficient for nephroprotection in patients without pre-existing risk factors for renal damage who undergo a restricted number of chemotherapy cycles.

The "fixed" charge of glomerular capillary wall as determinant of permselectivity.

The determinants of glomerular capillary wall (GCW) permeability to proteins have been subject of controversial discussion. To study this question we have developed a modified isolated perfused rat kidney model in which tubular transport processes are completely blocked by perfusion fixation with glutaraldehyde. This model allows to directly titrate the charge density of the GCW using albumin solutions buffered over a wide pH-range, a manipulation that cannot be performed in the intact kidney. Analyzing the results of these experiments helped to determine a fixed charge density of the GCW of 43 mEq/L. In the present work, we used the isolated perfused fixed rat kidney model to study the influence of this fixed charge on the transglomerular passage of proteins. To do this, the fixed kidney was perfused with albumin solutions containing different isoforms of horseradish peroxidase. The lowest sieving coefficient was obtained with the acidic isoform (0.035+/-0.008, n = 7), while the isoforms at pI 6.85 and 8.45 showed higher sieving coefficients (0.059+/-0.008, n = 7 and 0.090+/-0.008, n = 4, respectively). The highest sieving coefficient (0.59+/-0.031, n = 6) was observed in perfusion experiments of the fixed kidney with cationic HRP (pI > or = 9.30). However, when comparing the sieving coefficients, the highly cationic isoform was excluded because it has a lower molecular weight than the other isoforms. The sieving coefficients of the other isoforms were significantly different (p < 0.05. ANOVA, Scheffé test). In conclusion, the presence of a discrete (even if lower than previously thought) "fixed" charge on the GCW of 43 mEq/L restricts the transglomerular passage of isoforms of horseradish peroxidase by a factor 2-3. These results imply that the influence of charge selectivity has been overstated in the literature.

A randomized trial of amifostine in patients with high-dose VIC chemotherapy plus autologous blood stem cell transplantation.

This pilot study evaluates the degree of side effects during high-dose chemotherapy (HD-VIC) plus autologous bone marrow transplant (HDCT) and its possible prevention by the cytoprotective thiol-derivate amifostine. Additionally, the in-patient medical costs of both treatment arms were compared. 40 patients with solid tumours were randomized to receive HD-VIC chemotherapy with or without amifostine (910 mg/m(2)at day 1-3) given as a short infusion prior to carboplatin and ifosfamide. Patients were stratified according to pretreatment. HDCT consisted of an 18 h infusion of carboplatin (500 mg/m(2/)d over 18 h), ifosfamide (4 g/m(2)/d over 4 h) and etoposide (500 mg/m(2)/d) all given for 3 consecutive days. All patients received prophylactic application of G-CSF (5 microg kg(-1)subcutaneously) to ameliorate neutropenia after treatment. Patients were monitored for nephrotoxicity, gastrointestinal side effects, haematopoietic recovery, as well as frequency of fever and infections. The median fall of the glomerular filtration rate (GFR) was 10% from baseline in the amifostine group (105 to 95 ml min(-1)) and 37% in the control patient group (107 to 67 ml min(-1)) (P< 0.01). Amifostine-treated patients revealed a less pronounced increase in albumin and low molecular weight protein urinary excretion. Stomatitis grade III/IV occurred in 25% without versus 0% of patients with amifostine (P = 0.01). Acute nausea/vomiting was frequently observed immediately during or after the application of amifostine despite intensive antiemetic prophylaxis consisting of 5-HT3-receptor antagonists/dexamethasone/trifluorpromazine. However, delayed emesis occurred more often in the control patients. Engraftment of neutrophil (> 500 microl(-1))and thrombocytes (> 25 000 microl(-1))were observed at days 9 versus 10 and 10 versus 12, respectively, both slightly in favour of the amifostine arm. In addition, a lower number of days with fever and a shortened duration of hospital stay were observed in the amifostine arm. The reduction of acute toxicity observed in the amifostine arm resulted in 30% savings in costs for supportive care (Euro 4396 versus Euro 3153 per patient). Taking into account the drug costs of amifostine, calculation of in-patient treatment costs from the start of chemotherapy to discharge revealed additional costs of Euro 540 per patient in the amifostine arm. This randomized pilot study indicates that both organ and haematotoxicity of HD-VIC chemotherapy can be ameliorated by the use of amifostine. Additionally, a nearly complete preservation of GFR was observed in amifostine-treated patients which may be advantageous if repetitive cycles of HDCT are planned. Larger randomized trials evaluating amifostine cytoprotection during high-dose chemotherapy are warranted.

Silibinin protects against cisplatin-induced nephrotoxicity without compromising cisplatin or ifosfamide anti-tumour activity.

Cisplatin is one of the most active cytotoxic agents in the treatment of testicular cancer, but its clinical use is associated with side-effects such as ototoxicity, neurotoxicity and nephrotoxicity. Long-term kidney damage from cisplatin particularly affects the proximal tubular apparatus and can be detected by increased urinary excretion of brush-border enzymes, such as L-alanine-aminopeptidase (AAP), and magnesium. In the current study, the flavonoid silibinin was used as a nephroprotectant for cisplatin-induced nephropathy in a rat animal model. Infusion of silibinin before cisplatin results in a significant decrease in glomerular (indicated by creatinine clearance and serum urea level) and tubular kidney toxicity (excretion of brush-border enzymes and magnesium). Silibinin given alone had no effect on renal function. In order to exclude an inhibition of the anti-tumour activity of cisplatin and 4-hydroperoxy-ifosfamide by co-administration of silibinin, in vitro studies were performed in three established human testicular cancer cell lines. Dose-response curves for cisplatin (3-30 000 nmol) combined with non-toxic silibinin doses (7.25 x 10(-6) or 7.25 x 10(-5) mol l-1) did not deviate significantly from those of cisplatin alone as measured by relative cell survival during a 5 day assay using the sulphorhodamine-B staining technique. Also silibinin did not influence the cytotoxic activity of 4-hydroperoxy-ifosfamide (30-10 000 nmol) in vitro. In summary, these in vitro data rule out a significant inhibition of the anti-tumour activity of the major nephrotoxic components, cisplatin and 4-hydroperoxy-ifosfamide, by co-administration of silibinin in a human germ cell tumour cell line model. Together with these demonstrated cytoprotection effects in the rat animal model, these data form the basis for a randomised clinical trial of silibinin for the protection of cisplatin-associated nephrotoxicity in patients with testicular cancer.

Adriamycin affects glomerular renal function: evidence for the involvement of oxygen radicals.

The early nephrotoxic effect of the antitumor drug adriamycin (ADR) is suggested to be related to the generation of oxygen free radicals. Therefore the O2-dependence and the influence of free radical scavengers were studied in the model of the isolated perfused single glomerulus of Myxine glutinosa and by histochemical demonstration of the glomerular ATP-ase. In Myxine, the glomerular ATP-ase activity was decreased after injection of ADR (5 mg/kg, i.v.). Both ADR-treated Myxine and controls were exposed for 48 h to an artificial atmosphere of 20% O2/80% N2 or 80% O2/20% N2, respectively. After 10 days a significant decrease of the hydraulic conductivity (k) was measured in the experimental group exposed to 80% O2 (k-values expressed as nl/s.mm Hg.mm2: controls (7): 0.059 +/- 0.017; ADR (7): 0.033 +/- 0.026). The reduction of k following the administration of ADR (20 mg/kg) could be prevented by the sulphydryl donor N-acetylcysteine (NAC). The sieving coefficient for albumin (phi) was significantly increased in ADR-treated animals, showing no O2-dependence (phi x 10(-2): controls (7) 1.3 +/- 0.2; ADR 20% O2 (8): 8.1 +/- 9.6; ADR 80% O2 (7): 6.9 +/- 6.7). phi was not affected by NAC. The lipid peroxide levels in liver, kidney and heart of Myxine increased after the administration of ADR, peaking by day 2 to 5. The circulation disorders of ADR-treated Myxine were not due to an accumulation of the drug in the heart, but rather to a lack of the intracellular antioxidant glutathione. It is concluded that the early nephrotoxic effect of ADR, as reflected by a decreased glomerular ATP-ase activity, is mediated by free radical formation. Oxidative stress on membrane compounds seems to reduce the water permeability of the glomerular barrier, while the ADR-induced sieving defect may be due to oxygen independent pathological mechanisms.