Elevated alpha-1-acid glycoprotein reduces the volume of distribution and systemic clearance of saquinavir.

The purpose of this study was to characterize the relationship between plasma protein binding and the pharmacokinetic disposition of saquinavir during a normal and elevated alpha-1-acid glycoprotein condition. The extent of plasma binding of [14C]saquinavir to human plasma, human albumin, and human alpha-1-acid glycoprotein was also assessed. Transgenic mice, which overexpress plasma alpha-1-acid glycoprotein, and control mice were given a single intravenous injection of saquinavir (10 mg/kg) and plasma samples were harvested as a function of time. The extent of [14C]saquinavir (0.5-30 microg/ml) plasma protein binding in each group of mice was determined by ultrafiltration. Plasma saquinavir concentrations from in vivo administration were determined by high performance liquid chromatography with tandem mass spectrometry. Saquinavir binding in human plasma and control mouse plasma was similar (approximately 3% unbound). In contrast, the extent of binding was significantly increased in transgenic mice (1.5% unbound). Furthermore, saquinavir was more extensively bound to alpha-1-acid glycoprotein than to albumin (2.1 versus 11.5% unbound). The systemic clearance and volume of distribution of saquinavir were significantly reduced in transgenic mice compared with control mice. The results of this study show that alpha-1-acid glycoprotein is the predominant plasma protein to which saquinavir binds. In addition, elevations in plasma alpha-1-acid glycoprotein considerably alter the pharmacokinetic disposition of saquinavir. This is consistent with the observations that systemic exposure to saquinavir in human immunodeficiency virus patients is greater than that in healthy volunteers and that alpha-1-acid glycoprotein levels increase with the degree of HIV infection.

Reduction of cisplatin nephrotoxicity by procainamide: does the formation of a cisplatin-procainamide complex play a role?

Procainamide protects mice bearing P388 leukemic cells against the toxicity of cisplatin without diminishing antitumor activity. The mechanism of action of procainamide protection was investigated both in vitro and in vivo. HPLC studies showed that procainamide forms a complex with cisplatin in vitro that has a UV spectrum similar to that of DPR, a triamine platinum complex that contains procaine as ligand. We report here the effect of the reaction product of cisplatin and procainamide on both cisplatin-induced DNA interstrand cross-links (ISCLs) and on the total DNA platination of isolated DNA. Total DNA platination in vitro of isolated DNA was increased by 113% (P <.01) and 17% (P <.05) after incubation times of 1.75 and 6 h, respectively, compared with products from the reaction of cisplatin with water. Furthermore, the reaction product of cisplatin and procainamide was bound to DNA to a significantly greater extent than was cisplatin itself. ISCLs were decreased by 41% when this drug combination was incubated with DNA for 1.75 h, but no changes were observed after incubation for 6 h. We also examined the influence of the time interval between administration of cisplatin and procainamide on normal kidney injury, the renal distribution and urinary excretion of platinum, and the formation of cisplatin-DNA adducts in renal tissue of Sprague-Dawley rats after i.p. administration of 7.5 mg/kg cisplatin either with or without procainamide. The plasma concentrations of urea and creatinine and kidney histology demonstrated that procainamide provided effective protection in vivo in the rat when administered either simultaneously or at 0.5 and 1 h before or after cisplatin. The protection was accompanied by both higher renal levels of platinum and cisplatin-DNA adducts and by an increase in the formation of ISCLs. Moreover, a dose-dependent reduction of urinary excretion and concentration of platinum was also observed. We propose that procainamide, after accumulation in the kidney, may coordinate with cisplatin to form a less toxic DPR-like complex that renders rats less susceptible to cisplatin-induced toxicity.

Measurement of platinum in saliva of patients treated with cisplatin.

A procedure for the measurement of platinum (Pt) in the saliva of patients treated with cisplatin has been developed. The saliva is collected and solubilized in hyamine hydroxide before analysis by graphite furnace atomic absorption spectrometry using assay by standard additions. The method has an analytical detection limit of 0.025 microgram/mL and is precise, with coefficients of variation of 3-10.0% over a range of 0.05-2.0 micrograms/mL. Platinum was measured in saliva collected during an 8-h infusion of cisplatin from five patients, at the end of a 30-min infusion in nine, and 24 and 48 h later from a further 15 patients, all of whom were treated with cisplatin for squamous cell carcinoma of the neck. The platinum concentration in saliva taken at the end of a 30-min infusion was 0.27 +/- 0.23 microgram/mL (mean +/- 1 SD) but was below the detection limit of 0.025 microgram/mL at 24 and 48 h. After an 8-h infusion the salivary Pt was significantly less (0.12 +/- 0.04 microgram/mL; P < 0.05). The plasma Pt concentrations after 30-min and 8-h infusions were 2.98 +/- 1.03 and 2.54 +/- 0.59 micrograms/mL, respectively, and were not significantly different. The results indicate higher concentrations of free platinum in plasma after 30 min compared with an 8-h infusion. The monitoring of salivary concentrations of platinum may therefore provide a non-invasive way to study the unbound fraction of cisplatin in blood and facilitate optimization of cisplatin treatment.

Cisplatin combined with tiopronin or sodium thiosulfate: cytotoxicity in vitro and antitumor activity in vivo.

We have previously reported that the thiol compound tiopronin protects rat kidneys in vitro against the toxic activity of cisplatin. The influence of tiopronin and sodium thiosulfate (STS) on the cytotoxicity of cisplatin has been investigated on P388 leukemic cells in vitro after 3 days. The combination has also been investigated in vivo in BDF1 mice bearing a P388 s.c. tumor. In contrast to STS, tiopronin did not significantly reduce the cytotoxic activity of cisplatin in vitro and nor did it affect the uptake of platinum (cisplatin-derived), binding to DNA or the percentage of interstrand cross-links (%ISCL) formation. The co-administration of cisplatin (4 mg/kg) and tiopronin (150 and 300 mg/kg) to BDF1 female mice bearing a s.c. P388 tumor produced a significant reduction in tumor growth similar to that of a single 6 mg/kg dose of cisplatin. Interestingly, pre-incubation in vitro of either tiopronin or STS for 2 h with the species formed from cisplatin by hydrolysis demonstrated their ability in inhibiting the cytotoxicity of these reactive platinum products. These results indicate that tiopronin does not reduce the cytotoxicity of cisplatin in vitro, as STS does. This may be, at least partly, because of a different effect of the two thiol compounds on the cellular uptake and binding of platinum to DNA. Notably, tiopronin substantially reduced tumor growth in mice treated with a non-toxic dose of cisplatin (p < or = 0.0277), suggesting some positive influence of this thiol compound on the antitumor properties of cisplatin. The ability of tiopronin to protect in vitro against the cytotoxicity of the aquation products of cisplatin may be related to its nephroprotective effect.

Tiopronin protects against the nephrotoxicity of cisplatin in the rat.

1. Tiopronin (N-(2-mercaptopropionyl)-glycine) is a drug with a free thiol (sulphydryl) group that is used clinically. We have reported previously that tiopronin protects rat kidney slices in vitro from the nephrotoxic effects of cisplatin and does not reduce the antitumour activity of cisplatin. Tiopronin has been investigated therefore for its protective effects in rats in vivo. 2. The extent of kidney damage was studied 5 days after the administration of cisplatin. A single injection (i.p.) of cisplatin (6 mg/kg; 20 micromol/kg) to female Wistar albino rats caused a sustained decrease in body weight and, after 5 days, plasma urea, creatinine and kidney weight were increased. Tiopronin (2.5 mmol/kg, p.o.) ameliorated cisplatin nephrotoxicity when given 1 h before cisplatin. Tiopronin provided marked protection against cisplatin-induced increases in urea (from 237+/-19 mg to 48+/-23 mg/100 ml; control: 17+/-1) and creatinine (from 6.5+/-0.5 to 1.7+/-0.5 mg/100 ml control: 1.0+/-0.1). Tiopronin did not, prevent the body weight loss caused by cisplatin. In addition, an intraperitoneal dose (1 mmol/kg) of tiopronin afforded similar protection to that of an oral dose. Rats that received an i.p. mixture of cisplatin (6 mg/kg) and tiopronin (65 mg/kg) displayed generally less toxicity, as indicated by a small fall in body weight and smaller increases in urea and creatinine and kidney weight. 3. The results show that tiopronin protects against cisplatin-induced nephrotoxicity. Oral administration of tiopronin may be a clinically useful way to prevent cisplatin nephrotoxicity.

Variability of mitomycin C adsorption by activated charcoal.

A saline suspension of mitomycin C adsorbed on activated charcoal and administered intraperitoneally has been reported to be safe and effective in the treatment of gastric carcinoma. Activated charcoal specifically targets tumour and lymph-node tissues and the sustained higher local drug concentration is thought to be beneficial. The charcoal particles used in these suspensions have varied in size from > 147 microm to < 20 nm in diameter, but no data have been published to show how this might affect drug adsorption and delivery. Any variability in drug adsorption could pose a serious clinical risk for drugs with a narrow therapeutic index. We have, therefore, investigated the adsorption of mitomycin C on activated charcoal in-vitro. Activated charcoal was ground and sieved to yield four size-fractions between 180 and 53 microm. Adsorption isotherms (n > or = 3) were constructed and applied to the Freundlich model with 0-l00 microg mL(-1) mitomycin C measured by HPLC with detection at 365 nm. Adsorption of mitomycin C by activated charcoal varied by a factor of three under identical conditions at room temperature (21 degrees C) and at 37 degrees C. The specific adsorption (microg mitomycin C (mg activated charcoal)(-1)) was generally higher at 37 degrees C than at room temperature. The variability of mitomycin C adsorption was greatly reduced by addition of the surface-active agent polyvinylpyrollidone, used to determine that adsorption of mitomycin C was independent of activated charcoal particle size. The characteristics of adsorption of mitomycin C by activated charcoal are complex and should be thoroughly investigated to discover the critical controlling factors before submitting the suspensions for further clinical evaluation.

Cisplatin-induced changes in adenine nucleotides in rat kidney slices: amelioration by tiopronin and procaine.

The adenine nucleotides (ATP, ADP and AMP) in rat renal cortical slices exposed in-vitro to cisplatin, an anticancer drug, were determined by HPLC. Cisplatin had no effect on total adenine nucleotides in the slices but caused a time- and concentration-dependent decrease in ATP levels with a concomitant increase in ADP and AMP levels. The decrease in ATP and increases in ADP and AMP concentrations became statistically significant after incubation with cisplatin (2 mM) for 90 min or after cisplatin (1 mM) for 120 min. Both tiopronin, a sulphydryl-containing drug, and procaine, an antioxidant, protected against cisplatin-induced changes in the adenine nucleotides. The results indicate a cisplatin-induced defect in cellular energetics that occurs at a relatively late stage in the process of toxicity to the slices in this in-vitro model. Cisplatin-induced depletion of ATP in the slices might result from an increase in catabolism of ATP to ADP and AMP. Maintenance of the normal concentration of ATP in the slices might be involved in the protection afforded by tiopronin and procaine against cisplatin-induced nephrotoxicity.

Cisplatin-induced nephrotoxicity in vitro: increases in cytosolic calcium concentration and the inhibition of cytosolic and mitochondrial protein kinase C.

Exposure of rat kidney cortical slices to cisplatin (2 mM) significantly increased the activity of cytosolic phosphorylase a, indicating that the concentration of cytosolic Ca2+ was increased. Dithiothreitol (DTT), a sulphydryl reducing agent, markedly reversed this effect but N,N'-diphenylphenylenediamine (DPPD), an antioxidant, did not. Cisplatin inhibited protein kinase C (PKC) activity in both mitochondrial and cytosolic fractions after the slices were exposed to cisplatin. Both DTT and DPPD prevented these inhibitory effects of cisplatin on PKC but diethylmaleate, a glutathione depletor, potentiated this inhibitory effect. These results suggest that the increase in cytosolic Ca2+ is related to depletion of SH-groups, but independent of lipid peroxidation, whereas inhibition of PKC may be associated with cisplatin-induced depletion of thiols and with lipid peroxidation.

Tiopronin protects against the nephrotoxicity of cisplatin in rat renal cortical slices in vitro.

The protective effect of N-(2-mercaptopropionyl)-glycine (tiopronin), a clinically used sulfhydryl-containing compound, on cisplatin-induced toxicity to rat renal cortical slices was investigated. Exposure of the slices to cisplatin (2 mM) resulted in toxicity, as shown by an increase in leakage of the two enzymes aspartate aminotransferase and lactate dehydrogenase into the incubation medium and a time-dependent decrease in the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the slices. Tiopronin (2 mM) completely prevented the cisplatin-induced increase in enzyme leakage and substantially blocked the decrease of MTT reduction caused by cisplatin. These protective effects were concentration-dependent and furthermore, the depletion of ATP, glutathione and induction of lipid peroxidation in the slices by cisplatin (2 mM) were reversed by 2 mM tiopronin. Pretreatment of slices with tiopronin for 60 min also significantly protected the renal slices from cisplatin-induced toxic effects. These protective effects, however, were abolished by p-aminohippuric acid, a compound with some structural similarity to tiopronin, which both undergoes and inhibits active transport in the cells of the proximal convoluted tubule. Cisplatin (1 mM) also depleted the free sulfhydryls of tiopronin (1 mM) in a second incubation medium system and PAH (2 mM) diminished the extent of this depletion somewhat. These observations suggest that tiopronin protects against cisplatin-induced nephrotoxicity by acting as an alternative target for cisplatin both intra- and extracellularly and thus protects against cisplatin-induced depletion of glutathione in the kidney cell.

Differential effects of cisplatin on the production of NADH-dependent superoxide and the activity of antioxidant enzymes in rat renal cortical slices in vitro.

Cisplatin-induced cytotoxicity has been investigated with rat renal cortical slices in vitro. Incubation of renal slices with cisplatin caused an unexpected decrease in NADH-dependent. lucigenin-enhanced chemiluminescence in the homogenate of the slices in a time- and concentration-dependent fashion. Cisplatin caused a concentration-related (0.2 1 mM) decrease in the formation of NADH-dependent superoxide anion. Cisplatin (2 mM) significantly suppressed the chemiluminescence to 70% of control as early as 15 min, and to 15% after 90 min. of incubation. A decrease was also observed 90 min. after incubation of slices with 0.25 mM cisplatin. In addition, the activities of superoxide dismutase (SOD) and catalase in the slices were significantly increased after 60 min. of exposure to cisplatin (2 mM) while decreases in the activities of glutathione (GSH) peroxidase and GSSG reductase became significant at 90 and 120 min. of incubation respectively. SOD and catalase activities were increased by 1.6 and 1.5 fold respectively after 90 min. of incubation and the activities of GSH peroxidase and GSSG reductase were decreased to 82% and 72% of control, respectively at 120 min. Both dithiothreitol (2 mM), a sulphydryl agent and diphenylphenylenediamine (5 microM), an antioxidant, protected against cisplatin-induced leakage of lactate dehydrogenase, lipid peroxidation and decreases of GSH peroxidase and GSSG reductase but had no effect on the decrease of chemiluminescence caused by cisplatin. The results suggest that neither an increase in the production of NADH-dependent superoxide anion nor a decrease in activity of several antioxidant enzymes were directly responsible for cisplatin-induced lipid peroxidation.

Synthesis and physicochemical properties of the furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, an inhibitor of plasma protein binding in uraemia.

The furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) accumulates in the plasma of patients with chronic renal failure and is a major contributor to the drug binding defect of uraemic plasma. This acid has also been implicated in several other aspects of the uraemic syndrome: anaemia, irregularities of thyroid function, neurological symptoms and inhibition of active tubular secretion. The acid is not commercially available and its synthesis, starting with Meldrum's acid and methyl succinyl chloride, is described. The pKa values were measured by titration and values of 3.2 and 3.6 respectively were assigned to the carboxylic acid groups attached directly to the ring at position 3 and at position 2 (on the side-chain). The partition coefficient (log P) between hydrochloric acid and octanol was 1.2 and the distribution coefficient (log D; octanol-phosphate buffer pH 7.4) was -0.59. The pKa values and the degree of hydrophobic character of 5-propyl FPA are consistent with those of other protein-bound acids which undergo active tubular secretion by the kidney and this substance may serve as an endogenous marker for the effects of drugs and disease on this process.

Retention of an albumin-bound furan dicarboxylic acid in patients with chronic renal failure or after a kidney transplant.

BACKGROUND: 3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) is a furan dicarboxylic acid which accumulates in the plasma of patients with renal impairment. 5-Propyl FPA is an inhibitor of the binding of drugs to albumin and is also implicated in other aspects of the uraemic syndrome. METHODS: Plasma concentrations of propyl FPA have been measured in non-dialysis-dependent, chronic renal failure patients and in renal transplant patients by high-performance liquid chromatography. Concentrations of haemoglobin, albumin and creatinine were also determined. RESULTS: There was a positive correlation between serum creatinine and 5-propyl FPA and a negative correlation between haemoglobin concentration and 5-propyl FPA in chronic renal failure patients. There was a negative correlation between 5-propyl FPA and duration of transplant only when the serum creatinine was >200 microM. The mean plasma concentration of 5-propyl FPA in chronic renal failure patients with plasma creatinine CONCLUSIONS: This retention of 5-propyl FPA may therefore reflect a specific tubular defect in renal transplant patients treated with cyclosporin and points to the possibility that 5-propyl FPA may serve as a marker of tubular dysfunction.

Plasma clearance in the rat of a furan dicarboxylic acid which accumulates in uremia.

The furan dicarboxylic acid 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) accumulates in the plasma of patients with chronic renal failure and has been implicated in several aspects of the uremic syndrome: the defective binding of organic acids in uremic plasma, inhibition of active tubular secretion, anemia and the severity of neurological symptoms. Evidence from experiments with rat kidney slices suggests that 5-propyl FPA undergoes active tubular secretion, and so its clearance after an intravenous bolus dose (5 mg/kg; 21 mumol/kg) was investigated in anaesthetized female Wistar albino rats in vivo. The effects of intravenous bolus doses of p-aminohippuric acid (PAH) and probenecid on the clearance of this dose of 5-propyl FPA were also studied. The mean values (N = 16) for plasma half-life, plasma clearance and apparent volume of distribution of 5-propyl FPA were 3.6 hours, 2.4 ml . min(-1) . kg(-1) and 0.69 liter . kg(-1), respectively. An equimolar dose of PAH did not affect the clearance of 5-propyl FPA, but a tenfold higher molar dose of PAH (40.4 mg/kg) increased the area under the plasma-concentration time curve of 5-propyl FPA, and there was a trend towards a decrease in the clearance and a prolongation of the half-life. Probenecid at a fivefold higher dose than 5-propyl FPA had a similar effect to PAH and increased the AUC of 5-propyl FPA. PAH and probenecid decreased the plasma clearance of 5-propyl FPA, which is evidence that this uremic metabolite undergoes active tubular secretion. It follows that 5-propyl FPA could therefore inhibit the secretion of other organic acids.

Comparison of the toxicities of cisplatin and a new cisplatin-procaine complex to rat renal cortical slices.

1. Procaine has previously been shown to diminish the nephrotoxicity of cisplatin and the nephrotoxic effects of cisplatin and a new cisplatin complex (cis-diamminechloro-[2-(diethylamino) ethyl-4-aminobenzoate, N4]-chlorideplatinum (II) monohydrochloride monohydrate; DPR), that contains procaine hydrochloride were compared with rat renal cortical slices. 2. Cisplatin at 1 mM caused toxicity to the slices, as shown by an increase in the leakage of aspartate aminotransferase and lactate dehydrogenase from the slices into the incubation medium and a decrease in the reduction of a tetrazolium dye (MTT assay). Addition of procaine (1 mM) protected against cisplatin-induced toxicity. DPR either at 1 mM or at 4 mM had no effect either on the enzyme leakage or MTT reduction by the renal slices, but DPR at 10 mM produced a similar magnitude of enzyme leakage to cisplatin (1 mM). 3. DPR lowered the concentration of ATP and glutathione (GSH) in the slices but was less potent than cisplatin. Thiobarbituric acid reactive substances, indicators of lipid peroxidation, released into the medium were increased by the highest concentration of DPR (10 mM), which suggests that DPR has the potential to cause oxidative stress. 4. The results suggest that DPR was far less toxic than either cisplatin alone or a mixture of cisplatin and procaine.

Hypothesis: is accumulation of a furan dicarboxylic acid (3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid) related to the neurological abnormalities in patients with renal failure?

The Plasma concentrations of a lipophilic furan dicarboxylic acid (3- carboxy-4-methyl-5-propyl-2-fluranpropanoic acid; 5-propyl FPA), which is highly bound to albumin and not removed by haemodialysis, have been measured in patients with renal impairment who were not dialysis dependent or who were treated by either haemodialysis or peritoneal dialysis. Neurological abnormalities were assessed as absent, moderate, or severe. A relationship was observed between the increasing severity of abnormalities attributable to the uraemic state and the higher plasma concentrations of 5-propyl FPA. There are theoretical grounds for believing that 5-propyl FPA contributes to these neurological abnormalities because of its structure and also because it inhibits the transport of organic acids in the kidney and could do likewise at the blood-brain barrier.

Role of calcium in cisplatin-induced cell toxicity in rat renal cortical slices.

The disruption of intracellular Ca(2+) homoeostasis is involved in cisplatin-induced nephrotoxicity. The role of Ca(2+) in cisplatin toxicity was studied by use of rat renal cortical slices. Cisplatin (2 mm) increased the leakage of aspartate aminotransferase (AST) from 1.4 +/- 0.5 units/g wet weight (mean +/- SE) with control slices to 3.4 +/- 0.5 units/g wet weight. The leakage of lactate dehydrogenase (LDH) was increased from 3.8 +/- 1.1 units/g wet weight to 13.7 +/- 1.0 units/g wet weight. Pretreatment of slices with ethylene glycol-bis(beta-aminoethylether)N,N,N'N'-tetraacetic acid (1 mm) to buffer intracellular Ca(2+) significantly decreased the cisplatin-induced leakage of these two enzymes to 65% and 53%, respectively, of levels with cisplatin alone. An increase in extracellular Ca(2+), or omission of Ca(2+) from the medium, had no effect on cisplatin-induced slice toxicity. Furthermore, the Ca(2+) channel blockers nifedipine and diltiazem did not protect against cytotoxicity by cisplatin, although verapamil gave mild protection and decreased the cisplatin-induced release of AST and LDH to 78% and 75%, respectively, of that caused by cisplatin alone. The results suggest that intracellular Ca(2+) is important in cisplatin-induced nephrotoxicity but that disruption of cytosolic Ca(2+) is not caused by opening of Ca(2+) channels of the plasma membrane or even by leakage through the injured membrane.

Effects of haemodialysis and continuous ambulatory peritoneal dialysis on the plasma clearance of an albumin-bound furan dicarboxylic acid.

Organic acids that are strongly bound to albumin are not removed by dialysis and the plasma concentrations of one such substance, a furan dicarboxylic acid (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid: 5-propyl FPA) have been measured by HPLC in healthy subjects (n = 21), patients on regular haemodialysis (n = 30), and patients treated by continuous ambulatory peritoneal dialysis (n = 21). The mean (+/- SD) concentrations of 5-propyl FPA were significantly higher in haemodialysis (95 +/- 44 microM) compared to CAPD patients (28 +/- 19 microM) and both were higher than in healthy individuals (14 +/- 7 microM). Haemoglobin concentrations in CAPD patients were significantly higher than in those on haemodialysis while these patients had significantly higher albumin concentrations than CAPD patients. The concentration of 5-propyl FPA was positively correlated with the duration of dialysis for haemodialysis patients but not for CAPD patients. The lower concentrations of 5-propyl FPA in CAPD patients may at least partly explain the higher haemoglobin levels found in these patients.

Cisplatin nephrotoxicity: decreases in mitochondrial protein sulphydryl concentration and calcium uptake by mitochondria from rat renal cortical slices.

The effects of cisplatin on several aspects of the function of mitochondria isolated from the rat renal cortex have been investigated in vitro. Incubation of renal cortical slices with cisplatin (2 mM) caused a rapid loss of mitochondrial protein-SH followed by a substantial decrease in Ca2+ uptake by the mitochondria and a decline in the mitochondrial membrane potential, which was assessed by rhodamine 123 uptake by the slices. Dithiothreitol, a glutathione (GSH)-reducing agent, significantly reversed the alterations in protein-SH, Ca2+ accumulation and rhodamine 123 uptake. There was also a marked amelioration of cisplatin-induced cytotoxicity, as shown by the decreased leakage of several enzymes from the slices. Diethylmaleate, a GSH depletor, enhanced both the cisplatin-induced increase in toxicity, as assessed by enzyme leakage, and also the decreases in protein-SH, Ca2+ accumulation and rhodamine 123 uptake. The antioxidant N,N'-diphenylphenylenediamine substantially alleviated cisplatin toxicity but did not protect against cisplatin-induced alterations to protein-SH and Ca2+ uptake. In addition, the cytotoxicity caused by cisplatin was not affected by cyclosporin A, an inhibitor of Ca2+ release from mitochondria and ruthenium red, an inhibitor of the reuptake of Ca2+. It was concluded that loss of mitochondrial protein-SH and a decrease of Ca2+ uptake are implicated in the toxicity of cisplatin and that mitochondrial GSH is an important factor in relation to oxidative stress to mitochondria and cytotoxicity.

Amelioration of cisplatin toxicity in rat renal cortical slices by dithiothreitol in vitro.

1. The protective effects of dithiothreitol (DTT) on cisplatin-induced nephrotoxicity were investigated with rat renal cortical slices. 2. The nephrotoxic effects of cisplatin (2 mmol l-1) were manifested in several ways: the Na+ and water content were increased while K+ was decreased. The malondialdehyde (MDA) concentration in the slices and the lactate dehydrogenase (LDH) released into the medium were increased. The uptake of p-aminohippurate (PAH), the synthesis of glucose and the glutathione (GSH) concentration in the slices were all decreased. 3. Despite a DTT-related increase in platinum (Pt) uptake by the slices, DTT (0.5-2 mmol l-1) ameliorated all these toxic effects of cisplatin in a concentration related manner. 4. The results suggest that the protective mechanism of DTT is its antioxidative action. DTT is also a metal chelator, however, and so a protective effect via chelation of Pt by DTT cannot be excluded.

Interpretation and analysis of receptor binding experiments which yield non-linear Scatchard plots and binding constants dependent upon receptor concentration.

Receptor-binding assays with radiolabelled ligands are widely used to evaluate the biological activity of drugs and hormones. The affinity, usually expressed as the dissociation constant (Kd), and the capacity (Bmax) of the receptor preparation for various ligands are determined in order to compare quantitatively various agonists and antagonists. Experiments with the same ligand and receptor, however, often yield rather disparate values for these binding parameters. One obvious reason for variability in Kd is that straight lines are fitted to data that are clearly curved. Another and more serious reason is that a ligand's apparent dissociation constant decreases when the receptor preparation is diluted and so experiments done at different receptor concentrations do not give identical results. We demonstrate that both of these observations, i.e. the effect of receptor concentration and the curvature of Scatchard plots, can be explained by the presence of a competitive inhibitor in the receptor preparation, a possibility which is not normally considered in the analysis and interpretation of receptor binding assays. We show that the apparent Kd obtained by the conventional one- or two-site analysis may be several orders of magnitude larger than the true dissociation constant and the affinity is therefore seriously underestimated. Application of a model, which assumes that an inhibitor is present in the receptor preparation, will improve the quantitative determination of Kd and Bmax significantly. As a simple alternative method we explain how the apparent binding parameters obtained by the conventional method should be interpreted and how they can be used to estimate the true affinity, provided sufficiently low concentration data are available.