Charles L. Litterst

Alterations in the toxicity of cis-Dichlorodiammineplatinum-II and in tissue localization of platinum as a function of NaCl concentration in the vehicle of administration

Abstract The lethality to mice of the anticancer drug cis -dichlorodiammineplatinum-II (cisPt) was greatly reduced when the drug was dissolved in hyperonic (4.5%) NaCl relative to preparation in distilled water (DW). Concomitant administration of 4.5% NaCl and cisPt prepared in DW failed to protect mice against cisPt toxicity. When other chloride salts were used as vehicles, partial protection was observed and when NaI was used as a vehicle, nearly complete protection was obtained. Similarly in rats, toxicity was reduced with the hypertonic vehicle when lethality, clinical chemistry, or histopathology criteria were evaluated. In vitro and in vivo binding to plasma proteins was greater with the DW vehicle. Tissue Pt levels were less and decayed more rapidly with the hypertonic NaCl vehicle. Antitumor effectiveness of cisPt was not altered when 4.5% NaCl was utilized as the vehicle. A postulated explanation for these results is a shift in the cisPt aquation reaction due to the high concentration of chloride ions in the vehicle.

Pharmacokinetics and protein binding of cis-dichlorodiammine platinum (II) administered as a one hour or as a twenty hour infusion

SummaryThe pharmacokinetics of cis-dichlorodiamminoplatinum (II) (cisplatin) have been studied in seven patients, of whom four received the drug as a one hour infusion and three received it as a 20 h infusion. The patients receiving the drug over one hour exhibited biphasic clearance of total platinum with a rapid initial phase (8.7–22.5 min) and a prolonged second phase (30.5–106 h). Free (ultrafilterable) cisplatin was readily detectable in this group and was rapidly cleared (half-life about 22 min). The volume of distribution of the drug was 50.3–65.6 liters and it was 26.6–50% excreted in the urine in 48h. In the patients receiving the 20 h infusion, a more complex plasma elimination curve was seen, with the appearance of a secondary peak. Free drug was not detectable in these patients and they showed less urinary excretion (21.4–25.9% at 48 h) than the one hour group. Cisplatin was bound to several plasma proteins, including albumin, transferrin, and γ-globulin. The data indicate that cisplatin is retained in the body more extensively after a 20 h infusion than after a one hour infusion.

Comparative alterations in extrahepatic drug metabolism by factors known to affect hepatic activity.

Abstract Various factors known to alter hepatic drug metabolism were examined for their effects on drug metabolism in certain extrahepatic organs, viz. lung and kidney. The prominent sex-related differences in drug metabolism in rat liver were not seen in either lung or kidney. Pretreatment of rats with phenobarbital produced the expected large increases in hepatic NADPH cytochrome c reductase, cytochrome P-450. aminopyrine demethylase and biphenyl hydroxylase activities without concomitant changes in any of these parameters in lung, and only scattered and smaller changes in kidney. 3-Methylcholanthrene (3-MC) pretreatment significantly increased cytochrome P-450 levels in all three organs. Pretreatment of rats with carbon tetrachloride (CCl4) produced consistent inhibition of mixedfunction oxidation in hepatic microsomes. but the extrahepatic effects were less predictable and were both organ- and enzyme-specific. An increase in renal UDP-glucuronyltransferase activity was observed after CCl4 treatment that paralleled a similar but larger increase observed in liver. Extrahepatic NADPH cytochrome c reductase and N-methyl-p-chloroaniline demethylase values were unaffected by CCl4. Lung and kidney responded in a like manner to liver to the additions in vitro of β-diethylaminoethyl diphenylpropylacetate (SKF-525A). Losses in enzyme activities in lung and kidney microsomes roughly paralleled those of liver when stored as pellets for up to 14 days at −70°. Two or 4 days of starvation produced substrate-specific changes in enzyme-specific activity in liver and kidney, with lung appearing resistant to the effect. When enzyme activity was expressed on a whole organ basis, however, lung cytochrome P-450 values decreased significantly and parameters from liver and kidney increased or decreased in a substrate-specific manner. It is concluded that some physiological and pharmacological factors that influence hepatic drug metabolism produce similar effects in lung and kidney, while other factors produce organ-specific effects.

Physiological model for the pharmacokinetics of cis-dichlorodiammineplatinum (II) (DDP) in the tumored rat.

A physiological model has been developed to describe the disposition of cisdichlorodiammine-platinum(II) (DDP) following i.v. dosing in the female rat bearing the Walker 256 carcinoma. The model simulates concentrations of DDP and its mobile and fixed metabolites in plasma, liver, gut, skin, muscle, tumor, carcass, and kidney, and DDP and mobile metabolite excretion following a 4 mg/kg dose. In the kinetic model, DDP binds irreversibly to low MW nucleophiles and macromolecules (largely proteins) within the plasma and tissue compartments to form mobile and fixed metabolites, respectively. Reaction rates for the formation of each metabolite are tissue/organ specific. The rate constant for the biotransformation of DDP to fixed metabolite in plasma (k2p=0.0082 min−) was determined from in vitro incubation studies. This rate was used as the basis for estimating the biotransformation rate constants for DDP to fixed and mobile metabolites in other compartments. Both DDP and mobile metabolite are assumed to follow flowlimited transport, to freely traverse compartmental barriers, and to partition equally in all compartments. Both are excreted in the urine, the major route of Pt elimination. Urinary excretion is modeled as a linear process involving filtration only; an assumption based on a calculated renal clearance of 1.1 ml/min, a value very similar to the estimated GFR. Biliary excretion is a minor route of mobile metabolite elimination and is modeled as a linear process occurring in the liver. Four hours after dosing, approximately 60% of the administered Pt remains in the tissues and plasma. Of this, over 75% of the plasma Pt and 90% of the metal ion in every other compartment is fixed (protein bound). Fixed Pt can be eliminated from a compartment only after its biotransformation to mobile metabolite. In most compertments this rate of elimination corresponds closely to the average rate of protein turnover in that compartment.

The effects of ascorbic acid deficiency and repletion on pulmonary, renal, and hepatic drug metabolism in the guinea pig

Abstract The effects of ascorbic acid (AA) deficiency on microsomal and soluble (postmicrosomal supernatant) enzymes which catalyze drug metabolism were studied in the guinea pig liver, lung, and kidney, (i) Twenty-one days of AA depletion produced a 50–60% decrease in hepatic cytochrome P -450 levels, 20–30% decreases in renal levels, but no significant changes in pulmonary cytochrome P -450 content. Upon repletion of ascorbic acid, recovery to control levels occurred within 7 days. (ii) The decreases in hepatic cytochrome P -450 in scurvy were not accompanied by a corresponding increase in cytochrome P -420. (iii) Aminopyrine N -demethylation decreased by 40% in livers of deficient animals, and recovered within 3 days, but there were no corresponding changes in lungs and kidneys. (iv) There were no significant alterations of NADPH-cytochrome c reductase activity in scorbutic animals in any of the three organs. (v) Activity of “native” UDP-glucuronyl transferase was increased in liver microsomes after 21 days of deficiency, but this apparent increase was not observed when the enzyme was fully activated in vitro with UDP N -acetylglucosamine. “Native” UDP-glucuronyl transferase was increased in kidneys of deficient animals and unchanged in lungs. (vi) In the postmicrosomal supernatant, glutathione S -aryl transferase activity in deficient livers decreased tc 50% of control and did not fully recover after 14 days of ascorbic acid repletion. These changes were not seen in kidney and lung. (vii) Also in the postmicrosomal supernatant, p -aminobenzoic acid (PABA) N -acetyl transferase activity increased in the kidneys of deficient animals, but was unchanged in liver and lungs. (viii) Addition of ascorbic acid in vitro to hepatic microsomes prepared from scorbutic animals had no effect on activities of aminopyrine N -demethylase, NADPH-cytochrome c reductase, PABA N -acetyl transferase, and glutathione S -aryl transferase.

The effect of cisplatin on renal ATPase activity in vivo and in vitro

SummaryThe effect of cisplatin on ATPase activity was determined in vitro and in vivo to investigate the correlation between nephrotoxicity and the inhibition of ATPase activity by cisplatin. Purified Na,K-ATPase was preincubated for 0–240 min with cisplatin at concentrations of 50–800 μM in vitro before the determination of enzyme activity. Although ATPase activity was reduced by cisplatin, either a high concentration of cisplatin (280 μM) or a long period of preincubation (160 min) with cisplatin was required to obtain 50% inhibition of ATPase activity. Similar in vitro experiments using kidney homogenate from female Sprague-Dawley rats instead of purified Na,K-ATPase were performed. Activity of Na,K-ATPase in rat kidney homogenate was inhibited by 50% after 110 min preincubation with 800 μM cisplatin or 160 min preincubation with 400 μM cisplatin. Female Sprague-Dawley rats were given 5, 7 or 10 mg/kg of cisplatin IV and BUN level, ATPase activity and Pt concentration in kidney homogenate were evaluated 1 h, 6 h, 1 day, 3 days, and 5 days after cisplatin injection. In rats given 10 mg/kg cisplatin a significant increase of BUN was observed on days 1, 3, and 5. In rats treated with 5 or 7 mg/kg of cisplatin BUN was increased on days 3 and 5. Normal ATPase activity, however, was preserved until day 3 at all doses. The highest concentration of Pt observed in kidney tissue was 19.3 μg/g tissue. This value was insufficient to inhibit ATPase activity significantly in vitro. Thus, it seems unlikely that the inhibition of ATPase activity is the cause of nephrotoxicity, although cisplatin can affect ATPase activity.

Profiling of prostaglandin biosynthesis in biopsy fragments of human lung carcinomas and normal human lung by capillary gas chromatography-negative ion chemical ionization mass spectrometry.

Methods for the profiling of prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) biosynthesis in tissue samples of clinical origin by capillary gas chromatography-negative ion chemical ionization mass spectrometry (CGC-NICIMS) are detailed. Aliquots (25 microliter 1) of incubates (1 ml volume) of human lung carcinoma and normal human lung tissue fragments (total protein content = 0.2 to 2.0 mg) were derivatized for vapor phase analysis in the presence of 0.75 to 1.60 ng of tetradeuterated analogs of PGE2, PGF2 alpha and 6KPGF1 alpha without prior extraction and/or chromatography. The derivatized analytes and internal standards were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The inter-sample and intra-sample coefficients of variation for the assay method were typically less than 12%. The analysis of biological samples was completed with less than 2.5% of each derivatized sample per injection. The samples were of adequate purity for the identification and quantitation of each of the eicosanoids. The methods described in this report are highly selective and highly sensitive with detection limits of 0.1 to 0.2 picograms per injection. The analytical procedures provide the basis for comparisons of the qualitative and quantitative profiles of prostaglandin biosynthesis and should be adaptable for use in a variety of biological and clinical studies.

Drug metabolism by microsomes from extrahepatic organs of rat and rabbit prepared by calcium aggregation

Abstract Microsomes were prepared from liver, lung and kidney of rats and rabbits using a Ca +2 aggregation method. Microsomal protein yield from the lung of both species was higher by this method of preparation as compared with ultracentrifuged samples. Specific activities of rat and rabbit pulmonary p-chloro-N-methylaniline (CMA) demethylase, biphenyl 4-hydroxylase and rat pulmonary TPNH cytochrome c reductase also were decreased. Specific activities of rabbit hepatic TPNH cytochrome c reductase, CMA N-demethylase, UDP-glucuronyltransferase and biphenyl hydroxylase were decreased by calcium aggregation Renal enzyme activities were unchanged by this method of preparation. These data indicate an apparent species and organ difference in microsomal enzyme response to calcium aggregation.

Alterations in hepatic and renal levels of glutathione and activities of glutathione S-transferases from rats treated with cis-dichlorodiamminelatinum-II

SummaryAdult female rats were treated intraperitoneally with 8 mg/kg of cis-dichlorodiammineplatinum (II). At various times after treatment 1, 3, 5, 8, 12 days replicate animals were killed and liver and kidney cytosols examined for activity of glutathione-dependent transferase activities and levels of glutathione. Hepatic levels of glutathione were depressed by 13–28% at 1, 3, 5 days after dosing. Renal levels of glutathione were increased by 3–5 fold at 8 and 12 days after drug administration. Renal levels of glutathione were decreased at nearly all times studied with a nadir at 5 days. Activity of glutathione s-acryl transferase was increased and S-epoxidetransferase was decreased at 5, 8, 12 days after dosing. When cisplatinum was added to incubation mixtures in vitro, no changes in enzyme activities were observed. When cisplatin and reduced glutathione were determined chromatographically in tissue cytosols from treated rats, 30% of the recovered platinum was associated with glutathione. In tissue cytosols, greater than 95% of the total platinum content was retained in the supernatant when protein was precipitated with trichloroacetic acid, while only 3–5% of the protein was retained.

Increased tissue deposition and decreased excretion of platinum following administration of cisplatin to cisplatin-pretreated animals.

SummaryGuinea pigs were pretreated IP with cisplatin (10 mg/kg) for various times before IV administration of 195mPtlabeled cisplatin. Concentrations of 195mplatinum were greater in tissues of pretreated animals than in those of control animals. Amounts of 195mplatinum in subcellular fractions from pretreated rabbits were similarly greater in pretreated animals. Amounts of radioactivity appeared to be greatest in animals receiving a larger number of pretreatment injections, even though the total amount of cisplatin administered was equal in all groups. BUN was elevated on day 1 after the radioactive dose only in those animals which had been pretreated. Urinary excretion of platinum was significantly less in pretreated than in control animals. It appears that pretreatment with cisplatin damages the kidney severely enough for subsequent doses of cisplatin not to be excreted as efficiently, thus leading to a greater tissue deposition of platinum in pretreated animals.