Persistence of platinum-ammine-DNA adducts in gonads and kidneys of rats and multiple tissues from cancer patients.

The persistence of platinum-DNA adducts was investigated using normal rats as well as tissues from cancer patients receiving either cisdiamminedichloroplatinum(II) (cisplatin) or diamminecyclobutanedicarboxylatoplatinum(II) (carboplatin) for cancer chemotherapy. These studies used an enzyme-linked immunosorbent assay, established with a rabbit anti-cisplatin-DNA that is specific for intrastrand platinum-DNA adducts. The gonads and kidneys of male and female rats, sites for antitumor activity and toxicity, respectively, were monitored for cisplatin-DNA adduct formation after a single dose of drug and during multiple-dose exposures (once a wk for 3 wk). DNA adducts were measured by enzyme-linked immunosorbent assay 4 h and 2, 4, 7, and 14 days after administering a single i.v. injection of 8 mg/kg of cisplatin. Adduct profiles in renal tissues were similar in both males and females with adduct levels increasing between 4 h and 2 days, decreasing between Days 2 and 7, and stable between Days 7 and 14. In both sexes, levels of kidney DNA adduct measured 7 to 14 days after cisplatin injection comprised about 30% of the highest (Day 2) value. In testes and ovaries, adduct removal was complete by 4 days, and 40 to 50% of adducts present at Day 2 persisted until Days 7 and 14. A study of multiple dosing showed that adducts in renal and testicular DNA from rats given three weekly doses of 5 mg/kg of cisplatin had different accumulation profiles. In the testis there was a 2-fold accumulation of adduct after the third dose, while in the kidney adducts dropped with repeated dosing. In humans, the persistence of platinum-DNA adducts was studied in tissues from eight cancer patients who received their last dose of cisplatin or carboplatin chemotherapy between 1 day and 15 mo before autopsy. The patients had either ovarian cancer, breast cancer, or lymphoma, and the tissues studied included ovarian tumor, bone marrow, kidney, liver, spleen, lymph node, peripheral nerve, and brain. When samples were available from tumor tissues and from bone marrow within the same patient, adduct levels were similar in the two tissues. In addition, adducts were persistent for many months, since half of the individuals received their most recent platinum-drug therapy 7 to 15 mo before death. Overall, these studies demonstrate a widespread distribution and high degree of platinum-DNA adduct persistence in both animal and human tissues subsequent to cisplatin or carboplatin treatment.

Characterization of the DNA damage recognized by an antiserum elicited against cis-diamminedichloroplatinum (II)-modified DNA.

A series of in vitro and in vivo studies were performed to characterize DNA damage recognized by an antiserum elicited against DNA modified with cis-diamminedichloroplatinum(II) (cisplatin). Adducts determined by the cisplatin-DNA enzyme-linked immunosorbent assay (ELISA) in human blood cell DNA have been shown to correlate well with positive clinical outcome in testicular and ovarian cancer patients receiving platinum drug-based chemotherapy (Reed et al. (1990) Proc. Natl. Acad. Sci., 84; 5024, and Reed et al. (1988) Carcinogenesis, 9, 1909). DNAs from calf thymus, salmon sperm, pBR322 and synthetic oligonucleotides were modified with cisplatin in vitro before or after specific DNA digestion steps to yield adducted samples of known size and/or chemical composition. These cisplatin modified DNAs were assayed by atomic absorption spectrometry (AAS) to assess absolute platinum content, and by ELISA to determine the antiserum specificity. The antiserum recognizes native cisplatin-modified calf thymus DNA, and native oligonucleotides containing intrastrand cis-Pt (NH3)2-d(pGpG) adducts (Pt-GG) and intrastrand cis-Pt (NH3)2-d(pApG) adducts (Pt-AG). Modified plasmid DNA fragments of varying sizes (down to 309 base pairs) are recognized similarly to cisplatin-modified calf thymus DNA. The antiserum does not cross-react with individual Pt-GG or Pt-AG adducts not bound to DNA. In experiments designed to assess the relationship between adduct measured by ELISA and total platinum bound to DNA as measured by AAS, male and female Sprague-Dawley rats were injected i.p. with cisplatin and a dose response for adduct formation was determined in kidney DNA samples. Values obtained by ELISA were substantially lower than those measured by AAS, and the two were directly related in DNA from kidney tissues of rodents but not in DNA from human nucleated blood cells. In rodent samples the ELISA measured a consistent 0.2% of the total DNA-bound platinum determined by AAS, with a correlation coefficient of 0.91. Among 54 blood cell DNA samples from human patients, which gave measurable adduct values in both ELISA and AAS, the ELISA measured a variable fraction (0.2-33.0%) of the total DNA-bound platinum measured by AAS. We conclude that the cisplatin-DNA ELISA measures a three dimensional lesion in DNA that is formed in direct proportion to total DNA-bound platinum in rat kidney, but that in human biological samples, interindividual variability precludes a relationship that conforms to simple mathematical algorithms.

Metabolic activation of 4-ipomeanol in human lung, primary pulmonary carcinomas, and established human pulmonary carcinoma cell lines.

4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [14C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [14C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 549 +/- 60). In lung cancer tissue, values ranged from 0 to 2.566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 60, P greater than .3). When patients were divided into smokers and current non-smokers (no tobacco products smoked for greater than 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P greater than .1 in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10; P greater than .30). The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted.

Covalent binding of platinum to renal protein from sensitive and resistant guinea pigs treated with cisplatin: possible role in nephrotoxicity.

Covalent binding of platinum from the anticancer drug cisplatin has been determined in subcellular organelles from kidney and liver of guinea pig strains sensitive and resistant to the systemic toxicity of cisplatin. Organ distribution of platinum was similar between the two strains, but the sensitive animals (pigmented) excreted only half as much platinum in 24 hr as did the resistent animals (albino). Subcellular distribution of platinum in mitochondria, microsomes, and cytosol was approximately equal for both strains, but the sensitive animals had twice as much platinum bound covalently to cytosolic acid-insoluble protein as did the resistant animals. In the albino guinea pigs, concentrations of total platinum and of covalently bound platinum were greater in kidney subcellular organelles than in either liver or lung organelles, which may help explain the sensitivity of the kidney to the toxic effects of cisplatin.

Profiles of prostaglandin biosynthesis in normal lung and tumor tissue from lung cancer patients.

prostaglandin (PG) biosynthetic profiles from endogenous arachidonic acid were determined by capillary gas chromatography-mass spectrometry in matched fresh normal lung (NL) and lung cancer (LC) tissue fragments obtained from 42 individual LC patients at the time of diagnostic thoracotomy. The histological diagnoses represented were squamous cell carcinoma (N = 20), adenocarcinoma (N = 7), small cell carcinoma (N = 4), mixed cell carcinoma (N = 2), bronchioloalveolar cell carcinoma (N = 2), large cell undifferentiated carcinoma (N = 3), bronchial carcinoid (N = 1), and metastatic tumors (N = 3). When PG biosynthesis was determined in NL tissue separately, low mean levels of PGE2 and PGF2 alpha (less than 2 pmol/mg protein/15 min), intermediate levels of PGD2 and 6-keto-PGF1 alpha (6KPGF1 alpha) (2-7 pmol/mg protein/15 min), and high levels of thromboxane B2 (TXB2) (greater than 7 pmol/mg protein/15 min) were observed. There was no particular correlation with cigarette smoking history and PG biosynthesis in NL. When PG production in LC tissue was evaluated separately, high levels of PGE2, PGF2 alpha, and 6KPGF1 alpha as well as TXB2 and low levels of PGD2 were noted. In addition, LC tissue from cigarette smokers demonstrated elevated levels of PGE2, 6KPGF1 alpha, and TXB2 when compared to current nonsmokers with LC (P less than 0.05 in all instances). Simultaneous comparison of PG production in matched LC and NL tissue from individual patients indicated increased biosynthesis of PGE2 and PGF2 alpha and low levels of PGD2 in LC compared to NL tissue (P less than 0.05 in all instances; paired, two-tailed, Student's t test). Individual comparison of PG biosynthesis according to LC histological cell type revealed that PGE2 and PGF2 alpha were consistently elevated in all four common primary LC histological cell types, the only exception being large cell undifferentiated carcinoma. Interestingly, this latter LC histological cell type presented a unique profile with lower levels of PGE2 and PGD2 in LC than in NL tissue (P less than 0.05 in both instances). In addition, the biosynthesis of all 5 PGs studied was consistently higher in primary than metastatic adenocarcinomas of the lung (P less than 0.05 in all instances). No differences were observed in NL and LC tissue for the major LC histological cell types when PGD2, TXB2, or 6KPGF1 alpha biosyntheses were compared. These findings indicate that the profiles of PG biosynthesis in LC and NL tissue from individual patients may differ substantially. These differences may reflect, in part, contributions to the PG biosynthetic profile unique to malignant cells.

The effects of platinum levels in plasma, urine, and kidney on blood urea nitrogen levels after cisplatin injection in rats.

To analyze the relationship between the kinetics of filterable Pt and nephrotoxicity after cisplatin injection, Sprague-Dawley rats were given 5 mg/kg of i.v. cisplatin during hypothermia (hypothermia group) or given 5 mg/kg of cisplatin preincubated with rat serum (preincubation group). Plasma Pt levels, kidney Pt contents, urinary excretion of Pt, and blood urea nitrogen (BUN) levels after cisplatin injection were then determined. These hypothermia and preincubation treatments were performed in order to change the kinetics of filterable Pt without changing the total dose of cisplatin. Plasma Pt levels were significantly changed by both hypothermia or preincubation. Urinary excretion of Pt was unaffected by hypothermia but was significantly reduced by preincubation. However, significant differences in BUN elevation were not observed either in the hypothermia group or the preincubation group relative to the control group. Kidney Pt contents were not affected by hypothermia or preincubation at 1 h, 6 h, and 24 h after cisplatin injection. When rats were injected i.v. with 5, 7, or 10 mg/kg cisplatin, dose-related increases were observed both in kidney Pt contents and in BUN levels. These results suggest that the degree of nephrotoxicity more accurately correlates with renal Pt content rather than with plasma Pt levels or urinary concentration of Pt, and that the role of filterable Pt species in producing nephrotoxicity may have to be reevaluated.

In vivo biochemical indices of nephrotoxicity of platinum analogs tetraplatin, CHIP, and cisplatin in the Fischer 344 rat.

In vivo biochemical indices of nephrotoxicity were investigated in Fischer 344 rats treated with a new platinum analog, tetraplatin [tetrachloro(dl-trans)1,2-diaminocyclohexane platinum(IV), NSC-363812], in comparison with rats receiving equimolar dosages of cisplatin and CHIP [cis-dichloro,trans-dihydroxybis-isopropylamine platinum(IV), NSC-256927]. The goals of this study were to assess the comparative nephrotoxicities and to determine which battery of tests might be useful for the assessment of platinum analog-induced nephrotoxicity in future clinical investigations of these drugs. An iv bolus injection of 6.7, 13.3, 26.7, and 53.3 mumol/kg of each drug in saline was administered and assessment of biochemical parameters was conducted for 15 days postinjection. A combination of urinary enzyme and protein excretion rates along with blood urea nitrogen (BUN) determinations was used to assess the nephrotoxicity of these compounds. At equimolar dosages, tetraplatin appeared to be less nephrotoxic than cisplatin, and CHIP was not nephrotoxic. At all dosages tested, cisplatin increased the rate of urinary excretion of protein, lactate dehydrogenase (LDH), and N-acetylglucosaminidase (NAG) between Days 1 and 5. Tetraplatin did not affect these parameters until the 13.3 mumol/kg dosage. Cisplatin had little effect on the excretion rates of the brush border enzymes alkaline phosphatase and maltase, whereas tetraplatin caused an initial elevation with delayed onset of peak excretion rates at 8 days postinjection. Changes in BUN were not evident until after the 13.3 mumol/kg dosage of cisplatin and the 26.7 mumol/kg dosage of tetraplatin. BUN was useful for ranking the relative toxicities of the three compounds tested, but was not as sensitive in detecting the onset of injury that correlated with early histopathological changes. Tetraplatin appeared to be less nephrotoxic than cisplatin on an equimolar basis and the specific manifestations of its toxicity were different from those observed with cisplatin. Urinary excretion rates for LDH, NAG, and protein proved to be sensitive indicators of platinum analog-induced nephrotoxicity. These indices, combined with BUN determinations and functional assessments, facilitated comparisons of the nephrotoxicity induced by cisplatin and tetraplatin in rats.

Comparative toxicity and renal distribution of the platinum analogs tetraplatin, CHIP, and cisplatin at equimolar doses in the Fischer 344 rat.

Tetraplatin [tetrachloro(dl-trans)1,2-diaminocyclohexane platinum(IV), NSC-363812] is a new anticancer platinum drug analog targeted for clinical development because of its effectiveness against cisplatin-resistant tumor cell lines and its improved formulation. The toxicity of tetraplatin was compared at equimolar doses to that of cisplatin [cis-diamminedichlorophatinum(II)] and CHIP [cis-dichloro,trans-dihydroxybis-isopropylamine platinum(IV), NSC-256927]. Adult male Fischer 344 rats received an iv bolus injection of 6.7, 13.3, 26.7, or 53.3 mumol/kg of one of these drugs in saline and were killed on Day 1, 3, 5, 8, or 15 postinjection for assessment of toxicity with emphasis on evaluation of nephrotoxicity. Rats to be killed on Day 15 were housed in metabolism cages for daily urine collection. Tetraplatin was less nephrotoxic than cisplatin at equimolar doses; CHIP was not nephrotoxic at these doses. Renal platinum contents were similar after all three drugs and did not appear to be related directly to the nephrotoxicity. Nephrotoxicity was detected 4-5 days after 6.7 mumol/kg cisplatin, was localized to the corticomedullary junction, and progressed with time and dose. Tetraplatin-induced alterations of renal function were first observed after 13.4 mumol/kg on Day 4 as an elevation of urine volume (up to 10-fold) and a smaller elevation of urinary glucose excretion. Tetraplatin lesions were localized in the mid- and outer cortex and, even at the highest dose, were less severe than those observed with cisplatin. There were other prominent toxic effects of tetraplatin, such as gastrointestinal toxicity and myelosuppression, which indicate that factors other than comparative nephrotoxicity may impact the clinical potential of this new agent.

Alterations in plasma pharmacokinetics of cisplatin in tumor-bearing rats.

Rats were inoculated s.c. with the Walker 256 solid carcinosarcoma, and when tumors reached a weight of approximately 2-3 g, pharmacokinetics, tissue distribution, and urinary excretion of 195mPt-labelled cisplatin were studied. Cisplatin was given i.v., blood was sampled through arterial cannulae, and data were fitted to a three-compartment model. Distribution half-times were prolonged two- to threefold in tumor-bearing animals, although there was no change in elimination half-time. Initial and steady-state volumes of distribution were also increased in tumor-bearing animals. There was no change in AUC, urinary excretion, tissue distribution, or plasma protein binding. The results indicate that a solid tumor represents an additional compartment for distribution of cisplatin and alters the rate at which cisplatin is distributed from the plasma.

The disposition and metabolism of 2',3'-dideoxycytidine, an in vitro inhibitor of human T-lymphotrophic virus type III infectivity, in mice and monkeys.

The pharmacokinetics and metabolism of the anti-human T-lymphotrophic virus type III/lymphadenopathy-associated virus agent 2',3-dideoxycytidine have been examined in BDF1 mice and rhesus monkeys, with ancillary enzyme studies carried out on tissue derived from both the latter species and also from human subjects. For the pharmacokinetic studies, 2',3-dideoxycytidine and its catabolic product 2',3-dideoxyuridine have been separated and measured in plasma, urine, and cerebrospinal fluid by a reverse HPLC method. For metabolic studies, tritium-labeled drug (labeled in the 5- and 6-positions of the pyrimidine ring) has been employed, utilizing an ion exchange HPLC analytical method suitable for the separation of the parent nucleoside from its mono-, di-, and triphosphates in cell extracts and in tissue homogenates. The drug is rapidly cleared from plasma in a biphasic manner (terminal t 1/2 in BDF1 mice and rhesus monkeys of 67 min and 109 min, respectively) following an iv bolus dose of 325 mg/m2. This two-compartment open model is predictive of plasma concentrations during long term ip infusions in mice. Dideoxycytidine is predominantly excreted in the urine as unchanged parent compound, although a minor urinary metabolite (2,3-dideoxyuridine) is detected in the monkey but not in the mouse. Oral absorption of 2',3'-dideoxycytidine is rapid, with plasma levels approaching those seen after iv administration within 45 min in the mouse. Entry to the central nervous system is also rapid, but the cerebrospinal fluid to plasma AUC ratio after iv administration is only 0.026-0.040 in rhesus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

cis-Diamminedichloroplatinum (II)-DNA adduct formation in renal, gonadal, and tumor tissues of male and female rats.

cis-Diamminedichloroplatinum(II) (cisplatin), a potent anticancer agent, is thought to exert its cytotoxic effects through DNA damage. Using a polyclonal rabbit antisera which recognizes intrastrand bidentate deoxy(ApG)- and deoxy(GpG)-N7-diammineplatinum adducts, an enzyme-linked immunosorbent assay has been developed to quantitate this adduct in cisplatin-exposed DNA. Cisplatin-DNA adducts were measured in renal, gonadal, and tumor (sarcoma) tissues of Sprague-Dawley rats following i.v. or i.p. administration of cisplatin. When drug was administered i.v. to animals fed ad libitum adduct levels were highest in kidneys, 50% lower in s.c. sarcoma, and substantially lower in gonads. Under these experimental conditions, a large interindividual variability in adduct formation was observed in renal and tumor tissues, and adduct levels in some samples were too low to measure. Higher values among individuals were obtained using tissues of animals fasted overnight and treated i.p. Adduct levels following i.p. injections of drug were higher in kidneys and gonads of male rats than in kidneys and gonads of female rats. Analysis of tissue platinum content demonstrated higher platinum levels in kidneys of male rats than in kidneys of female rats, but the magnitude of this gender difference in total tissue platinum was not as great as that observed for adduct formation. When the influence of castration on adduct formation was investigated, adduct levels in kidneys of castrated females were higher than those in sham-operated females, but adduct levels in kidneys of the castrated male animals were not substantively different from those seen in sham-operated male controls. We conclude that the route of drug administration, diet, and hormonal status of the animal are factors that may influence cisplatin-DNA adduct formation in the rat.

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.

The effect of sodium thiosulfate on subcellular localization of platinum in rat kidney after treatment with cisplatin.

Female Sprague-Dawley rats were given 10 mg/kg of i.v. cis-diammine-dichloroplatinum(II) (cisplatin) simultaneously with i.v. sodium thiosulfate (STS) at a 200-fold molar excess to cisplatin, then subcellular (nuclei, mitochondria, microsomes, cytosol) distribution of platinum within rat kidney cells was determined 15 min, 1 h, 8 h and 24 h after cisplatin injection. Blood urea nitrogen levels were measured in rats treated in the same manner described above. STS was found to block cisplatin-induced nephrotoxicity. However, differences in platinum concentrations in total homogenate or each subcellular fraction between STS-treated rats and controls were not significant enough to fully account for the drastic protective effect of STS against cisplatin nephrotoxicity.

Polyclonal antibodies to quantitate cis-diamminedichloroplatinum(II)--DNA adducts in cancer patients and animal models.

cis-Diamminedichloroplatinum (II) (cis-DDP), the antitumor drug, is cytotoxic in vitro primarily by binding to DNA and disrupting its normal functions. We have studied cis-DDP modification of DNA in nucleated peripheral blood cells (buffy coat cells) of testicular and ovarian cancer patients receiving cis-DDP chemotherapy, and of untreated controls. Using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with an antiserum specific for the bidentate intrastrand N7-deoxyguanosine adduct, blood cell DNA was assayed at multiple times during courses of cis-DDP treatment. A total of 138 samples were analyzed from 54 individuals. Of these, all samples from 18 untreated controls were negative, while 44 out of 120 samples from cis-DDP patients were positive. Testicular and ovarian cancer patients receiving chemotherapy on the first course, and given cis-DDP in 21- or 28-day cycles (five days of drug infusion followed by two or three drug-free weeks) accumulated cis-DDP-DNA adducts in blood cell DNA as a function of dose. Patients receiving their first course of cis-DDP on 56-day cycles and those given high doses of this drug after failing other chemotherapy showed much slower adduct accumulation than patients receiving their first course on 21- or 28-day cycles. Adduct accumulation, in positive patients, occurred both as a function of total cumulative dose and with increasing cycle number, suggesting that adduct removal took at least a month in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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 cis-dichlorodiammine-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-1) 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 flow-limited 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 compartments this rate of elimination corresponds closely to the average rate of protein turnover in that compartment.

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

The 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 microM in vitro before the determination of enzyme activity. Although ATPase activity was reduced by cisplatin, either a high concentration of cisplatin (280 microM) 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 microM cisplatin or 160 min preincubation with 400 microM 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 micrograms/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.

Distribution, elimination, metabolism and bioavailability of hexamethylenebisacetamide in rats.

Hexamethylenebisacetamide (HMBA), an in vitro differentiating agent, was studied for its pharmacodynamic actions in animals. Plasma stability, organ distribution, excretion, oral bioavailability, and estimates of pharmacokinetic parameters and acute lethality were determined in rats. The single dose intraperitoneal LD50 was greater than 3000 mg/kg in both mice and rats. The drug was stable in plasma from several different species during an 8 h in vitro incubation at 37 degrees C. Following a single intravenous (iv) bolus injection (1000 mg/kg) to rats, HMBA was removed from the plasma with a half time of 2.2 +/- 0.5 h, and 65 +/- 8% of the dose was excreted unchanged in the urine during the first 24 h after dosing. During an 8 h iv infusion, plasma concentrations of 4 mM were easily maintained with no apparent adverse effects. Drug was uniformly distributed, with highest concentrations found in thymus, kidney, liver, and lymph node throughout the first 24 h after a single iv bolus dose. In vivo metabolism was very small, and the presence of apparent metabolites was undetectable until 48 h after iv administration. Oral bioavailability was good (32%), with peak plasma concentrations of 2 mM achieved one hour after oral administration. After oral dosing urinary excretion and plasma decay were comparable to similar data obtained after iv dosing.

Amelioration of cisplatin-induced nephrotoxicity by the diuretic acetazolamide in F344 rats.

Renal toxicity following a single iv nonlethal dose (6 mg/kg) of cisplatin (CDDP) was investigated in male F344 rats pretreated with 20 mg/kg of acetazolamide (ACZ) sc 30 minutes before CDDP administration. There was a decrease in nephrotoxicity as indicated by a smaller elevation of BUN, a milder histopathologic lesion, and a more rapid recovery of BUN in ACZ-pretreated animals. Renal platinum content of rats receiving ACZ was significantly lower than that in animals treated with CDDP alone. There was no change in platinum level in the plasma or the liver but there was a decreased platinum excretion in urine during 24 hours after treatment. Reduction of platinum concentration in the kidney might account for protection against the renal toxicity. The diuretic ACZ might be useful in the hydration regimens used clinically to prevent renal toxicity from CDDP.