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Certain DNA minor groove binding agents, distamycin, netropsin, and a series of anticancer bis-benzimidazoles can block DNA helicase activity by binding to duplex DNA at specific base sequences. DNA helicases are crucial to cell DNA replication, transcription and repair because these enzymes separate double-stranded DNA, thereby preparing the strands for enzymatic manipulation. From our studies we have developed a hypothesis that focuses on cellular DNA helicase action as a mechanistic site where these minor groove binders can act. A crucial aspect for modulation of DNA activity by drugs is for specificity and selectivity. A series of DNA-interactive bis-benzimidazole analogues of Hoechst 33258 was also prepared to explore the potential for anticancer activity mediated for certain of the drugs via bioreductive activation by endogenous NADH or NADPH. The biological endpoints examined included intracellular distribution in euoxic and hypoxic conditions observed by fluorescence microscopy; relative efficacy as antimetabolites determined by the MTT [tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay in euoxic and hypoxic conditions; and relative inhibitory activities on human DNA helicase, as determined by degree of dissociation of GC B6486 DNA. The intracellular distribution was unique to each of the test compounds. Compounds V-93 and V-153, the respective semiquinone and quinone derivatives, demonstrated the predicted enhanced cytotoxicity and anti-helicase activities, supporting the concept that preferential binding of DNA at 5'-CG and TG sequences provides a novel approach to anticancer drug development.

We previously showed that anthracycline antibiotics potently block SV40 large T antigen helicase; in the present study, we describe the kinetics and the structure-activity characteristics of this process. The concentration vs effect data for helicase blockade were fitted by the Hill equation to yield nearly parallel log-concentration effect curves for a series of active anthracycline antibiotics. The effective concentration for 50% helicase blockade (EC50) values ranged from 0.34 microM for daunorubicin to 40.8 microM for 3'-deaminodaunorubicin. Clinically inactive 3'-N-acyl anthracyclines produced no blockade. The Hill constants for the blockade ranged from 1.1 to 1.6 for the entire series of active anthracyclines, indicating no positive cooperativity and suggesting that a single molecule of bound drug is sufficient to block helicase action. The EC50 values for several clinically effective anthracyclines showed a relationship to the average DNA binding constants for these drugs, and Lineweaver-Burk analysis of the blockade kinetics indicated non-competitive inhibition. The kinetics of the blockade indicated that the anthracycline, DNA, and helicase form a ternary complex that is irreversible under the reaction conditions. This mechanism may be central to the cytotoxic and anti-cancer activities of anthracycline antibiotics and may be useful in understanding the enzymatic mechanism of DNA helicase action.

CI-958, a new DNA-intercalating drug derived from a series of substituted 2H-[1] benzothiopyrano[4,3,2-cd]indazoles, is being tested in clinical trails because of its curative properties against murine solid tumor models and because it has demonstrated activity in a pilot phase II study of patients with hormone-refractory prostate cancer. However, the mechanism of anticancer action of CI-958 has not been established. Because CI-958 binds to DNA and DNA helicases are profoundly affected by DNA-binding drugs, we examined the effects of CI-958 on human DNA helicase action. DNA helicase activity was measured by strand dissociation of double-stranded (ds) DNA with a gel electrophoresis assay, and ATPase activities were determined on thin-layer chromatography by measurement of the conversion of ATP to ADP. For human helicase blockade, CI-958 is slightly more potent than doxorubicin (EC50 values 0.17 and 0.26 microM, respectively). We observed no difference in helicase-blockade EC50 values recorded for three helicase substrates containing A-T rich, G-C rich, and both types of oligonucleotide sequences. The effects of CI-958 helicase blockade and DNA-dependent ATPase activities were similar for the two reactions. The kinetics of the blockade by CI-958 of the human DNA helicase indicates that it involves a reversible ternary complex of helicase-drug-dsDNA. CI-958 produces potent blockade of human DNA helicases with no apparent strong DNA sequence-binding preference. Similar potency against helicase strand dissociation and DNA-dependent ATPase suggests that the mechanism against these reactions is the same. The blockade of DNA helicases by CI-958 may be central in its mechanism of action as an anticancer drug.

DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.

Helicases are essential to both DNA replication and transcription because they separate double-stranded DNA, preparing the single strands for replication or transcription. Because the anti-cancer anthracycline antibiotics stabilize double-stranded DNA primarily by their intercalative binding, we expected the intercalated antibiotics to interfere with helicase action. We examined anthracycline antibiotic effects on SV40 large T antigen helicase activity, using a duplex DNA helicase substrate of 32P-labeled 17-mer annealed to complementary M13mp19(+) circular single-stranded DNA. The T antigen helicase activity was potently inhibited by the anthracycline antibiotics. The T antigen helicase IC50 values for the anthracycline antibiotics were as follows: nogalamycin, 2 x 10(-7) M; daunorubicin, 4 x 10(-7) M; doxorubicin, 4 x 10(-7) M; idarubicin, 1.8 x 10(-6) M; 4'-epidoxorubicin, 2 x 10(-6) M; aclacinomycin, 4 x 10(-6) M; and menogaril, 6 x 10(-6) M. Partially purified helicases from HeLa cells and murine mammary carcinoma FM3A cells also were potently inhibited by doxorubicin, with IC50 values of 4 x 10(-7) M and 9 x 10(-7) M, respectively. Because the abundance, specificities, and types of helicases vary in the cell, this site of action for anthracycline antibiotics may help explain anthracycline potency, drug specificity for DNA or RNA inhibition, and some types of cellular resistance to these drugs.

Forty-six patients with refractory solid malignancies received the new platinum complex [2,2-bis(aminomethyl)-1,3-propanediol-N-N'] [1,1-cyclobutanedicarboxylato] [(2-)0,0')] platinum (zeniplatin). Zeniplatin was given, without hydration or mannitol, as a 60- to 90-min i.v. infusion every 3 weeks at doses ranging from 8 to 145 mg/m2. The maximum tolerated dose of zeniplatin was 145 mg/m2. The dose-limiting toxicity of zeniplatin was dose-related leukopenia and neutropenia, with the nadir usually observed between 1 and 2 weeks after therapy and recovery usually occurring by 3 weeks after therapy. Thrombocytopenia was rare. The most prominent non-hematological side-effect of zeniplatin was nausea and vomiting. Other non-hematological side-effects were mild or absent. Zeniplatin did not induce significant neurological or auditory toxicity. Zeniplatin was not nephrotoxic at doses less than or equal to 120 mg/m2. At 145 mg/m2, the clearance decreased by a mean of 40% after 2 cycles of therapy. Two patients, one with malignant melanoma and one with renal cell cancer, achieved a partial response. Pharmacokinetics of free (plasma ultrafiltrates) and total platinum in plasma were determined in 5 patients. An in vitro study of the rate and extent of zeniplatin binding to protein in human plasma was also performed. Free and total platinum were measured by flameless atomic absorption spectrometry; free zeniplatin was measured in ultrafiltrate by HPLC. Total and free plasma platinum concentrations were co-modelled using the information from the in vitro study.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.

The metabolism and tissue distribution of aclacinomycin A (ACL), marcellomycin (MCM), and musettamycin (MST), three new anthracycline antibiotics, were compared after IV administration to mice. In plasma, total MCM- and ACL-derived fluorescence declined according to first-order kinetics, whereas an initial decline followed by a rebound was observed for MST. In plasma, MCM remained the predominant compound. ACL was eliminated more quickly, and was replaced by two metabolites, the reduced glycoside M1, and an aglycone. In the case of MST, two unidentified metabolites were observed in concentrations equivalent to that of the parent drug. The three drugs were distributed widely to organs, but only ACL achieved measurable concentrations in the brain. Initially, high concentrations of all three drugs were present in the lungs, but these decreased quickly to values similar to those present in the liver and kidneys. Intermediate concentrations of the three drugs were measured in heart and skeletal muscle. Splenic concentrations of all three drugs rose progressively, reaching a maximum at 8 h after injection in the case of ACL and MST, and at 24 h after injection in the case of MCM. Concentrations of the metabolites of MCM and MST were low in all organs except liver and kidney, where the aglycones 7-deoxypyrromycinone and bisanhydropyrromycinone were seen. The metabolism of ACL was extensive. Aglycones were dominant in the liver and kidneys, whereas reduced glycosides predominated in the spleen. These observations indicate that the murine pharmacology of these three structurally similar drugs differs markedly.

Rat liver cytosol and buttermilk xanthine oxidase both converted 7-deoxypyrromycinone, the 7-deoxyaglycone of marcellomycin, a new anthracycline antibiotic, to a nonfluorescent compound under anaerobic conditions and in the presence of an electron donor. Reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate were equally effective electron donors for liver cytosol, and xanthine was the best cofactor for xanthine oxidase. However, xanthine was inactive with liver cytosol. Reactions with xanthine oxidase obeyed Menten-Michaelis kinetics and were inhibited by allopurinol. No xanthine oxidase activity was detected in liver cytosol. Xanthine oxidase also induced a loss of fluorescence when incubated with 7-deoxydaunorubicin aglycone. The nonfluorescent metabolite of 7-deoxypyrromycinone was tentatively identified as the dihydroquinonic derivative of the parent deoxyaglycone on the basis of its spectrophotometric, fluorescent, thin layer chromatographic, and mass spectral characteristics. Our data demonstrate that more than one enzymatic activity, xanthine oxidase, and an unidentified rat liver cytosolic enzyme convert the 7-deoxyaglycones of anthracycline antibiotics to nonfluorescent metabolites.

K562 cell cultures were obtained from three laboratories (A, B and C) outside our institution, and were designated according to source as K562A, K562B or K562C. The cultures obtained were constitutive or "wild type" K562 cell cultures, not cloned sublines. These cell cultures were compared with respect to growth kinetics, cell surface protein markers, surface antigens, cytogenetics and hemoglobin production. Morphology, growth kinetics in liquid suspension culture, cloning efficiency in soft agar culture, binding of anti-K562 monoclonal antibodies, and the majority of cell surface proteins were generally similar. In contrast, several important differences were observed: (1) hemoglobin synthesis induced by hemin was significantly different among K562A, B and C, K562A being most sensitive (P less than 0.05); (2) whereas more than 90% of K562A or C cells appeared to be Philadelphia chromosome (Ph1)-positive, less than 15% of K562B cells contained a Ph1; (3) membrane proteins (93 and 85 kilodalton) were identified in K562A, whereas only the 93 kilodalton protein was detected in K562B and neither of the proteins were detected in K562C. Our results indicate that K562 cells maintained in different laboratories can undergo tangible changes which may influence experimental results obtained in studies using these cells.

After anaerobic reductive activation by either NADPH cytochrome P-450 reductase (EC 1.6.2.4) or xanthine oxidase (EC 1.2.3.2), mitomycin C readily alkylated DNA. When the mitomycin C-alkylated DNA is digested by DNase, snake venom phosphodiasterase, and alkaline phosphatase, only partial release of the monofunctionally linked mitomycin C nucleotide adduct occurs. Cross-linked adducts are not released into dinucleotides but resist nuclease digestion and remain in oligonucleotides and insoluble precipitates. Kinetic analyses show that the nuclease-resistant fraction which is indicative of DNA cross-linking by mitomycin C takes place quite readily. This nuclease-resistant fraction is particularly significant when the amount of total bound mitomycin C is less than 15 mumol/mmol of DNA. The cross-linked mitomycin C product accounts for more than half of the total alkylation under all pH conditions tested. Our data suggest that particular DNA sites are available for DNA cross-linking by mitomycin C, and these sites are probably the preferred and immediate alkylating targets. Furthermore, DNA cross-links by mitomycin C are not the secondary product of monofunctional adducts. Activity of both flavoenzymes is pH dependent, hence, mitomycin C activation and the rate of DNA alkylation are pH dependent. At elevated mitomycin C alkylation of DNA, the highest amount of cross-linking occurs at neutral pH. High pressure liquid chromatographic separation of the nuclease-digested DNA detected one major and two less prominent mitomycin C adducts. These were verified to be mononucleotide mitosene types by UV spectra showing maximum absorbance at 312 and 250 nm. The major adduct was purified and identified as O6-(2'-deoxyguanosyl)-2,7-diaminomitosene by NMR, indicating that the O6 position of guanine is a preferred site in DNA for at least monofunctional linkage formation.

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.

Adriamycin free radical anion concentrations have been correlated with production of 7-deoxyadriamycin aglycone in a reaction catalyzed by NADPH-cytochrome c reductase. The free radical species is detected by electron spin resonance (ESR) spectroscopy and quantified by double integrations. The 7-deoxyaglycone product is isolated by thin-layer chromatography (TLC) and quantified by fluorometry. As the concentration of adriamycin increases, a concomitant increase in aglycone and free radical levels occurs. These results as well as those with inhibitors Vitamin K3, Vitamin E, and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) point to an obligatory free radical intermediate in the metabolism of adriamycin. DMPO inhibits the reaction under aerobic conditions only, and shows no effect under anaerobiosis at the concentrations studied here. Vitamin E and aerobic DMPO act as free radical scavangers, while Vitamin K3 competes for the reducing power of NADPH in the NADPH-cytochrome c reductase system.

This laboratory has reported previously that a cytoplasmic extract of weanling or regenerating adult rat liver (but not normal rat liver) will produce a 2.5-fold increase in the incorporation of tritiated thymidine ([3H]dThd) into liver DNA of a 34%-hepatectomized test animal. (J. Physiol. London 248: 273-284, 1975). The present study showed that hepatic stimulator substance (HSS) will stimulate DNA synthesis in normal adult rats and CF1 mice as well. The increased incorporation of [3H]dThd into DNA produced in the normal, nonhepatectomized adult rat was comparable with that induced by a 34% hepatectomy. Autoradiographic studies revealed that the [3H]dThd was incorporated into nuclear DNA and that the stimulation occurred almost exclusively in parenchymal cells. HSS was shown to be heat stable (100 degrees C for 15 min) and was precipitated but not inactivated by alcohol. Ultrafiltration and dialysis studies suggested a molecular weight slightly greater than 10,000. HSS proved to be organ specific, stimulating the liver but not the kidney, bone marrow, or spleen. HSS was found to contain no insulin, glucagon, epidermal growth factor, or peptides of the nonsuppressible insulinlike/multiplication-stimulating activity (somatomedin) group.

We investigated the in vitro interaction with and antitumor effect on several murine and human leukemic cell lines of diaziquone (AZQ). L1210 cells accumulated AZQ from Roswell Park Memorial Institute Medium 1640 with or without newborn calf serum by a temperature-dependent and sodium azide-resistant process. AZQ inhibited, in a dose-dependent fashion, [3H]thymidine incorporation into L1210 cells, but this inhibition was slow to develop, requiring approximately 6 hr to become apparent. The minimal inhibitory concentration of AZQ for this process was 0.05 to 0.25 nmol/ml. AZQ was a much less effective inhibitor of L1210 cell [3H]uridine and [14C]valine incorporation. In suspension cultures, AZQ inhibited growth of L1210 and HL-60 cells at minimal inhibitory concentrations of 0.5 to 1 nmol/ml. In soft agar cultures, AZQ inhibited HL-60 cell cloning at minimal inhibitory concentrations of 0.1 to 0.3 nmol/ml. AZQ provoked a dose-dependent increase in oxygen consumption when added to intact L1210, HL-60, and K562 cells and was converted to an AZQ anion free radical by these cells. When the aziridine rings of AZQ were opened by acid treatment, the resulting molecule was not accumulated by L1210 cells, did not provoke O2 consumption, did not form free radicals when added to L1210 cells, and was a much less effective inhibitor of [3H]thymidine incorporation by L1210 cells than was AZQ.

The metabolism and disposition of marcellomycin (MCM), a new anthracycline antitumor antibiotic, were studied after iv administration to mice. In plasma, total drug fluroescence decreased according to first order kinetics and was mainly comprised of parent drug. In addition to MCM, five compounds (M2, P1, P2, G1, G2) were seen. M2 reflected the presence of a contaminant in the parent drug. P1 and P2 represented polar conjugates of MCM and M2, respectively; and G1 and G2 proved to be aglycones. P1 and G1 were observed during the first hours after injection, and G2 was more persistent and represented 35-50% of total drug fluorescence by 8 hr after injection. MCM was distributed widely to organs, except the brain. High MCM concentrations were measured in the lungs initially but they decreased quickly and, by 60 min, reached values similar to those present in the liver and kidneys. Low MCM concentrations were observed in heart and muscles. The splenic concentration of fluorescence rose progressively and, by 16 hr, was higher than that in all other organs. The concentrations of MCM metabolites were small in all tissues except in the liver, where an aglycone identified as 7-deoxypyrromycinone, was seen during the first 4 hr after injection. Our results indicate that the metabolism of MCM in the mouse is qualitatively similar to that in man, but that important quantitative differences exist. The significance of this observation as a possible explanation of the differences in observed toxicities between the two species remains to be established.

Diaziquone (also called "aziridinyl benzoquinone," or AZQ), an antitumor drug designed to penetrate the blood-brain barrier, has demonstrated activity against central nervous system (CNS) neoplasms. Four-hour infusions of carbon-14 (14C)-labeled AZQ (0.8 mg/kg) were given via the left common carotid artery or left brachial vein to two groups of puppies. A third group, harboring a transplantable canine glioma, received 14C-AZQ by intravenous infusion. Levels of chloroform (CHCl3)-extractable 14C (AZQ only) and total 14C (AZQ and metabolites) were determined in serial samples of plasma and cerebrospinal fluid (CSF). At the end of the infusion time, total and CHCl3-extractable 14C levels were determined in brain and tumor. Intra-arterial infusion of AZQ caused no histological abnormalities in the retina or brain. For the intravenous infusion group, the concentrations of CHCl3-extractable 14C (in nmol/ml or nmol/gm) were 0.68, 0.35, and 0.84 for plasma, brain, and CSF, respectively. For the intra-arterial infusion group, the concentrations were 0.25, 0.13, and 0.32 for plasma, brain, and CSF, respectively. Comparison of right and left hemispheres following intra-arterial infusion showed a slightly higher concentration of 14C in the ipsilateral (left) hemisphere, with concentrations (nmol/gm) of CHCl3-extractable 14C/total of 14C of 0.15/0.87 on the left and 0.12/0.65 on the right. Concentrations (nmol/gm) of CHCl3-extractable 14C/total 14C in brain and tumor were 0.60/1.24 and 0.58/1.65, respectively. In tumor-bearing animals, tumor and surrounding brain contained similar concentrations of AZQ, but there were higher concentrations of metabolites in tumor. This may reflect different metabolism of AZQ within brain and tumor or different permeability to metabolites. This study revealed that AZQ enters the CNS and brain-tumor tissue in substantial concentrations and that there is no significant advantage to intracarotid infusion of AZQ.

The dose-limiting toxic effect of high-dose (100 mg/m2) cisplatin is renal insufficiency. Hydration with furosemide- or mannitol-induced diuresis has been reported to ameliorate this toxicity. Animal studies suggest that mannitol may be superior to furosemide in this regard. Twenty-two patients with advanced neoplasms refractory to conventional therapy were treated with cisplatin at a dose of 100 mg/m2 every 21--28 days. Patients were randomized to receive 37.5 g of mannitol by 6-hour infusion with cisplatin or 40 mg of furosemide prior to cisplatin therapy. Hydration with at least 1 liter of normal saline was given prior to cisplatin. Nephrotoxicity (creatinine greater than 2 mg/100 ml, creatinine clearance greater than 50 ml/minute) occurred in 19% of courses in the furosemide-treated group and in 28% of courses in the mannitol-treated group. Peak plasma platinum concentration, terminal half-life, urinary excretion, and percent protein-bound plasma platinum were similar in both groups. The use of cisplatin at this dose schedule resulted in similar toxicity and pharmacokinetics when using hydration with either furosemide or mannitol.

Nuclei isolated from rat liver, heart, and kidney catalyze oxygen consumption in the presence of reduced pyridine nucleotide (NADPH) and quinone or quinone-imine antibiotics such as Adriamycin, daunorubicin, actinomycin D, mitomycin C, and streptonigrin. The Km and Vmax values for NADPH were 2.4 x 10(-3) M and 3 x 10(-8) mol O2 per min per mg protein and Km values for the antibiotics ranged from 1.4 x 10(-4) M to 5.9 x 10(-6) M. Metabolism of the anthracycline antibiotics, i.e., reductive glycosidase reaction, occurs in reaction mixtures after all oxygen is consumed. During the reaction, free-radical species of Adriamycin and daunorubicin are detectable by electron paramagnetic reasonance spectrometry. These observations indicate that some cytotoxic antibiotics can be activated to a free-radical state at the site where damage to nuclear DNA may result.

Ten patients with brain tumors and indwelling ventricular reservoirs were pretreated with 5% to 10% dimethyl sulfoxide (DMSO) (intravenous, oral, or both) and were then treated with 1.0 to 1.25 gm/m2 cyclophosphamide (CYC). All patients were also on anticonvulsants and dexamethasone. CYC and alkylating activity (alk act) in plasma and concomitant ventricular cerebrospinal fluid (CSF) were measured by gas chromatography and p-nitrobenzyl pyridine assay. CYC entered the CSF without difficulty and was lost from CSF more slowly than from plasma. Alk act did not enter CSF as well as did CYC. DMSO did not alter any measured aspect of CYC or alk act disposition. Specifically, it did not alter the CYC plasma half-life (t1/2), CSF t1/2, peak CSF: peak plasma CYC concentration ratio, or the urinary excretion of CYC. DMSO did not alter the plasma t1/2 or urinary excretion of alk act or the peak CSF:peak plasma concentration ratio of alk act. Our data show reduced plasma t1/2 of CYC and increased plasma and urinary alk act. This may reflect tht effect of long-term therapy with anticonvulsants or steroids.

Aldehyde and ketone xenobiotic substances are preferentially reduced to alcohols by cytoplasmic enzymes in mammals. These enzymes are widely distributed in the tissues, have broad substrate specificities, have similar physical-chemical characteristics, and require reduced nicotinamide adenine dinucleotide as cofactor for the reductions. These reductases define a system of detoxification for aldehyde and ketone groups.

A patient with a choledochal T tube and normal liver function received adriamycin as therapy for abdominal histiocytic lymphoma. Plasma, urine, and bile samples were collected after drug administration. Adriamycin and its metabolites were extracted from the samples and separated by thin-layer chromatography. The pharmacokinetics of adriamycin and metabolites in plasma urine resembled those of previous patients, with a plasma elimination half-life for adriamycin of 25.2 hr. Bile contained adriamycin and 11 metabolites, 4 of which were not present in plasma or urine. Forty-one percent of the administered dose of adriamycin appeared in the bile in 7 days; of this amount, 42% was adriamycin, 22% was adriamycinol, the major metabolite, and 36% was other metabolites. Hepatic clearances of adriamycin and adriamycinol showed early, rapid removal of drug by the liver, with subsequent slowing of removal rate as plasma drug concentration declined. Adriamycin was more efficiently cleared than adriamycinol by both liver and kidney. Disease states which impair the capacity of the liver to excrete adriamycin may result in prolonged, high drug levels and increased toxicity.

Plasma and cerebrospinal fluid (CSF) kinetics of diaziquone, 2,5-diaziridinyl-3,6-biscarboethoxyamino-1,4-benzoquinone (AZQ), were evaluated after intravenous injection in patients with implanted Ommaya reservoirs. After a 30-min infusion plasma disappearance was very rapid, with a disposition half-life of 2 to 6 min and on elimination half-life of 25 to 35 min. Area under the concentration-time curve for CSF was 22% to 42% of the corresponding plasma area. Based upon total plasma AZQ concentration, volume of distribution for AZQ was 2.0 to 10.0 l/m2. Plasma binding of AZQ was 79% in one normal subject. When the effect of plasma binding is considered, distribution volumes are more plausible and it appears that free plasma AZQ is completely available to the CSF.

We studied the effect of deoxyguanosine (dGuo) on cellular cytarabine (ara-C) nucleotide accumulation of the human leukemia cell line K562 and of bone marrow blast cells derived from patients with acute nonlymphocytic leukemia. Exposure of cells in culture to dGuo increased ara-C nucleotide accumulation measured in cell lysate, with an average increase of 386% (range, 242%-537%) of control in the presence of 500 microM dGuo. Maximal elevation occurred after 8 hours of exposure and remained constant through 48 hours. dGuo also enhanced deoxycytidine nucleotide accumulation, but dGuo enhancement favored accumulation of ara-C nucleotides over dCyd nucleotides. In cell cycle kinetic studies using flow cytometry, dGuo slowed accumulation of cells with apparent S-phase DNA content in a concentration-dependent fashion. However, neither the rate nor the magnitude of this effect correlated with the increase in ara-C nucleotide accumulation. Since the increase in ara-C nucleotide accumulation caused by dGuo could be prevented by 5 micrograms/ml of cycloheximide, this process appears to require new protein synthesis. Although these data suggest that the elevation of ara-C nucleotide accumulation caused by dGuo may represent induction of enzyme synthesis, other possibilities are discussed. Exposure of bone marrow blast cells obtained from patients with acute nonlymphocytic leukemia to dGuo for 16 hours in liquid culture also increased ara-C nucleotide accumulation. In six of seven studies, exposure to dGuo in concentrations from 50 to 500 microM increased ara-C nucleotide accumulation from 160% to 3400%. These data suggest that dGuo may alter ara-C metabolism in a clinically useful fashion.

Cyclo(His-Pro) is believed to be a metabolite of TRH. A specific antiserum directed against cyclo(His-Pro) was used to detect immunoreactive material in human serum. Gel filtration column chromatography was used to establish that cyclo(His-Pro)-like immunoreactivity was found not only in free form established to be identical to cyclo(His-Pro) by high pressure liquid chromatography analysis on reverse phase and cation exchange columns, but also in a fraction of about 100,000 mol wt. Free cyclo(His-Pro) was released by heating column fractions of 70,000 mol wt that were not immunoreactive before heating. A procedure for estimation of the content of free cyclo(His-Pro) in human serum was developed. Normal levels of cyclo(His-Pro) determined by this procedure were in the range of 11-33 pmol/ml. The level of cyclo(His-Pro) in sera of individuals with hypothyroidism, hyperthyroidism, or alcoholic cirrhosis was in the normal range, while patients with renal failure had approximately 3-fold elevated levels of the peptide. Analysis of human sera drawn at 3-h intervals over a 24-h period suggested a circadian rhythm of cyclo(His-Pro) levels.

Plasma and milk concentrations of doxorubicin (DOX) and cisplatin were measured after iv administration of these agents to a lactating patient with ovarian cancer. Cisplatin was undetectable in human milk. Milk concentrations of DOX often exceeded those detected in concomitant plasma samples. For DOX, the highest milk:plasma concentration ratio was 4.43:1 and was observed 24 hours after administration of the drug. The area under concentration versus time curve (AUC) for DOX was approximately the same in plasma and milk. Doxorubicinol was the major metabolite of DOX in plasma and in milk. The AUC for doxorubicinol was ten times higher in milk than in plasma. However, the total amount of anthracycline antibiotic delivered in the milk (maximum concentration of active anthracycline antibiotic: 0.24 mg/L) was negligible.

The effect of hyperthermia on the uptake and metabolism of doxorubicin (ADM) was studied in in vitro systems. ADM uptake in rat liver slices was not affected by increasing the temperature from 37 degrees C to 43 degrees C. In rat liver homogenates, the aerobic transformation of ADM was low and was not affected by hyperthermia. Approximately 90% of the parent drug remained unchanged after 60 minutes of incubation, and two metabolites, adriamycinol and a polar metabolite, were formed in small amounts. Under anaerobic conditions, ADM was quickly and extensively converted to two metabolites identified as 7-deoxyadriamycinol aglycone and 7-deoxyadriamycin aglycone. Whereas the disappearance of ADM and the formation of 7-deoxyadriamycin aglycone were not modified by the hyperthermic conditions, there was a slight but significant increase of the formation of 7-deoxyadriamycinol alycone (area under the concentration versus time curve in microM X minute: 216 +/- 24 at 37 degrees C; 235 +/- 24 at 39.5 degrees C; and 271 +/- 8 at 42 degrees C; P less than or equal to 0.05). However, the percentage of dA3 was not significantly different at the end of the incubation.

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.

Rat liver microsomes contain a phenobarbital inducible, NADPH dependent, reductive glycosidase capable of cleaving several anthracycline antibiotics, including adriamycin and daunorubicin, to deoxyaglycone products. The pH optimum for the reaction ranges from 7 to 7.4, and no metal requirements are noted. Molecular oxygen reversibly inhibits the microsomal enzyme greater than 95% at 20% oxygen partial pressure. Carbon monoxide, SKF 525A and sulfhydryl reagents are not inhibitory to the reaction, but the enzyme is sensitive to Cu++ and Zn++. Since the intact glycoside is necessary for conversion to the deoxyaglycone and a possible intermediate hydroxylated aglycone is not reduced to the deoxyaglycone, a concerted reaction mechanism is proposed. The reductive glycosidase activity is also present in rat brain, kidney and other tissues. Sensitivity of this enzyme to molecular oxygen suggests a possible regulatory role for the enzyme in vivo.

The aziridinyl quinone 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone is an antitumor agent that enhances electron flow from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen in rat liver microsomes, rat liver nuclei, and purified NADPH-cytochrome c reductase. The process is enzymatic and has an optimum pH of 7.5; NADPH is the preferred cofactor. Electron spin resonance studies show the presence of a free radical semiquinone characterized by five hyperfine lines at g = 2.0046.

Thirty-six patients with disseminated sarcomas were treated with adriamycin according to an intermittent single high-dose schedule based on our prior pharmacologic observations. The dose of 60 mg/M2 IV q 3 weeks was reduced 50-75% in the presence of liver disease. Complete remission occurred in 9%, parital remission in 32%, and improvement in 21% for median durations of 37, 18, and 15 weeks, respectively. Survival was prolonged in responding patients. Hematologic suppression was mild in the absence of liver disease or prior extensive radiotherapy. Congestive heart failure, a well-known long-term complication of therapy with anthracycline drugs, occurred in two out of four patients who had cumulative doses of 600 mg/M2 or more. Mucous membrane toxicity was not observed. Adriamycin is an effective new agent in the therapy of disseminated sarcomas and can be used with relative safety by following pharmacologic principles.

The one-electron electrochemical reduction of diaziquone (AZQ) and 12 analogs is analyzed using ESR spectroscopy and cyclic voltammetry. The hyperfine coupling constants arising from the interaction of the unpaired electron with the aziridine nitrogen nuclei fall within 1.20 and 2.26 G. Smaller couplings are observed arising from the protons and nitrogens in the carboethoxyamino groups. The in vitro activity of AZQ and its analogs is examined. Methyl groups in the aziridine rings increase the activity of some analogs. In the absence of aziridines, a chloroquinone compound with only carboethoxyamino groups was surprisingly active. This compound has a more positive cathodic peak than diaziquone.

6-thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic methods. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50-100% recovery efficiency. The neutralized extracts were chromatographed on a muBondapak C18 column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5-10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate-25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.