New TFO conjugates containing a carminomycinone-derived chromophore.

Conjugates obtained by linking the anthracycline intercalating chromophore to triple helix forming oligonucleotides (TFOs) have been used in a physicochemical study of the stability of triple helices with DNA sequences of pharmacological relevance. The intercalating moiety is represented by carminomycinone derivatives obtained upon O-demethylation and hydrolysis of the glycosidic linkage of daunomycin followed by the introduction of an alkylating residue at two different positions. Results of experiments with a polypurinic region present in the multidrug resistance (MDR) gene indicate that the stability of the triple helix is significantly enhanced by replacement of C's with (5-Me)C's in the TFO sequences tested. The stability is not changed when a 3'-TpT is present in place of a 3'-CpG at the presumed intercalation site of the anthraquinone chromophore. The same carminomycinone derivatives were used for the preparation of conjugates able to form triple helices with the polypurine tract (PPT) present in the human integrated genome of HIV-1 infected cells. Three different TFOs (T(4)(Me)CT(4)(Me)CC, C2; T(4)(Me)CT(4)(Me)CC(Me)CC(Me)CCT, C6; and T(4)(Me)CT(4)G(6), G6) were designed and linked to the anthraquinone moiety. These conjugates showed a significantly enhanced ability to bind the PPT region of HIV with respect to the nonconjugated TFOs.

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA.

We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: 3389-3395, 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the epsilon-rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin.

Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5.

A methyltransferase that acts on carminomycin and 13-dihydrocarminomycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycin- and baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5% yield with a purification of 114-fold in specific activity over the sample precipitated with 30-50% ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M(r) of about 41,000. The enzyme was shown by gel filtration chromatography to have an M(r) of approximately 166,000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-L-methionine typical of antibiotic methyltransferases; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-L-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, epsilon-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.

Determination of the new morpholino anthracycline MX2.HCl and its metabolites in biological samples by high-performance liquid chromatography.

Methods for determining concentrations of a new morpholino anthracycline MX2.HCl and its metabolites in biological samples using reversed-phase high-performance liquid chromatography and fluorescence detection are described. The limits of detection were less than 1 ng/ml for all compounds after extraction from 0.5 ml of plasma using C18 Sep-Pak cartridges and consecutive solvent extraction. The recoveries from rat plasma ranged from 72.0 to 89.3%. The peak-height ratio of the fluorescence intensities of these compounds versus internal standard showed a linear correlation for concentrations up to at least 500 ng/ml in the plasma (correlation coefficient r greater than 0.999). The within-day and between-day precisions of this assay were in the range 0.8-8.7% (n = 5) and 2.0-3.5% (n = 5), respectively. The concentrations of these compounds in the blood and urine can be also determined by a slight modification of the extraction procedure.

Establishment of a human leukemia subline resistant to the growth-inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) and showing non-P-glycoprotein-mediated multi-drug resistance.

We have previously reported that K562/ADM, a typical P-glycoprotein-mediated multi-drug-resistant cell line, is cross-resistant to the growth-inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) and non-TPA type tumor promoters. To elucidate the mechanism of cross-resistance to tumor promoters in K562/ADM, we have established a K562 subline resistant to TPA-induced growth inhibition by exposing K562 cells to N-methyl-N'-nitro-N-nitrosoguanidine for 24 hr followed by continuous exposure to TPA. A K562 subline resistant to the TPA-induced growth inhibition, termed K562/TPA, was selected by a limiting dilution technique. K562/TPA was more than 500-fold resistant to TPA compared with parental K562 cells. K562/TPA showed cross-resistance to etoposide, teniposide, adriamycin (ADM), vincristine, vindesine and 3-[(4-amino-2-methyl-5-pyrimidinyl)] methyl-1-(2-chloroethyl)-1-nitrosourea, but showed collateral sensitivity to cisplatin. Although K562/ADM was not cross-resistant to 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2), an anthracycline derivative, K562/TPA was cross-resistant to MX2. By Northern blot analysis, K562/TPA did not express MDR-1. Accumulation of ADM by K562/TPA was no lower than that of K562 although that of K562/ADM was 5-fold lower than K562. We examined the subcellular distribution of ADM by fluorescence microscopy. The fluorescence of ADM was located in the nucleus of K562 and mainly in the cytoplasm of K562/TPA and K562/ADM. The distribution of ADM in K562/TPA, however, was different from that in K562/ADM. These results suggested that K562/TPA had a non-P-glycoprotein-mediated multi-drug-resistance phenotype and that the mechanism of drug-resistance in this cell line might be explained by an alteration in the intracellular drug distribution.

Cellular pharmacology of MX2, a new morpholino anthracycline, in human pleiotropic drug-resistant cells.

We previously reported that MX2, a new morpholino anthracycline, showed marked effects on pleiotropic drug-resistant sublines of murine P388 leukemia in vivo as well as in vitro. In this study we examine the in vitro cytotoxicity against pleiotropic drug-resistant sublines of human tumor cell lines. MX2 was effective against multidrug-resistant sublines of four human tumor cell lines; these cells, having a 4.8- to 200-fold cross-resistance to Adriamycin (ADM) showed only a 0.7- to 2.3-fold resistance to MX2 compared with the sensitive cells. To elucidate the mechanism by which MX2 overcomes multidrug resistance, the intracellular pharmacology of MX2 in human myelogenous leukemia K562 and its ADM-resistant subline (K562/ADM) was examined. Both K562 and K562/ADM cells accumulated MX2 more easily than ADM, and the intracellular accumulation of MX2 attained a steady state in both cell lines within 30 min of incubation at 37 degrees C. The amount of MX2 that accumulated in K562/ADM at a steady state was only 1.3 times lower than that in K562. However, ADM was accumulated slowly in both cell lines compared with MX2, and the intercellular concentration reached a steady state in K562/ADM after 90 min of incubation and in K562 after more than 120 min. K562/ADM cells accumulated a 3.3-fold lower concentration of ADM than K562 after 120 min of exposure. The steady-state concentration of ADM in K562/ADM was 8.3 times lower than that of MX2. In addition, greater than 70% of MX2 was retained in both cell lines after 150 min of incubation in the absence of this drug. Verapamil, a calcium antagonist, hardly augmented the cytotoxicity of MX2 against K562/ADM, and no distinct effect of this drug on both the time course and the maximal level of accumulation of MX2 was observed. Interestingly, MX2 effectively inhibited ATP/Mg2(+)-dependent [3H]vincristine binding to K562/ADM membrane preparations, indicating that MX2 could be transported outside the cell by an active efflux pump. The high intracellular accumulation and retention of MX2 in K562/ADM through the rapid influx of the drug into the cells may be one of the reasons why MX2 circumvents pleiotropic drug resistance.

3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks.

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.

Role of DNA-binding in the cytotoxicity of an anthracycline, R20X2 and its morpholino analog, MX2.

3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2), a morpholino anthracycline derived from 13-deoxo-10-hydroxycarminomycin (R20X2) was 16 times less cytotoxic than R20X2 against cultured P388 leukemia cells. The reduced cytotoxicity of MX2 was not explainable by intracellular or intranuclear concentration of the drug or by its DNA-intercalating activity. Binding of MX2 and R20X2 to DNA was measured, after isolating the DNA fraction from an incubation mixture of the drugs with P388 cells or with calf thymus DNA. The amount of R20X2 bound to the DNA was obviously larger than that of MX2, and was dependent on incubation time. These data suggest that the poor binding activity of MX2 to DNA contributes to its reduced cytotoxicity.

[Interaction of carminomycin with DNA according to data of laser polarized fluorescence]. / Vzaimodeistvie karminomitsina s DNK po dannym lazernoi poliarizovannoi fluorestsentsii.

Anti-tumour antibiotic carminomycin interaction with chicken erythrocyte DNA is studied in aqueous-salt solutions by the laser polarized fluorescence method. Fluorescence quenches almost equally effectively during the antibiotic absorption on native (nDNA) and denatured (dDNA) DNAs, but the polarization degree of residual fluorescence differs about two times. Carminomycin binding to dDNA is characterized by one interaction type with a large density of occupancy sites - one antibiotic molecule per base pair. Carminomycin forms two types of complexes with nDNA, differing significantly with binding constants. Strong binding, intercalation, is saturated at one carminomycin molecule per 3 base pairs independently on the solution ionic strength. The weaker, external, interaction is characterized by the binding constant being by two orders of magnitude lower than that for intercalation, and the external interaction contribution is negligible.

["Chimeric" antibiotics, daunorubicin and its analogs N-acylated with bruneomycin (Streptonigrin)]. / "Khimernye antibiotiki"--daunorubitsin i ego analogi, N-atsilirovannye bruneomitsinom (Streptonigrinom).

Anthracycline antibiotics (daunorubicin, carminomycin and doxorubicin) N-acylated with antibiotic bruneomycin (streptonigrin) have been obtained from the parent compounds upon treatment with N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide. These "chimeric" antibiotics are less active both in vitro and in vivo than the parent antibiotics. This demonstrates the stability of the intermolecular amide linkage in these compounds towards chemical and enzymatic hydrolysis as well as their inability to interact with corresponding receptors in contrast to less hindered derivatives of the parent antibiotics.

In-beam light absorption measurement of anthracyclines in cells with a flow-through system.

A flow-through cuvette in which cells attach as a monolayer to a quartz plate was developed for measurement of the light absorbance of anthracyclines in cells. Despite the drawback of a short path-length (of the order of the cell diameter), a dynamic flow-through set-up and baseline storage made it possible to measure intracellular absorbance and obtain spectral data for daunomycin and carminomycin. Stopping the flow and allowing the drug to equilibrate between medium and cells led to a 20% decrease of molar light absorption of cellular anthracycline, which permitted measurement of the total cellular concentration. Furthermore, accumulation and efflux kinetics were determined for H35 rat hepatoma cells. On the basis of the reported formation constant of the iron-complex of carminomycin, which is of the order of 10(34), we expected to find this complex within the cells. However, the spectrum of cellular drug did not show absorbance bands characteristic of the complex. A red shift and hypochromism were found in the daunomycin spectrum after intracellular binding, which corresponds with the spectral change observed after intercalation of daunomycin into DNA.

An unusual NAD(P)H-dependent O2-.-enerating redox system in hepatoma 22a nuclei.

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.

[Formation of free radicals during interaction of adriamycin and carminomycin with xanthine oxidase]. / Obrazovanie svobodnykh radikalov pri vzaimodeistvii adriamitsina i karminomitsina s ksantinoksidazoi.

Xanthine oxidase reduces carminomycin and adriamycin to the semiquinones which have been detected by ESR technique. The steady state carminomycin semiquinone concentration is some tens times higher than the corresponding value for adriamycin. This effect appears to be a result of carminomycin semiquinone stabilization due to internal hydrogen bonding.

Cell surface effects of adriamycin and carminomycin immobilized on cross-linked polyvinyl alcohol.

Previous reports have claimed Adriamycin to be cytotoxic to cultured tumor cells when the drug is covalently immobilized on a solid support, thus suggesting a cell surface mechanism of action for the drug. Although these previous reports attempted to rule out released drug or endocytosis of drug-support particles as alternative explanations for the observed cytotoxicity, a more thorough analysis is necessary to substantiate fully the cell surface idea. In the present work, the stability of the drug-support linkage was increased by use of cross-linked polyvinyl alcohol as the support and cyanuric chloride or a diazonium salt for attachment of the drug. Different anthracycline orientations were tested by coupling Adriamycin at the amino sugar and carminomycin at the D-ring. The Adriamycin cross-linked polyvinyl alcohol and carminomycin cross-linked polyvinyl alcohol preparations had much lower drug release rates than did the earlier used carbamate-linked Adriamycin cross-linked agarose materials. All three immobilized drug preparations inhibited the growth of L1210 or S180 clones following 2- or 20-h incubation with cells at 37 degrees C. The results strongly support the concept that immobilized anthracyclines can be cytotoxic to cultured cells, for at least two different orientations of the drug on the support.

[Intracellular distribution of a DNA and carminomycin complex]. / Issledovanie vnutrikletochnogo raspredeleniia kompleksa DNK s karminomitsinom.

The complex of the tritium labeled DNA with carminomycin was injected intravenously to mice. Five h after the complex injection the rats were sacrificed. The liver was isolated and homogenized. The cells were consecutively divided into fractions: intercellular liquid, cytoplasm, nuclear juice and chromatin. The content of high-molecular exogenous DNA and the products of its degradation, as well as the quantity of free and DNA bound carminomycin were determined radiometrically and spectrofluorometrically in every of the above fractions. It was found that the carminomycin complex located in the liver remained unchanged for a long time after injection and penetrated into the cell in the form of the complex. In the cytoplasm, the complex disintegrated to liberate carminomycin which penetrated into the nucleus where it was bound to chromatin DNA.

Ligand self-association at the surface of liposomes: a complication during equilibrium-binding studies.

Daunomycin and carminomycin, two anthracycline antibiotics known to bind phospholipid bilayers, appear to self-associate at the surface of liposomes at high bound drug/lipid ratios (r). Fluorescence intensity, lifetime, and anisotropy measurements have been used to monitor the equilibrium binding of these drugs to small unilamellar solid-phase dipalmitoylphosphatidylcholine vesicles. Association of an anthracycline with excess liposome (low r) resulted in an increase in both the observed intensity and the fluorescence lifetime. At low vesicle concentrations (high r), a decrease in the total emission intensity was observed which was not paralleled by the excited-state lifetime. The data from these experiments are consistent with the formation of nonfluorescent anthracycline complexes at the surface of liposomes. Such ligand self-association is a potential complication in any studies on the interaction of amphipathic molecules with liposomes conducted at high r values. Because ligand self-association limits the collection of binding data over certain concentration ranges, this consequently results in greater uncertainty in the determination of the maximum value of r (n) in equilibrium binding studies.

Pharmacokinetics of carminomycin in man: biweekly schedule vs single dose every three weeks.

Carminomycin was administered to five patients at a dose of 7.5 mg/m2 twice weekly. Plasma and urine samples were obtained during two subsequent 72-hr periods following drug administration, and assayed for carminomycin (C) and carminomycinol (Col) by HPLC with fluorescence detection. Distribution of carminomycin was rapid and drug levels decreased below the detection limit (5 X 10(-9)M) within 24 hr. Carminomycinol appeared very quickly and surpassed carminomycin levels in 10 min-4 hr, disappearing very slowly, with a half-life of 40-98 hr. No major differences in pharmacokinetic behavior were found when comparing the five patients in this study with patients who received 18 mg/m2, as described in a previous report. After the second dose of carminomycin in the 7.5 mg/m2 twice weekly schedule, however, carminomycin pharmacokinetics were found to be altered in comparison with the first dose, the most pronounced difference being an increase in the t1/2 for Col from 65 +/- 28 to 173 +/- 81 hr.

Rapid quantitative determination of seven anthracyclines and their hydroxy metabolites in body fluids.

A reversed-phase high-performance liquid chromatographic method is described for the determination of seven anthracycline analogues and their hydroxy metabolites. The method is suitable for analysis of 30 plasma samples a day. The detection limit is 0.5 ng/ml for all compounds. Examples of the pharmacokinetics of adriamycin and carminomycin in man are shown.