Copper(II) interaction with 3,5-diisopropylsalicylic acid (Dips): new insights on its role as a potential *OH inactivating ligand.

The purpose of this study was to identify the low molecular mass complexes formed between copper(II) and 3,5-diisopropylsalicylic acid (Dips) in physiological conditions. Copper(II)-Dips complex equilibria were determined using glass electrode potentiometry and their solution structures checked by UV-visible (UV-vis) spectrophotometry. Because of the low solubility of Dips in water, the equilibria were investigated in different water/ethanol mixtures. Formation constants were extrapolated to 100% water and then compared with the values obtained for the other anti-inflammatory drugs previously studied. Given the prime role of histidine as the copper(II) ligand in blood plasma, copper(II)-histidine-Dips ternary equilibria were studied under similar experimental conditions. Computer simulations of copper(II) distribution relative to different biofluids, gastrointestinal (g.i.) fluid and blood plasma, show that like salicylic and anthranilic acids, Dips favors g.i. copper absorption, but cannot exert any significant influence on plasma copper distribution. Moreover, Dips can mobilize increasing fractions of copper(II) as the pH decreases. In conclusion, Dips seems to correspond to the notion of *OH-inactivating ligand (OIL) as determined for anthranilic acid.

Hydroxyapatite bioceramic with large porosity.

Calcium phosphate based biomaterials have been used as bone graft with great success in the last decade. This material is employed in orthopedic and dental applications depending on their specific properties. In this work, we made a bioceramic with a large porosity, then we measured porosity, density and compressive strength of HA bioceramic. The SEM analysis was performed to show the morphology of the structure. The mechanical properties depend on the sintering of the HA bioceramic and the amount of pores. Thus, properties can be controlled by designing bioceramics with the appropriate porosities and calcination temperatures. One advantage of using gelatin is the formation of solids of any desired shape following a short time period, as the gelatin absorbs the water and expands into a solid composite form.

Interaction of adriamycin with cardiolipin-containing vesicles. Evidence of an embedded site for the dihydroanthraquinone moiety.

In the presence of cardiolipin-containing small unilamellar vesicles, the antitumor compound adriamycin loses its ability to catalyse the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. The data strongly suggest that in the presence of cardiolipin the dihydroanthraquinone moiety is embedded in the phospholipid bilayer and thus inaccessible to the enzyme.

Physicochemical studies of the iron(III)-carminomycin complex and evidence of the lack of stimulated superoxide production by NADH dehydrogenase.

Fe(III) complex of an antitumoral antibiotic carminomycin has been studied. Using potentiometric and spectroscopic measurements we have shown that carminomycin forms with Fe(III) a well-defined species in which three molecules of drug are chelated to one Fe(III) ion. This occurs with the release of one proton per molecule of drug. Magnetic susceptibility measurements suggest that six oxygen atoms are bound to iron. The stability constant is 3 X 10(34). The in vitro inhibition of P 388 leukemia cell growth by this complex compares with that of the free drug. This complex, unlike the free drug, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase.

Comparison of the interaction of doxorubicin, daunorubicin, idarubicin and idarubicinol with large unilamellar vesicles. Circular dichroism study.

Doxorubicin, daunorubicin and other anthracycline antibiotics constitute one of the most important groups of drugs used today in cancer chemotherapy. The details of the drug interactions with membranes are of particular importance in the understanding of their kinetics of passive diffusion through the membrane which is itself basic in the context of multidrug resistance (MDR) of cancer cells. Anthracyclines are amphiphilic molecules possessing dihydroxyanthraquinone ring system which is neutral under the physiological conditions. Their lipophilicity depends on the substituents. The amino sugar moiety bears the positive electrostatic charge localised at the protonated amino nitrogen. The four anthracyclines used in this study doxorubicin, daunorubicin, idarubicin and idarubicinol (an idarubicin metabolite readily formed inside the cells) have the same amino sugar moiety, daunosamine, with pKa of 8.4. Thus, all drugs studied will exhibit very similar electrostatic interactions with membranes, while the major differences in overall drug-membrane behaviour will result from their hydrophobic features. Circular dichroism (CD) spectroscopy was used to understand more precisely the conformational aspects of the drug-membrane systems. Large unilamellar vesicles (LUV) consisting of phosphatidylcholine, phosphatidic acid (PA) and cholesterol, were used. The anthracycline-LUV interactions depend on the molar ratio of phospholipids per drug. At low molar ratios drug:PA, these interactions depend also on the anthracycline lipophilicity. Thus, both doxorubicin and daunorubicin bind to membranes as monomers and their CD signal in the visible is positive. However, doxorubicin with its very low lipophilicity binds to the LUV through electrostatic interactions, with the dihydroxyanthraquinone moiety being in the aqueous phase, while daunorubicin, which is more lipophilic is unable to bind only through electrostatic interactions and actually the hydrophobic interactions are the only detected. The highly hydrophobic idarubicin, forms within the bilayer a rather complex entity involving 2-3 molecules of idarubicin associated in the right-handed conformation, one cholesterol molecule and also molecule(s) of phosphatidic acid, as this special oligomeric species is not detected in the absence of negatively-charged phospholipids. Idarubicinol differs from idarubicin with CH(13)-OH instead of C(13)=O and its interactions with LUV are distinctly different. Its CD signal in the visible becomes negative and no self associations of the molecule within the bilayer could be detected. The variation of the sign of the Cotton effect (positive to negative) may derive from the changes in the C(6a)-C(7)-O(7)-C(1') dihedral angle. It is noteworthy that C(13)-OH group, which strongly favours formation of the dimeric species in aqueous solutions when compared to idarubicin prevent association inside the LUV bilayer. At high ratios of phospholipids per drug all of them are embedded within the bilayer as monomer.

Non-competitive inhibition of P-glycoprotein-associated efflux of THP-adriamycin by verapamil in living K562 leukemia cells.

The decrease of the intracellular concentration of drug in resistant cells as compared to sensitive cells is, in most of cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein (P-gp) responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to follow the P-gp-associated efflux of these drugs in the absence or presence of verapamil. In the present study, 4'-o-tetrahydro-pyranyladriamycin (THP-adriamycin) was used. Two different methods were used to determine the kinetics of active efflux of THP-adriamycin: (1) at the steady-state, (2) directly, after the addition of glucose to cells first incubated with THP-adriamycin in the presence of N3- and in the absence of glucose. Kinetic analysis indicates: (1) a saturation of the active efflux when the cytosolic free drug concentration increased (the Michaelis constant Km = 0.5 +/- 0.3 microM) and (2) that the inhibitory effect of verapamil on P-gp-associated efflux of THP-adriamycin in living cells is non-competitive.

Interactions of iron-anthracycline complexes with living cells: a microspectrofluorometric study.

The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed. The three daunorubicin molecules that bind to one Fe(III) are not chemically modified through complexation with iron. In the case of the Fe(III)-adriamycin and Fe(III)-4'-O-tetrahydropyranyladriamycin complexes, about one of the three anthracycline molecules is chemically modified, yielding a highly lipophilic derivative, the 7,8-dehydro-9,10-desacetyladriamycin. The others molecules remain unchanged, i.e., highly hydrophilic in the case of adriamycin. These two species have a different fluorescent spectrum and can be identified inside the cell, using microspectrofluorometry. In the case of the Fe(III)-adriamycin complex, the lipophilic derivative is more rapidly internalized in the cell than the hydrophilic one. Diffusion into the plasmic membrane is the limiting step for the uptake of anthracycline by cells; this means that the plasmic membrane speeds up the dissociation of the Fe(III)-anthracycline complex.

P-glycoprotein-mediated efflux of hydroxyrubicin, a neutral anthracycline derivative, in resistant K562 cells.

Hydroxyrubin (OH-Dox), a neutral doxorubicin derivative that is only slightly cross-resistant to doxorubicin (Dox), can be actively pumped out of resistant K562 cells by P-glycoprotein (P-gp). This efflux is saturable and can be inhibited by verapamil. The Michaelis constant is equal to 2 +/- 0.5 microM. However, the efficiency of P-gp in pumping out the drugs is 2.5 times less for OH-Dox than for Dox. This shows that in order to be pumped out by P-gp a molecule does not necessarily have to have a basic center. The mean influx coefficient for the drug is 5 times higher for OH-Dox than for Dox. In conclusion, the degree of resistance of analogs is related not only to their ability to be recognized and transported by P-gp but also, and probably essentially, to their kinetics of uptake. Both parameters have to be taken into account in the rational design of new compounds capable of overcoming multidrug resistance.

Bifunctional antitumor compounds: interaction of adriamycin with metallocene dichlorides.

In order to synthesize bifunctional antitumor compounds, the interactions of adriamycin with metallocene dichlorides, Cp2MCl2, where M = Zr, Ti, V, have been studied. Using absorption, fluorescence, and circular dichroism measurements, we have shown that adriamycin is able to coordinate to the three metal ions. The interaction of Cp2ZrCl2 and Cp2VCl2 with adriamycin leads to compounds of 1:2 metal:drug stoichiometry, whereas the interaction of Cp2TiCl2 with adriamycin leads to two types of compounds of 1:2 and 1:1 stoichiometry. The Zr-adriamycin complex, which is unable to dissociate, even at a pH lower than 1, does not display antitumor activity against P-388 leukemia. However Ti-adriamycin complexes, which are more susceptible to dissociation in acidic media, exhibit antitumor activity that compares with that of the free drug. These complexes, unlike adriamycin, do not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. In addition, the presence of metal ions promote the binding of the drug to DNA and erythrocyte ghosts.

Solution structure of iron(III)-anthracycline complexes.

The interaction of Fe(3+) with the anthracycline anticancer drug idarubicin (Ida) was studied by absorption, CD, Mössbauer, and EPR spectroscopy. The formation of two major Fe(3+)-Ida complexes, labeled I and II, was observed. In complex I, Fe(3+) ion was bound to anthracycline at the {C(12)=O; C(11)-O(-)} coordination site. In complex II, two Fe(3+) ions were bound at sites {C(5)=O; C(6)-O(-)} and {C(12)=O; C(11)-O(-)}, respectively. Complex I was an equimolar monomeric species with a 1:1 Fe(3+):Ida stoichiometry (beta(1) = 4.8 x 10(11) M(-1)), whereas in complex II the anthracycline ligand was bridging two metal ions, alternatively bound to both anthracycline ring chelating sites with the assumption that the ratio of Fe(3+):Ida in complex II was 2:1 (beta(2) = 5.3 x 10(24) M(-2)). Alternatively, complex II may be oligomeric with Fe(3+):Ida = 1:1 and with each Fe(3+) bridging two Ida molecules. Our findings could be important in understanding the biological effects of the anthracycline-ferric complexes. Thus, providing information about the nature of the Fe(3+)-Ida system, we suggest that the formal 1:3 Fe(3+):anthracycline complexes, reported in the previous literature, could be a mixture of species I, II, and free ligand.

The effect of crown ethers, tetraalkylammonium salts, and polyoxyethylene amphiphiles on pirarubicin incorporation in K562 resistant cells.

The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance (P-gp-MDR) is a decrease in steady-state accumulation drug levels as compared to drug-sensitive controls. In an attempt to identify mechanism(s) by which MDR can be circumvented, we examined the cellular accumulation, in resistant cells, of 4'-O-tetrahydropyranyl-doxorubicin (pirarubicin) alone and in conjunction with various molecules belonging to three different classes: the crown ethers, the tetraalkylammonium salts, and the polyoxethylene amphiphiles. The present study was performed using a spectrofluorometric method which enabled us to follow the uptake and release of fluorescent molecules by living cells while the cells were being incubated with the drug. Erythroleukemia K562 cell lines were used. Our data show that the compounds of these three completely different classes were able to increase the incorporation of pirarubicin provided they had a minimum degree of lipophilicity. Study of the growth inhibitory activity of these compounds revealed that cross-resistance to the tetraalkyl ammonium salt increased with the lipophilicity and was equal to 58 for tetraoctylammonium salt, the most lipophilic compound of this series. This demonstrates that neither the presence of a positive charge nor an aromatic moiety is required for MDR recognition.

Accumulation of degradation products of doxorubicin and pirarubicin formed in cell culture medium within sensitive and resistant cells.

Quantitative study of doxorubicin (Adriamycin), pirarubicin (4'-o-tetrahypyranyladriamycin) and daunorubicin in the nucleus of living cells was performed using microspectrofluorometry. As for the cytotoxic assays, drug-sensitive and drug-resistant K562 cells were incubated for 3 days with concentrations of drug ranging from 4 nM to 1 mM. When drug-sensitive cells were incubated with pirarubicin, the spectrum recorded from inside the nucleus was characteristic of anthracycline intercalated between the base pairs in the nucleus. However, when drug-sensitive cells were incubated with doxorubicin and drug-resistant cells with pirarubicin or doxorubicin, a new fluorescent spectrum was obtained which was due to 7,8-dehydro-9,10-desacetyldoxorubicinone, a pirarubicin and doxorubicin degradation product that is formed in the medium. This compound which is highly lipophilic is taken up rapidly into both sensitive and resistant cells.

How Fe3+ binds anthracycline antitumour compounds. The myth and the reality of a chemical sphinx.

The interaction of Fe3+ with several anthracycline antitumour antibiotics has been reinvestigated. Absorption and circular dichroism (CD) measurements were carried out (i) in aqueous solution and (ii) in semi-aqueous MeOH to avoid the stacking of the anthracycline molecules. The Fe3+ binding to anthracycline was dependent on the metal-to-ligand molar ratio, antibiotic concentration, ionic strength, and pH. The formation of two major Fe3(+)-anthracycline complexes, I and II, was observed for all the drugs. These species differed in their coordination modes to the anthracycline ligands. Complex I was a monomeric species, where Fe3+ was bound to the anthracycline through the {C(11)-O-; C(12) = O} chelating site. In complex II, Fe3+ was also bound through the {C(5) = O; C(6)-O-} coordination site. Thus, the antibiotic ligand was acting as a bridge between two metal ions, forming oligomeric (or polymeric) structures. The different degree of association of the anthracyclines could be responsible for the reactivity of the metal ion. In fact, complexes I and II could constitute mononuclear, binuclear or polynuclear Fe3+ species depending on the competitive kinetics of both coordination and hydrolysis of the metal ion.

Spectral shape modifications of anthracyclines bound to cell nuclei: a microspectrofluorometric study.

Anthracyclines remain today the medications of choice against a wide spectrum of human cancers. Anthracyclines are fluorescent molecules and microfluorimetric methods are often used to determine their cellular distribution. The use of microspectrofluorometric techniques yields additional information because not only the fluorescence intensity but also the spectral modifications of the chromophore can be used to assess the intracellular drug concentration, its localisation and also eventually its metabolisation. It is well-documented that the shape of the fluorescence spectrum of anthracyclines changes markedly with the hydrophobicity of their environment. This change can be quantitatively measured by the ratio rho of the fluorescence emission intensities at 560 and 590 nm. We have observed that the shape of the fluorescent spectrum of adriamycin, daunorubicin and 4'-O-tetrahydropyranyladriamycin recorded from a small volume inside the cell nucleus was strongly dependent on the drug concentration and that the rho value decreases as the drug concentration increases. These data were compared with the rho variations when the drugs were either dissolved in different solvents or intercalated between the base pairs of DNA. We arrived at the conclusion that the shape variation of the drug spectra was not due to a change in their hydrophobicity environment but to an excitonic coupling of the electric dipolar transition moments of the pi --> pi* transition.

Circular dichroism studies on anthracycline antitumor compounds. Relationship between the molecular structure and the spectroscopic data.

The band assignment of the circular dichroism (CD) spectra of anthracyclines can provide us with the tools to study the interaction of these molecules with biomolecules, such as DNA and membranes, and also with metal ions. This paper reports the CD spectra of 17 anthracycline derivatives and the tentative assignment of the bands to specific electronic transitions. The deprotonation of some anthracyclines, such as doxorubicin, daunorubicin, and idarubicin, have been also studied in order to characterize the electronic transitions involved in the acid-base process. Our evidence suggests the following assignment. The position of the band assigned to pi-->pi transition, polarized along the short axis of the molecule ( approximately 290 nm), does not depend on the hydroxyl group at C(11) (presence and/or ionization state), whereas the position of the band assigned to the pi-->pi transition ( approximately 480 nm), polarized along the long axis, is strongly dependent on it. Concerning the n-->pi transitions, the bands at approximately 320 and approximately 350 nm have a strong contribution of the n-->pi C(12)=O transition and the n-->pi C(5)=O transition, respectively.

Mitomycin antitumor compounds. Part 1. CD studies on their molecular structure.

The UV-Vis and circular dichroism (CD) spectra of several mitomycin antitumor compounds and some of their derivatives were analyzed in order to attribute the proper assignment to their electronic transitions. The lowest energy pi-->pi* transition was found to depend on the effect of the auxochromic group in the aromatic ring, whereas the three n-->pi* transitions, present at around 240, 400 and 560 nm, are related to the C(9)==O of the carbamoyl group and to the C(8)==O and the C(5)==O of the quinone, respectively. The chirality of the C(9) is responsible for the sign of the Cotton effect (CE) at around 240 nm, whereas the substituents of the chromophore for mitosane derivatives and the conformation of the carbamoyloxymethyl group at C(9) determine the CE sign of the (1)A-->(1)L(b) transition. When the aziridine ring was opened and mitosenes derivatives were obtained, CD spectra did not differ significantly among the compounds and the bands associated to the different transitions had similar Cotton effect. Our findings suggest that the differences in the CD spectra, observed between mitosanes and mitosenes, are probably related to the more rigid molecular structure of the mitosene derivatives and the different conformations in solution of the C(9) side chain.

Metal anthracycline complexes as a new class of anthracycline derivatives. Pd(II)-adriamycin and Pd(II)-daunorubicin complexes: physicochemical characteristics and antitumor activity.

Pd(II) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric absorption, fluorescence, and circular dichroism measurements, we have shown that adriamycin can form two complexes with Pd(II). The first complex (I) involves two molecules of drug per Pd(II) ion; one of the molecules is chelated to Pd(II) through the carbonyl oxygen on C12 and the phenolate oxygen on C11, and the other one is bound to Pd(II) through the nitrogen of the amino sugar. This complexation induces a stacking of the two molecules of drug. In the second complex (II), two Pd(II) ions are bound to two molecules of drug (A1 and A2). One Pd(II) is bound to the oxygen on the carbons C11 and C12 of molecule A1 and the amino sugar of molecule A2 whereas the second Pd(II) ion is bound to the oxygen on C11 and C12 of molecule A2 and the amino sugar of molecule A1. The same complexes are formed between Pd(II) and daunorubicin. The stability constant for complex II is beta = (1.3 +/- 0.5) X 10(22). Interaction with DNA has been studied, showing that almost no modification of the complex occurred. This complex displays antitumor activity against P-388 leukemia that compares with that of the free drug. Complex II, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase.

Degradation of anthracycline antitumor compounds catalysed by metal ions.

The influence of some metal ions on the degradation of anthracyclines was examined. One of the degradation products is the 7,8-dehydro-9,10-desacetyldoxorubicinone, D* ( yen), usually formed by hydrolysis at slightly basic pH. D* is a lipophilic compound with no cytostatic properties. Its formation could be responsible for the lack of antitumor activity of the parent compound. The coordination of metal ions to anthracycline derivatives is required to have degradation products. Cations such as Na(+), K(+), or Ca(2+) do not induce the D* formation however metals which can form stable complexes with doxorubicin afford D*. Iron(III) and copper(II) form appreciable amount of D* at slightly acidic pH. Terbium(III) forms D* but its complex is stable only at slightly basic pH. Palladium(II) which does not form D*. The influence of the coordination mode of metal ions to anthracycline on the D* formation is discussed.

Spectroscopic studies on iron complexes of different anthracyclines in aprotic solvent systems.

Iron complexes of daunorubicin, idarubicin, pirarubicin, and doxorubicin in anhydrous DMF were studied by UV/vis, CD, fluorescence, Mössbauer, and EPR spectroscopy. Titration studies of the metal-free anthracyclines showed one (UV-detectable) deprotonation step requiring 2 equiv of base, compared to 1 equiv for quinizarine. Metal complexation was studied at three different metal/ligand ratios, and with increasing amounts of base. The results obtained from optical spectroscopy show the existence of two different complex species and give clear indications for the requirements of metal complexation. Complex species I, formed at a low iron-to-ligand ratio, is less dependent on base addition than complex species II formed with equimolar ferric ion. EPR and Mössbauer experiments provide further insight into the structures of both complex species. Lack of spin density of the Mössbauer samples in EPR indicates spin coupling between the metal centers. Mössbauer spectra consist of single quadrupole doublets with values typical for high-spin ferric ion in an octahedral arrangement. The Mössbauer spectroscopic features at 7 T exclude the presence of S = 0 dimers. Complex I represents a monomeric ferric iron complex whereas complex II is consistent with a more or less aggregrated oligomeric Fe-anthracycline system.

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids.

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point. The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions. Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles. The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3'-hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13-hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol. We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.