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Quantitation of the molecular mechanisms of biological synergism in a mixture of DNA-acting aromatic drugs
Abstract
It is suggested that the widely reported biological synergism of a mixture of DNA-targeting aromatic drug molecules both in vivo and in vitro can be explained, in part, at the molecular level by competition between two basic mechanisms: the 'interceptor' (hetero-association between Drug1 and Drug2) and 'protector' mechanisms (complexation of Drug1 and Drug2 on DNA-binding sites). In the present work a complete analytical methodology has been developed to quantify these processes, providing an estimate of the relative importance of the interceptor/protector mechanisms using just a set of equilibrium association constants. The general methodology may be applied to other molecules with receptors for aromatic drugs.
... To the best of our knowledge this property was first noted in ref (Dashwood and Guo, 1993). with respect to drug-chlorophyllin systems and further confirmed for other interceptors (Skamrova et al., 2014;Evstigneev et al., 2005Evstigneev et al., , 2006aEvstigneev et al., , 2006bEvstigneev et al., , 2008Evstigneev et al., , 2011aHernandez Santiago et al., 2009;Buchelnikov et al., 2012;. ...
... However, the fundamental similarity of the mechanisms was noted, originating from the physical interaction of aromatic molecules in mixed solution either at the nucleic acid level or at the level of direct interaction between the solute molecules. These mechanisms were formulated in the form of two molecular processes ( Fig. 1): d protector (Skamrova et al., 2014;Traganos et al., 1991aTraganos et al., , 1991bEvstigneev et al., 2005Evstigneev et al., , 2006aEvstigneev et al., , 2006bEvstigneev et al., , 2008Evstigneev et al., , 2011aHernandez Santiago et al., 2009;Buchelnikov et al., 2012;Davies et al., 2001;Chin et al., 2012;Maria Johnson et al., 2003a), i.e. competition of the drug and interceptor for DNA binding sites leading to removal of the drug from DNA as a result of interceptor-DNA binding, and d interceptor (Traganos et al., 1991a(Traganos et al., , 1991b(Traganos et al., , 1993Hill et al., 2011;Skamrova et al., 2014;Woziwodzka et al., 2011Woziwodzka et al., , 2013bKapuscinski et al., 2002;Ganapathi et al., 1986;Lyles and Cameron, 2002;Bedner et al., 2001;Borowik et al., 2018;Guo, 1992, 1993;Evstigneev et al., 2005Evstigneev et al., , 2006aEvstigneev et al., , 2006bEvstigneev et al., , 2008Evstigneev et al., , 2011aHernandez Santiago et al., 2009;Buchelnikov et al., 2012;Davies et al., 2001;Chin et al., 2012;Maria Johnson et al., 2003a;Piosik et al., 2002Piosik et al., , 2003Piosik et al., , 2005Piosik et al., , 2010Ulanowska et al., 2005Ulanowska et al., , 2007Tachino et al., 1994;Pietrzak et al., 2006Pietrzak et al., , 2008Gołu nski et al., , 2015Banerjee et al., 2011;Kharasch and Novak, 1981;Raiczyk and Pinto, 1988;Ramu et al., 2000;Arimoto et al., 1993;Ardelt et al., 2001;Ridge et al., 1994), i.e. hetero-association between the Drug and Interceptor leading to formation of inactive hetero-complexes unable to bind with DNA (an analogue of host-guest interaction). ...
... However, the fundamental similarity of the mechanisms was noted, originating from the physical interaction of aromatic molecules in mixed solution either at the nucleic acid level or at the level of direct interaction between the solute molecules. These mechanisms were formulated in the form of two molecular processes ( Fig. 1): d protector (Skamrova et al., 2014;Traganos et al., 1991aTraganos et al., , 1991bEvstigneev et al., 2005Evstigneev et al., , 2006aEvstigneev et al., , 2006bEvstigneev et al., , 2008Evstigneev et al., , 2011aHernandez Santiago et al., 2009;Buchelnikov et al., 2012;Davies et al., 2001;Chin et al., 2012;Maria Johnson et al., 2003a), i.e. competition of the drug and interceptor for DNA binding sites leading to removal of the drug from DNA as a result of interceptor-DNA binding, and d interceptor (Traganos et al., 1991a(Traganos et al., , 1991b(Traganos et al., , 1993Hill et al., 2011;Skamrova et al., 2014;Woziwodzka et al., 2011Woziwodzka et al., , 2013bKapuscinski et al., 2002;Ganapathi et al., 1986;Lyles and Cameron, 2002;Bedner et al., 2001;Borowik et al., 2018;Guo, 1992, 1993;Evstigneev et al., 2005Evstigneev et al., , 2006aEvstigneev et al., , 2006bEvstigneev et al., , 2008Evstigneev et al., , 2011aHernandez Santiago et al., 2009;Buchelnikov et al., 2012;Davies et al., 2001;Chin et al., 2012;Maria Johnson et al., 2003a;Piosik et al., 2002Piosik et al., , 2003Piosik et al., , 2005Piosik et al., , 2010Ulanowska et al., 2005Ulanowska et al., , 2007Tachino et al., 1994;Pietrzak et al., 2006Pietrzak et al., , 2008Gołu nski et al., , 2015Banerjee et al., 2011;Kharasch and Novak, 1981;Raiczyk and Pinto, 1988;Ramu et al., 2000;Arimoto et al., 1993;Ardelt et al., 2001;Ridge et al., 1994), i.e. hetero-association between the Drug and Interceptor leading to formation of inactive hetero-complexes unable to bind with DNA (an analogue of host-guest interaction). ...
The review discusses the theory of interceptor-protector action (the IPA theory) as the new self-consistent biophysical theory establishing a quantitative interrelation between parameters measured in independent physico-chemical experiment and in vitro biological experiment for the class of DNA binding drugs. The elements of the theory provide complete algorithm of analysis, which may potentially be applied to any system of DNA targeting aromatic drugs. Such analytical schemes, apart from extension of current scientific knowledge, are important in the context of rational drug design for managing drug's response by changing the physico-chemical parameters of molecular complexation.
... Two mechanisms have been proposed for the modulation of DNA intercalating drugs [14,15], which involve equilibria of complexed drug and caffeine, caffeine and DNA, and drug and DNA. The mechanism proposed works within a system that consists of two ligands in the presence of DNA. ...
... Significant attenuation (P < 0.001) was observed for all intercalating drugs in this study; large attenuations were observed for berberine, camptothecin, doxorubicin and ellipticine, while chelerythrine and sanguinarine were marginally attenuated. The increased in IC 50 due to caffeine may be attributed to either caffeine competing for intercalation sites in DNA (the "protector" scheme), or that the caffeine has formed a π-π complex with the intercalating drug (the "interceptor" scheme, Fig. 1) [15]. ...
... "Interceptor" scheme "Protector" scheme Modulation of DNA intercalation by caffeine via "interceptor" (left) or "protector" (right) interactions [15]. ...
... Це дає пiдставу розглядати гетероасоцiацiю як технологi. реґуляцiї медикобiологiчної активностi лiкарських сполук при комбiнованому використаннi [9][10][11][12][13]. Окрiм цього, нековалентнi взаємодiї ароматичних систем також стають об'єктом iнтенсивного вивчення в рiзних галузях нанотехнологiї, зокрема для створення молекулярних магнiтiв i нового класу провiдникiв [14,15]. ...
... Розрахунок зроблено для двох груп комбiнацiй ароматичних сполук: кофеїн-лiґанд i вiтамiн-лiґанд, для яких ранiше була доведена можливiсть реґуляцiї медично-бiологiчної активностi за рахунок механiзму гетероасоцiацiї (див. огляд [13]). У ролi "лiґанду" використовували антипухлиннi антибiотики: актиномiцин D (AMD), дауномiцин (DAU), доксорубiцин (DOX), ногаламiцин (NOG), норфлоксацин (NOR), новантрон (NOV) i мутагени: акридиновий оранжевий (АО), бромистий етидiй (EB), профлавiн (PF), йодистий пропидiй (PI). ...
... Для всiх розглянутих сполук ранiше методом ЯМР-спектроскопiї ми одержали чисельнi значення термодинамiчних параметрiв реакцiї (1) в єдиних експериментальних умовах (див. огляд [13] та посилання в ньому). ...
In the present work the method of calculation of thermodynamic potentials (Gibbs free energy, enthalpy and entropy) and analysis of contribution of change of translational, rotational and vibrational degrees of freedom to the energy of heteroassociation of aromatic drug molecules with caffeine and flavin mononucleotide is developed. The obtained values of change of Gibbs energy and entropy of translational, rotational and vibrational degrees of freedom are required for complete energetic analysis of the heteroassociation reactions.
... competition of drug and interceptor for DNAbinding sites) mechanisms. The generalization of this view has been accomplished within the framework of the theory of interceptor-protector action (the IPA theory) (Evstigneev, 2010;Evstigneev et al., 2008;Buchelnikov et al., 2012), which aims to find a quantitative link between the in vitro biological data and parameters of physico-chemical interactions (concentrations and equilibrium complexation constants). ...
... So far the IPA theory has been successfully applied to quantification of relative change in apoptosis in human leukemia cell lines induced by administration of antitumor antibiotics together with caffeine (Evstigneev et al., 2006(Evstigneev et al., , 2008(Evstigneev et al., , 2011. However, to date one more set of biological data, well matching the basic postulates of the IPA theory, has been accumulated, viz. the antimutagenic action of the interceptor molecules towards aromatic mutagens in bacteria cell systems (see for review Piosik et al. (2003); Woziwodzka et al. (2011); Dashwood and Guo (1993)). ...
... which stands for relative change in biological effect of X on addition of Y (examples of linking the A D factor to biological data are given in Evstigneev et al. (2006Evstigneev et al. ( , 2008Evstigneev et al. ( , 2011). ...
... Taking into account that C 60 fullerene has a saturated π-electron system and therefore can form complexes with aromatic Dna intercalators, including antineoplastic antibiotics and mutagens Evstigneev et al. 2013;Mchedlov-Petrossyan et al. 2001), it was suggested that a non-covalent complexation in the 'drug-fullerene' system (also referred to as hetero-association) is a key mechanism causing the observed biological synergism . It has been known for quite a long time and so far been observed in proliferating cells or bacterial systems [see for review (Evstigneev et al. 2008;Evstigneev 2010Evstigneev , 2013] that hetero-association regulates the action of the biologically active compounds during simultaneous administration of combinations of different aromatic molecules, one of which is the main drug of interest, and the other one is an interceptor molecule. This phenomenon is referred to as an 'interceptor' mechanism. ...
... However, the study of such a mechanism is complicated by possible influence from other molecular processes, one of which is most often referred to as a 'protector' mechanism, i.e., the drug/interceptor competition for the binding sites on Dna (Buchelnikov et al. 2012;Evstigneev 2010Evstigneev , 2013Evstigneev et al. 2011;gołuński et al. 2013). Investigation of the interceptor and protector mechanisms for Dna-binding drugs used in combination is the subject of the theory of interceptor/protector action (Buchelnikov et al. 2012;Evstigneev et al. 2008Evstigneev et al. , 2011Evstigneev 2010). Unfortunately, the applicability of the protector mechanism within the system containing C 60 fullerene cannot be estimated on the basis of the currently available literature data. ...
... a range of typical Dna-binding aromatic BaCs has been used in the present study: the antitumor antibiotic doxorubicin (DOX), aromatic mutagen proflavine (PF) and ethidium bromide (EB). These drugs form stable non-covalent complexes with C 60 fullerene Evstigneev et al. 2013) and were previously characterized as the ligands that follow the mechanism of interceptor/ protector action when used in combination with various types of aromatic interceptor molecules (Buchelnikov et al. 2012;Evstigneev et al. 2006Evstigneev et al. , 2008Evstigneev 2010Evstigneev , 2013larsen et al. 1996;Piosik et al. 2010;Traganos et al. 1991). ...
C60 fullerenes are spherical molecules composed purely of carbon atoms. They inspire a particularly strong scientific interest because of their specific physico-chemical properties and potential medical and nanotechnological applications. In this work we are focusing on studying the influence of the pristine C60 fullerene on biological activity of some aromatic drug molecules in human buccal epithelial cells. Assessment of the heterochromatin structure in the cell nucleus as well as the barrier function of the cell membrane was performed. The methods of cell microelectrophoresis and atomic force microscopy were also applied. A concentration-dependent restoration of the functional activity of the cellular nucleus after exposure to DNA-binding drugs (doxorubicin, proflavine and ethidium bromide) has been observed in human buccal epithelial cells upon addition of C60 fullerene at a concentration of ~10(-5 )M. The results were shown to follow the framework of interceptor/protector action theory, assuming that non-covalent complexation between C60 fullerene and the drugs (i.e., hetero-association) is the major process responsible for the observed biological effects. An independent confirmation of this hypothesis was obtained via investigation of the cellular response of buccal epithelium to the coadministration of the aromatic drugs and caffeine, and it is based on the well-established role of hetero-association in drug-caffeine systems. The results indicate that C60 fullerene may reverse the effects caused by the aromatic drugs, thereby pointing out the potential possibility of the use of aromatic drugs in combination with C60 fullerene for regulation of their medico-biological action.
... The correlation provides some 'modelindependent' insight into the principal contributions of different physical factors to aromatic-aromatic stacking interactions and may be used to estimate the enthalpy from the structural properties of aromatic stacking interactions or vice versa. [12][13][14][15][16][17] and references therein). The selection of the molecules and their complexes was primarily dictated by the quality of the structural and thermodynamic information available. ...
... In Refs. [12][13][14][15][16][17], all results were obtained using similar experimental conditions and numerical analysis, which is crucial in the search for statistical correlations. ...
... Calculation of the overlap area in the complexes of aromatic molecules Calculation of the overlap area of aromatic chromophores in each 1:1 complex was made on the basis of the published energy-minimized structures [12][13][14][15][16][17] and accomplished using the GEUP 3 software [20]. For selected systems the results were independently verified by means of manual estimation of the calculated areas. ...
An approximately linear correlation has been found between the enthalpy of complexation and the area of overlap of the chromophores using published structural and thermodynamical data on the self- and hetero-association of aromatic molecules measured under similar solution conditions. This finding is consistent with the assumption that short-range van-der-Waals forces dominate over other contributions to the enthalpy of stacking of aromatic molecules. It provides a 'model-independent' approach for a priori estimation of the enthalpy of aromatic-aromatic stacking interactions from knowledge of the structural properties or vice versa.
... Hence, the fraction of CAF molecules bound with DNA may be of the same order of magnitude as the fraction of the heterocomplexes; that is, the "interceptor" and "protector" mechanisms may, in principle, be equally effective. The interceptor/protector approach was developed in [6,49,53,62,65,67,69,76] and resulted in the separation of the contribution of the interceptor and protector mechanisms into the net effect of drug removal from DNA on the addition of methylxanthine. Moreover, these studies have demonstrated the possibility of quantitative estimation of the in vitro change of biological effect in drug-methylxanthine systems [6,67,76]. ...
... The interceptor/protector approach was developed in [6,49,53,62,65,67,69,76] and resulted in the separation of the contribution of the interceptor and protector mechanisms into the net effect of drug removal from DNA on the addition of methylxanthine. Moreover, these studies have demonstrated the possibility of quantitative estimation of the in vitro change of biological effect in drug-methylxanthine systems [6,67,76]. Unfortunately, direct experimental measurement of the contribution of these mechanisms is currently not possible, although recent investigations [69] have shown that the exclusion of the protector mechanism leads to underestimation of experimentally observed molar absorption in three-component drug-interceptor-DNA system. Importantly, the above-formulated molecular mechanisms of methylxanthine action, as well as the cytotoxic effects, have been reported specifically for the group of aromatic intercalators and are commonly not observed for nonaromatic compounds. ...
... These results were further transferred to three-component systems drug-riboflavin-DNA where riboflavin acts as an interceptor molecule. Although the RBF/FMN binding with DNA is thought to be relatively small and so far observed only on the level of oligonucleotides [92], theoretical evaluation of the contribution of interceptor and protector mechanisms into change of biological effect of the drug on addition of FMN was accomplished [6,76,92]. At this point it is worth noting that there are many reports in the literature (see, e.g., [79,85]), on anticarcinogenic or antimutagenic effect of riboflavin against nonaromatic drugs, though in these publications totally different mechanisms of the observed synergetic effects, different for different drugs, are commonly discussed. ...
The mechanisms of synergistic biological effects observed in the simultaneous use of aromatic heterocyclic compounds in combination are reviewed, and the specific biological role of heteroassociation of aromatic molecules is discussed.
... During the past decade a number of reports have appeared regarding the possibility of manipulating the biological effect of DNA-binding aromatic drugs in three-component drug–interceptor–DNA systems in vitro (Piosik et al. 2002; Evstigneev et al. 2008; Osowski et al. 2010; Woziwodzka et al. 2011). This effect may be achieved through change of concentration of the interceptor molecule (Bedner et al. 2001; Evstigneev et al. 2005 Evstigneev et al. , 2006a , b) and special selection of the ligands in the drug–interceptor pair which form stable heterocomplexes in solution (Hernandez). ...
... This effect may be achieved through change of concentration of the interceptor molecule (Bedner et al. 2001; Evstigneev et al. 2005 Evstigneev et al. , 2006a , b) and special selection of the ligands in the drug–interceptor pair which form stable heterocomplexes in solution (Hernandez). Typical well-known interceptor molecules are xanthines (Piosik et al. 2002; Hernandez Santiago et al. 2009; Osowski et al. 2010; Woziwodzka et al. 2011), aromatic vitamin B 2 (Evstigneev et al. 2005Evstigneev et al. , 2008), and chlorophyllin (Pietrzak et al. 2006Pietrzak et al. , 2008). Within the framework of the theory of interceptor/protector action (Evstigneev et al. 2008; Evstigneev 2010), it has been suggested that two molecular mechanisms are responsible for the in vitro change of the biological effect of a drug, viz. the protector (i.e., competition between the drug and interceptor for DNA binding sites) and interceptor (i.e., hetero-association between the drug and interceptor) mechanisms. ...
... Typical well-known interceptor molecules are xanthines (Piosik et al. 2002; Hernandez Santiago et al. 2009; Osowski et al. 2010; Woziwodzka et al. 2011), aromatic vitamin B 2 (Evstigneev et al. 2005Evstigneev et al. , 2008), and chlorophyllin (Pietrzak et al. 2006Pietrzak et al. , 2008). Within the framework of the theory of interceptor/protector action (Evstigneev et al. 2008; Evstigneev 2010), it has been suggested that two molecular mechanisms are responsible for the in vitro change of the biological effect of a drug, viz. the protector (i.e., competition between the drug and interceptor for DNA binding sites) and interceptor (i.e., hetero-association between the drug and interceptor) mechanisms. It was shown that such a view provides a potential possibility for quantitation of the relative change of biological effect (the so-called A D factor) in the given drug–interceptor system, if the magnitudes of equilibrium complexation constants are known and the concentrations of the components in the drug–interceptor–DNA mixture are specifically adjusted (the so-called quasiphysiological conditions) (Evstigneev et al. 2008Evstigneev et al. , 2011 ). ...
Relative insensitivity of theoretical estimation of biological effect in drug-interceptor-DNA systems is found with respect to variation of parameters of quasiphysiological conditions. The "inertness" of the biological response, in part, justifies the use of parameters of intermolecular interaction, derived from independent physicochemical experiments, in estimation of relative biological effect in the theory of interceptor/protector action.
... So far, several protocols have been suggested in order to quantify the three-component system of drug-interceptor-DNA (see the models developed in Pietrzak et al. 2006;Evstigneev et al. 2008), and the link has been established between the observed biological effect of the drug on administration of the interceptor and the relevant equilibrium complexation constants (Evstigneev et al. , 2011a. So far, these developments contain some drawbacks, which commonly fall outside the scope of various scientific papers and are usually omitted as scientific assumptions or simplifications. ...
... The principal difficulty is the necessity to take into consideration the full set of interactions among the drug, interceptor and DNA, particularly the statistical distribution of the bound ligands over the DNA sequence. Models solving the three-(or more) component systems of competing drugs are known in the literature, although they all ignore some types of interactions; for example, the model in Wang et al. (1998) takes into account the full set of competitive complexations with a biopolymer, but ignores the hetero-association between the ligands (i.e., ignores the interceptor mechanism); the model in Pietrzak et al. (2006) ignores the competition for the binding sites, but treats indefinite hetero-association (i.e., ignores the protector mechanism); and, finally, the model in Evstigneev et al. (2008) takes into account both protector and interceptor mechanisms, but does it for DNA fragments, i.e., ignoring the statistical effects of ligand binding with polymeric DNA. In this work we developed the general model of competitive binding in drug-interceptor-DNA systems removing all these limitations. ...
... We shall now adapt this method to polymeric DNA within the framework of the general model. It was suggested (Evstigneev 2010;Evstigneev et al. 2005Evstigneev et al. , 2006aEvstigneev et al. , 2008) that the 'protector' and 'interceptor' mechanisms could be quantified using the criterion, R D , which is the relative decrease in proportion of X-DNA complexes on addition of the ligand Y as summarized in Eq. 15. R D is calculated for two limiting situations: ...
A general model of competitive binding in drug-interceptor-DNA systems has been developed in order to quantify both the interceptor and protector mechanisms. The model involves full parameterization of the basic equations governing the mutual competition between drugs binding to DNA and incorporates as partial cases various similar models existing in the literature. The generality of the model results from strict accounting of the statistical effects of the binding of the drug and interceptor with DNA according to the McGhee-von Hippel formalism, and to the strict treatment of hetero-association between the drug and interceptor, which includes formation of all possible types of self- and hetero-complexes in solution. Indirect experimental evidence is provided for the importance of the protector mechanism in drug-caffeine-DNA systems, which is sometimes ignored in the literature because of the small magnitude of the CAF-DNA binding constant.
... Two mechanisms have been proposed for the modulation of DNA intercalating drugs [14,15], which involve equilibria of complexed drug and caffeine, caffeine and DNA, and drug and DNA. The mechanism proposed works within a system that consists of two ligands in the presence of DNA. ...
... Significant attenuation (P < 0.001) was observed for all intercalating drugs in this study; large attenuations were observed for berberine, camptothecin, doxorubicin and ellipticine, while chelerythrine and sanguinarine were marginally attenuated. The increased in IC 50 due to caffeine may be attributed to either caffeine competing for intercalation sites in DNA (the "protector" scheme), or that the caffeine has formed a π-π complex with the intercalating drug (the "interceptor" scheme, Fig. 1) [15]. ...
... "Interceptor" scheme "Protector" scheme Modulation of DNA intercalation by caffeine via "interceptor" (left) or "protector" (right) interactions [15]. ...
Many anti-tumor drugs function by intercalating into DNA. The xanthine alkaloid caffeine can also intercalate into DNA as well as form π-π molecular complexes with other planar alkaloids and anti-tumor drugs. The presence of caffeine could interfere with the intercalating anti-tumor drug by forming π-π molecular complexes with the drug, thereby blocking the planar aromatic drugs from intercalating into the DNA and ultimately lowering the toxicity of the drug to the cancer cells. The cytotoxic activities of several known DNA intercalators (berberine, camptothecin, chelerythrine, doxorubicin, ellipticine, and sanguinarine) on MCF-7 breast cancer cells, both with and without caffeine present (200 μg/mL) were determined. Significant attenuation of the cytotoxicities by caffeine was found. Computational molecular modeling studies involving the intercalating anti-tumor drugs with caffeine were also carried out using density functional theory (DFT) and the recently developed M06 functional. Relatively strong π-π interaction energies between caffeine and the intercalators were found, suggesting an "interceptor" role of caffeine protecting the DNA from intercalation.
... The latter may be used to regulate the toxicity level of anti-tumour agents or carcinogens (Piosik et al. 2002; Evstigneev et al. 2006a), regulate the rate of drug M. P. Evstigneev (&) Á A. A. Mosunov Á V. P. Evstigneev Department of Physics, Sevastopol National Technical University, Universitetskaya str., 33, Sevastopol 99053, Ukraine e-mail: max_evstigneev@mail.ru degradation (Evstigneev et al. 2006b ), optimize the solubility of the drug (Evstigneev et al. 2006c) or solve specific biochemical tasks (Bedner et al. 2001 ). Recently we proposed that relative biological effect in a combination of aromatic drugs may be quantified (Evstigneev et al. 2008), which may make it possible to regulate the activity of a drug by a predictable change in the synergistic effect. The anti-tumour agent topotecan (TPT) has long been known to interact synergistically with caffeine (CAF), a xanthine-based aromatic molecule, which was interpreted in terms of TPT–CAF hetero-association and interference with the binding of TPT to the DNA–enzyme complex (Traganos et al. 1993). ...
... This stimulated a series of investigations of TPT-DNA systems [ (2008)] eventually leading to general recognition that TPT binding with DNA has its own significance. Hence, the principal aim of the present work is to explore the possibility of describing the experimental apoptosis–concentration curve from Traganos et al. (1993) using a model (Evstigneev et al. 2008; Evstigneev 2010) based on the assumption that non-covalent complexations between TPT, CAF and DNA are the main determinants of the observed change in biological effect of the antibiotic on addition of CAF. ...
... Eur Biophys J (2011) 40:969–980 973 derived from drug–tetranucleotide studies and also used previously in analysis of CAF–drug–DNA systems (Davies et al. 2001; Evstigneev et al. 2006a Evstigneev et al. , 2008 ). The appropriateness of such an approximation is also supported by the fact that the hetero-association constants between CAF and aromatic drugs, mimicking the unstacked DNA bases in the nicked site, on average, are close in value to K YN = 246 M -1 (Evstigneev 2010). ...
Using published in vitro data on the dependence of the percentage of apoptosis induced by the anti-cancer drug topotecan in a leukaemia cell line on the concentration of added caffeine, and a general model of competitive binding in a system containing two aromatic drugs and DNA, it has been shown to be possible to quantify the relative change in the biological effect just using a set of component concentrations and equilibrium constants of the complexation of the drugs. It is also proposed that a general model of competitive binding and parameterization of that model may potentially be applied to any system of DNA-targeting aromatic drugs under in vitro conditions. The main reasons underpinning the proposal are the general feature of the complexation of aromatic drugs with DNA and their interaction in physiological media via hetero-association.
... On the other hand, when CAF is administered together with aromatic cytotoxic drugs (such as doxorubicin, ethidium bromide, novantrone), there is a remarkable reduction in the in vitro toxicity of the drugs acting on nuclear DNA6789. The effect was investigated in detail using 1 H NMR measurements101112, and the results were interpreted in terms of the competing action of two basic molecular processes, viz. the interceptor (hetero-association of CAF and drug) and protector (competition of CAF and drug for DNA binding sites) mechanisms of action of caffeine on DNA-binding aromatic ligands. This analysis led to a series of investigations of the hetero-association of CAF with various aromatic BACs which has provided detailed structural and thermodynamic information on the complexation process [7, 10, 11,1314151617. ...
... The relative importance of the protector and interceptor mechanisms of the action of THP on the complexation of aromatic drugs with DNA is investigated by proton nuclear magnetic resonance (NMR) spectroscopy in the present work. This necessitates determination of the equilibrium hetero-association constants for complexation of THP with a range of aromatic drug compounds (such as daunomycin, novantrone, ethidium bromide, proflavine, norfloxacin) and with the same DNA oligomer, d(TGCA), as measured previously for the same set of drug molecules101112 17]. The results are compared with the effect of CAF under similar conditions. ...
... Hetero-association in the THP–drug system was analysed as previously101112 using concentration dependences of proton chemical shifts. An example of the experimental2 Experimental dependence of the proton chemical shifts of proflavine (PF ) and theophylline (THP) in mixed solutions (T = 298 K, pD 7.1, 0.1 M Na phosphate buffer) at constant concentration of THP (C THP = 2 mM) and varying concentrations of PF(C curves for each system is given inFig. ...
(1)H NMR measurements (500 MHz) have been used to determine the equilibrium hetero-association constants of theophylline (THP) with various biologically active aromatic compounds (daunomycin, novantrone, ethidium bromide, proflavine, norfloxacin) and the complexation constants of THP with both single- and double-stranded oligonucleotides in solution. The results provide a quantitative estimation of the effect of THP on the binding of aromatic ligands with DNA, and a determination of the fraction of aromatic ligand removed from DNA on addition of THP.
... Generally, the values of the hetero-association constants in Table 1 appear to be very close to those obtained from NMR data [324 M −1 for NOV-CAF and 49,000 M −1 for NOV-FMN (Evstigneev et al. 2008)], indicating that the analytical approach used here is adequate. ...
... The dots show the experimental biological data and the lines show the A D factor profiles calculated using the IPA theory concentration is measured in moles of base pairs per liter. To describe non-cooperative DNA binding we use standard approach taken from Ref. (Evstigneev et al. 2008). For such a system, the mass balance equations are written in general form as follows : ...
We performed a qualitative and quantitative analysis of intermolecular interactions in aqueous solution between the antitumor antibiotic mitoxantrone and C60 fullerene in comparison with interactions between the antibiotic and well-known aromatic molecules such as caffeine and flavin mononucleotide, commonly referred to as interceptor molecules. For these purposes, we obtained equilibrium hetero-association constants of these interactions using a UV/Vis titration experiment. Special attention was paid to the interaction of C60 fullerene with mitoxantrone, which has been quantified for the first time. Based on the theory of interceptor-protector action and using a set of measured equilibrium constants we managed to estimate the relative biological effect of these mixtures in a model living system, taking human buccal epithelium cells as an example. We demonstrated that C60 fullerene is able to restore the functional activity of the buccal epithelium cell nucleus after exposure to mitoxantrone, which makes it possible to use C60 fullerene as regulator of medico-biological activity of the antibiotic.
... The hetero-association constants K h of hypericin and acridine orange were estimated numerically using MATLAB R2013a. The model of determination of the association constants, taking into account all possible self-and hetero-association and formation of higher-order complexes in the tested mixtures, was suggested by [16][17][18][19]. The proposed postulates were used, however some simplifications resulting from the spectral properties the analyzed were introduced. ...
... Buffers with the maximum DMSO content of 50% were used in the main part of the research concerning the hetero-association of hypericin and acridine orange. Taking into account the above results, the proposals of [16][17][18] were simplified, because dimerization of acridine orange was not observed. Only self-association of hypericin and hetero-association of hypericin and acridine orange in different configuration and formation of higher-order were taken into account. ...
The present study was designed to estimate the ability of hypericin to interact with a model mutagen – acridine orange. The hetero-association of hypericin and acridine orange was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO. The data indicate that hypericin forms complexes with acridine orange and that the association constants are relatively high and depend on DMSO concentration. The absorption spectra of the hypericin – acridine orange complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule.
... Many authors [10,11,12] have postulated that during determining a correlation between the interceptor molecule and the intercepted molecules consideration should be given to any possible objects formed in the analyzed system, including dimers and higher order aggregates of both the trapping and the trapped compound. When discussing heteroassociation of hypericin and other molecules it is also necessary to take account of the self-association mechanism and to determine association constants at specified experimental conditions, considering the specific character of the measuring methods applied. ...
... 6,7 This mechanism can also be effectively used in chemotherapy since CNTs can act as interceptors of biologically active aromatic compounds. The interceptor properties of aromatic compounds have been studied quite well to date in molecular biophysics 8,9 and can be used for manipulation of the active drugs' concentrations as well as their biological effects. 10,11 In particular, it has been suggested that the complexation between CNTs and an antitumor antibiotic can regulate the medico-biological activity of the drug. ...
Despite the fact that non-covalent interactions between various aromatic compounds and carbon nanotubes are being extensively investigated now, there is still a lack of understanding about the nature of such interactions. The present paper sheds light on one of the possible mechanisms of interaction between the typical aromatic dye proflavine and the carbon nanotube surface, namely, π-stacking between aromatic rings of these compounds. To investigate such a complexation, a qualitative analysis was performed by means of ultraviolet visible, infrared, and nuclear magnetic resonance spectroscopy. The data obtained suggest that π-stacking brings the major contribution to the stabilization of the complex between proflavine and the carbon nanotube.
... Caffeine concentration (M × 10 4 ) k obs × 10 3 (min −1 ) ± SD k1 × 10 3 (min −1 ) ± SD k2 × 10 3 (min −1 ) ± SD k1/k2 very similar to well-known regulatory action of CF with respect to aromatic drugs binding with DNA [44,45] and antibiotic-vitamin interaction [46], which is also considered as a consequence of the hetero-association between CF and other molecular species in solution. The likely reason for the influence of the hetero-complex formation on the kinetics of RF photodegradation is the formation of low dielectric environment within the volume of hetero-complex as a consequence of water exclusion, altering the energetic barriers for chemical transformation of RF. ...
tA study of the photodegradation of 5 × 10−5M riboflavin (RF) in 0.2–1.0 M phosphate buffer in the pres-ence and absence of 2.50 × 10−4M caffeine at pH 6.0–8.0 has been carried out. RF in phosphate buffer isphotodegraded simultaneously by normal photolysis (photoreduction) and photoaddition reactions giv-ing rise to lumichrome (LC) and cyclodehydroriboflavin (CDRF) as the main final products, respectively.RF and its photoproducts, formylmethylflavin (FMF), lumiflavin (LF), LC and CDRF in degraded solutionhave been determined by a specific multicomponent spectrophotometric method with an accuracy of±5%. The apparent first-order rate constants for the photodegradation of RF and for the formation of LCand CDRF are 5.47–15.05 × 10−3min−1, 1.06–8.30 × 10−3min−1and 4.31–8.05 × 10−3min−1, respectively.An increase in phosphate concentration leads to an increase in the rate of formation of CDRF and altersthe photodegradation of RF in favor of the photoaddition reaction. This photoaddition reaction is furtherenhanced in the presence of caffeine which results in a further decrease of the fluorescence of RF in phos-phate buffer. Caffeine may facilitate the photoaddition reaction by suppression of the photoreductionpathway of RF.
... Piosik et al. (2005) showed that methylxanthines are able to protect cells against the cytostatic and cytotoxic effects of some aromatic compounds. Caffeine and theophylline were reported to markedly reduce the mutagenic activity of some aromatic compounds, including daunomycin, doxorubicin, and mitoxanthron (Traganos et al., 1991a(Traganos et al., , 1991bEvstigneev et al., 2006Evstigneev et al., , 2008. The concentration of active drug forms was reduced as the concentration of methylxanthines increased. ...
Human diet may contain many mutagenic or carcinogenic aromatic compounds as well as some beneficial physiologically active dietary components, especially plant food phytochemicals, which act as mutagenesis or carcinogenesis inhibitors. This study compared the binding properties of natural compounds in the human diet (caffeine, theophylline, theobromine, and resveratrol) with a water-soluble derivative of chlorophyll to bind to acridine orange, a known mutagen. An analysis was conducted to determine which substances were effective binding agents and may thus be useful in prevention of chemical-induced mutagenesis and carcinogenesis. Data indicated that in order to bind 50% of the mutagen in a complex, less than twice the concentration of chlorophyllin was needed, the resveratrol concentration was 20-fold higher, while a 1000-fold or even 10,000-fold excess of xanthines were required to bind acridine orange.
There is a range of experimental proofs that biologically relevant compounds change their activity in the presence of C60 fullerene clusters in aqueous solution, which most frequently act as a nanoplatform for drug delivery. Inspired by this evidence, we made an effort to investigate the interaction of fullerene clusters with the antibiotic topotecan (TPT). This study proceeded in three steps, namely, UV/vis titration to confirm complexation and in vitro assays on proliferating and nonproliferating cells to elucidate the role of C60 fullerene in the putative change in TPT activity. Surprisingly, although the nonproliferating cell assay is consistent with the titration data and confirms complex formation, it contradicted the results of the proliferating cell assay. The latter showed that the mixture of TPT and fullerene affects the cells in the same way as pure TPT, as if there were no fullerenes in solution at all, whereas the action of TPT was expected to be enhanced. We explained this contradiction by the specific stabilization of the biologically inactive carboxylate form of the antibiotic adsorbed in the alkaline shell of large fullerene clusters, which leads to neutralization of the drug delivery function and almost zero net biological effect of the antibiotic in vitro. The practical outcome of the work is that fullerene clusters can be used for the selective delivery of pH-sensitive drug forms.
Представлено аналіз енергетики нековалентних взаємодій при самоасоціації 12 ароматичних молекул, різних за структурою та зарядом. Розроблено методику обчислення внесків різних фізичних чинників у повну енергію Гіббса. Виявлено, що внески водневих зв'язків та ентропійні чинники завжди сприятливі, тоді як ван-дер-ваальсівські, електростатичні та (або) гідрофобні взаємодії можуть бути стабілізуючими чи дестабілізуючими чинниками залежно від досліджуваної системи. Аналіз, який проведено у даній роботі, дає відповідь на питання: які чинники стабілізують/дестабілізують стекінг ароматичних молекул у розчині та яка їх відносна важливість.
The energetics of noncovalent interactions at the self-association of aromatic molecules with various structures and charges has been analyzed. Twelve different molecules have been examined. Amethod to compute the contributions made by various physical factors to the total Gibbs energy has been developed. The contributionsgiven by hydrogen bonds and entropic factors were foundto be always favorable, whereas the contributions made by van der Waals, electrostatic, and/or hydrophobic effects may be stabilizingor destabilizing, depending on the specific system under consideration. The issues concerning the factors that stabilize/destabilizethe stacking of aromatic molecules in the solution and their relative importance have been elucidated.
We have shown that the main factors affecting the equilibrium constant of self-association of aromatic molecules during the growth of an aggregate are the loss of translational and rotational degrees of freedom on the formation of a complex, the ordering of molecules (the entropy of mixing) into aggregates, and the electrostatic interaction (if molecules possess a charge). On the basis of these ideas, we have first obtained the formula for the equilibrium constant of self-association of aromatic compounds as a function of the number of molecules in the aggregate and drawn conclusion that, in the frame of the model in use, the profile of the constant is decaying.
The hetero-association of theophylline (THP) with other biologically-active aromatic molecules (e.g. the anti-cancer drugs daunomycin and novantrone, the antibiotic norfloxacin, the vitamin flavin-mononucleotide and two mutagens ethidium bromide and proflavine) has been studied by NMR in aqueous-salt solution (0.1 M Na-phosphate buffer, pD 7.1). It was found that THP shows an essentially similar hetero-association ability as caffeine (CAF) towards aromatic drugs, except for novantrone (NOV), which has much less affinity to THP than CAF as a result of energetically unfavourable orthogonal orientation of the chromophores of THP and NOV in the hetero-complex.
We report an analysis of the energetics of aromatic–aromatic stacking interactions for 39 non-covalent reactions of self- and hetero-association of 12 aromatic molecules with different structures and charge states. A protocol for computation of the contributions to the total energy from various energetic terms has been developed and the results are consistent with experiment in 92% of all the systems studied. It is found that the contributions from hydrogen bonds and entropic factors are always unfavorable, whereas contributions from van-der-Waals, electrostatic and/or hydrophobic effects may lead to stabilizing or destabilizing factors depending on the system studied. The analysis carried out in this work provides an answer to the questions “What forces stabilize/destabilize the stacking of aromatic molecules in aqueous-salt solution and what are their relative importance?”
With an aim of searching efficient interceptors of aromatic drugs, the self- and hetero-association of dim-
ethylxanthine derivatives with different structures, selected according to Strategy 1 (variation of the
position of methyl groups) and Strategy 2 (variation of the length of
A
(CH
2
)
n
A
COOH group), with aro-
matic drug molecules: Ethidium Bromide, Proflavine and Daunomycin, were studied using
1
H NMR spec-
troscopy. It was found that the association proceeds in a form of stacking-type complexation and its
energetics is relatively independent on the structure of the dimethylxanthines. However, on average,
the dimethylxanthines possess higher hetero-association constant and, hence, higher interceptor ability
as compared to the trimethylxanthine, Caffeine, used during the past two decades as a typical interceptor
molecule.
The analysis of heteroassociation of antibiotic topotecan (TPT) with aromatic biologically active compounds (BAC): caffeine, mutagens ethidium bromide and proflavine, antibiotic daunomycin, vitamins flavin-mononucleotide and nicotinamide, has been carried out in the work using 1H NMR spectroscopy data. The equilibrium constants of heteroassociation and induced chemical shifts of the protons have been obtained in the complexes with BAC. It is found that the complex formation TPT-BAC has the nature of stacking of the chromophores, additionally stabilized in the case of proflavine by intermolecular hydrogen bond. Calculation of the basic components of the Gibbs free energy of the complexation reactions is carried out, and the factors which stabilize and destabilize the heterocomplexes of molecules are revealed.
On the ground of the basic hetero-association model derived previously, a generalized statistical-thermodynamical model of hetero-association of aromatic molecules has been developed for the NMR data interpretation. In the proposed model, unlike the basic one, the edge effects are taken into consideration, i.e. the dependence of the proton chemical shift on the position of the molecule situated inside, at the edge of the aggregate or in the hetero-stack. Both basic and generalized models were used for the analysis of hetero-association of acridine dye, proflavine (PF), and phenan-thridinium dye, ethidium bromide (EB), in aqueous solution. The calculation of the association parameters of the molecules has been carried out using H-NMR (500 MHz) experimental data. The experimental concentration and temperature dependence s of the proton chemical shifts of interacting aromatic molecules have been studied. The parameters calculated according to the basic and generalized models are found to differ approximately by 30 % and depend substantially on the magnitude of the equilibrium hetero-association constant KC - the larger the KC value the higher the discrepancy between two models. The analysis of the structural and thermodynamical characteristics of the PF-EB complexation indicates the major role of dispersive interactions in stabilization of the PF-EB hetero-comptex.
The hetero-association of the vitamin B2 derivative, flavin-mononucleotide (FMN), with a mutagenic dye, ethidium bromide (EB) or proflavine (PF), has been studied by 1D and 2D 500 MHz 1H NMR spectroscopy. The variations of proton chemical shifts of both the vitamin and dye as a function of concentration and temperature were analysed in terms of the structural and thermodynamical properties of the FMN-EB and FMN-PF complexes in solution. The structures of the complexes were also investigated by observed intermolecular ROE contacts and molecular mechanics calculations. The results show that the 1 : 1 hetero-association complexes in solution are more stable than the self-association complexes, which is consistent with formation of an intermolecular hydrogen-bond in the hetero-complexes of FMN-EB and FMN-PF. Hence it is possible that the toxicity of aromatic molecules such as EB and PF may be reduced in vitro by the presence of FMN, partly because of the known antimutagenic action of FMN and partly because it has been shown in this work that there is an effective intermolecular association between the mutagens and the vitamin.
The self-association of the antitumour drug, novatrone, NOV (mitoxantrone) and its hetero-association with caffeine (CAF) have been investigated by 1D and 2D 500 MHz 1H NMR spectroscopy. Two-dimensional homonuclear correlation NMR spectroscopy (2D TOCSY and 2D ROESY) has been used for complete assignment of proton signals and for a qualitative analysis of the mutual arrangements of the aromatic drug molecules in the aggregates. The structural and thermodynamical parameters of molecular self- and hetero-association of the aromatic compounds have been determined from measurements of the NMR chemical shifts of the drug protons as a function of concentration and temperature. The self-association of NOV has been analysed using both the indefinite cooperative and non-cooperative models, and the hetero-association of NOV and CAF has been analysed in terms of a statistical-thermodynamical model, in which molecules form indefinite aggregates for both self- and hetero-association. The magnitudes of parameters (equilibrium reaction constants, enthalpy (ΔH) and entropy (ΔS)) have been calculated for self-association of NOV and its complexation with CAF; at 318 K the equilibrium constant for self-association of NOV is 12400 (±4000) l mol−1 and for hetero-association with CAF is 256 (±30) l mol−1. The most favourable structures of the NOV dimer and the 1∶1 NOV–CAF hetero-association complexes have been determined from the calculated limiting values of the induced chemical shifts of the drug protons.
A general nuclear magnetic resonance analysis of a statistical-thermodynamical model of hetero-association of aromatic molecules in solution has been developed to take “edge effects” into consideration, i.e., the dependence of proton chemical shifts on the position of the molecule situated inside or at the edge of the aggregate. This generalized approach is compared with a previously published model, where an average contribution to proton shielding is considered irrespective of the position of the molecule in the stack. Association parameters have been determined from experimental concentration and temperature dependences of 500 MHz proton chemical shifts of the hetero-association of the acridine dye, proflavine, and the phenanthridinium dye, ethidium bromide, in aqueous solution. Differences in the parameters in the range 10%–30% calculated using the basic and generalized approaches have been found to depend substantially on the magnitude of the equilibrium hetero-association constant Khet—the larger the value of Khet, the higher the discrepancy between the two methods. © 2001 American Institute of Physics.
Hetero-association of the anthracycline drug, daunomycin (DAU), with typical mutagens, the acridine dyes proflavine (PF) and acridine orange (AO), has been studied by 500 MHz 1H NMR spectroscopy as a function of concentration and temperature in 0.1 mol dm−3 phosphate buffered aqueous solutions at pD = 7.1. The results have been analysed in terms of a statistical-thermodynamical model of hetero-association of aromatic molecules, described previously [Davies, D. B., Veselkov, D. A., and Veselkov, A. N., 1999, Molec. Phys., 97, 439], but generalized in this work, so that there is no limitation on the magnitudes of the self-association constants of the interacting molecules. Expressions suitable for the analysis of NMR parameters of both components in the mixed solution have been developed enabling both the structural and thermodynamic properties of hetero-association to be determined. The magnitude of the equilibrium constant for hetero-association of PF + DAU is found to be substantially higher than the self-association constants of these molecules, whereas that for hetero-association of AO + DAU is intermediate between the equilibrium constants of self-association of AO and DAU. Intermolecular cross-peaks observed in 2D-ROESY spectra of PF + DAU mixed solutions are consistent with formation of a hetero-association complex in which an intermolecular hydrogen bond can form between either of the 3,6-diamino groups of the PF chromophore and the 9-MeCO group of DAU, which is in contrast to AO + DAU hetero-association, where such hydrogen bonds are unable to form. Quantitative structural and thermodynamical analysis of PF + DAU complexation is consistent with an intermolecular hydrogen bond contributing to the stability of the hetero-complex in aqueous solution. The NMR results show that hydrophobic interactions play a substantial role in the stabilization of the AO-DAU complex, characterized by a relatively small entropy change on complexation, compared to the PF-DAU hetero-complex, which is mainly stabilized by hydrogen bond and dispersive van der Waals interactions.
A heteroassociation of the antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied
by one- and two-dimentional 1H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and the
vitamin. The equilibrium reaction constants, the induced proton chemical shifts, and the thermodynamic parameters (ΔH and ΔS) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical
shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method
of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results
suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores.
An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules
of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous
solutions, which could result in a modulation of their medical and biological action.
Weekly low dose mitoxantrone (3 mg/m2) plus doxorubicin (8 mg/m2) was administered as second-line chemotherapy to 33 patients with advanced breast cancer. Four out of 28 evaluable patients (14%) obtained a partial response with a median duration of 34 weeks (range 18-67+ weeks), while 8 patients (29%) showed stable disease with a median duration of 28 weeks (range 11+-60 weeks). Gastrointestinal toxicity and alopecia were mild. Grade II and III leukopenia occurred in 63% of the courses without serious infectious disease. Four patients experienced an asymptomatic drop of 16-20% in the left ventricular ejection fraction (LVEF) after relatively low cumulative doses of each drug, and one patient with a history of pericarditis carcinomatosa and mediastinal irradiation developed a heart failure. In conclusion, this second-line combination treatment had moderate activity in breast cancer and caused only few subjective side effects, especially with respect to gastrointestinal symptoms.
Exposure of L1210 cells to DNA-intercalating antitumor drugs Novantrone (mitoxantrone; 20 ng/ml), doxorubicin (0.5 micrograms/ml), ellipticine (5 micrograms/ml), or the doxorubicin analogue AD198 (0.4 micrograms/ml), for 1 h, results in inhibition of cell proliferation, arrest of cells in the G2 phase of the cell cycle, and an increase in the number of cells entering higher DNA ploidy. These effects are significantly reduced when 5 mM concentrations of the methylxanthines caffeine or pentoxifylline are present either simultaneous with, or, in some cases, when added for 1 h immediately following pulse exposure to the drug. Both caffeine and pentoxifylline alone (5 mM) have little effect on cell growth or cell cycle progression. The possible mechanism of cell protection against intercalating drugs provided by caffeine was studied spectrophotometrically by measuring the interaction between Novantrone and the caffeine chromophore and in a model system using permeabilized L1210 cells and measuring the effect of caffeine in reducing binding of the intercalating dye acridine orange to cellular DNA and RNA. The data indicate that the observed protection of cells against intercalating drugs by caffeine or pentoxifylline is most likely a consequence of the direct interaction between the methylxanthines and the planar aromatic molecules of the intercalating drugs: formation of caffeine-drug complexes in solution effectively lowers the concentration of the free drug and thereby reduces its pharmacological activity. The principle of selective entrapment of the intercalator by compounds like caffeine may be considered in designing strategies to modulate the activity of intercalating drugs in vivo, e.g., in lowering drug toxicity when inadvertently applied at too high doses.
Adriamycin-treated rats were monitored for survivorship while consuming a normal diet adequate in riboflavin, a normal diet and receiving daily high-dose injections of riboflavin-5'-phosphate (flavin mononucleotide, FMN), or a riboflavin-deficient diet. Each animal was compared to a corresponding pair-fed, saline-treated control. In Adriamycin-treated rats fed the normal chow diet alone, survivorship declined within 7 days and remained constant after 12 days to about 50%. Adriamycin-treated rats consuming the normal diet and injected with FMN initially showed similar survivorship; however, after 20 days survival fell to 14%. Adriamycin-treated, riboflavin-deficient rats showed within 5 days a precipitous decline in survivorship which leveled to 5%. These results suggest that during Adriamycin treatment, proper riboflavin nutriture may be a crucial determinant of survival.
Riboflavin 5'-phosphate (flavin mononucleotide; FMN) inhibits the mutagenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide), the only known ultimate carcinogenic metabolite of benzo[a]pyrene. Coincubation of 10, 25, and 50 nmol of FMN with strain TA100 of histidine-dependent Salmonella typhimurium inhibits the mutagenicity of 0.05 nmol of the diol epoxide by 50, 70, and 90%, respectively. Ribose 5-phosphate and riboflavin show no significant effects at comparable doses. Reaction of B[a]P diol epoxide with FMN in aqueous solution at neutral pH produces only tetraols, with no evidence for covalent adducts. At pH 7 the rate of hydrolysis of B[a]P diol epoxide in dioxane/water, 1:9 (vol/vol), at 25 degrees C is increased more than 10-fold in the presence of 100 muM FMN. Spectrophotometric studies and quantitative rate data for the reaction of the diol epoxide with FMN indicate that a complex is formed between the diol epoxide and the flavin moiety of FMN (Ke = 1,400-3,400 M-1) prior to general acid-catalyzed hydrolysis of the epoxide to tetraols by the phosphate monoanion of FMN. Comparable concentrations of ribose 5-phosphate and riboflavin do not significantly increase the rate of hydrolysis, although evidence for complex formation between riboflavin and the diol epoxide is observed. General acid-catalyzed hydrolysis of bay-region polycyclic hydrocarbon diol epoxides by compounds that have a high affinity for these ultimate carcinogens represents a potentially useful way of inhibiting their carcinogenic activity.
Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine-6,6-dione; CAF) is known to potentiate the cytotoxic effects of DNA damaging agents such as ionizing radiation and alkylating agents. In contrast, however, the cytotoxic and cytostatic activity of aromatic, DNA-intercalating, DNA topoisomerase II inhibitors such as Adriamycin, ellipticine, or mitoxantrone are diminished in the presence of CAF. To resolve whether the protective effect of CAF is associated with a particular mechanism of drug interaction (e.g., intercalation into DNA, inhibition of DNA topoisomerase II), or the aromatic nature of the drug structure, per se, we have presently studied the effects of CAF on the cytostatic and cytotoxic action of camptothecin (CAM) and its less toxic but more water soluble derivative topotecan (TPT) on HL-60 human myelogenous leukemia cells: both drugs have aromatic structures but are nonintercalating inhibitors of DNA topoisomerase I. By using spectroscopy and titration microcalorimetry, we have also studied the direct interaction between CAF and TPT in solution. Low (20 nM) concentrations of CAM or TPT perturbed progression of HL-60 cells through S-phase, whereas higher concentrations (0.15 microM) of these drugs induced apoptosis; both effects were easily demonstrable after 4 h of treatment. When added simultaneously with CAM or TPT, CAF prevented both effects. The protective effect of CAF was concentration dependent and evident within the concentration range of 1-5 mM; nearly total protection was seen at a CAF concentration of 5 mM. The bathochromic and hypochromic shift in the absorption spectrum of the water soluble compound TPT upon addition of CAF indicated that CAF and TPT interact (stack) in a fashion similar to that previously observed for CAF and DNA intercalators. Microcalorimetric measurements of TPT titration with CAF indicate an exothermic reaction between these compounds (the enthalpy change was delta H degree = -4.2 kcal/mol), which is consistent with a stacking model of CAF-TPT interaction. Thus, the ability of CAF to protect HL-60 cells against the cell kinetic effects of CAM or TPT, as in the case of DNA intercalating topoisomerase II inhibitors, is most likely due to formation of complexes between CAF and these aromatic molecules, which result in reducing the effective concentration of the free form of these drugs available to the cells.
The molecular mechanism of the combined action of antibiotic and vitamin was studied by NMR spectroscopy. The heteroassociation of the antitumor antibiotic actinomycin D and flavin mononucleotide was investigated as a function of concentration and temperature by 500 MHz H-1 NMR spectroscopy. The equilibrium association constant, the thermodynamic parameters (DeltaH, DeltaS) of heteroassociation of actinomycin D with flavin mononucleotide, and the limiting values of proton chemical shifts in the heterocomplex were determined from the concentration and temperature dependences of proton chemical shifts of molecules. The most favorable structure of the 1:1 actinomycin D-flavin mononucleotide heteroassociation complex was determined using both the molecular mechanics methods (X-PLOR software) and the limiting values of proton chemical shifts of the molecules. In the calculated structure, the planes of the chromophores of actinomycin D and flavin mononucleotide molecules in the 1:1 heterocomplex are parallel and separated from each other by a distance of about 0.34 nm. At the same time, there is a probability of formation of intermolecular hydrogen bonds in the calculated structure of 1:1 actinomycin D-flavin mononucleotide complex. The analysis of the results obtained suggests that aromatic molecules of vitamins, e.g., flavin mononucleotide, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solution, modulating thereby the efficacy of their medical and biological action.
The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5′-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) 1H NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters ΔH and ΔS of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)2 play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.
One of the central problems in the study of the mechanism of DNA-antitumor drug interactions is the existence and nature of sequence specificity with respect to the base pairs of DNA. Results of a theoretical exploration of this problem are presented for a particularly important group of such drugs, namely the groove binding ligands. The great majority of the investigated antitumor agents of this category show a marked specificity for the minor groove of AT sequences of B-DNA. Contrary to current concepts and some proposals, hydrogen bond formation between the ligands and the receptor bases of DNA or contacts between specific hydrogen atoms on the ligand and on the bases do not appear to be the main determinants of this specificity. The essential factor responsible for this preference is the strong concentration of the electrostatic molecular potential in this groove of these sequences in B-DNA. This distribution of the potential renders difficult the conception of drugs specific for the minor groove of GC sequences and is responsible for the failure in this respect of lexitropsins and isolexins. Nevertheless, for the first time in theoretical computations, a particular isolexin was conceived which should bind preferentially to the minor groove of GC sequence: this is a neutral ligand composed of three furan or imidazole rings linked by NH groups. An increased GC specificity is, moreover, predicted for vinylexins (analogs of isolexins with C=C linkers) and an increased binding energy, with the specificity conserved, may be obtained for monocationic such ligands. In fact, monocationic vinylexins should form a family of universal groove binding ligands, the AT or GC specificity of which may be dictated by the arrangement of their hydrogen bond donor or acceptor heteratomic ring systems.
1H NMR spectroscopy at 500 Mhz has been used to determine the structures and thermodynamics in aqueous salt solution of the hetero-association of Daunomycin (DAU) with a series of phenanthridine dyes having different numbers of amino/azido groups in the chromophore, together with the self-association of the phenthridine dyes under the same solution conditions (0.1 M phosphate buffer, pD 7.1, 298 K). The NMR measurements have been analyzed using statistical-thermodynamical models of both self-association and hetero- association in which no limitation is set on the size of molecular stacks. In this work the magnitudes of the self-association parameters of Ethidium Bromide (EB) and its azido-derivatives, 8-azido-Ethidium Bromide (EMB) and 3,8-diazido-Ethidium Chloride (EDC), show a successive decrease with mono- and di-substitition of the 3,8-amino groups of EB. A similar pattern is observed for the equilibrium constants for hetero-association of the phenanthridines with DAU. The thermodynamical and structural parameters of hetero-association of the phenanthridines with DAU are consistent with an intermolecular hydrogen bond between the 3,8 amino-groups of EB and the 9MeCO group of DAU contributing to the stability of the hetero-complex in aqueous solution.
Self-association of aromatic drug molecules, proflavine (PF), Acridine Orange (AO), ethidium bromide (EB) and actinomycin D (ActD), in aqueous salt solution has been studied by one- and two-dimensional 500 MHz 1H NMR spectroscopy. 2D-COSY and 2D-NOESY measurements were used for complete assignment of proton signals of EB and ActD in solution and for a qualitative determination of their self-association, i.e. the mutual arrangement of the drug molecules in the complexes. Concentration and temperature dependences of proton chemical shifts of the drugs have been measured. Experimental results have been analysed using indefinite non-cooperative and cooperative models of molecular self-association, enabling the determination of equilibrium reaction constants, parameters of cooperativity, the thermodynamical parameters (enthalpy and entropy) of the self-association reactions and the limiting values of drug proton chemical shifts in the complexes. The most favourable dimer structures of PF, AO, EB and ActD have been constructed using calculated values of induced chemical shifts of drug protons in conjunction with intermolecular NOEs. The results show that the drug chromophores have an antiparallel orientation in all the dimers studied.
The interactions of small molecules with nucleic acids have provoked considerable interest in the field of anticancer drug design over the past three decades; however, critical information linking the physical-chemical properties associated with these complexes with their biological effectiveness remains unclear. Significant progress has been made towards unraveling the structural and dynamic properties of many ligand-DNA complexes which has provided pivotal insight into the design and development of more effective second and third generation chemotherapeutic agents for the successful treatment of many types of cancer. Interactions of ligands with DNA are studied by a variety of physical and biochemical methods in an effort to determine the chemical and physical basis of novel binding phenomena such as DNA base sequence selectivity, correlation of structure-activity relationships, linkages between the geometry and thermodynamic properties describing drug-DNA complexes, and influences of substituent modifications on the physical, chemical, and biological properties of the drug-DNA complex.
The self-association of 6-methylpurine and 4-methylpyrimidine and their hetero-association with caffeine and theophylline in deuterium oxide at 35°C were studied by measuring the concentration-dependent changes in proton chemical shift. The association parameters were calculated using simple and competitive dimer models. The self-association constants for 6-methylpurine and 4-methylpyrimidine were found to be 2.24 ± 0.07 and 0.200 ± 0.007 1 mol−1, respectively, and the hetero-association constants could be ordered in the decreasing series caffeine-6-methylpurine (4.85 ± 0.12 1 mol−1) > theophylline-6-methylpurine (4.11 ± 0.15 1 mol−1) > caffeine-4-methylpyrimidine (1.45 ± 0.13 1 mol−1) > theophylline-4-methylpyrimidine (0.98 ± 0.10 1 mol−1). The equilibrium constants imply that methylation enhances the association ability and the upfield dimer shifts can be interpreted in terms of a stacking-like interaction.
The matrix method of statistical mechanics is used to calculate equilibria for the binding of small molecules to polymers. When there is only one kind of binding site the problem is simple; some examples are given for illustrative purposes. If, however, the binding sites are not all equivalent and the bound molecules interact or interfere with each other, the problem is no longer trivial, being formally analogous with calculation of the helix–coil transition equilibrium in a heterogeneous polypeptide. Particular difficulties arise when the sequence of binding sites is aperiodic; most naturally occurring materials fall in this class. The purpose of this paper is to point out that problems of this type are readily solved with good accuracy by use of random-number methods on a high-speed digital computer. One such calculation is presented for illustration. The methods developed are applicable to such systems as the binding of actinomycin, Hg–, and acridine dyes to DNA.
The complexation of antitumour antibiotics novatrone (NOV) and daunomycin (DAU) in aqueous solution has been studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz) in order to elucidate the probable molecular mechanism of the action of aromatic antitumour drugs in combination chemotherapy.The equilibrium reaction constants, thermodynamical parameters (ΔH, ΔS) of hetero-association of NOV with DAU and the limiting values of proton chemical shifts of the molecules in the hetero-complexes have been determined from the experimental concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favourable structure of the 1:1 NOV–DAU hetero-association complex has been determined using both the molecular mechanics methods (X-PLOR software) and the limiting values of proton chemical shifts of the molecules. The obtained results have shown that intermolecular complexes between NOV and DAU molecules are mainly stabilized by stacking interactions of the aromatic chromophores. It is likely that there is an additional stabilization of the NOV–DAU hetero-complexes by intermolecular hydrogen bonds. It is concluded that aromatic molecules of antibiotics may form energetically stable hetero-association complexes in aqueous solution and hence effect their medical–biological (and probably toxic) activity.
The aim of this study was to determine the intracellular pharmacokinetics of mitoxantrone in vivo and to use these results to establish how leukemic cells should be incubated to perform clinically relevant in vitro studies of this drug.Blood samples were obtained from 11 patients with acute nonlymphoblastic leukemia at certain intervals up to 20 h after the infusion of mitoxantrone 12 mg/m2. Plasma and leukemic cells were separated and the drug concentrations were determined with HPLC. Before treatment, leukemic cells from 12 patients were incubated with 0.02, 0.05, 0.1, 0.2 and 1.0 μM mitoxantrone for 1–4 h and thereafter cultured in suspension culture for 20 h; during this time cell samples were taken at certain intervals for drug determination. In cells incubated with 0.05 and 0.2 μM mitoxantrone the cytotoxic effect was measured with the DiSC assay after cultivation for 4–5 days. In vivo, the intracellular levels exceeded the plasma concentrations already at the end of infusion and after 2 h the intracellular concentrations were 200–300 times higher than in plasma. In vitro, the intracellular steady state level of mitoxantrone was reached after 1–2 h and there was a pronounced intracellular retention even after 20 h culture in drug-free medium. Incubation with 0.05 μM during 1 h gave intracellular concentrations of mitoxantrone similar to those achieved in vivo. This incubation concentration gave a mean cytotoxic effect of 53% living cells measured with the DiSC assay, which gives good possibilities to discriminate between mitoxantrone-sensitive and unsensitive cells. We believe that exposing leukemic cells in vitro for in vivo mimicking mitoxantrone concentrations could increase the clinical relevance of predictive assays.
Significant progress has been made over the past few years in studies of drug—DNA interactions. Structure-based design strategies have yielded new DNA-binding agents with clinical promise. The hairpin polyamides represent the result of a design strategy with outstanding potential. One specific molecule of this class has now been proven to inhibit the expression of a specific gene in vivo. A new bisintercalating anthracycline antibiotic binds with high affinity to DNA, and appears to overcome a specific form of multidrug resistance. Progress in fundamental studies of drug binding to DNA continues, with detailed thermodynamic studies providing new insights into the forces that drive complex formation. New tools have been developed in order to characterize both the binding mode and the sequence specificity of drug binding to DNA, tools that will enable the fundamental aspects of these biologically important reactions to be understood in more detail.
Doxorubicin is a major anticancer agent introduced to extended clinical use in the early 1970s. The fulfillment of a wide program of analogue synthesis led to the development of the better tolerated epirubicin and of a highly potent antileukemic drug, idarubicin. In recent years, on the basis of the available information on the molecular requirements for action, a new synthetic program, coupled with target-oriented pharmacological experiments, was carried out. Various interesting derivatives, namely, the 8- and 10-fluoro compounds and the disaccharides, were obtained. The latter compounds exhibited a strong dependence of biological activity on the orientation (axial vs. equatorial) of the second sugar moiety, daunosamine. A member of this group, namely, , is presently undergoing clinical trials as a third generation antitumor anthracycline.
To investigate the effects of mitoxantrone in combination with other anticancer agents, a human T-cell leukemia cell line, MOLT-3, was incubated for 3 days in the presence of two drugs (mitoxantrone and the combined drug) and cell growth inhibition was determined by assay with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazonium bromide. The effects of drug combinations at doses giving 80% inhibition (ID80) were analyzed by an improved isobologram method. A supra-additive (synergistic) effect was observed for mitoxantrone in combination with amsacrine, cisplatin, or cytosine arabinoside. An additive effect was observed for its combination with bleomycin, doxorubicin, etoposide, 5-fluorouracil, mitomycin C, 6-mercaptopurine, or vincristine. A sub-additive (antagonistic) effect was observed for its combination with methotrexate. These data suggest that mitoxantrone, administered simultaneously with any one of a majority of anticancer agents we studied, is advantageous for cytokilling. Of the anticancer agents tested, amsacrine, cisplatin, and cytosine arabinoside are the most suitable for combination with mitoxantrone, and these combinations are worthy of clinical investigation. Methotrexate in our system is inappropriate for simultaneous administration with mitoxantrone. These data should provide useful information for the establishment of clinical protocols involving mitoxantrone.
Caffeine is known to potentiate the cytotoxicity of a variety of DNA damaging agents presumably by reducing the ability of the cells to repair potentially lethal lesions. However, in the present study we observe that 5 mM caffeine reverses the cell kinetic and cytotoxic effects of the intercalating drug Novantrone (mitoxantrone) on L1210, HL-60 and CHO cells. Novantrone alone, at a concentration of 20-30 ng/ml, given to cultures for 1 h, inhibits cell growth by about 50% and causes cells to accumulate in S and G2 phase and to enter a higher DNA ploidy level. Treatment of these cell lines with 5 mM caffeine alone for 1 h has a minimal effect on cell proliferation; suppression of cell growth varies from 5 to 10%. Exposure of cells to Novantrone for 1 h in the presence of caffeine results in a significant reduction of the Novantrone effects; the cell growth rate is partially restored (e.g. caffeine reduces suppression of L1210 cell growth from 48 to 83% of control) and in each of the cell lines studied, the Novantrone-induced cell accumulation in S and G2 is abolished. Combined treatment with caffeine and Novantrone also increases the clonogenicity of CHO cells 8.5 times over that seen in cultures treated with Novantrone alone. In contrast to the combined treatment with caffeine + Novantrone, pretreatment of cells with caffeine provides no protection. Likewise, post-treatment with caffeine provides little reversal of growth inhibition and G2 cell accumulation, especially if the post-treatment is delayed in time. The present data, in conjunction with evidence in the literature that caffeine protects cells against the cytotoxic effects of doxorubicin, suggest that caffeine may play a more general role in protecting cells against planar aromatic molecules such as intercalating agents.
In this review recent publications are cited for a number of antimutagens. The molecules surveyed are potential or proven "desmutagens" or "interceptors." These are biologically prevalent or synthetic molecules that are most often small metabolites proficient in binding to, or reacting with, mutagenic chemicals and free radicals. Many of this class of "blocking agents" are "soft" and "hard" nucleophiles with consequently varying abilities to react with particular classes of electrophiles, the major classes of direct-acting mutagens. Although they serve as a first line of defense against mutagens and carcinogens, many interceptor molecules are under-investigated with regard to their spectra of activity and their possible relevance to prophylaxis or treatment of human disease states.
In a prospective phase I trial involving 35 patients with metastatic carcinoma, we tested a pharmacokinetic strategy for guiding dose escalation of the anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX), a new analogue reported to be more potent and less toxic than doxorubicin. This strategy is potentially a safe and more rapid way of determining the maximum tolerated dose (MTD) of anticancer agents. Retrospective studies have shown that the total plasma drug exposure after a dose lethal to 10% of mice (LD10) is approximately equivalent to the total exposure produced in humans by the MTD. Thus, we intended to aim dose escalation in humans to achieve the area under the curve for I-DOX plasma concentration x time (AUC) equivalent to that produced in mice by an LD10. However, differences in I-DOX pharmacokinetics and metabolism in BDF1 mice and humans at the initial dose prevented immediate application of this strategy. Therefore, we escalated the dose by the modified Fibonacci scheme while investigating the pharmacology of I-DOX and its major plasma metabolite 4'-iodo-4'-deoxy-13-dihydrodoxorubicin (I-DOXOL). Plasma pharmacokinetics was characterized by rapid elimination and extensive metabolism of I-DOX to I-DOXOL. The ratio of I-DOXOL to I-DOX plasma AUC was 12.8 +/- 7.3 SD. The plasma pharmacokinetics of I-DOX and I-DOXOL were linear in the range of tested doses (2-90 mg/m2). The LD10 in mice was 6.8 mg/kg for I-DOXOL and 6 mg/kg for I-DOX, and the concentration of drug that inhibited by 50% (IC50) the growth of human granulocyte-macrophage colony-forming units (CFU-GM) was 80 nM for I-DOXOL and 50 nM for I-DOX. From these findings, we concluded that the toxic effects of I-DOX and I-DOXOL are equivalent and reset the pharmacokinetic target of escalation to the sum of I-DOX and I-DOXOL AUCs at I-DOX LD10. Then we safely applied pharmacokinetically guided escalation to determine the MTD (80 mg/m2). The plasma AUC of I-DOX and I-DOXOL at the human MTD is 71% of the AUC at mouse LD10. The only dose-limiting toxic effect was severe granulocytopenia.
The sensitivity of Chinese hamster ovary cells to ethidium bromide, a deoxyribonucleic acid (DNA)-intercalating cationic dye, was reduced in the presence of caffeine, dimethylsulfoxide (DMSO) or 3-aminobenzamide (3AB). As drug resistance is frequently attained by decreased permeability of the drug, the effects of caffeine, DMSO and 3 AB on the intracellular accumulation of ethidium were examined. These chemicals were found to decrease the intracellular dose of ethidium via their suppressing effects on the dye uptake with no effect on the efflux. The dye uptake was also suppressed by ouabain and K+, suggesting that ethidium was taken up by the K+ transport system. Although the exact mechanism implicated in the inhibitory effects of caffeine. DMSO and 3AB on the dye uptake is not yet clear, these chemicals may interfere with this transport system. The rate of decrease in intracellular dose caused by caffeine, DMSO and 3 AB correlated well with that of the reduction in respective cellular sensitivity. This strongly suggests that the decrease in intracellular dye dose in the presence of these chemicals is mainly responsible for the reduced sensitivity. Caffeine, DMSO and 3 AB caused a decrease in the binding of ethidium to DNA in vitro. The release of ethidium from DNA molecules may lessen DNA damage, thereby contributing to the reduction in cellular sensitivity to ethidium. However, correlation between inhibitory effects of these chemicals on the dye binding and those on cellular sensitivity is not as good as that between the uptake and the sensitivity. This indicates that effects on dye binding to DNA may not be a major reason for the reduction of cellular sensitivity to ethidium.
Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.
The term soft tissue sarcoma refers to a large variety of malignant tumors arising in extraskeletal connective tissues that connect, support, and surround discrete anatomic structures. All visceral organs also contain a connective stroma that can undergo malignant transformation. Because of the histological similarities of this group of tumors and their relative rarity, treatment prescriptions for patients that have disseminated disease are most often uniform. In this study, we asked the question whether adding a third drug (cyclophosphamide or actinomycin D) to Adriamycin (Adr [Adria Laboratories, Columbus, OH])-(3,3-dimethyl-1-triazeno)- imidazole-4-carboxamide (DTIC) would improve the response rate and/or survival. A unique feature of this cooperative group clinical trial was the mandatory pathology review of the histological material. All patients of the Southwest Oncology Group between June 1, 1976, and November 17, 1979, who had a biopsy-confirmed diagnosis of a soft tissue sarcoma with convincing clinical or biopsy-documented evidence of metastatic disease were eligible for the study. Patients were randomized to receive (1) Adr, 60 mg/m2 intravenously, day 1, and DTIC, 250 mg/m2 every 3 weeks (104 patients); (2) Adr and DTIC as in (1) and cyclophosphamide, 500 mg/m2, day 1 (112 patients); or (3) Adr and DTIC as in (1) and actinomycin D, 1.2 mg/m2, day 1, (119 patients). There was no statistically significant difference in response rates (33%, 34%, and 24%) (P = .25). Median durations of response were 31 weeks in the Adr-DTIC arm, 26 weeks in the cyclophosphamide-DTIC-Adr arm, and 23 weeks in the Adr-DTIC-Actinomycin D arm (P = .78). Median durations of survival were 37, 42, and 50 weeks, respectively. Again, no statistically significant differences were observed (P = .59). Toxicities from each of these treatment arms were formidable and were equivalent. Prognostic factor analysis showed a prognosis based on bone marrow reserve, sex, and pathology subtype favorable to patients.
Concepts elucidated from preclinical pharmacology studies have made a substantial impact on the clinical use of anticancer drugs. However, the majority of animal pharmacology results have not been available until after drugs have entered clinical trials. Since clinical pharmacokinetic measurements are already part of many phase I trials, human data could be directly compared with mouse data if mouse pharmacology studies were completed before clinical trials were initiated. Once the starting dose in a phase I clinical trial has been evaluated, subsequent doses are escalated until the maximum tolerated dose is reached. The rate of escalation is empirically defined by a modified Fibonacci series. This universal escalation scheme is applied to all drugs, with no modifications based upon pharmacology or other factors. If the starting dose is far removed from the maximum tolerated dose, a large number of dose escalations are required. Consequently, most patients receive subtherapeutic doses, and the amount of resources allocated to each drug increases. We are exploring potential strategies for controlling the rate of dose escalation based upon pharmacokinetic determinations in mouse and man. Retrospective analyses indicate that 20%-50% savings in the total number of dose escalations are possible.
Exponentially growing Chinese-hamster V79-cells were treated with various doses of adriamycin (ADR) for 1 hr in the presence or absence of 2 mM caffeine and were subsequently incubated for 24 hr in fresh medium with or without caffeine (2 mM) before plating to assay for survival. The results indicated a reduction in killing when caffeine was present during treatment with ADR (e.g., reduction in killing from 0.03 to 0.3 after exposure to 0.5 microgram/ml ADR). This reduction in killing was even more pronounced after a 24 hr treatment with ADR in the presence of caffeine (e.g., reduction from 0.005 to 0.5 after exposure to 0.08 microgram/ml ADR). Incubation with caffeine after ADR treatment (1 hr) caused only a comparably small increase in cell survival. Presence of caffeine either simultaneously or after treatment with ADR caused a reduction of the inhibition of growth and mean-cell-volume increase, and a reduction of the accumulation of cells in G2-phase. Qualitatively similar results were also obtained after continuous treatment with ADR in the presence or absence of caffeine. Reduction in growth inhibition and accumulation of cells in G2-phase was observed under conditions only slightly affecting cell survival, thus suggesting that caffeine may affect these two phenomena by independent mechanisms. Flow cytometry measurement of the intracellular ADR content indicated a reduction in the presence of caffeine. Furthermore, post-treatment incubation with caffeine was found to increase the rate of decay of ADR-related fluorescence. Quantitative comparison between the effect of caffeine in the intracellular ADR accumulation and cell survival suggested that the observed reduction in killing could be attributed to a decrease in the intracellular drug levels. The reduction by caffeine of the ADR-induced cell cycle delays is attributed to either the decrease in the intracellular ADR dose in the presence of caffeine, or to an effect of caffeine similar to that exerted after exposure of cells to ionizing radiation. Trifluoperazine, which had only a small effect on cell survival of cells treated with ADR alone, potentiated killing when cells were treated with ADR in the presence of caffeine. This effect can be partly attributed to the observed modification in the intracellular ADR content under these conditions but, as a quantitative comparison suggests, other effects might also be involved.
The arrangement of the protein component on the DNA of the chromatin complex was studied by comparing the rate of release of oligonucleotides and of protein after addition of deoxyribonuclease I and deoxyribonuclease II to rat thymus chromatin. Also the action of deoxyribonuclease I on normal chromatin and on chromatin depleted of non-histone protein was compared, to elucidate the role of the latter protein in chromatin structure. As a preliminary to the above, the rate of action of deoxyribonuclease I on DNA and on chromatin at the same DNA concentration, and the dependence of the action of this enzyme on the Mg(2+) concentration, were studied. It was found that: (1) little if any DNA in chromatin is present in extensive, truly ;free' zones, i.e. completely uncovered by protein; (2) at relatively low concentrations of added Mg(2+), deoxyribonuclease I degrades chromatin more rapidly than DNA; (3) the non-histone protein is not attached directly to the DNA in chromatin.
The binding of deoxyribonucleoprotein to Toluidine Blue, to cetylpyridinium chloride and to polylysine of various molecular weights was studied to determine the percentage of free DNA phosphate groups in deoxyribonucleoprotein. Binding was measured by addition of these reagents to deoxyribonucleoprotein at a range of concentrations such that complete precipitation of the deoxyribonucleoprotein occurred. With Toluidine Blue the binding corresponded to about 48% of the DNA phosphates in deoxyribonucleoprotein. The dye did not cause appreciable displacement of protein from the DNA. With cetylpyridinium chloride the binding corresponded to about 41% of the DNA phosphates. With polylysine preparations of molecular weight 1250 and 7790 the binding values for deoxyribonucleoprotein were 46 and 38% respectively. The results suggest that the free phosphates lie in stretches sufficiently long to accommodate most of each polylysine molecule. With polylysine of molecular weight 62000 cross-linking of free stretches of DNA on different deoxyribonucleoprotein molecules probably occurs. It is concluded that although most of the free phosphates are probably ;hidden' beneath covering histone, corresponding perhaps to runs of non-basic residues in the latter, they are surprisingly accessible to very large molecules. The relevance of this finding to the problem of gene repression is discussed.
1) The self-association of both caffeine (Cf) and 5'-adenosine monophosphate (AMP) in aqueous solution has been reinvestigated by 1H NMR. The self-association process is characterized by an isodesmic model. The apparent self-association constants of the vertical stacking process are KCf = (10.6 +/- 1.0) M-1 and KAMP = (1.67 +/- 0.17) M-1. The arrangement of the monomeric units in the stacked aggregates is discussed in terms of isoshielding curves theoretically calculated by Giessner-Prettre and Pullman. Models are proposed which are consistent with these and further previous NMR data. 2) The interaction of Cf and AMP has been studied by 1H NMR. The apparent association constant of the complex Cf-AMP is KC-A = (7.3 +/- 1.2) M-1. Two models of the mutual arrangement of AMP and Cf in the complex are proposed on the basis of the calculated isoshielding curves considering both ring current and local atomic diamagnetic anisotropy effects. 3) The interaction of Cf and poly(riboadenylate), (rA)n, is indicated by a downfield shift of the H-8 line but an upfield shifts of the H-2 line in the 1H NMR spectra of (rA)n. The concentration dependence of the 1H NMR shifts of both Cf and (rA)n can be explained by the existence of two binding mechanisms. We suggest (i) partial insertion of Cf between adjacent base residues of ordered single-stranded regions of (rA)n and (ii) outside binding of Cf in form of monomeric Cf as well as of self-associated aggregates. The complex geometry of insertion proposed on the basis of the calculated isoshielding curves is characterized by a stronger overlapping of the Cf ring and the H-2 proton of (rA)n as compared to the H-8 proton.
The relative orientation of caffeine in stacks formed by self-association in aqueous solution has been evaluated by both 1H- and 13C-nmr spectroscopy. The data, which were interpreted through the calculation of ring-current and atomic diamagnetic anisotropic effects of the caffeine molecule, suggest two caffeine bases may stack in a nearly orthogonal manner. The interactions between caffeine and adenylyl-3′,5′-adenosine, polyadenylic acid, and ribo-(A-A-G-C-U-U)2 helix were also studied by nmr at different caffeine/base ratios and at varying temperatures. The results show that caffeine tends to stack on the top of the terminal purine bases or to insert (the single-stranded) or to intercalate (the double-stranded) between purine bases.
Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 microM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase (Ki approximately 120 microM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.
Chlorophyllin is known to inhibit the mutagenicity of a variety of compounds. Using highly purified samples of chlorophyllin and its family compounds, we studied the mechanism of the inhibition. Since mutagens with polycyclic planar structures are particularly strongly inhibited, it seemed likely that the inhibition arises by trapping of the mutagens by chlorophyllin through complex formation at the planar surfaces of these molecules. To explore this possibility, we prepared a Sepharose bearing covalently linked chlorophyllin as ligand, and the absorption of mutagens to this Sepharose was measured. Three different chlorophyllin derivatives were used, i.e., copper-chlorin, iron-chlorin and chlorin, to investigate the role of metal in the center of the chlorophyllin chromophore. Adsorption of 37 different compounds, mostly mutagens, in 0.02 M Tris-HCl buffer at pH 8.0 to these chlorophyllin-Sepharose preparations was studied in a quantitative manner. The results showed that most of the compounds having three or more fused rings were strongly adsorbed with apparent dissociation constants of 10⁻⁵−10⁻⁶ M, whereas those having two fused rings or one ring were only poorly adsorbed. Since the three Sepharose adsorbents gave similar adsorption profiles, it appeared that the central metal in the chlorophyllin molecule does not play a crucial role in the adsorption. We also measured the inhibitory effect of copper-chlorin against the mutagenicity of some of these compounds using the Salmonella assay. The results showed that those mutagens that were strongly adsorbable to copper-chlorin-Sepharose were subject to efficient inhibition by copper-chlorin, whereas many of those only poorly adsorbed were inhibited only weakly. We concluded that trapping by complex formation plays a role in the antimutagenic actions of chlorophyllin against many mutagens, particularly notable being the actions against ICR-170, quinacrine, aflatoxin B 1, Trp-P-1 and Trp-P-2.
Pulse treatments of U-937 human promonocytic leukemia cells with the DNA topoisomerase-II inhibitors 4'-(9-acridynilamino)methanesulfon-m-anisidide (amsacrine, mAMSA) or etoposide (VP-16) caused growth inhibition, G2-arrest, increase in cell size and expression of differentiation markers. All these effects were greatly reduced by the presence of 5-10 mM caffeine. In addition, caffeine partially prevented the increase in the number of topoisomerase-DNA cleavable complexes caused by the topoisomerase inhibitors, as determined by SDS/CIK precipitation assays; it caused chromatin condensation, as determined by flow cytometry assays, and interacted with mAMSA in solution, as suggested by spectrophotometric assays. Pulse treatment with caffeine greatly inhibited RNA synthesis but not DNA or protein synthesis, as indicated by labelled precursor incorporation assays. The transcription inhibitor 5,6-dichloro-I-beta-D-ribofuranosylbenzymidazole reduced the mAMSA- and VP-16-produced growth inhibition in a similar manner. It is concluded that RNA synthesis inhibition is one of the possible mechanisms by which caffeine protects cells from the action of topoisomerase-II inhibitors.
A study was conducted to assess the effects of riboflavin deficiency and riboflavin supplementation on carcinogen-DNA binding. After 12 wk on a riboflavin-sufficient or a riboflavin-deficient diet male Wistar rats were administered 3H-labelled benzo[a]pyrene (BP) ip. [3H]BP was given either at a uniform dose of 450 muCi/rat irrespective of body weight or at a dose adjusted to body weight. After 17 hr the animals were killed, various organs were dissected and the level of [3H]BP bound to DNA was quantified in organs that are known to be the seats of drug metabolism (i.e. the liver, lungs and intestinal mucosa). In a separate experiment, the effect of riboflavin supplementation on BP-DNA binding was also investigated. When [3H]BP was administered at 450 microCi/rat, BP-DNA binding was markedly increased in the livers and intestinal mucosae of the pair-fed and deficient groups compared with controls. With the administration of [3H]BP adjusted to body weight, no differences in BP-DNA binding between groups were observed in any tissue. However, on administration of riboflavin there was a decrease in the level of [3H]BP bound to DNA in almost all tissues, especially in the lungs, where the reduction was significant. The results suggest that undernutrition/riboflavin deficiency may increase the risk of carcinogenesis by way of an increase in carcinogen binding, which however can be reversed by riboflavin supplementation.
The protective effects of clinically used drugs on the toxicity and microsomal lipid peroxidation induced by doxorubicin (adriamycin, ADM), an anthracycline type antitumor agent, were studied in mice and rats. Regarding the effects of anthracyclines (aclarubicin, ACL; daunorubicin, DAU; ADM; epirubicin, EPI; pirarubicin, PIR) on rat liver microsomal lipid peroxidation, ACL had the smallest effect, and effectiveness increased in the order of PIR, ADM, DAU and EPI. The increasing effect of lipid peroxidation induced by these drugs was closely correlated with the decrease in the body weight of mice administered intraperitoneally at a dose of 20 mg/kg and in rats at LD50 of the drugs. The survival times of ADM-administered mice (which were injected 15 mg/kg of ADM twice) treated with the following drugs, expressed as a percent of that of the control group, were 236% for adenosine triphosphate, 224% for coenzyme Q10 (Co Q), 235% for dextran sulfate (DS), 123% for dipyridamole, 121% for flavin adenine dinucleotide, 213% for reduced glutathion, 155% for inositol nicotinate, 157% for nicardipin and 297% for nicomol. The rat heart microsomal lipid peroxidation levels in vivo may be one of the indications of ADM-induced toxicity. The levels treated with DS correlated well with the development of ADM-induced toxicity: mouse survival time, change of body weight and tissue wet weight loss. Another type of drug, such as Co Q, may improve the myocardiac mitochondrial functions compared to those of ADM-administered mice.
Formation of single strand breaks in nuclear DNA induced by hepatocarcinogens aflatoxin B1 and N-nitrosodimethylamine was observed to be more pronounced in rats maintained on a riboflavin-deficient diet compared to that on a normal diet. This increased damage was reversed on riboflavin supplementation. The induction of repair enzymes poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was significantly higher in riboflavin-deficient rats following DNA damage caused by the administration of carcinogens. Riboflavin supplementation brought down the induction to the levels found in rats maintained on normal diet. Since damage to DNA and its altered repair may relate to carcinogenesis, modulation of these parameters by riboflavin suggests a potential chemopreventive role of this vitamin.
The interactions of a set of structurally selected betacarbolines (BC), (9H-pyrido[3,4-b]indoles) and indoles (IND) with two representative flavins (FN): riboflavin (RFN) and flavin mononucleotide (FMN) have been investigated by absorption and fluorescence spectroscopies. Spectral results provided evidence on the formation of 1:1 non-fluorescent molecular complexes, whose stability constants and other related thermodynamic parameters have been estimated from Stern-Volmer quenching analysis. The FMN complexes are somewhat more stable than the RFN complexes. The stabilities of the IND and BC complexes for a given FN follow approximately the order IND approximately tetrahydro BC < dehydro BC < fully aromatic BC. Protonation of the pyridinic nitrogen atom of BCs has a destabilizing effect, which is more pronounced for fully aromatic than for tetrahydro derivatives. The influence of structural factors on the stability of the complexes has been discussed and, aided by theoretical AM1 calculations, a qualitative model for the structure (stacking) and binding forces (cooperative localized charge transfer and dispersion forces) of the complexes has been proposed.