Pharmacodynamic PD properties

In vitro antitumor activity

All anthracyclines discussed so far have demonstrated cytotoxicity against a wide range of animal and human tumor cell lines. Cytotoxicity increases exponentially with both drug concentration and duration of exposure, and maximal lethal effects were demonstrated in the S- and G2-phases of the cell cycle and less or no cell kill in the G1 and M phases. However, at high concentrations cytotoxic effects can be observed in G1 and M phases as well [10].

The cytotoxicity of DOX and EPI in tumor cell cultures (e.g., liver, lung, colon, breast) were nearly identical at equimolar concentrations. No advantage was found with respect to a broader spectrum of activity for EPI except for gastric cancer cells, which were found to be more sensitive to EPI than to DOX [11]. A number of in vitro studies with DNR and IDA in animal and human tumor cell lines have demonstrated a higher potency of IDA when cytotoxici-ty was measured and compared with DNR. IDA was always more potent than DNR at equimolar dose [12]. Interestingly, IDA was tested in vitro in several solid tumor cell lines with remarkable cytotoxic efficacy. It was found that idarubicinol, the major metabolite of IDA, had similar activity as the parent drug in these experiments. This phenomenon was not observed for doxorubi-cinol, epirubicinol or daunorubicinol. It is well known that in vitro studies with antitumor agents, and in particular with anthracyclines, do not always predict the antitumor activity in vivo. The relevance of the numerous in vitro studies with anthracyclines for in vivo studies is therefore debatable.

In vivo antitumor activity

In general, the antitumor activity of doxorubicin and epirubicin appears to be similar in various orthotopic tumor models as well as in human tumor xenografts in nude mice. Differences in the spectra of antitumor activity have been noted but it appears that the predictive value for clinical use remains uncertain. Both drugs, DOX and EPI, showed activity against breast carcinoma, small cell lung cancer, and sarcoma and were not active in colon tumors [13]. In non-small cell lung cancer the in vivo results showed activity in three quarters of tumors transplanted into nude mice with both anthracyclines, a result which does not correlate with clinical results. The same holds true for melanoma. For this reason in vivo evaluations in a large panel of human tumors in nude mice can only give a first indication for future clinical development. There is clearly a limitation of tumor in vivo models which do not reflect correctly the tumor biology in humans, e.g., host-tumor interactions in man are not addressed sufficiently in the available models.

For IDA it was shown that this drug has a 4-to-8-fold greater potency than DOX and DNR in leukemias and lymphomas [14]. The evaluation of the antitumor activity of IDA in solid tumors is limited to only a few orthotopic murine tumor models including mammary carcinoma and sarcoma and to human tumor xenografts in nude mice: i.e., breast, lung, melanoma, ovarian and sarcoma. In these in vivo models, IDA and DNR showed similar activity. Idarubicinol demonstrated antitumor activity equivalent to that of IDA [15].

Mode of action and molecular biology

The precise mechanism of antitumor action for the anthracyclines is not fully understood. The following chapter summarizes the proposed modes of action of anthracyclines.

Drug-cell membrane interactions

Each drug which is administered iv or po is present with a certain concentration in the central compartment where the amount can be determined (see section pharmacokinetics). To enter the tumor cell, the anthracycline must leave the blood vessels, enter the interstitial tissue und penetrate and cross the cell membrane in order to reach the inner compartment of the cell. The transmembrane movement of the anthracyclines occurs by free diffusion of the non-ionized drug [16]. No active drug carrier is known for the anthracyclines. The daunosamine sugar is partly protonated within the physiologic pH range and therefore both extracellular and intracellular pH has a significant impact on tumor cell uptake of anthracyclines [17]. The uptake of anthracyclines from the extracellular space into the tumor cell is hampered by a pH of 6-6.5 which is often found in tumor masses as small as 1 cm because a protonated anthra-cycline cannot rapidly diffuse through the cell membrane. If the pH is in the physiologically range in the extracellular space, the anthracycline can cross the cell membrane very easily as non-ionized drug and is then trapped in the cyto-plasma/nucleus of the tumor cell by intracellular acidosis as well as rapid binding to intracellular components such as DNA. Interestingly, two other phe-nomenons with respect to drug-cell membrane interactions are noteworthy. Several tumor cells as well as normal cells feature an efflux pump system, with which several natural products are efficiently pumped out of the cell. This protein, called P170-glycoprotein, is integrated into the cell membrane and has an ATP-binding site in the cell and is an important drug carrier system (from inside to outside) and has been widely discussed as one of the reasons for anthracycline resistance [18]. The second phenomenon is the fact that even anthracyclines which cannot cross the tumor cell membrane show cytotoxic activity. Doxorubicin was covalently coupled to large agarose beads which were unable to enter cells but still exerted strong antitumor effects in cell culture systems. Within this model the antitumor effects are produced at the cell membrane level and could be explained by the generation of reactive oxygen species (ROS) at the cell membrane, which in turn damage the membrane by lipid peroxidation thereby activating important signalling pathways [20]. A semiquinone free radical that is produced by daunorubicin incorporated into the cellular membrane of intact cells has been described [21].

Drug-DNA intercalation

Cytotoxicity mediated by anthracyclines is generally thought to be the result of drug-induced damage to the DNA. Because the drug concentrates in the cell nucleus and is a good intercalator of DNA [22], the drug was thought to exert its activity by DNA intercalation, but this simple explanation is not sufficient to explain the whole spectra of different actions of the anthracyclines. The planar aglycon (without the daunosamine sugar) intercalates with DNA as well, but no antitumor activity was found [23]. The intercalation of anthracyclines with DNA is reversible, no covalent binding is necessary. Hydrophobic interactions, hydrogen bonds to the phosphate groups of the DNA and the insertion of the daunosamine sugar into the small groove of the DNA with an affinity to the CpG-complex and transcriptional active sites of the DNA lead to a fixed drug-DNA-complex with a long half-life [24].

Drug-topoisomerase-II interaction

It has been shown that anthracyclines cause protein-associated breaks and these breaks correlate with cytotoxicity [25]. The reason for these protein-associated breaks are due to fine interactions of the anthracyclines with the topoisomerase-II (TOPO-II), an enzyme that promotes DNA strand breaks and is involved in resealing the breaks [26]. It is possible that the intercalation of anthracyclines induce an alteration in the three-dimensional conformation of DNA that arrests the cycle of TOPO-II action at the point of DNA cleavage, but it may well be that anthracyclines also stimulate TOPO-II-mediated DNA cleavage by nonintercalative mechanisms. A number of studies have shown that anthracyclines induce topoisomerase-II-mediated DNA damage at drug concentrations that are clinically relevant. Furthermore, a good correlation between cytotoxicity and DNA damage was observed. Cell lines which have altered TOPO-II activity exhibit resistance to anthracyclines [27]. Other TOPO-II inhibitors such as VP-16 showed a relative constant relationship between cytotoxicity and protein-associated DNA break frequency, and the anthracyclines exhibits more cytotoxicity per break. Therefore, the interaction of anthracyclines with TOPO-II is an important factor for the cytotoxicity but other mechanisms of action might be important as well. With respect to DNA intercalation and inhibition of topoisomerase-II, the anthracyclines act as chemically inert compounds by their ability to distort the three-dimensional geometry of the targets DNA and TOPO-II. Despite these important modes of actions induced by the unchanged drug, the anthracyclines are chemically very reactive compounds with an extraordinary and fantastic chemistry, not understood in all details yet [8, 28].

One- and two-electron reduction

Free radical formation after anthracycline administration is a major issue for understanding some of the side effects of this class of drugs. The one-electron reduction is crucial for cardiac toxicity. All anthracyclines in clinical use are anthraquinones that can undergo a one- and two-electron reduction to reactive compounds that are able to damage DNA and cell membranes (under certain conditions) [29]. In complex biological systems these reactions are catalyzed by enzymes. Several enzyme systems accept anthracyclines as substrates for a one-electron reduction: NADPH-cytochrome-P-450-reductase in the endo-plasmatic reticulum, NADH-dehydrogenase in the mitochondria, xanthinoxi-dase in the cytoplasma and not identified enzymes in the nucleus. Figure 2 depicts the reaction cascade of this electron transfer.

The one-electron reduction leads to the formation of the semi-quinone free radical which in the presence of oxygen donates its electron to oxygen thus generating a superoxide anion. At neutral pH the main reaction of the super-

Anthracycline Semiquinone

Figure 2. Free radical formation pathway for doxorubicin.

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