Introduction

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In 1966, Wall et al. (1966) discovered that camptothecin (CPT; Fig. 1) was the component in the extract from the stem of the Chinese tree Camptotheca acuminata active against L1210 murine leukemia cells. Early clinical trials with the sodium salt of CPT in the late 1960s showed that this plant alkaloid had activity against a variety of solid tumors (Gottlieb et al., 1970; Creaven et al., 1972; Muggia et al., 1972). However, further clinical trials were discontinued because of unpredictable and severe myelosuppression, gastrointestinal toxicity, and hemorrhagic cystitis.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The molecular structure of the camptothecin analogs used in these studies. CPT (top): R1 = H, R2 = H. C1CPT through C4CPT, R1 = H, R2 = methyl, ethyl, propyl, or butyl. OHCPT, R1 = OH, R2 = H. OHC1CPT through OHC4CPT, R1 = OH, R2 = methyl, ethyl, propyl, or butyl. OMeCPT, R1 = OCH3, R2 = H. OMeC1CPT through OMeC4CPT, R1 = OHC3, R2 = methyl, ethyl, propyl, or butyl. MDCPT (bottom), R2 = H. MDC1CPT through MDC4CPT, R2 = methyl, ethyl, propyl, or butyl.

Renewed interest in CPT was stimulated by the characterization of CPT as a specific topoisomerase (topo) I inhibitor (Hsiang et al., 1985, 1989). Topo I relaxes DNA supercoiling by making transient single-strand breaks (Champoux, 1990; Wang, 1991). These breaks are coupled with the transient formation of a covalent DNA-enzyme intermediate termed the cleavable complex (Hsiang et al., 1985, 1989). CPT and analogs specifically and reversibly stabilize cleavable complexes by inhibiting their religation [reviewed in Chen and Liu (1994) and Pommier et al. (1998)]. The mechanism of CPT cytotoxicity is thought to be the consequence of a collision between moving replication forks and CPT-stabilized cleavable DNA-topo I complexes (Holm et al., 1989; Hsiang et al., 1989; Pommier et al., 1994).

There are now several topo I inhibitors at various stages of clinical development. One of these, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-CPT (CPT-11; irinotecan), has recently been approved by the U.S. Food and Drug Administration for the second-line treatment of metastatic colorectal cancer. CPT-11 is a pro-drug and is converted to the active 7-ethyl-10-hydroxy-CPT (OHC2CPT; SN-38) by carboxylesterases to exert its antitumor activity (Kunimoto et al., 1987; Kawato et al., 1991).

The potent activity of OHC2CPT has led to the development of a number of 7- and 10-substituted camptothecins (Jaxel et al., 1989; Wall et al., 1993; Emerson et al., 1995; Luzzio et al., 1995; Valenti et al., 1997). These include the highly potent 10,11-methylenedioxy-CPT (MDCPT) and 7-chloromethyl-10,11-methylenedioxy-CPT (CMMD; Fig. 1). The latter compound is of particular interest because it is capable of forming a covalent complex with DNA through nucleophilic displacement of the chlorine moiety by DNA while in the cleavable complex (Pommier et al., 1995; Valenti et al., 1997).

It has been suggested that the property of the 7- and 10-substituted analogs thought to be most relevant to their potent antitumor activity is the slow reversal of the cleavable complexes formed with these drugs (Tanizawa et al., 1994; Tanizawa et al., 1995; Valenti et al., 1997). Compared with CPT, the 10-hydroxy-CPT (OHCPT), OHC2CPT, MDCPT, and CMMD all seemed to have a longer-lived cleavable complex. These compounds are all substantially more cytotoxic than CPT; therefore, it has been postulated that, because CPT toxicity is a time-dependent phenomenon, the persistence of cleavable complexes may be an essential property for increasing the likelihood of a collision between the replication fork and a cleavable complex, giving rise to lethal DNA lesions (Tanizawa et al., 1995; Valenti et al., 1997).

To understand the relationship between CPT substitution, cytotoxicity, and cleavable complex reversibility, we have systematically synthesized and evaluated a series of 7-alkyl-CPT, 7-alkyl-10-hydroxy-CPT, 7-alkyl-10-methoxy-CPT, and 7-alkyl-10,11-methylenedioxy-CPT that have incremental lengths in the 7-alkyl chains. These analogs have been characterized with respect to: 1) their ability to induce topo I-mediated cleavage of plasmid DNA and DNA-protein crosslinks in CEM cell nuclei; 2) the reversibility of the cleavable complexes formed with plasmid DNA and in CEM cell nuclei; and 3) the growth inhibitory activity of the analogs to selected cancer cell lines. Our results indicate that the potency of the 7- and 10-substituted analogs does not reflect the lifetime for reversal of the cleavable complex formed with these compoundseven using analogs capable of forming covalent complexes with DNA. We demonstrate that rate constants for reversal of cleavable complexes are similar among the analogs tested. Instead, the in vitro biological activity of the drugs seems to more closely parallel the concentration of drug required to produce cleavable complexes in plasmid DNA under steady-state conditions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemistry. The results for the CPT analogs are given in Table 1 and their structures given in Fig. 1. All CPT analogs used were the synthetic 20(S)-stereoisomer. CPT, OHCPT, and 10-methoxy-CPT (OMeCPT) were obtained as pure natural products (Wall et al., 1966; Wani and Wall, 1969). 7-Alkyl-CPT (C1CPT through C4CPT) were synthesized according to the method of Sawada et al. (1991). 7-Alkyl-OHCPT analogs (OHC1CPT through OHC4CPT) were synthesized from OHCPT by the procedure of Sawada et al. (1995). The 10-methoxy analogs OMeC1CPT through OMeC4CPT were synthesized according to the procedure for the synthesis of 10-methoxycamptothecin (Wani et al., 1980). The synthetic scheme for the preparation of MDCPT, MDC1CPT through MDC4CPT, and CMMD has also been reported previously (Wall et al., 1993; Luzzio et al., 1995). See the abbreviation list for definitions of these CPT analogs.

In Vitro Growth Inhibitory Activity. Human breast carcinoma lines MDA-231 and BT-20 were maintained in improved minimal essential media supplemented with 10% fetal bovine serum at 37°C in 5% CO₂ incubators until plated for use in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. The human cervical carcinoma line HeLa adapted for growth in serum-free media (HeLa/SF) was obtained from ATCC (Manassas, VA). The HeLa/SF cells were maintained in RPMI-1640 supplemented with the proprietary serum replacement TCH (Celox Laboratories, Hopkins, MN) as directed by ATCC. The total protein in the TCH-supplemented growth media was <0.1%.

Cleavable Complex Formation by CPT Analogs in Plasmid DNA. CPT analog-induced cleavable complex formation was performed in 10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol. A 250-ng sample of pBR322 plasmid DNA (Life Technologies, Gaithersburg, MD) was mixed with the drug of interest, and 4 U of human topoisomerase I enzyme (Topogen, Inc., Columbus, OH) was subsequently added to a 20-µl total mixture volume. Reaction mixtures were assembled on ice.

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Reversal of Cleavable Complex Formation in Plasmid DNA. Reversal of the topo I cleavage activity of the pBR322 plasmid DNA was accomplished by the method of Hertzberg et al. (1989). Cleavable complexes were formed in the presence of sufficient analog to induce >90% nicked DNA (as determined from EC₅₀ curves). The reaction protocol consisted in the preparation of one 75-µl reaction mixture constructed as described above. The reaction mixture was incubated for 30 min at 23°C. A 100-fold excess of sonicated salmon sperm linear DNA (10 mg/ml; Life Technologies) was added to the reaction mixture. Aliquots were then removed at 0.5-, 1-, 2-, 5-, 10-, 15-, 30-, 45-, and 60-min intervals. The reaction was stopped by the addition of 11 µl of water, 2 µl of 10% SDS, and 2 µl of Proteinase K, then incubated at 23°C for another 30 min. The resulting samples were further mixed with loading solution and analyzed using 1% agarose gels, as above. The percentage of cleaved DNA with respect to time was determined based on the amount of Form I (nicked) DNA present in the 0.5-s lane.

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DNA-Protein Crosslinks (DPC) in Isolated Nuclei. Trapping of the cleavable complexes of endogenous topoisomerase I in nuclei were assessed based on DPC induction measured by a standard K⁺/dodecyl sulfate precipitation technique (McHugh et al., 1989; Woynarowski et al., 1994). Briefly, human leukemia CEM cells, grown in Jokklik's minimal essential medium with 10% fetal calf serum were prelabeled with [¹⁴C]thymidine as described elsewhere (Woynarowski et al., 1997). Cells were lysed in isotonic Nuclei Isolation Buffer (2 mM KH₂PO₄, 5 mM MgCl₂, 150 mM NaCl, 1 mM EGTA, pH 6.9) with 0.3% (v/v) Triton X-100 followed by centrifugation (300g for 13 min) and resuspension in the isolation buffer at 0.5 × 10⁶ nuclei/ml. Aliquots of nuclear suspension were treated with drugs as indicated. In the reversal experiments, samples were centrifuged (500g for 5 min), the nuclear pellets resuspended in a fresh nuclei isolation buffer followed by the additional incubation as indicated. All the reactions were terminated by addition of equal volume of 3% SDS, 40 mM EDTA, and 0.4 mg/ml DNA, pH 8.0, preheated to 65°C. Further steps were performed exactly as described elsewhere (McHugh et al., 1989; Woynarowski et al., 1994). Separate aliquots of nuclear suspension were used to determine total radioactivity in each individual suspension (McHugh et al., 1989; Woynarowski et al., 1994). The results are expressed as percentage of total DNA that coprecipitated with proteins corrected for the background precipitation in control samples. The latter value typically amounted to 2 to 5% of total radioactivity. For the reversal experiments, the results are normalized for the DPC levels at the end of drug treatment.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Growth Inhibition by Camptothecin Analogs. The IC₅₀ values for growth inhibition of the synthesized compounds are given in Table 1. The CPT analogs were examined for their ability to inhibit growth of two breast carcinoma cell lines, BT-20 and MDA-231. Because of concerns about the partitioning of CPT analogs onto serum proteins and the effect of this phenomenon on availability of the compounds in solution, growth inhibition was also determined using a human ovarian carcinoma cell, HeLa/SF, line grown under serum-free conditions.

Induction of Cleavable Complexes by Camptothecin Analogs. Similar trends seen in the growth inhibition assays were also seen with the ability of the CPT analogs to act as topo I poisons. All compounds examined exhibited a simple hyperbolic dose response in their ability to induce cleavable complexes (Fig. 2A), and this response that could be fitted well by a simple E_max model (eq. 1). The EC₅₀ values obtained from the curve fits, such as that shown for OHCPT in Fig. 2A, are recorded in Table 1 for each compound tested.

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Induction of cleavable complexes and their reversal by 10-hydroxycamptothecin. A, concentration dependence of cleavable complex formation. Human topo I and pBR322 supercoiled plasmid DNA were incubated with 10-hydroxycamptothecin at concentrations of 50, 10, 5, 1, 0.7, 0.5, 0.3, 0.1, and 0 µM concentrations (lanes 1-9, respectively). Lane 10 contained no drug or topo I. Form I (supercoiled) and Form IR (relaxed, closed circular) DNA are indicated by F I and F IR, respectively. The upper DNA band (nicked; Form II or F II) was quantified and expressed as percentage of total DNA after subtracting the amount of nicked DNA observed in the absence of drugs or topo I (i.e., lane 10). The solid curve is to the EC50 equation (eq. 1 in the text). The EC50 values determined in this manner for all analogs are recorded in Table 1. B, time course for reversal of cleavable complexes formed with 10-hydroxycamptothecin (OHCPT). Complexes were formed with 20 µM OHCPT, and then 100-fold linear salmon sperm DNA was added. Aliquots were withdrawn before DNA addition (lane 1) and 0.5, 1, 2, 5, 10, 15, 30, 45, and 60 min after DNA addition (lanes 2-10, respectively). Nicked DNA (Form II) was quantified and plotted versus time, with the solid line being the fit to eq. 4 in the text. The rate constants, , were determined for all analogs by this method, and are given in Table 2.

Induction of Protein-DNA Crosslinks in CEM Cell Nuclei. To extend the findings with purified enzyme and plasmid DNA, we examined the effects of selected CPT analogs (CPT, MDCPT, MDC2CPT, and CMMD) in isolated human leukemic CEM cell nuclei. Drug-induced trapping of the cleavable complexes in nuclei was assessed based on induction of DNA-protein crosslinks (DPC). This system allows one to monitor drug effects on endogenous topo I on its natural targetnuclear chromatin (McHugh et al., 1989).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Time course for DNA-protein crosslinks induced in CEM nuclei by CPT analogs. CPT at 1 µM () is shown and compared with 0.1 µM CMMD (). Crosslinks induced by MDCPT () and MDC2CPT () at 30 min are shown for comparison. Error bars are positive for filled symbols and negative for open symbols.

Reversal of Cleavable Complexes Formed with CPT Analogs on Plasmid DNA. We used the competitive DNA approach (Hertzberg et al., 1989) to measuring the religation of DNA single-strand breaks on plasmid DNA. An example of the data obtained for OHCPT and its analysis is given in Fig. 2B. The assumption of this method is that once topo I is dissociated from the cleavable complex, the excess of linear DNA competes for the topo I and inhibits it from reattaching to the same or other plasmids (eqs. 2 and 3). Hence, the process should be a pseudo first-order dissociation of topo I from the cleavable complex, and therefore well described by a single exponential decay. This approach characterizes the stability of the complex induced by the drug, because drug dissociation is the limiting step in the process [i.e., religation in the absence of drug is rapid (Wang et al., 1998)]. Other groups have used salt-induced denaturation of the DNA to produce similar decay curves (Tanizawa et al., 1994, 1995; Pommier et al., 1995; Valenti et al., 1997; Wang et al., 1998), where the salt acts to affect the equilibria given in eq. 2 by decreasing k₁₂. However, because of the unknown solubilities of the hydrophobic CPT analogs in 0.35 to 0.5 M NaCl, we chose to keep the ionic strength constant and use competitive DNA. We show below that the data obtained here is consistent with that obtained using the salt-induce release of cleavable complexes.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Kinetics of competitive DNA-induced religation of cleavable complexes formed on plasmid DNA with human topoisomerase I and CPT analogs. The rate constants for the disappearance of cleavable complexes for all of the compounds are given in Table 2. In all panels, error bars are negative for filled symbols and positive for open symbols. A, CPT () and the 7-alkyl CPT analogs C1CPT (), C2CPT (), C3CPT (), and C4CPT (). The solid line is for the decay in the presence of CPT. B, 10-Hydroxy-CPT OHCPT (), and the 7-alkyl-10-hydroxy-CPT analogs OHC1CPT (), OHC2CPT (), OHC3CPT (), and OHC4CPT (). The solid line is for the decay of OHCPT and the dashed line is for OHC2CPT. C, 10-Methoxy CPT OMeCPT () and the 7-alkyl-10-methoxy CPT analogs OMeC1CPT (), OMeC2CPT (), OMeC3CPT (), and OMeC4CPT (). The solid line is for the decay of OMeCPT, whereas the dashed line is for OMeC2CPT. D, 10,11-Methylenedioxy CPT analog MDCPT (), and the 7-alkyl-10,11-methylenedioxy-CPT analogs MDC1CPT (), MDC2CPT (), MDC3CPT (), and MDC4CPT (). The dashed line is for MDC2CPT.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Comparison of cleavable complex reversal on plasmid DNA with reversal of DNA-protein crosslinks in CEM cell nuclei. A, reversal of cleavable complexes formed on pBR322 plasmids with CPT (), MDCPT (), CMMD (), and MDC2CPT (). B, reversal of DNA-protein crosslinks induced by the drugs in CEM cell nuclei (same symbols as in A). Rate constants for reversal of DNA-protein crosslinks with the four compounds shown are given in Table 2.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Persistence of cleavable complexes on plasmid DNA after addition of competitive DNA. Data are shown for CPT (), OHC2CPT (), MDCPT (), MDC2CPT (), and CMMD (). Note that although the percentage of cleavable complexes remaining after 1 h is markedly different, the rate constants for the complex reversal process are similar for all the analogs examined (Table 2).

Reversal of Protein-DNA Crosslinks in CEM Cell Nuclei. A comparison of reversal data from plasmid DNA versus cell nuclei is given in Fig. 5 for selected CPT analogs. These data are not normalized to the final concentration of cleavable complexes to indicate the relative amplitudes for complex decay. CMMD, the MDCPT analog with the potential for alkylation of DNA, shows an initially rapid, exponential reversal in plasmid DNA (Fig. 5A). However, only approximately 40% of the CMMD complexes religate during this decay compared with almost complete reversal by CPT. This reduced reversal for CMMD may not be caused by covalent linkage of the drug, because the data for the nonalkylating analog MDC2CPT reveal a slower relaxation of the complexes formed by this drugeven slower than those observed for CMMD (Fig. 5A). MDC2CPT does not seem to alkylate DNA (Pommier et al., 1995; Valenti et al., 1997).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The evaluation of the CPT analogs reported here has been performed to advance the development of "third generation" CPT analogs, with topo I poisoning activities higher than topotecan (TPT), OHC2CPT, or MDCPT. Recent reports (Tanizawa et al., 1995; Valenti et al., 1997) have suggested that CPT analogs with greater potency than CPT or TPT in forming cleavable complexes might produce this enhanced activity by forming complexes that are slow to reverse. Such persistent complexes would be more likely to be present during replication of a given DNA segment and would therefore be more toxic to cells. Thus, structural features of CPT analogs lending themselves to slowly-reversing cleavable complexes are important for generating improved CPT analogs with activities that might surpass CPT-11 and TPT much as CPT-11 and TPT surpassed the parent CPT.

In this report, we examined 7-alkyl- and 7-alkyl-10-substituted CPT analogs for their mechanism of in vitro topo I poisoning and their ability to inhibit tumor growth in cell culture. Our data indicate that all 7- or 7, 10-substituted CPT analogs are more potent than CPT in inducing topo I-mediated cleavable complexes with purified enzyme and plasmid DNA (Table 1). The MDCPT analogs of CPT are among the most potent compounds known for poisoning topo I. However, the kinetic stability of the ternary complexes is independent of the potency of the compounds to both poison topo I on plasmid DNA or inhibit cell growth (compare Tables 1 and 2). CPT and its 7-alkyl derivatives show rapid, exponential reversibility of the complexes formed (Fig. 4A), despite the much higher potency of the 7-alkyl derivatives to poison topo I and to inhibit tumor cell growth. Similar results were observed for the OHCPT, OMeCPT, and MDCPT analogs of CPT: no correlation between the potency of the drug to trap topo I cleavable complexes or inhibit cell growth and its ability to kinetically stabilize cleavable complexes. Our work is in agreement with a recent paper on 9,10-disubstituted CPT analogs by Hecht and colleagues (Wang et al., 1998). Using salt-induced religation and dissociation of topo I from a defined oligonucleotide, these authors also found no correlation between the potency of these analogs in poisoning topo I and their ability to kinetically stabilize cleavable complexes.

Our results, and those of Hecht and colleagues (Wang et al., 1998), point out a critical facet of topo I-DNA poisoning that may not be readily apparent when considering "longer-lived" cleavable complexes. In Fig. 6, we have recast data from Fig. 4 on a semilog plot using the CPT analogs and symbols taken from Fig. 3B of (Valenti et al., 1997). Comparison of our data, using competitive DNA, and their data, using salt-induced topo I dissociation, indicates that the data are remarkably similar with regard to the "stability" of the cleavable complexes. That is, in both of our studies, after a 1-h period, more complexes exist when MDC2CPT and CMMD are used; these in turn are greater in number than MDCPT- and OHC2CPT- induced complexes, with CPT-induced complexes even lower. However, note from Table 2 that the rate constants  for all of these compounds are similar, and the  value for OHC2CPT is identical with that for CPT. Hence, the "stability" of the complexes, as defined by the kinetic rate constants, are not correlated with the potency of the analogs in poisoning topo I or inhibiting cell growth. It should be acknowledged that our data represent average topo I cleavage sites on plasmid DNA. The possibility that selected topo I-binding DNA sequences have different kinetic behavior with more potent CPT analogs cannot be entirely ruled out.

Both the competitive DNA method used here and the salt-induced religation methods used elsewhere are examples of chemical relaxation experiments, where a steady-state is rapidly perturbed by addition of some component, and the system under study relaxes to a new steady-state [these methods are exhaustively reviewed in (Gutfreund, 1995)]. The concentration difference in components between the new steady state and the old gives rise to the amplitude of the perturbation (i.e., A in eq. 4). The decay time is given by . Hence, the highly potent CPT analogs may have a smaller A, because they poison topo I at very low concentrations, but generate cleavable complexes with identical kinetic "stabilities." In both the pBR322 cleavage assays and the cell growth assays, we are examining steady-state situations (i.e., constant amounts of drug present). Hence, it is not surprising that, in these assays, the potency of the drug in inhibiting cell growth more closely relates to the potency in poisoning topo I on plasmid DNA. In both situations, there is little or no dependence on kinetic parameters of the drug-DNA-topo I ternary complex. It should be noted, however, that although the EC₅₀ values for topo I poisoning are more reflective of the in vitro growth inhibition IC₅₀ values by the CPT analogs (that is compounds with lower EC₅₀ values also had lower IC₅₀ values), the actual correlation coefficient for these two parameters is low (r = 0.5 for BT-20 cells) or nonexistent (r = 0.1 for MDA-231 cells). This is most likely caused by differences in accumulation of the analogs within cells. In any event, our data with purified topo I and plasmid DNA, as well as isolated cell nuclei, suggest that the reason that OHCPT and MDCPT analogs are more active than CPT in inhibiting cell growth is that these compounds are highly potent under steady-state conditions. That is, at equivalent concentrations, the OHCPT and MDCPT analogs generate more cleavable complexes than CPT and OMeCPT analogs, not more stable cleavable complexes.

In contrast to the usual topo I poisoning assays and cell-growth inhibition assays described above, the clinical use of CPT analogs does involve a kinetic component. During a typical administration of CPT-11 or TPT, the blood concentration of OHC2CPT or TPT will rise then fall with time. Because cell kill in S-phase is also time-dependent (i.e., it requires collision of the replication fork with topo I-DNA complexes), clearly CPT analogs that form slow decaying ternary complexes might be desirable to maximize cancer cell killing. As suggested recently (Tanizawa et al., 1994, 1995; Valenti et al., 1997), development of a new generation of CPT analogs with antitumor activities greater than CPT-11 or TPT may include drugs designed to stabilize cleavable complexes to a much greater extent than the 7- and 10-substituted analogs examined here. Ideally, compounds such as the CMMD analog, which can alkylate DNA, may be most useful in forming stabile, irreversible cleavable complexes. However, only a small fraction of the available CMMD has been shown to covalently bind to DNA (Tanizawa et al., 1995; Valenti et al., 1997) and therefore these complexes rapidly reverse (Fig. 5). Hence, new, more reactive alkylating CPT analogs may prove more effective in their cytotoxic activity. We anticipate that the data presented here should aid in development of such compounds.