Synthesis and cytotoxicity of water soluble quaternary salt derivatives of camptothecin

In an effort to improve the water solubility of camptothecin, 16 water soluble 10-substituted quaternary ammonium salt derivatives of camptothecin were prepared. Their antitumor activity was evaluated on cancer cells in vitro. All of these salts possess lower cytotoxicities than CPT in comparison. The camptothecin salts 16, 20 showed similar cytotoxic activity to topotecan. Especially the salts 21 showed similar cytotoxic activity to CPT in vitro.     

Synthesis of new camptothecin analogs with improved antitumor activities

Novel hexacyclic camptothecin analogs containing cyclic amidine, urea, or thiourea moiety were designed and synthesized based on the proposed 3D-structure of the topoisomerase I (Topo I)/DNA/camptothecin ternary complex. The analogs were prepared from 9-nitrocamptothecin via 7,9-diaminocamptothecin derivatives as a key intermediate. Among them, 7c exhibited in vivo antitumor activities superior to CPT-11 in human cancer xenograft models in mice at their maximum tolerated doses though its in vitro antiproliferative activity was comparable to SN-38 against corresponding cell lines.     

Synthesis and antitumor activity of novel 10-substituted camptothecin analogues

In an attempt to improve the antitumor activity and decrease the cytotoxicity of camptothecin, 18 new 10-substituted camptothecin derivatives were prepared. The cytotoxicity in vitro on cancer cell lines and antitumor activity in vivo, and inhibitory properties of topoisomerase I of these derivatives were evaluated. Most of these derivatives possessed lower cytotoxicities than CPT, and the compounds 13, 21, 22, 23, and 24 showed similar topoisomerase I inhibitory activity to CPT. Analogues 13 exhibited the best antitumor activity in vivo among all derivatives we prepared.     

Synthesis and antitumor activity of novel 20s-camptothecin analogues

In an effort to decrease the toxicity and improve the stability of labile lactone ring of camptothecin, nitrogenous heterocyclic aromatic groups were introduced into 20-position of camptothecin and seventeen new 20s-camptothecin derivatives were obtained in quantitative yield. The cytotoxicity in vitro on three cancer cell lines and the stability of the lactone in phosphate-buffered solution (PBS) of these derivatives were evaluated. Most of these tested derivatives possessed better cytotoxicity than topotecan. Analogues 6, 12 exhibited the best antitumor activity in vivo in all derivatives we prepared. The results suggested that introduction of pyrazole in 10- or 20-position of camptothecin could promote antitumor activity in vitro and in vivo, simultaneously bring much increase of the stability of lactone.     

A novel method of eliminating non-neuronal proliferating cells from cultures of mouse dorsal root ganglia

1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin) during culture.2. Exposure to 20 M camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells.3. Following camptothecin treatment, neurons survived and continued to extend neurites for several weeks without obvious defects in morphology or viability.4. Transient camptothecin exposure is therefore an efficient and fast-acting method to purify DRG neurons in culture.     

Novel camptothecin derivatives. Part 1: oxyalkanoic acid esters of camptothecin and their in vitro and in vivo antitumor activity

A series of oxyalkanoic acid esters of (20S)-camptothecin derivatives was prepared by the method of acylation. Their antitumor activity was evaluated on cancer cells in vitro by the colony formation assay and in vivo. These newly synthesized derivatives show a dramatically higher chemotherapeutic activity in killing human cancer cells than the parent drug, camptothecin, and clinically available drugs, irinotecan and taxol.     

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

Induction of cleavage in topoisomerase I c-DNA by topoisomerase I enzymes from calf thymus and wheat germ in the presence and absence of camptothecin.

In this study, we further examined the sequence selectivity of camptothecin in mammalian topoisomerase I cDNA from human and Chinese hamster. In the absence of camptothecin, almost all the bases at the 3'-terminus of cleavage sites are T for calf thymus and wheat germ topoisomerase I. In addition, wheat germ topoisomerase I exhibits preference for C (or not T) at -3 and for T at -2 position. As for camptothecin-stimulated cleavage with topoisomerase I, G (or not T) at +1 is an additional strong preference. This sequence selectivity of camptothecin is similar to that previously found in SV40 DNA, suggesting that camptothecin preferentially interacts with topoisomerase I-mediated cleavage sites where G is the base at the 5'-terminus. These results support the stacking model of camptothecin (Jaxel et al. (1991) J. Biol. Chem. 266, 20418-20423). Comparison of calf thymus and wheat germ topoisomerase I-mediated cleavage sites in the presence of camptothecin shows that many major cleavage sites are similar. However, the relative intensities are often different. One of the differences was attributable to a bias at position -3 where calf thymus topoisomerase I prefers G and wheat germ topoisomerase I prefers C. This difference may explain the unique patterns of cleavage sites induced by the two enzymes. Sequencing analysis of camptothecin-stimulated cleavage sites in the surrounding regions of point mutations in topoisomerase I cDNA, which were found in camptothecin-resistant cell lines, reveals no direct relationship between DNA cleavage sites in vitro and mutation sites.     

Analysis of stereoelectronic properties of camptothecin analogues in relation to biological activity

Camptothecin and four of its 10,11-methylenedioxy analogues were examined for their activity against the pathogenic protozoan Leishmania donovani in vitro. The methylenedioxy analogues were 36- to 180-fold more potent than the parent camptothecin, possessing IC50 values ranging from 160 to 32 nM against the parasite. Our finding that the methylenedioxy camptothecins possess greater activity than camptothecin, which is also the case for other cell types and for the generation of cleavable complex in the presence of DNA and purified mammalian topoisomerase I, prompted us to examine the molecular features of camptothecin and methylenedioxy camptothecin analogues. A delocalization of positive potential was observed in the methylenedioxy camptothecin analogues, which could increase the affinity of these molecules for DNA. In addition, geometrical and electronic differences between the E ring of camptothecin and its methylenedioxy analogues were noted. One or both of these factors may contribute to the superior biological activity of the methylenedioxy camptothecin analogues.     

Synthesis and biological assays of E-ring analogs of camptothecin and homocamptothecin

Analogs of the anti-tumor agent camptothecin with both closed E-rings (lactone and ether) and open E-rings (reduced acid, hydrazide, and protected Weinreb amide) have been prepared and tested in topoisomerase and cellular assays. The results provide insights into the structural features of the camptothecin E-ring that affect biological activity.     

Oxidative stimuli-responsive nanoprodrug of camptothecin kills glioblastoma cells

The purpose of this study was to prepare and characterize nanometer-sized prodrug (nanoprodrug) of camptothecin. The camptothecin prodrug was synthesized using tetraethylene glycol spacer linked via carbonate bond to camptothecin and via ester bond to α-lipoic acid. The nanoprodrug was prepared through the spontaneous emulsification mechanism using the mixture of camptothecin prodrug and α-tocopherol which served as a structural matrix. The nanoprodrug was activated readily by porcine liver esterase and, with a much slower rate, by hydrolytic degradation. Upon longterm storage, the α-lipoic acid moiety of the camptothecin prodrug gradually oxidized without loss of structural integrity and therapeutic efficacy. Interestingly, the hydrolytic activation was negligible before the oxidation, but was significantly accelerated after the oxidation of the α-lipoic acid moiety, suggesting an oxidative stimuli-responsive activation of the prodrug. The camptothecin nanoprodrug was found to possess significant inhibitory effect on the proliferation of U87-MG glioma cells with an IC₅₀ of 20 nM.     

4-(Aminoalkylamino)-3-benzimidazole-quinolinones as potent CHK-1 inhibitors

CHK-1 is one of the key enzymes regulating checkpoints in cellular growth cycles. Novel 4-(amino-alkylamino)-3-benzimidazole-quinolinones were prepared and assayed for their ability to inhibit CHK-1. These compounds are potent cell permeable CHK-1 inhibitors and showed synergistic effect with a DNA-damaging agent, camptothecin.     

Antitumor agents 216. Synthesis and evaluation of paclitaxel-camptothecin conjugates as novel cytotoxic agents

Five conjugates (16-20) composed of a paclitaxel and a camptothecin derivative joined by an imine linkage were synthesized and evaluated as cytotoxic agents and as inhibitors of DNA topoisomerase I. All of the conjugates were potent inhibitors of tumor cell replication with improved activity relative to camptothecin. Significantly, compounds 16-18 were more active than paclitaxel and camptothecin against HCT-8 (colon adenocarcinoma) cell replication, and the spectrum of activity was different from a simple mixture of paclitaxel and camptothecin. All of the conjugates were significantly less potent than camptothecin as inhibitors of human topoisomerase I in vitro with 16, 18, and 19 showing only marginal activity at 50 microM. Based on activity against drug-resistant cell line replication, one could conclude that the conjugates are simply acting as 'weak taxanes', but the spectrum of activity, particularly against MCF-7 and HCT-8, strongly suggests that a novel mechanism of action has been achieved through conjugation.     

Analysis of resonance chemotherapy in leukemia treatment via multi-staged population balance models

An age-structured population balance model that explicitly models cell cycle phases is developed to investigate the effects of cell cycle specific (CCS) drugs. In particular, the benefits of timing CCS drug treatments in resonance chemotherapy are predicted and measured directly in vitro before evaluating likely in vivo scenarios. The phase transition rates are measured in vitro for the HL60 leukemia cell line and are used to predict the transient phase dynamics after exposure to the S phase specific drug, camptothecin. The phase oscillations predicted by the model are observed experimentally and the timing of a second camptothecin pulse is shown to significantly alter the overall treatment effectiveness. To explore the feasibility of designing resonance chemotherapeutic treatments to preferentially eliminate one cell type over another, Jurkat and HL60 leukemia cells are exposed to the same dual-pulse camptothecin treatment regimen. With the model framework validated for simplified cases, the model is used to extrapolate the effectiveness of resonance chemotherapy considering in vivo effects such as quiescence, drug metabolism, drug properties, and transport considerations that were not included in the in vitro experiments. While resonance chemotherapy is intuitive and looks promising in vitro, when in vivo considerations are included in the model, the phenomenon is dampened and the window of applicability becomes narrower.     

Synthesis and antitumor activity of 7-ethyl-9-alkyl derivatives of camptothecin

A series of new camptothecin derivatives, as topoisomerase I inhibitor, were synthesized to identify potent antitumor agents. The synthesis method was based on the Claisen rearrangement of 10-allyloxy-7-ethylcamptothecin. All of the compounds were assayed for cytotoxicity against two human tumor cell lines, Bel7402, HCT116, and showed good potency in vitro. Compounds 2, 4, 9, were assessed for the stability of lactone in human plasma. And then compound 2 was tested for antitumor activity in vitro against mouse tumor sarcoma-180. The results suggested that the small alkyl groups in the both 7- and 9-positions of camptothecin could promote liposolubility, antitumor activity in vitro and vivo, though did not bring much increase of the stability of lactone.     

Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay

The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.     

Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants

Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anti-cancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventitious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0???M 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0???M BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4???M) and IAA (5.7???M). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40???mol?m^?2?s^?1). The camptothecin content was determined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a considerable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction.     

Total and semisynthesis and in vitro studies of both enantiomers of 20-fluorocamptothecin

Both enantiomers of 20-fluorocamptothecin and the racemate have been prepared by total synthesis. The (R)-enantiomer is essentially inactive in a topoisomerase-I/DNA assay, while the (S)-enantiomer is much less active than (20S)-camptothecin. The lactone ring of 20-fluorocamptothecin hydrolyzes more rapidly than that of camptothecin in PBS. The results provide insight into the role of the 20-hydroxy group in the binding of camptothecin to topoisomerase-I and DNA.     

Effects of recombinant human PLD2 on proliferation and apoptosis of HL-60 cells

In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.     

Feasible production of camptothecin by hairy root culture of Ophiorrhiza pumila

Camptothecin derivatives are used clinically as anti-tumor alkaloids. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but transformed plant cell cultures may be an alternative source of production. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) transformed by Agrobacterium rhizogenes strain 15834. This hairy root culture grew well, increasing by 16-fold during 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of the cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amounts. Camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20) that absorbed camptothecin. Camptothecin was easily recovered from the resin. Our method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture.