Enhanced cytotoxicity to lung cancer cells by mitochondrial delivery of camptothecin.

Delivering traditional DNA-damaging anticancer drugs into mitochondria to damage mitochondria is a promising chemotherapy strategy. The impermeability of this mitochondrial inner membrane, however, impedes the delivery of drug molecules that could impact other important biological roles of mitochondria. Herein, the prodrug camptothecin (CPT)-triphenylphosphine (TPP) modified with hyaluronic acid (HA) via electrostatic adsorption (HA/CPT-TPP, HCT) was used to mediate the mitochondrial accumulation of CPT. These nanoparticles (NPs) showed enhanced drug accumulation in cancer cells through tumor targeting. HCT entered acidic lysosomes through endosomal transport, HA was degraded by hyaluronidase (HAase) in acidic lysosomes, and the positively charged CPT-TPP was exposed and accumulated fully in the mitochondria. Subsequently, CPT-TPP significantly disrupted the mitochondrial structure and damaged mitochondrial function, leading to increased reactive oxygen species (ROS) levels and energy depletion. Finally, HCT enhanced lung cancer cell apoptosis via the activation of caspase-3 and caspase-9. Furthermore, greatly increased tumor growth inhibition was observed in nude mice bearing A549 xenograft tumors after the administration of HCT via tail injection. This study demonstrated that the mitochondria-targeted delivery of CPT may be a promising antitumor therapeutic strategy.

Phytochemical Analysis of Nothapodytes tomentosa and Distribution and Content of Camptothecin and its Analogues in Four Plants.

Camptothecin (CPT) and its derivatives have attracted worldwide attention because of their notable anticancer activity. However, the growing demand for CPT in the global pharmaceutical industry has caused a severe shortage of CPT-producing plant resources. In this study, phytochemical analysis of Nothapodytes tomentosa results in the isolation and identification of CPT (13: ) and 16 analogues (1:  - 12, 14:  - 17: ), including a new (1: ) and five known (9, 10, 12, 15: , and 17: ) CPT analogues with an open E-ring. In view of the potential anticancer activity of CPT analogues with an open E-ring, the fragmentation pathways and mass spectra profiles of these six CPT analogues (1, 9, 10, 12, 15: , and 17: ) are investigated, providing a reference for the rapid detection of these compounds in other plants. Furthermore, based on the fragmentation patterns of CPT (13: ) and known analogues (2:  - 8, 11, 14, 16, 18:  - 26: ), the distribution and content of these compounds in different tissues of N. tomentosa, N. nimmoniana, Camptotheca acuminata, and Ophiorrhiza japonica are further studied. Our findings not only provide an alternative plant resource for further expanding the development and utilization of CPT and its analogues, but also lay a foundation for improving the utilization of known CPT-producing plant resources.

Integrated transcriptome and proteome analysis provides new insights into camptothecin biosynthesis and regulation in Camptotheca acuminata.

Camptotheca acuminata Decne., the main source of camptothecin (CPT), has received increasing attention for its remarkable antitumor activity. Many CPT derivatives are clinically used as effective anticancer agents worldwide. However, their biosynthesis mechanism remains unclear, and uncovering this pathway would greatly facilitate development of alternative CPT production methods to replace current inefficient plant-derived ones. The expression of >30,000 genes was accurately quantified using unique molecular identifier RNA sequencing in 10 C. acuminata tissues, and 7854 proteins from five tissues were quantified with label-free quantitative proteomics. Fifteen full-length transcriptomes were sequenced with long-read Oxford Nanopore Technologies, and 5692 alternative splicing events were discovered among 4746 genes. Integrated transcriptome and proteome analysis provided novel insights into CPT biosynthesis and its hierarchical regulation. Five cytochrome P450s and three O-methyltransferases were considered as candidates involved in the biosynthesis of CPT and its derivatives, while 15 transcription factors potentially regulating CPT biosynthesis were screened. These findings provide important clues for elucidating the biosynthetic mechanisms of CPT and its derivatives and substantially contribute to the future production of these anticancer agents with synthetic biology. The generated large-scale multiomics data also provide valuable resources for investigating the functional genomics of the most important CPT-producing plant species-C. acuminata.

Production and bioprocessing of camptothecin from Aspergillus terreus, an endophyte of Cestrum parqui, restoring their biosynthetic potency by Citrus limonum peel extracts.

The metabolic potency of fungi as camptothecin producer elevates their prospective use as an industrial platform for commercial production, however, the loss of camptothecin productivity by fungi with the storage and subculturing are the major obstacle. Thus, screening for endophytic fungal isolates inhabiting ethnopharmacological plants with an obvious metabolic stability and sustainability for camptothecin biosynthesis could be one of the most feasible paradigms. Aspergillus terreus ON908494.1, an endophyte of Cestrum parqui was morphologically and molecularly verified, displaying the most potent camptothecin biosynthetic potency. The chemical identity of A. terreus camptothecin was confirmed from the HPLC, FTIR and LC-MS/MS analyses, gave the same molecular structure and mass fragmentation patterns of authentic one. The purified putative camptothecin displayed a strong anticancer activity towards HepG-2 and MCF-7 with IC50 values 0.96 and 1.4 µM, respectively, with no toxicity to OEC normal cells. As well as, the purified camptothecin displayed a significant antifungal activity towards fungal human pathogen Candida albicans, Aspergillus flavus, and A. parasiticus, ensuring the unique structural activity relationships of A. terreus camptothecin, as a powerful dually active anticancer and antimicrobial agent. The camptothecin productivity of A. terreus was maximized by bioprocessing with Plackett-Burman design, with an overall 1.5 folds increment (170.5 µg/L), comparing to control culture. So, the optimal medium components for maximum yield of camptothecin by A. terreus was acid why (2.0 mL/L), Diaion HP20 (2.0 g/L), Amberlite XAD (2.0 g/L), dextrin (5.0 g/L), glucose (10.0 g/L), salicylic acid (2.0 g/L), serine (4.0 g/L), cysteine (4.0 g/L) and glutamate (10.0 g/L), at pH 6 for 15 days incubation. By the 5th generation of A. terreus, the camptothecin yield was reduced by 60%, comparing to zero culture. Interestingly, the productivity of camptothecin by A. terreus has been completely restored and over increased (210 µg/L), comparing to the 3rd generation A. terreus (90 µg/L) upon addition of methanolic extracts of Citrus limonum peels, revealing the presence of some chemical signals that triggers the camptothecin biosynthetic machinery. The feasibility of complete restoring of camptothecin biosynthetic-machinery of A. terreus for stable and sustainable production of camptothecin, pave the way for using this fungal isolate as new platform for scaling-up the camptothecin production.

Camptothecin Effectively Regulates Germline Differentiation through Bam-Cyclin A Axis in Drosophila melanogaster.

Camptothecin (CPT), first isolated from Chinese tree Camptotheca acuminate, produces rapid and prolonged inhibition of DNA synthesis and induction of DNA damage by targeting topoisomerase I (top1), which is highly activated in cancer cells. CPT thus exhibits remarkable anticancer activities in various cancer types, and is a promising therapeutic agent for the treatment of cancers. However, it remains to be uncovered underlying its cytotoxicity toward germ cells. In this study we found that CPT, a cell cycle-specific anticancer agent, reduced fecundity and exhibited significant cytotoxicity toward GSCs and two-cell cysts. We showed that CPT induced GSC loss and retarded two-cell cysts differentiation in a niche- or apoptosis-independent manner. Instead, CPT induced ectopic expression of a differentiation factor, bag of marbles (Bam), and regulated the expression of cyclin A, which contributed to GSC loss. In addition, CPT compromised two-cell cysts differentiation by decreasing the expression of Bam and inducing cell arrest at G1/S phase via cyclin A, eventually resulting in two-cell accumulation. Collectively, this study demonstrates, for the first time in vivo, that the Bam-cyclin A axis is involved in CPT-mediated germline stem cell loss and two-cell cysts differentiation defects via inducing cell cycle arrest, which could provide information underlying toxicological effects of CPT in the productive system, and feature its potential to develop as a pharmacology-based germline stem cell regulation agent.

Effects of common genetic variants of human uridine diphosphate glucuronosyltransferase subfamilies on irinotecan glucuronidation.

The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6-UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects.

Recent studies on chemical constituents of Ophiorrhiza plants.

Ophiorrhiza plants (Family Rubiaceae) are known to produce diverse monoterpenoid indole alkaloids including camptothecin with potent antitumor activity. This review contains a summary of recent chemical studies reported over the past 10 years regarding alkaloids (monoterpenoid indole and tetrahydroisoquinoline alkaloids, and cyclopeptide) in Ophiorrhiza plants. In addition, the alkaloid biosynthetic pathways based on their reported structures were proposed.

Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers.

Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (Papp AP-BL) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10-6 cm/s and (2.48 ± 0.18) × 10-6 cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.

Isolation and Phytochemical Screening of Endophytic Fungi Isolated from Medicinal Plant Mappia foetida and Evaluation of Its In Vitro Cytotoxicity in Cancer.

Isolated endophyte fungi from Mappia foetida have been explored as a potential source for the mass production of anticancer drug lead compounds in the current study. Since medical plants are not feasible economically for mass production of bioactive pharmaceutical important molecules using plant tissue culture due to factors like media design and fungal contamination, endophyte fungal mass culture have been an alternative for the relatively easy and inexpensive production. Two endophytic fungi isolated, Alternaria alternata and Fusarium species were mass cultured and their prepared alcoholic extract subjected to standard procedures to identify the phytochemical screening by gas chromatography-mass spectrometry (GCMS), high-performance liquid chromatography (HPLC), UV visible spectrophotometry (UV-VIS), and Fourier transform infrared spectroscopy (FTIR). GC-MS analysis revealed the presence of three major compounds in the extracts. The phytochemical screening confirmed the presence of an anticancer compound (camptothecin) in their extract. Moreover, the dose-dependent anticancer activity of ethanol extract was demonstrated against cervical carcinoma (HeLa), breast carcinoma (MCF-7), non-small cell lung carcinoma (H1975), and hepatocellular carcinoma cell line (Hep G2) by MTT assay where doxorubicin was used as the positive control. Furthermore, the microscopic examination also confirmed the cytotoxic effect of extract of endophytic fungi Alternaria alternata and Fusarium species against tested cancer cells. Hence, endophytic fungi Alternaria alternata and Fusarium species might be exploited for mass production of phytochemicals having anticancer activity.

Production, Bioprocessing and Anti-Proliferative Activity of Camptothecin from Penicillium chrysogenum, "An Endozoic of Marine Sponge, Cliona sp.", as a Metabolically Stable Camptothecin Producing Isolate.

Exploring the metabolic potency of fungi as camptothecin producers raises the hope of their usage as an industrial source of camptothecin, due to their short-life span and the feasibility of metabolic engineering. However, the tiny yield and loss of camptothecin productivity of fungi during storage and sub-culturing are challenges that counteract this approach. Marine fungi could be a novel source for camptothecin production, with higher yield and reliable metabolic sustainability. The marine fungal isolate Penicillium chrysogenum EFBL # OL597937.1 derived from the sponge "Cliona sp." has been morphologically identified and molecularly confirmed, based on the Internal Transcribed Spacer sequence, exhibiting the highest yield of camptothecin (110 µg/L). The molecular structure and chemical identity of P. chrysogenum derived camptothecin has been resolved by HPLC, FTIR and LC-MS/MS analyses, giving the same spectroscopic profiles and mass fragmentation patterns as authentic camptothecin. The extracted camptothecin displayed a strong anti-proliferative activity towards HEP-2 and HCT-116 (IC50 values 0.33-0.35 µM). The yield of camptothecin was maximized by nutritional optimization of P. chrysogenum with a Plackett-Burman design, and the productivity of camptothecin increased by 1.8 fold (200 µg/L), compared to control fungal cultures. Upon storage at 4 °C as slope culture for 8 months, the productivity of camptothecin for P. chrysogenum was reduced by 40% compared to the initial culture. Visual fading of the mycelial pigmentation of P. chrysogenum was observed during fungal storage, matched with loss of camptothecin productivity. Methylene chloride extracts of Cliona sp. had the potency to completely restore the camptothecin productivity of P. chrysogenum, ensuring the partial dependence of the expression of the camptothecin biosynthetic machinery of P. chrysogenum on the chemical signals derived from the sponge, or the associated microbial flora. This is the first report describing the feasibility of P. chrysogenum, endozoic of Cliona sp., for camptothecin production, along with reliable metabolic biosynthetic stability, which could be a new platform for scaling-up camptothecin production.

Camptothecin, triptolide, and apoptosis inducer kit have differential effects on mitochondria in colorectal carcinoma cells.

Mitochondrial fission and fusion are required for cell survival, and several studies have shown an imbalance between fission and fusion in cancer. High levels of mitochondrial fusion are observed in drug-resistant tumor cells, whereas mitochondrial fission may be important in sensitizing tumor cells to chemotherapy drugs. Based on current knowledge, we hypothesized that different chemotherapeutics might differentially affect mitochondrial dynamics and energy production. Thus, we selected chemotherapeutics with different mechanisms of action (camptothecin, triptolide and apoptosis inducer kit) and investigated their effect on mitochondria in colorectal carcinoma cells. We report that these chemotherapeutics decreased the activity of complex I and reduced the mitochondrial membrane potential, and also decreased the size of mitochondria in the colorectal carcinoma cell lines DLD1 and HCT-116. Treatment with camptothecin, triptolide and/or apoptosis inducer kit results in differential effects of fission on apoptosis in these cells. Our results suggest that fission is an important process in apoptosis induced by chemotherapeutics.

Cellular Uptake and Transport Characteristics of FL118 Derivatives in Caco-2 Cell Monolayers.

In the evaluation of the druggability of candidate compounds, it was vital to predict the oral bioavailability of compounds from apparent permeability (Papp) across Caco-2 cell-culture model of intestinal epithelium cultured on commercial transwell plate inserts. The study was to investigate the transport characteristics and permeability of FL118 (10, 11-Methylenedioxy-20(S)-camptothecin) derivatives 7-Q6 (7-(4-Ethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin) and 7-Q20 (7-(4-Trifluoromethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin). Transport characteristics and permeability of the tested compounds to the small intestine were assessed at different concentrations (0.5, 1 µM) via Caco-2 cell monolayers model in vitro. Uptake studies based on Caco-2 cells, including temperatures, concentrations, and the influence of efflux transporters, were combined to confirm the transport characteristics of the tested compounds. Furthermore, cytotoxicity results showed that the concentrations used in the experiments were non-toxic and harmless to cells. In addition, The Papp of 7-Q6 was (3.69 ± 1.07) × 10-6 cm/s with efflux ratio (ER) 0.98, while the Papp of 7-Q20 was (7.78 ± 0.89) × 10-6 cm/s with ER 1.05 for apical-to-basolateral (AP→BL) at 0.5 µM, suggesting that 7-Q20 might possess higher oral bioavailability in vivo. Furthermore, P-glycoprotein (P-gp) was proved to slightly affect the accumulations of 7-Q20, while the absorption of 7-Q6 was irrelevant with P-gp and breast cancer resistant protein (BCRP) based on the cellular uptake assays. Accordingly, 7-Q6 was completely absorbed by passive diffusion, and 7-Q20 was mainly dependent on passive diffusion with being effluxed by P-gp slightly. Meanwhile, both 7-Q6 and 7-Q20 were potential antitumor drugs that might exhibit high oral bioavailability in the body.

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

Camptothecin's journey from discovery to WHO Essential Medicine: Fifty years of promise.

Nature represents a rich source of compounds used for the treatment of many diseases. Camptothecin (CPT), isolated from the bark of Camptotheca acuminata, is a cytotoxic alkaloid that attenuates cancer cell replication by inhibiting DNA topoisomerase 1. Despite its promising and wide spectrum antiproliferative activity, its use is limited due to low solubility, instability, acquired tumour cell resistance, and remarkable toxicity. This has led to the development of numerous CPT analogues with improved pharmacodynamic and pharmacokinetic profiles. Three natural product-inspired drugs, namely, topotecan, irinotecan, and belotecan, are clinically approved and prescribed drugs for the treatment of several types of cancer, whereas other derivatives are in clinical trials. In this review, which covers literature from 2015 to 2020, we aim to provide a comprehensive overview and describe efforts that led to the development of a variety of CPT analogues. These efforts have led to the discovery of potent, first-in-class chemotherapeutic agents inspired by CPT. In addition, the mechanism of action, SAR studies, and recent advances of novel CPT drug delivery systems and antibody drug conjugates are discussed.

A chromosome-level Camptotheca acuminata genome assembly provides insights into the evolutionary origin of camptothecin biosynthesis.

Camptothecin and its derivatives are widely used for treating malignant tumors. Previous studies revealed only a limited number of candidate genes for camptothecin biosynthesis in Camptotheca acuminata, and it is still poorly understood how its biosynthesis of camptothecin has evolved. Here, we report a high-quality, chromosome-level C. acuminata genome assembly. We find that C. acuminata experiences an independent whole-genome duplication and numerous genes derive from it are related to camptothecin biosynthesis. Comparing with Catharanthus roseus, the loganic acid O-methyltransferase (LAMT) in C. acuminata fails to convert loganic acid into loganin. Instead, two secologanic acid synthases (SLASs) convert loganic acid to secologanic acid. The functional divergence of the LAMT gene and positive evolution of two SLAS genes, therefore, both contribute greatly to the camptothecin biosynthesis in C. acuminata. Our results emphasize the importance of high-quality genome assembly in identifying genetic changes in the evolutionary origin of a secondary metabolite.

A 4-aminonaphthalimide-based fluorescent traceable prodrug with excellent photoinduced cytotoxicity.

A blue light activated anti-cancer prodrug, NST, was designed based on a photoactive 4-aminonaphthalimide derivative and an anticancer drug, 10-hydroxycamptothecin. NST was hard to be taken up by living cells and showed negligible dark cytotoxicity. The irradiation caused photocleavage of NST and resulted in high cytotoxicity.

Screening and selection of camptothecin producing endophytes from Nothapodytes nimmoniana.

Endophytic fungi with the ability to produce plant based secondary metabolites are a potential alternative for producing the host plant metabolite and to prevent natural plants from extinction. To isolate a high metabolite yielding endophytic strain from plants, hundreds of endophytic strains are screened and tested for product yield separately under axenic state, before shortlisting the potential endophyte, which involves huge time consumption. In this study, strategies for screening and selection of high camptothecin yielding endophytes from their natural habitat were proposed. A correlation was built between the camptothecin yield in the explants and the endophytes isolated from them. In addition, camptothecin yield was compared between the endophytes isolated from young and matured plants. Further, camptothecin producers and non-producers strains were compared for their tolerance toward camptothecin. The study indicates that high camptothecin yielding endophytes were isolated from high yielding explants and younger plants and they were more tolerant to camptothecin in comparison to non-camptothecin yielding endophytes. Thus, choosing a young and high yielding explant for endophyte isolation, and use of camptothecin as a selective agent in the growth medium, can be instrumental in screening and selection of high camptothecin yielding endophytes from nature in relatively less time.

In Vitro Metabolism of E2, G2: Novel Bile Acid-Coupling Camptothecin Analogues, in Rat Liver Microsomes.

BACKGROUND: E2 (Camptothecin - 20 (S) - O- glycine - deoxycholic acid), and G2 (Camptothecin - 20 (S) - O - acetate - deoxycholic acid) are two novel bile acid-derived camptothecin analogues, modified deoxycholic acid at 20-position of CPT(camptothecin) with greater anticancer activity and lower systematic toxicity in vivo. OBJECTIVE: We aimed to investigate the metabolism of E2 and G2 by Rat Liver Microsomes (RLM). METHODS: Phase I and Phase II metabolism of E2 and G2 in rat liver microsomes were performed, respectively, and the mixed incubation of phase I and phase II metabolism of E2 and G2 was also processed. Metabolites were identified by liquid chromatographic/mass spectrometry. RESULTS: The results showed that phase I metabolism was the major biotransformation route for both E2 and G2. The isoenzyme involved in their metabolism had some difference. The intrinsic clearance of G2 was 174.7 mL/min. mg protein, more than three times that of E2 (51.3 mL/min . mg protein), indicating a greater metabolism stability of E2. 10 metabolites of E2 and 14 metabolites of G2 were detected, including phase I metabolites (mainly via hydroxylations and hydrolysis) and their further glucuronidation products. CONCLUSION: These findings suggested that E2 and G2 have similar biotransformation pathways except for some differences in the hydrolysis ability of the ester bond and amino bond from the parent compounds, which may result in the diversity of their metabolism stability and responsible CYPs(Cytochrome P450 proteins).

A NMR study of binding the metabolite of SN38 derivatives to a model nicked DNA decamer mimicking target of Topo I inhibitors.

In this account we present NMR based results of the interaction of 7-ethyl-9-hydroxymethyl-10-hydroxycamptothecin (1), a derivative of SN38, with a model nicked DNA decamer mimicking the wild type DNA target of Topoisomerase I inhibitors from the camptothecin family. The title compound 1 can be considered a main metabolite of phase I in the metabolic pathway of camptothecin derivatives bearing the alkylamino substituent. Therefore, its pharmacodynamic properties are of interest. It was established by DOSY (Diffusion Ordered Spectroscopy) that compound 1 forms a fairly stable molecular complex with a model nicked DNA decamer with affinity constant Ka 3.02 mM-1. The analysis of NOESY experiments revealed intermolecular cross peaks and mutual induced shifts on both interacting components allowing the conclusion that guest molecule 1 is stacking the nitrogen bases inside the nick. MD (Molecular Dynamics) analysis of four possible inclusions of 1 inside the nick allows establishing the detailed geometry of a complex. Two conformations are suggested as the ones best representing the results of molecular modeling reconciled with experimental NOESY results. The aromatic core of both structures is stacking the nitrogen bases in a nick facing the unbroken strand with ring A. The protons in ring E interact with ribose protons of edge bases of a nick. In conclusion, it can be asserted that SN38 derivative 1 can effectively bind the molecular target of Topo I enzyme and play a role as a Topo I inhibitor.

Sustainable production of camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana.

Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host-plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 µg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.