Levels Of Camptothecin Research Articles

Sequestration of Camptothecin, an Anticancer Alkaloid, by Chrysomelid Beetles

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic toposiomerase-I. Several derivatives of CPT are in clinical use against ovarian and lung cancers. CPT has been reported from several plant species belonging to the order Asterids, with the highest concentration in Nothapodytes nimmoniana (family Icacinaceae). In this paper, we report an intriguing observation of chrysomelid beetles (Kanarella unicolor Jacobby) feeding on the leaves of N. nimmoniana without any apparent adverse effect. LC-MS/MS analysis of the beetles indicated that 54.9% of the ingested CPT's was recovered from the wings, followed by lesser amounts in the head and abdomen. LC-HRMS analysis revealed that most of the CPT in the insect body was in the parental form available in the plants without any major metabolizable products, including sulfated and glucuronilated forms. The mechanism by which the beetles are able to tolerate substantially high levels of CPT in their body tissue is under investigation.

Camptothecin accumulation in various organ cultures of Camptotheca acuminata Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g−1 DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g−1 DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g−1 DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g−1 FW were determined at 4 and 8 immersion cycles daily (IC d−1), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.

Propagation and chlorophyll fluorescence of Camptotheca acuminata cuttings

The Chinese happy tree, Camptotheca acuminata Decne, is grown extensively in plantations for harvest and extraction of its anti-cancer and potentially anti-viral compound, camptothecin. This study determined whether C. acuminata was amenable to clonal propagation as rooted cuttings and whether application of the rooting hormone, indole-3-butyric acid (IBA), affected chlorophyll fluorescence (FV/FM) or the percentage of cuttings that formed roots. The species was found highly amenable to vegetative propagation because a consistently high percentage of cuttings formed roots when treated with 3 or 8 g IBA / kg powder. IBA application was unnecessary in spring when more than 90% of untreated cuttings formed roots, but the highest IBA dose increased the number of plants produced by 18 and 82% (from tip cuttings) and by 46 and 102% (from node cuttings) in two later collections when rooting of untreated cuttings had declined. IBA treatments did not affect chlorophyll fluorescence. Industrial deployment of C. acuminata is entirely feasible using rooted cuttings, allowing clonal multiplication of genotypes that contain high levels of camptothecin.

Response of Camptotheca acuminata calli stimulated by mechanical vibration

Plants in their natural environment are usually affected by mechanical stress because of their sessile growth habit. This work was aimed at determining the effects of mechanical vibration on the growth rate, physiological indexes, and secondary metabolite biosynthesis of Camptotheca acuminata calli. In this study, mechanical vibrations of 1–4 Hz in frequency were applied to stimulate the C. acuminata calli; we found that a mechanical vibration of moderate frequency (2 Hz) can clearly promote the growth rate and increase the soluble protein content and superoxide dismutase (SOD) activity. Under a mechanical vibration of a 2 Hz frequency, camptothecin (CPT), the main secondary metabolite produced by C. acuminata, was increased by about fourfold compared to the control group, In contrast, the increased accumulation disappears at higher frequencies. The optimal vibration time for obtaining the highest levels of biomass and CPT was 60 min. This study showed that there are neutral frequencies and optimal periods of mechanical vibration on C. acuminata calli.

Phase I study of XMT-1001 given IV every 3 weeks to patients with advanced solid tumors.

e13121 Background: XMT-1001 is a water soluble macromolecular conjugate of camptothecin (CPT). In this novel CPT prodrug, CPT is conjugated with a 70 kDa biodegradable hydrophilic polyacetal, poly (1-hydroxymethylethylene hydroxymethylformal). XMT-1001 has an improved therapeutic window compared with irinotecan in human tumor xenograft models, providing a compelling rationale for its clinical development. Methods: This is a dose-escalation study of XMT-1001 given as an IV infusion once every 3 weeks. The objectives are to determine the maximum tolerated dose (MTD) and assess safety and pharmacokinetics (PK) of XMT-1001. Three patients (pts) are entered at each dose level, with expansion to 6 pts if there is dose-limiting toxicity. Analyses of plasma and urine were performed for XMT-1001 (conjugated CPT), 2 major drug release products, and for unconjugated (free) CPT. Results: To date, 54 pts have received 160 cycles of XMT-1001 at dose levels from 1.0 to 113 mg CPT equivalents (eq.)/m2. Two pts had Gr 3 infusion reactions to study drug. After the introduction of clinical trial material with an improved formulation, no infusion reactions suggestive of hypersensitivity have occurred (33 pts, 98 cycles). No hemorrhagic cystitis or ≥ Gr 3 diarrhea was noted. Gr 2-4 neutropenia occurred in 3 pts treated at 85 mg CPT eq. /m2. Stable disease was observed in 14 of 50 evaluable pts with the following tumor types and doses in mg CPT eq./m2: sarcoma (85 mg/m2, 6 wks), NSCLC (1.0 mg/m2, 6 wks; 20.5 mg/m2, 36 wks; 36.3 mg/m2, 18 wks), nasopharyngeal (36.3 mg/m2, 12 wks), ovarian (4.9 mg/m2, 12 wks), pancreas (4.9 mg/m2, 36 wks), appendiceal (7.3 mg/m2, 12 wks), bile duct (9.1 mg/m2, 18 wks), basal cell (11.6 mg/m2, 6 wks), colon (15.4 mg/m2, 18 wks), gastric (20.5 mg/m2, 12 wks), prostate (85 mg/m2, 6 wks), and rectal (27.3 mg/m2, 6 wks). Tumor shrinkage was noted in a pt with colon cancer at the 15.4 mg/m2 dose level. The exposure to XMT-1001 and its release products, as measured by Cmax and AUC, demonstrated dose proportional increases with respect to dose of XMT-1001. Levels of free CPT in urine have been low. Conclusions: XMT-1001 can be given safely to patients; XMT-1001 has a favorable PK profile; and prolonged stable disease ≥ 12 wks was seen in 9 pts at doses below the MTD. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Mersana Pharmaceuticals Mersana Pharmaceuticals, Mersana Therapeutics, Inc. Mersana Therapeutics, Inc.

Abstract B52: Phase 1 study of XMT-1001, a novel water soluble camptothecin conjugate, given as an intravenous infusion once every three weeks to patients with advanced solid tumors

Abstract Introduction: XMT-1001 is a water soluble macromolecular conjugate of camptothecin (CPT). In this novel CPT pro-drug, CPT is conjugated with a 70 kDa biodegradable hydrophilic polyacetal, poly (1-hydroxymethylethylene hydroxymethylformal). XMT-1001 has an improved therapeutic window compared with CPT and irinotecan in human tumor xenograft models, providing a compelling rationale for its clinical development. Methods: This is an open label, dose escalation study of XMT-1001 given as an IV infusion once every 3 weeks. The objectives of the Phase 1 study are to determine the maximum tolerated dose (MTD) and to assess safety and pharmacokinetics (PK) of XMT-1001. Initially 3 patients (pts) are entered at each dose level, with expansion to 6 pts if there is a dose limiting toxicity. Analyses of plasma and urine were performed for XMT-1001 (conjugated CPT), 2 major drug release products, and for unconjugated (free) CPT. Results: Forty six patients with refractory solid tumors have received 141 cycles of XMT-1001 at 13 dose levels ranging from 1.0–64.2 mg CPT equivalents (equiv.)/m2. Two pts had Grade (Gr) 3 infusion reactions consistent with hypersensitivity to study drug. Symptoms reversed upon discontinuation of study drug. Upon introduction of new clinical trial material with an improved formulation, no infusion reactions suggestive of hypersensitivity have occurred (25 pts, 79 cycles). No hemorraghic cystitis or ≥ Gr 3 diarrhea was noted. Gr 2 neutropenia occurred in 1 pt treated at 15.4 mg CPT equiv./m2. Stable disease was observed in 12 of 43 evaluable pts with the following tumor types and dose levels expressed in mg CPT equiv./m2: NSCLC (1.0 mg/m2, 6 wks; 20.5 mg/m2, 30 wks to date; 36.3 mg/m2, 18 wks to date), nasopharyngeal (36.3 mg/m2, 12 wks to date), ovarian (4.9 mg/m2, 12 wks), pancreas (4.9 mg/m2, 36 wks), appendiceal (7.3 mg/m2, 12 wks), bile duct (9.1 mg/m2, 18 wks), basal cell (11.6mg/m2, 6 wks), colon (15.4 mg/m2, 18 wks), gastric (20.5 mg/m2, 12 wks), and rectal (27.3mg/m2, 6 wks). The PK of XMT-1001 (conjugated CPT) demonstrates dose proportional increases in exposure XMT-1001 and confirms the formation of its expected release products. Levels of free CPT in urine have been low. Conclusions: 1. XMT-1001 can be given safely to patients; 2. XMT-1001 has a favorable PK profile; 3. Prolonged stable disease lasting ≥ 12 weeks was seen in 9 patients with advanced, refractory tumors at doses below MTD. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B52.

A phase I study of the safety and pharmacokinetics (PK) of XMT-1001 given as an intravenous (IV) infusion once every three weeks to patients with advanced solid tumors

2574 Background: XMT-1001 is a water soluble macromolecular conjugate of camptothecin (CPT). In this novel CPT pro- drug, CPT is conjugated with a 70 kDa biodegradable hydrophilic polyacetal, poly (1-hydroxymethylethylene hydroxymethylformal). XMT-1001 demonstrated an improved therapeutic window as compared with CPT and irinotecan in human tumor xenograft models, providing a compelling rationale for its clinical development. Methods: This is an open label, dose escalation study of XMT- 1001 administered as an IV infusion once every 3 weeks. The objectives of this phase I study are to determine the maximum tolerated dose and to assess safety and PK of XMT-1001. Initially 3 patients (pts) are entered at each dose level. The cohort is expanded to 6 pts if a patient experiences a dose limiting toxicity. Analyses of plasma and urine were performed for XMT-1001 (conjugated CPT), 2 major drug release products, and for unconjugated (free) CPT. Results: Thirty two pts with refractory solid tumors have received 82 cycles of XMT-1001 at 8 dose levels ranging from 1.0 to 20.5 mg CPT equivalents/m2. Two pts had Gr 3 infusion reactions consistent with hypersensitivity to study drug. Symptoms reversed upon discontinuation of study drug. After the introduction of new clinical trial material with an improved formulation, to date, no infusion reactions suggestive of hypersensitivity have occurred (11 patients, 22 cycles). No hemorrhagic cystitis or ≥ Gr 3 diarrhea was noted. Myelosuppression was observed in 3 pts treated at 15.4 mg CPT equiv/m2 dose level. Stable disease was observed in 7 pts with the following tumor types and dose levels expressed in mg CPT equivalents/m2: NSCLC (1.0 mg/m2, 6 wks), ovarian (4.9 mg/m2, 12 wks), pancreas (4.9 mg/m2, 36 wks), appendiceal (7.3 mg/m2, 12 wks), bile duct (9.1 mg/m2, 18 wks), basal cell (11.6 mg/m2, 6 wks), and colon (15.4 mg/m2, 6 wks). PK demonstrates dose proportional increases in plasma levels of XMT-1001 (conjugated CPT) and confirms the formation of its expected release products. To date, levels of free CPT in urine are low. Conclusions: 1. XMT-1001 can be given safely to patients; 2. XMT-1001 has a favorable PK profile; 3. Prolonged stable disease was observed in patients with refractory tumors. [Table: see text]

Prospecting for Camptothecines from Nothapodytes nimmoniana in the Western Ghats, South India: Identification of High-Yielding Sources of Camptothecin and New Families of Camptothecines

Camptothecin (CPT), a monoterpene alkaloid, is an important anti-cancer compound obtained from several plant sources including Camptotheca acuminta (from China) and Nothapodytes nimmoniana (from India). Currently, by far the highest levels of CPT (approximately 0.3% w/w) are reported from Nothapodytes nimmoniana, a small tree distributed in the Western Ghats, India. In recent years because of the heavy demand, there has been a serious threat of extinction of the populations of the tree in the Western Ghats forest of south India. Several studies have chemically profiled populations of the species in the Western Ghats to identify sources of high yield and therefore to enable the sustainable production and harvesting of CPT. In this study, using both high-performance liquid chromatography and liquid chromatography-mass spectrometry, we report for the first time the identification of trees that produce at least 5- to 8-fold more CPT than hitherto reported. Furthermore, we show for the first time the production of a few minor camptothecines, including 10-hydroxy camptothecin, in the stem and root bark extracts of the tree. These results have important implications for not only harnessing the high-yielding individuals for clonal multiplication but also for exploiting some of the minor camptothecines, which also have been shown to have important anti-cancer and anti-viral activity.

Tissue-specific and Developmental Regulation of Camptothecin and 10-hydroxycamptothecin Levels in Camptotheca acuminate

Camptothecin (CPT) and its analogue 10-hydroxycamptothecin (HCPT) are two naturally occurring monoterpene indole alkaloids in Camptotheca acuminata. They both show potential efficacy as anti-cancer compounds as well as act as defensive chemicals in plants. Here we report the regulation of CPT and HCPT contents in C. acuminata during seed imbibition and seedling development. The levels of CPT in endosperm was 2 to 3 fold higher than in other tissues, and clearly decreased during the imbibition period. In other tissues, CPT contents exhibited slight fluctuations. In comparison, HCPT contents in all the tissues were much lower. HCPT was less abundant in cotyledon and embryonal axis than in endosperm and seed coat. During seedling development, CPT and HCPT contents in cotyledons reached maximum at 9-15 days stage. In embryonal axis, CPT contents decreased logarithmically, while HCPT content followed the same pattern as in cotyledons. CPT and HCPT levels in radicles were very low, with CPT levels remaining constant and HCPT levels decreasing during development. In leaves, the contents of CPT showed linear reduction during seedling growth. These results demonstrate that the levels of CPT and HCPT are tissue- specifically and developmentally regulated. The physiological implications and significance of the regulation are discussed.

Somatic embryogenesis, plant regeneration, and the evaluation of camptothecin content in Nothapodytes foetida

Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.

High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C 8 analytical column, using a mixture of acetonitrile–25 m M phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.

Sensitivity to topoisomerase I inhibitors and cisplatin is associated with epidermal growth factor receptor expression in human cervical squamous carcinoma ME180 sublines

The relationship between expression and function of the epidermal growth factor (EGF) family of receptors and chemosensitivity remains controversial. We studied the chemosensitivity to various anticancer agents of human cervical squamous carcinoma ME180 cells, and two resistant subclones, ME180/TNF and ME180/Pt, which also differ in their EGF receptor (EGFR) expression. Compared with ME180 cells, EGFR is overexpressed sixfold in ME180/TNF cells and is barely detectable in ME 180/Pt cells. Cell cycle analysis by flow cytometry and BrdU incorporation into DNA showed a correlation between EGFR expression and percentage of cells in S phase and active DNA replication (35% in high EGFR-expressing ME180/TNF cells, 19% in non-EGFR-expressing ME180/Pt cells and 23% in parental, intermediate-level EGFR-expressing ME 180 cells). By MTT assay and compared with parental, intermediate-level EGFR-expressing ME180 cells, high EGFR-expressing ME180/TNF cells had a three- to fourfold increased sensitivity to cisplatin, camptothecin (CPT), and topotecan, and low EGFR-expressing ME180/Pt cells had a five- to ninefold reduced sensitivity to the same agents. In contrast, the degree of cross-resistance with the topoisomerase II inhibitors doxorubicin and etoposide was minimal and the pattern of sensitivity to the anti-microtubulin agents vinblastine and paclitaxel was different, with a two- to fourfold decreased sensitivity in the high EGFR-expressing ME180/TNF cells and only a 1.5-fold decreased sensitivity in the low EGFR-expressing ME180/Pt cells. Neither alterations in intracellular CPT levels nor changes in topoisomerase I expression or activity, measured as ability to form DNA-protein complexes, were found to explain the differences in sensitivity to CPT among the three cell lines. Co-treatment with CP358774, a specific EGFR tyrosine kinase inhibitor, reduced the enhanced sensitivity of high EGFR-expressing ME180/TNF cells to the values observed in intermediate EGFR-expressing ME180 cells, but only reduced modestly the sensitivity of intermediate expressing ME180 cells. As a result, the resistance index of low EGFR-expressing ME180/Pt cells compared with intermediate EGFR-expressing ME180 cells was reduced only from five- to fourfold for cisplatin and from seven- to fourfold for CPT when ME180 cells were exposed to CP358774. CP358774 did not affect the sensitivity to either agent in low EGFR-expressing ME180/Pt cells. These results provide evidence that changes in EGFR expression or function may play a role in determining chemosensitivity to platinum and topoisomerase I poisons in some human tumor systems, and that the EGFR-related changes in chemosensitivity may vary depending on the level of EGFR expression and/or function.

Influence of soil fertilization, plant spacing, and cokppicing on growth, stomatal conductance, abscisic acid, and camptothecin levels in Camptotheca acuminata seedlings

Changes in amount of the plant alkaloid, camptothecin (CPT), were investigated in field‐grown Camptotheca acuminata seedlings in response to fertilization, variation in plant density, and cokppicing. The relationship between CPT and growth, and abscisic acid (ABA) levels were also examined. Fertilization caused positive responses in growth of both the slow‐ and the fast‐growing C. acuminata plants. It lowered stomatal conductance (g), reduced leaf CPT level in slow‐growing but not fast‐growing seedlings in mid‐summer (July), and increased leaf ABA level in dense plantings in early fall (September). Shoots induced to develop as a result of cokppicing were significantly higher in leaf CPT level than the original non‐cokppiced shoots. These results suggest that in the C. acuminata plantation management, cokppicing should be employed to produce high concentrations of CPT, but that soil fertilization should be used cautiously so that greater growth is not achieved at the cost of lower CPT biosynthesis.

Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress.

Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata. Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2, two autonomously regulated TDC genes from Camptotheca. When expressed in Escherichia coli, the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa), tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1, tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca, while tdc2 serves as part of a defense system induced during pathogen challenge.

Sites of Accumulation of the Antitumor Alkaloid Camptothecin inCamptotheca acuminata

Camptothecin is an anticancer and anti-viral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae). Despite previous reports of low levels of anticancer activity in leaves of Camptotheca acuminata, we have discovered that camptothecin accumulates to approximately 0.4% of the dry weight of young leaves. This level is 1.5-fold higher than that of the seeds and 2.5-fold higher than that of the bark, the two currently used sources of the drug. As the leaves mature, the concentration and absolute amount of camptothecin decreases rapidly. The high levels of camptothecin in young leaves could provide an easily harvested, non-destructive source of this important drug.

Inhibition of Human Neurotropic Virus (JCV) DNA Replication in Glial Cells by Camptothecin

Progressive multifocal leukoencephalopathy is a subacute demyelinating disease of the central nervous system (CNS) resulting from opportunistic infections in immunocompromized patients infected with a common polyomavirus, JC virus (JCV). Unlike other polyomaviruses, JCV exhibits an unusually narrow tissue tropism by primarily infecting glial cells of the CNS. JCV DNA replication is similar to that of the well-characterized papovavirus, SV40, which requires the viral early protein T-antigen and host-replication factors including DNA polymerases and DNA topoisomerase I. In this study we have been able to effectively block replication of viral DNA in glial cells using camptothecin, a drug which inhibits DNA topoisomerase activity. Pulse-treatment of cells with non-toxic levels of camptothecin specifically blocks viral DNA replication with no inhibitory effect on host transcription and translation processes as examined by viral gene expression in the transfected cells. Furthermore, drug treatment of the cells exhibits no significant effect on DNA topoisomerase I gene transcription. We further demonstrate that repeated pulse-treatment of cells with the drug is required for complete blockage of viral DNA replication. The importance of these findings in the treatment of AIDS encephalopathy is discussed.