BALB/c nude mice 6 weeks old were inoculated with glioma C6 cell-line and the efficacy of the different amount of Etanidazole-discs and Taxol-microspheres was investigated. Poly (D,L-lactic-co-glycolic acid) (PLGA) was used as the main encapsulating polymer and polyethylene glycol was added to increase the porosity. The 1% drug loading microspheres of each drug were produced by spray drying and the discs were obtained by compressing the Etanidazole-microspheres. Intra-tumoral injection followed by irradiation resulted in high systemic dosage and thus systemic toxicity. Tumors grown for 6 days, 9 days and 16 days were implanted with 0.5 mg or 1.0 mg or 1.5 mg of the drug. A radiation dosage of 2 Gy each time for a number of times was given for animals implanted with Etanidazole and no irradiation was given for animals implanted with Taxol. Increasing the number of doses clearly decreased the rate of tumor growth. The increase in the amount of drug on smaller sized tumors controlled the tumor better and there was agglomeration of the microspheres resulting in deviation of release profile of the drug as compared to the in vitro studies. It was observed that 1.0 mg of Taxol given to a tumor grown for 6 days was able to suppress the tumor for a total period of approximately two months and no tumor resurrection was observed during the second month. / Singapore-MIT Alliance (SMA)

Abstract In recent years, Taxus species have attracted a great interest because Paclitaxel, an anticancer agent, was isolated from T.brevifolia. Since the discovery of Paclitaxel many new taxane diterpenes from various Taxus species were isolated. In order to discover new taxoids, twigs and leaves of Taxus sumatrana (Miq.) de Laub. Growing in Taiwan were employed. Chromatographic separation of acetone extract of the leaves and twigs of the plant has yielded four taxane diterpenes and one derivative, designated tasumatrol M (1), tasumatrol N (2), tasumatrol O (3) and 7-deacetyl-canadensene (4). All structures were established primarily on the basis of 1D and 2D NMR techniques, including DEPT, COSY, HMBC and NOESY experiments. Compound 1, 2 and 3 were inactive as tested against human KB, Hepa59T, Hela, DLD-1 and WiDr tumor cell lines. Compounds 1 and 2, possessing a bicyclic skeleton, together with compounds 3 were new compounds from natural source. The possible biosynthetic pathway of compounds 1 and 2 were derived from geranylgeranyl pyrophosphate and verticillene.

Thesis (doctoral)--Ludwig-Maximilians Universität München, 2002.

Freie Universiẗat, Diss., 2001--Berlin.

<P> Taxol, a highly functionalized and complex diterpene belonging to the taxane group, possesses strong antitumor activity against various cancers, especially in cases of advanced ovarian and breast cancers. Because of the unique mechanism of action and the unusual chemical structure, taxol may represent the prototype of a new class of chemotherapeutic agents. The non-clinical work up to date on taxol is described in this thesis, including natural resources, chemical synthesis and chemical manipulation of taxol. </p> <p> The biosynthesis of taxol has not yet been studied. The hypothesis proposed for the biosynthetic sequence involves cyclization of geranylgeranyl pyrophosphate (GGPP) into hydrocarbon intermediates, which are then further transformed into taxol. In order to study the biosynthesis of taxol, [^14C]-labelled GGPP was prepared. Through the use of this labelled precursor in incubations with cell-free extract of yew labelled biosynthetic intermediates which are formed can be isolated and identified, hence leading to further understanding of the biosynthesis of taxol. </p> <p> The synthesis of [16,17-^14C]geranylgeranyl pyrophosphate was achieved in eight steps starting from commercially available geranylgeraniol. The alcohol was protected as the acetate derivative and the terminal double bond selectively epoxidized. The epoxide was opened to the diol, which was then cleaved. The resulting aldehyde was coupled to [^14C] isopropyltriphenylphosphonium ylid in a Wittig reaction, giving [16,17-14C]geranylgeraniol after deprotection of the acetate group. The alcohol was converted into the chloride derivative and subsequently to [16,17-14C]geranylgeranyl pyrophosphate. </p> / Thesis / Master of Science (MSc)

Paclitaxel is a natural occurring diterpene alkaloid originally isolated from the bark of Taxus brevifolia. It is now one of the most important chemotherapeutic agents for clinical treatment of ovarian and breast cancers. Recent clinical trials have also shown paclitaxel's potential for the treatment of non-small-cell lung cancer, head and neck cancer, and other types of cancers. While tremendous chemical research efforts have been made in the past years, which established the fundamental structure-activity relationships of the paclitaxel molecule, and provided analogs for biochemical studies to elucidate the precise mechanism of action and for the development of second-generation agents, many areas remain to be explored. In continuation of our efforts in the structure-activity relationships study of A-norpaclitaxel, five new analogs modified at the C-1 substituent and analogs with expanded B-ring or contracted C-ring have now been prepared. Preliminary biological studies indicated that the volume rather than functionality at the C-1 position plays a role in determining the anticancer activity by controlling the relative position of the tetracyclic ring system, which in turn controls the positions of the most critical functionalities such as the C-2 benzoyl, the C-4 acetate, and the C-13 side chain. The optimum conformation could possibly be modulated by ring contraction or expansion, as suggested by the improved activity of a B-lactone-A-norpaclitaxel analog. Chemical investigations were also carried out in the C-6 and C-7 positions and led to the synthesis of five new analogs. Of particular importance, 6a-hydroxy-paclitaxel, the major human metabolite of paclitaxel, was synthesized for the first time through a C-7 epimerization reaction. The availability of the major human metabolite through synthesis makes it possible to perform in vivo biological investigations on the metabolite, and it also offers an important opportunity for the production of standard HPLC samples of the metabolites which could be useful in the clinical monitoring of paclitaxel's disposition in human patients. Previous modifications at the C-4 position suggested that analogs with an acyl group other than an acetate at C-4 may exert similar activity to paclitaxel. Little was known, however, on the conformation-activity relationships of the C-4 position. In order to further explore the C-4 chemistry, a mild C-4 acylation method using acid as the acyl source was successfully developed. The new method was exemplified by the synthesis of water-soluble paclitaxel analogs with hydrophilic functional groups at the terminal of the C-4 acyl moiety. This method should be applicable to a variety of similar carboxylic acids and offer an alternative or even better approach for the preparation of C-4 modified paclitaxel analogs. Lastly, in addition to the extension of paclitaxel analog library, specially designed analogs have been sought to probe the active conformation of paclitaxel. An analog that has a bridge to tie up the C-4 acyl group with an inert position would be useful for this purpose. With successful demonstration of the above C-4 acylation method, combined with the well established C-6 chemistry, the synthesis of such a novel C-4 and C-6 bridged paclitaxel analog was completed. / Ph. D.

Taxol (1) was first isolated from the bark of the Pacific yew about 35 years ago by Drs. Wall and Wani. Although its development as an anticancer agent was delayed by numerous reasons, including its scarcity and insolubility, the discovery of its tubulin-assembly activity and other factors motivated oncologists to overcome these problems. It has since become one of the most important current drugs for the treatment of several cancers, including breast and ovarian cancers. Like almost all anticancer drugs taxol does have some toxic side effects and many tumors also show significant resistance to therapy with taxol. Drug targeting studies aimed at improving its selectivity and efficacy is described. Two targeting methods, the estrogen receptor (ER) directed targeting and colloidal gold (cAu)directed targeting, were used in our research. In this dissertation, a series of estradiol-taxol conjugates (ETCs) were synthesized. They were active in four cytotoxicity assays and tubulin polymerization assay, but less active than taxol. One of them showed the desired selectivity for ER positive cancer cells. Recently, several studies have attempted to elucidate the bioactive binding conformation of taxol on microtubules. Three models have been proposed for this conformation. The T-taxol conformation was proposed by Dr. Snyder based on electron crystallographic density and molecular modeling. In this dessertation, a series of cyclopropyl-containing taxol analogs and macrocyclic taxol lactones were synthesized. The bioassay results showed they are less active than taxol. The molecular modeling studies suggested that the cyclopropyl-containing taxol analogs could not adopt the T-taxol conformation, which would result in the loss of bioactivities. It is an indirect evidence to support T-taxol conformation. As for macrocyclic taxol lactones, it is proposed that they would have a close contact between the ester moiety on the C-3' phenyl ring and Phe272 of the β-tubulin protein when they adopt T-taxol conformation. It will push the macrocyclics out of the binding pocket and lead to the lost of bioactivities. / Ph. D.

Paclitaxel was isolated from the bark of <i>Taxus brevifolia</i> in the late 1960s. It exerts its biological effect by promoting tubulin polymerization and stabilizing the resulting microtubules. Paclitaxel has become one of the most important current drugs for the treatment of breast and ovarian cancers. Studies aimed at understanding the biologically active conformation of paclitaxel bound on β–tubulin are described. In this work, the synthesis of isotopically labeled taxol analogs is described and the REDOR studies of this compound complexed to tubulin agrees with the hypothesis that palictaxel adopts T-taxol conformation. Based on T-taxol conformation, macrocyclic analogs of taxol have been prepared and their biological activities were evaluated. The results show a direct evidence to support T-taxol conformation. (+) Discodermolide is a polyketide isolated from the Caribbean deep sea sponge <i>Discodermia dissoluta</i> in 1990. Similar to paclitaxel, discodermolide interacts with tubulin and stabilizes the microtubule <i>in vivo</i>. Studies aimed at understanding the biologically active conformation of discodermolide bound on β–tubulin are described. In this work, the synthesis of fluorescent labeled discodermolide analogs is described and their biological activities were evaluated. Synthetic approaches to fluorescent labeled and isotopically labeled discodermolide analogs discodermolide are also described. / Ph. D.

Paclitaxel is a very important anticancer drug commonly called Taxol®. We know from previous work (Hoffman, et al., 1998) that Taxol exists in filbert, Corylus avellana L. material. Quantification of Taxol in filbert plant material is painstaking and hitherto was accomplished by rapidly processing single batches in a complicated procedure (Hoffman, et al., 1998), which seemingly unavoidably, was accompanied by some degradation. However for extraction, testing, plant physiological and horticultural purposes a simplified method of determining Taxol yield is required. All simplified methods tested were found limited by rapid degradation of Taxol. Under these conditions not only is the sought product broken down, but we were unable to distinguish Taxol precursors from degradation products. Thus it was decided to go back to first principles and study the degradation of Taxol in vitro. Degradation kinetics of Taxol was studied using electrospray ionization mass spectrometry (ESIMS) to identify possible Taxol adducts and degradation products. Our preparation for ESIMS analysis by experimental necessity involved various other components. Since some of these components interacted with the degradation products, we developed a program to distinguish these putative adducts from spurious components found in the system and we were able to plot the pH dependence of Taxol degradation in this system at room temperature (approximately 25oC). The results of the mass spectrometric analysis of these degradants were found dependent on pH and time. Our results show major degradation at pH 9 and beyond, plus minor degradation at pH 5. Two optimum pHs for stability were found at pH 4 and pH 7. This data varies slightly from the published results Dordunoo and Burt (1996). Our smoother curves define two pHs events in this pH range which to our knowledge, have not been reported and our temperature was lower. We hope that this information will help us extract Taxol more efficiently with greater yield from novel plant sources, e.g. hazel (filbert) tree, Corylus avellana L. The possibility of Taxol dimer formation in solution and perhaps in vivo can be inferred, but not proven, in this work. This dimer, may exist, in dynamic equilibrium with parent compound, Taxol. / Graduation date: 2006

This thesis presents an investigation into the transcriptional regulation of TaxolTM biosynthsis in Taxus cuspidata cell suspension cultures. The potent anticancer drug TaxolTM has been shown to be successful in the treatment of breast, lung and ovarian cancer and the acquired immunodeficiency syndrome (AIDS) related Kaposi’s sarcoma. Produced by all species of yew, TaxolTM belongs to the class of taxane diterpenoids and is of huge pharmaceutical importance. The plant material utilised in this thesis is a cell suspension culture initiated from isolated procambium cells of T. cuspidata. The latter is a meristematic tissue giving rise to the conductive tissue of plants. This un-differentiated cell suspension culture exhibits an increased and stable production of TaxolTM in response to the plant hormone elicitor methyljasmonate, limited cell aggregation and fast growth when compared to a cell suspension culture initiated from differentiated cells (somatic) of T. cuspidata. In order to assess the stem cell characteristics of the employed procambium cell suspension culture, the transcriptome of T. cuspidata was sequenced utilising Roche/ 454 and Illumina/ Solexa NlaIII tag sequencing technoloxiv gies. Statistical analysis uncovered differential expression profiles of 563 genes present within the procambium cell derived transcriptome by comparison with the somatic cell derived transcriptome. Gene ontology analysis of the latter identified that genes associated with response to stress and defence response were upregulated in the differentially expressed portion within the procambium cell suspension culture. This is consistent with the characteristics of animal stem cells which exhibit robust defence strategies to environmental stress. Furthermore PHLOEM INTERCALATED WITH XYLEM (PXY ) and TRACHEARY ELEMENT DIFFERENTIATION 2 (TED2), which are essential for ordered procambium cell division and differentiation into trachaery elements respectively in A. thaliana and Z. elegans, are up-regulated in the T. cuspidata procambium cell suspension culture. Further T. cuspidata homologues of the jasmonate signalling components JASMONATE ZINC FINGER LIKE ZIM DOMAIN 2 (JAZ2) and JAZ3 were identified among up-regulated transcripts in response to jasmonate treatment in both the procambium and the somatic cell line. Blast analysis identified 211 transcription factors within the APETELA 2 (AP2), BASIC-HELIX-LOOPHELIX (bHLH), WRKY, MYB and BASIC-LEUCIN-ZIPPER (bZIP) families. Further characterisation established 21 transcription factors which are significantly up-regulated in response to jasmonate treatment and show a higher expression level in procambium cells. These provide promising targets for further functional characterisation to elucidate their involvement within TaxolTM biosynthesis. In order to investigate transcriptional regulation of the TaxolTM structural genes, a 513 bp fragment corresponding to the TAXADIENE SYNTHASE (TASY ) promoter was cloned by genome walking. In-silico analysis of the TASY and 3’-N-DEBENZOYLTAXOL N-BENZOYLTRANSFERASE (DBTNBT) promoter resulted in the identification of methyljasmonate and pathogen-responsive elements which may significantly contribute to jasmonate mediated accumulation of TaxolTM. Analysis of a chimeric promoter construct driving the reporter gene β-GLUCURONIDASE (GUS) in N. benthamiana confirmed jasmonate-responsiveness of the TASY promoter. Finally, comparison of the expression level of genes coding for potentially rate-limiting enzymes within the TaxolTM pathway established a significantly increased expression of BACCATIN II PHENYLPROPANOYLTRANSFERASE (BAPT) in response to jasmonate treatment within the procambium cell suspension culture. Furthermore transcripts of TASY, PHENYLALANINE AMINOMUTASE (PAM) and DBTNBT show an overall higher expression and prolonged transcript accumulation in procambium compared to somatic cells. In this thesis jasmonate-signalling components, jasmonate-responsive transcription factors and differential gene expression profiles of TaxolTM structural genes were identified which, may contribute to an increased TaxolTM production in the utilised procambium cell suspension culture. Furthermore the T. cuspidata procambium cell suspension culture was found to have an increased level of stress- and defence-response reflected by differential gene expression profiles and content of phenolic compounds and TaxolTM.