KRAS pathway expression changes in pancreatic cancer models by conventional and experimental taxanes.

The KRAS signalling pathway is pivotal for pancreatic ductal adenocarcinoma (PDAC) development. After the failure of most conventional cytotoxic and targeted therapeutics tested so far, the combination of taxane nab-paclitaxel (Abraxane) with gemcitabine recently demonstrated promising improvements in the survival of PDAC patients. This study aimed to explore interactions of conventional paclitaxel and experimental taxane SB-T-1216 with the KRAS signalling pathway expression in in vivo and in vitro PDAC models in order to decipher potential predictive biomarkers or targets for future individualised therapy. Mouse PDAC PaCa-44 xenograft model was used for evaluation of changes in transcript and protein levels of the KRAS signalling pathway caused by administration of experimental taxane SB-T-1216 in vivo. Subsequently, KRAS wild-type (BxPc-3) and mutated (MiaPaCa-2 and PaCa-44) cell line models were treated with paclitaxel to verify dysregulation of the KRAS signalling pathway gene expression profile in vitro and investigate the role of KRAS mutation status. By comparing the gene expression profiles, this study observed for the first time that in vitro cell models differ in the basal transcriptional profile of the KRAS signalling pathway, but there were no differences between KRAS mutated and wild-type cells in sensitivity to taxanes. Generally, the taxane administration caused a downregulation of the KRAS signalling pathway both in vitro and in vivo, but this effect was not dependent on the KRAS mutation status. In conclusion, putative biomarkers for prediction of taxane activity or targets for stimulation of taxane anticancer effects were not discovered by the KRAS signalling pathway profiling in various PDAC models.

Novel T9 loop conformation of filamenting temperature-sensitive mutant Z from Mycobacterium tuberculosis.

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Šresolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.

Hedgehog pathway overexpression in pancreatic cancer is abrogated by new-generation taxoid SB-T-1216.

The Hedgehog pathway is one of the major driver pathways in pancreatic ductal adenocarcinoma. This study investigated prognostic importance of Hedgehog signaling pathway in pancreatic cancer patients who underwent a radical resection. Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 45 patients with histologically verified pancreatic cancer. The effect of experimental taxane chemotherapy on the expression of Hedgehog pathway was evaluated in vivo using a mouse xenograft model prepared using pancreatic cancer cell line Paca-44. Mice were treated by experimental Stony Brook Taxane SB-T-1216. The transcript profile of 34 Hedgehog pathway genes in patients and xenografts was assessed using quantitative PCR. The Hedgehog pathway was strongly overexpressed in pancreatic tumors and upregulation of SHH, IHH, HHAT and PTCH1 was associated with a trend toward decreased patient survival. No association of Hedgehog pathway expression with KRAS mutation status was found in tumors. Sonic hedgehog ligand was overexpressed, but all other downstream genes were downregulated by SB-T-1216 treatment in vivo. Suppression of HH pathway expression in vivo by taxane-based chemotherapy suggests a new mechanism of action for treatment of this aggressive tumor.

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Metabolism of new-generation taxanes in human, pig, minipig and rat liver microsomes.

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.

Recent advances in the new generation taxane anticancer agents.

Recent advances in the design and preclinical evaluations of promising new generation taxane anticancer agents are reviewed in this article. Paclitaxel and docetaxel are two of the most important anticancer drugs today. However, recent reports have shown that treatment with these drugs often encounters undesirable side effects as well as drug resistance. Therefore, it is important to develop new taxane anticancer agents with fewer side effects, superior pharmacological properties, and improved activity against drug-resistant human cancers. Structure-activity relationship (SAR) studies led to the discovery of a series of highly active second-generation taxanes. One of them, "Ortataxel" (SB-T-101131, IDN5109, BAY59-8862), exhibits excellent activity against a variety of drug-sensitive and drug-resistant cancer cell lines, as well as human tumor xenografts in mice. It is orally active and is currently in phase II clinical trials. Photoaffinity labeling of microtubules and P-glycoprotein using photoreactive radiolabeled taxoids has disclosed the drug-binding domain of tubulin as well as Pgp. Together with information on microtubule-bound fluorine-labeled taxoids obtained by solid-state NMR studies, the bioactive conformation of paclitaxel and taxoids appears to emerge. Novel taxane-monoclonal antibody (mAb) immunoconjugates, have shown highly promising results for the tumor-specific delivery and release of an extremely cytotoxic, second-generation taxane. Also, another novel series of second generation taxanes conjugated with n-3 polyunsaturated fatty acids, e.g. decosahexaenoic acid (DHA), has exhibited impressive antitumor activity with minimum general toxicity against the highly drug-resistant DLD-1 human colon cancer xenografts in SCID mice.

Tumor specific novel taxoid-monoclonal antibody conjugates.

The basic principle of the targeted delivery approach is that the conjugation of a drug to a tumor-specific molecule renders the drug inactive until it reaches the target site. Monoclonal antibodies (mAbs), which have shown high binding specificity for tumor-specific antigens, could be used as targeting agents. Paclitaxel has brought significant impact on the current cancer chemotherapy, but seriously suffers from the lack of tumor specificity. A series of paclitaxel-monoclonal antibody conjugates via C-2' ester linkage were reported. Taking into account the fact that the cytotoxicity of paclitaxel is not good enough and thus not applicable to this target delivery prodrug approach, new taxoids bearing methyldisulfanyl(alkanoyl) groups were designed, synthesized, and their activities evaluated. A highly cytotoxic C-10 methyldisulfanyl-propanoyl taxoid, was conjugated to monoclonal antibodies recognizing the epidermal growth factor receptor (EGFR). These conjugates were shown to possess remarkable target-specific antitumor activity in vivo against EGFR-expressing A431 tumor xenografts in SCID mice, resulting in complete inhibition of tumor growth in all the treated mice without any noticeable toxicity to the animals.

%VO2max as an indicator of prosthetic rehabilitation outcome after dysvascular amputation.

In this study, independent ambulation of at least 100 metres with/without a cane was regarded as successful prosthetic rehabilitation. The subjects were classified into two groups according to this criterion at the time of discharge. The successful group attained this performance, the other group failed to reach this level. The successful group included 8 unilateral trans-femoral amputees aged 72.2 +/- 2.1 years who underwent amputation at more than 70 years, and succeeded in walking with a prosthesis. The group which failed included 9 unilateral trans-femoral amputees aged 63.2 +/- 2.1 years who underwent amputation between the ages of 60-65 years, and had great difficulty in walking with a prosthesis. The purpose of this research was to investigate whether or not %VO2max as an indicator of physical fitness is useful in predicting prosthetic rehabilitation outcome after dysvascular amputation by comparing these two groups. Evaluation of physical fitness was conducted before the subjects began prosthetic rehabilitation. Information about each subject before fitting with a prosthesis was collected retrospectively from clinical charts made during admission. The successful group were capable of strenuous exercise, reaching the intensity of 50% VO2max or more. In the group which failed only one reached the intensity of 50% VO2max. The working capacity of 50% VO2max or greater would appear to be a valid initial guideline level of physical fitness at which an amputee can expect to succeed in walking with a prosthesis. Apart from physical fitness, a lesser number of comorbidity, good ability to stand on the remaining leg, and a strong will to walk were found to be important factors contributing to successful prosthetic rehabilitation. This study also showed that age alone was not an important factor.

Modulation of human mammary cell sensitivity to paclitaxel by new quinoline sulfonamides.

Sulfonamide derivatives of chloroquine and primaquine were synthesised and evaluated against both paclitaxel-sensitive and paclitaxel-resistant mammarian cancer cell lines. All derivatives exhibited at least 96% MDR reversal activity when co-administered with paclitaxel at 5 microM. The best compound, a chloroquine derivative, exhibited 99% MDR reversal activity when co-administered with paclitaxel at 1 microM. Molecular modelling studies reveal that these derivatives share a common pharmacophore with taxane MDR reversal agents.

Tubulins in the primate retina: evidence that xanthophylls may be endogenous ligands for the paclitaxel-binding site.

The xanthophylls-lutein, zeaxanthin, and meso-zeaxanthin (L&Z)-are found in the central region of the primate retina, which is called the macula lutea (yellow spot). How they are anchored there and what their function is has been debated for over 50 years. Here, we present evidence that they may be bound to the paclitaxel (Taxol) binding site of the beta-tubulin subunit of microtubules and that a major function may be to modulate the dynamic instability of microtubules in the macula. Also, we compare nucleic acid and amino acid sequences of tubulins that are in human brain with those we have isolated from human-retina and monkey-macula cDNA libraries. In so doing, we suggest that in primates, class I beta-tubulin consists of at least two subtypes (beta(Ia) and beta(Ib)). Alignment analysis of the sequences of the genes for beta(Ia) and beta(Ib) indicates that the corresponding mRNAs may have other functions in addition to that of coding for proteins. Furthermore, we show that there are at least five different types of beta-tubulin in the macula lutea of rhesus monkey.

Effects of orally active taxanes on P-glycoprotein modulation and colon and breast carcinoma drug resistance.

BACKGROUND: The taxane paclitaxel (Taxol) is often of limited efficacy in chemotherapeutic regimens because some cancer cells express high levels of the efflux pump, P-glycoprotein (Pgp), which removes the drug from the cells. The orally active paclitaxel analog IDN-5109 has been reported to overcome Pgp-mediated drug resistance. We tested whether IDN-5109 acts by modulating Pgp activity. METHODS: Human MDA435/LCC6mdr1 and MDA435/LCC6 breast carcinoma cells, which express and do not express Pgp, respectively, were incubated with [3H]IDN-5109 and paclitaxel to determine intracellular drug accumulation. Flow cytometry was used to analyze intracellular retention of two Pgp substrates, rhodamine 123 (Rh-123) and doxorubicin, in both breast carcinoma cell lines and in human colon carcinoma cells (SW-620, DLD1, and HCT-15, whose Pgp levels vary) treated with different taxanes. The effects of IDN-5109 and paclitaxel on tumor growth in vivo were studied with the use of tumors established through xenografts of Pgp-expressing SW-620 and DLD1 cells in severe combined immunodeficiency mice. All statistical tests were two-sided. RESULTS: Pgp-expressing cells treated with IDN-5109 or with the taxane-based drug resistance reversal agent tRA96023, which blocks Pgp activity, retained 8.1- and 9.4-fold more Rh-123 (P =.0001), respectively, and 1.7- and 1.9-fold more doxorubicin (P =.001), respectively, than cells treated with paclitaxel. Non-Pgp-expressing cells treated similarly demonstrated no increased retention of either substrate. MDA435/LCC6mdr1 cells retained 5.3-fold more [3H]IDN-5109 than [3H]paclitaxel after 2 hours (P =.01). IDN-5109 showed statistically significantly higher tumor growth inhibition than paclitaxel against the SW-620 xenograft (P =.003). CONCLUSIONS: IDN-5109 modulates Pgp activity, resulting in superior tumor growth inhibition against Pgp-expressing tumors as compared with paclitaxel. IDN-5109 may broaden the spectrum of taxane use to include colon tumors.

Novel DMAP-catalyzed skeletal rearrangement of 5-exo-(2-hydroxy-ethylene)oxasilacyclopentanes.

[reaction: see text] 5-exo-(Hydroxyethylene)-2-oxa-1-silacyclopentanes are found to undergo a novel DMAP-catalyzed skeletal rearrangement through silicon-oxygen exchange during acetylation to yield the corresponding 5-(2-acetoxyalkyl)-2-oxa-1-silacyclopent-4-enes exclusively. The mechanism for this unprecedented rearrangement is proposed.

The use of a novel taxane-based P-glycoprotein inhibitor to identify mutations that alter the interaction of the protein with paclitaxel.

Murine thymoma cell lines expressing mutated forms of the mdr1b P-glycoprotein were isolated using a novel taxane-based P-glycoprotein inhibitor tRA-96023 (SB-RA-31012). The selection strategy required resistance to a combination of tRA-96023 and colchicine. Five mutations were identified (N350I, I862F, L865F, L868W, and A933T) that reduce the capacity of tRA-96023 to inhibit P-glycoprotein-dependent drug resistance. These mutations also result in a loss of paclitaxel resistance ranging from 47 to 100%. Four mutations are located in the second half of the protein, within or near the proposed transmembrane segment (TMS) 10--11 regions. The fifth mutation (N350I) is within the first half of the protein, proximal (cytoplasmic) to TMS 6. The variant cell line expressing the L868W mutation was subjected to a second round of selection involving tRA-96023 and the toxic drug puromycin. This resulted in the isolation of a cell line expressing a P-glycoprotein with a double mutation. The additional mutation (N988D) is located within TMS 12 and conveys further decreases in resistance to paclitaxel and the capacity of tRA-96023 to inhibit drug resistance. Taken together, the results indicate a significant contribution by the TMS 10--12 portion of the protein to the recognition and transport of taxanes and give evidence that the cytoplasmic region proximal to TMS 6 also plays a role in taxane interactions with P-glycoproteins. Interestingly, mutations within TMS 6 and 12 were found to cause a partial loss of PSC-833 inhibitor activity, suggesting that these regions participate in the interactions with cyclosporin and its derivatives.

Desymmetrization of 4-dimethylsiloxy-1,6-heptadiynes through sequential double silylformylation.

[reaction in text] Desymmetrization of dimethylsilyloxyalkadiynes (1) by Rh-catalyzed intramolecular silylformylation affords 5-exo-(formylmethylene)oxasilacyclopentanes 2 in high yields. Novel sequential double silylformylation of 1a also provides desymmetrization, giving 3-(3-silyl-2-formylprop-2-enyl)-5-exo-(formylmethylene)oxasilacyclopentanes 4 in excellent yields. Reduction of 2a and 4 with NaBH(4) gives the corresponding 5-exo-(hydroxymethylmethylene)oxasilacyclopentanes 3a and 5, respectively.

Effect of endurance training program based on anaerobic threshold (AT) for lower limb amputees.

We have already reported that the one-leg cycling test driven by the subject's sound leg as the exercise load test is effective in measuring the anaerobic threshold (AT) of unilateral lower limb amputees. The aim of this research is to investigate whether or not endurance training based on each subject's AT gained from the one-leg cycling test is useful in improving the physical fitness of lower limb amputees. The test subjects were all unilateral transfemoral amputees comprising a group of 14 undertaking endurance training and a control group of 10. The form of endurance training is driving an ergometer with the sound limb only in the same way as the load test. The training program was designed so that the subjects would exercise at a target heart rate corresponding to AT point for 30 minutes per day, 3-5 days each week for 6 weeks. After the training periods, in the training subjects the AT and maximum oxygen uptake (__O2max) increased significantly. The rate of increase averaged 36.5%, 26.0%, respectively, compared to their levels before the training. On the contrary, no changes occurred in the control subjects. These results suggest that our chosen training program based on each subject's AT is effective in improving the physical fitness of lower limb amputees.

Chemistry and chemical biology of taxane anticancer agents.

Taxol (paclitaxel) and Taxotere (docetaxel) are currently considered to be among the most important anticancer drugs in cancer chemotherapy. The anticancer activity of these drugs is ascribed to their unique mechanism of action, i.e., causing mitotic arrest in cancer cells, leading to apoptosis through inhibition of the depolymerization of microtubules. Although both paclitaxel and docetaxel possess potent antitumor activity, treatment with these drugs often results in a number of undesirable side effects, as well as multidrug resistance (MDR). Therefore, it has become essential to develop new anticancer agents with superior pharmacological properties, improved activity against various classes of tumors, and fewer side effects. This paper describes an account of our research on the chemistry of paclitaxel and taxoid anticancer agents at the biomedical interface, including: 1. The structure-activity relationship (SAR) study of taxoids leading to the development of the "second-generation" taxoids, which possess exceptional activity against drug-resistant cancer cells expressing the MDR phenotype. 2. Development of fluorinated taxoids to study the bioactive conformation of paclitaxel and photoaffinity labeling taxoids for mapping of the drug-binding domain on both microtubules and P-glycoprotein. 3. The synthesis of novel macrocyclic taxoids for the investigation into the common pharmacophore for microtubule stabilizing anticancer agents.

Antitumour activity of novel taxanes that act at the same time as cytotoxic agents and P-glycoprotein inhibitors.

Taxanes antitumour agents such as paclitaxel and docetaxel represent a successful family of chemotherapeutic drugs. Unfortunately, acquired and innate resistance represents a clinical problem for these drugs. We investigated, on a panel of 7 human cancer cell lines, the growth inhibition effect of 3 newly developed taxanes (SB-T-1213, SB-T-1250 and SB-T-101187) with modification at the C10 and C3' positions of the taxane framework. These positions have been previously characterized as critical to make taxanes highly active against cells overexpressing the efflux pump P-glycoprotein (P-gp). Paclitaxel and docetaxel were used as reference compounds. Results unambiguously indicate the exceptional activity of the novel taxanes toward P-gp positive cells (up to >400 fold higher potency than that of paclitaxel). SB-T-1213 and SB-T-1250 are also substantially more active than the reference compounds against P-gp negative cells. To better understand the mechanisms underlying the enhanced activity of the newly developed taxanes, we performed cell cycle and apoptosis analysis. This study demonstrates that the striking growth inhibition effect exhibited by the novel taxanes is ascribed to their increased ability in inducing apoptosis and G(2)/M cell cycle block. SB-T-1213 and SB-T-1250 are also more active than reference compounds in inducing intracellular accumulation of the beta-tubulin subunits. Finally, it is revealed that these novel taxanes have ability to inhibit the function of the P-gp efflux pump on the basis of the Rhodamine 123 assay. These findings strongly suggest that SB-T-1213, SB-T-1250 and SB-T-101187 represent a new tool to overcome innate or acquired P-gp mediated taxane-resistance.

Syntheses and biological activity of C-3'-difluoromethyl-taxoids.

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40-70 times better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative cancer cells.

Structural significance of the acyl group at the C-10 position and the A ring of the taxane core of paclitaxel for inducing nitric oxide and tumor necrosis factor production by murine macrophages.

The antitumor agent, paclitaxel (Taxol), mimics the actions of lipopolysaccharide (LPS) on murine macrophages (Mphi). Various synthetic analogs of paclitaxel were examined for their potencies to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by murine peritoneal Mphi, and by human peripheral blood cells. The benzoyl group at C-2, the hydroxy group at C-7 and the acetyl group at C-10 were found to be critically important sites to activate murine Mphi. Nor-seco-taxoid analogs lacking the A ring of the taxane core of paclitaxel were inactive, but inhibit paclitaxel- or LPS-induced NO production. All the compounds tested did not induce TNF production by human blood cells.

Synthesis and evaluation of 6-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine as a PET tracer for nicotinic acetylcholine receptors.

Both ABT-594 ((R)-2-chloro-5-(2-azetidinylmethoxy)pyridine) and A-85380 (3-[2(S)-2-azetidinylmethoxy]pyridine), novel nicotinic agonists that possess potent non-opioid analgesic properties, have high affinity for neuronal nicotinic acetylcholine receptors (nAChR) but do not elicit the pronounced toxicity of epibatidine. 6-[(18)F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[(18)F]fluoro-A-85380), a F-18 labeled analogue of these two compounds, is therefore a promising radioligand for positron emission tomography (PET) studies in humans. The use of trimethylammonium as a leaving group in nucleophilic aromatic substitution reactions has proven to be a versatile and efficient strategy, and offers several advantages over other leaving groups. Here, we report the synthetic strategy for the preparation of a precursor, as a trimethylammonium iodide salt, and its use in the radiosynthesis to 6-[(18)F]fluoro-A-85380. Preliminary compartative PET studies of 6-[(18)F]fluoro-A-85380 and 2-[(18)F]fluoro-A-85380 were carried out in baboon to examine their suitability as tracers for studying nAChR system.