In situ blood-brain barrier permeability of a C-10 paclitaxel carbamate

We report the synthesis and blood-brain barrier (BBB)-permeability of (14)C-CNDR-29, a paclitaxel C-10 carbamate derivative shown to be devoid of P-glycoprotein (Pgp)-interactions, in an in situ mouse brain perfusion model, in comparison with (14)C-paclitaxel. The results presented reveal a 3- to 4-fold higher BBB-permeability for the C-10 modified taxane compared to paclitaxel. These results support the notion that circumvention of Pgp-mediated efflux can lead to higher BBB-permeability. Further studies however are needed to evaluate the therapeutic potential of the C-10 carbamates paclitaxel derivatives for the treatment of CNS diseases.     

Paclitaxel succinate analogs: Anionic and amide introduction as a strategy to impart blood-brain barrier permeability

A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers, esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interactions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. Calculations of logD and cross-sectional areas revealed that these analogs are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.     

A facile route to paclitaxel C-10 carbamates

A general protocol for the synthesis of paclitaxel C-10 carbamates is described. The method entails MeI-mediated activation of 2'-O-TBS-7-O-TES-10-O-deacetyl-paclitaxel-10-O-carbonylimidazole prior to reaction with amines. This method is effective for the synthesis of paclitaxel C-10 derivatives, including bifunctional molecules.     

NSC23925, Identified in a High-Throughput Cell-Based Screen,Reverses Multidrug Resistance

Background

Multidrug resistance (MDR) is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.

Methodology/Principal Findings

To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1) but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 µM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.

Conclusions/Significance

The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.     

The synthesis of novel taxoids for oral administration

A group of novel taxoids, with modifications at C-7, C-10, C-3′ and C-14 positions of paclitaxel, was synthesized in order to improve their biological profile by decreasing their affinity with P-glycoprotein (P-gp) and increasing cellular permeability. Most of the new taxoids demonstrated the similar potent cytotoxic activities in MCF-7 human tumor cell line as paclitaxel in vitro. In the permeability assay with monolayers of Caco-2 cells, most of the compounds demonstrated an increased trans-cellular transport in A-to-B direction in comparison with paclitaxel. Among them the compounds T-13, T-15 and T-26 showed the highest permeability, and with efflux ratios better than that of ortataxel. The interaction of the compounds T-13 and T-26 with P-gp was evaluated using Madin-Darby canine kidney (MDCK)-multidrug resistance-1(MDR1) and MDCK-wild-type (WT). The results indicated that T-13 and T-26 were poor substrates for P-gp and possessed inhibiting effects of P-gp mediated efflux. It was thus clear that simultaneous modifications at the C-7, C-10 and C-3′ positions of paclitaxel significantly impaired its interactions with P-gp and interfered with P-gp mediated efflux.     

Synthesis and biological evaluation of C-3'NH/C-10 and C-2/C-10 modified paclitaxel analogues

Concurrent modifications on the C-3'NH/C-10, and C-2/C-10 positions on paclitaxel were carried out as a way of investigating possible synergistic effects. The biological activities of these analogues were evaluated in both a microtubule assembly assay and human ovarian cancer (A2780) and prostate cancer (PC3) cytotoxicity assay. In some cases the doubly modified analogues were more active than would have been predicted based on the activity of the singly modified analogues, indicating probable synergistic effects.     

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 microtubule binding of fluorescent paclitaxel derivatives

The preparation of two new fluorescent derivatives of paclitaxel in which the fluorophore is bonded to paclitaxel at the C-10 position is reported. Both analogues, 10-deacetyl-10-(m-aminobenzoyl)paclitaxel (1, BTax) and 10-deacetyl-10-[7-(diethylamino) coumarin-3-carbonyl]paclitaxel (2, CTax) retain good activity as promoters of in vitro tubulin assembly. Microtubule binding enhances the emission intensity of both probes.     

Synthesis and structure-activity relationships of new second-generation taxoids

A series of second-generation taxoids bearing a substitutent on the C-2-benzoyl group and modifications at C-3′/C-10 positions was synthesized. These taxoids exhibited 2–3 orders of magnitude higher potency than that of paclitaxel against drug-resistant human breast cancer cell lines. It is also noteworthy that three taxoids showed almost no difference in activity against drug-resistant and drug-sensitive cell lines, which are categorized as “advanced second generation taxoids“.     

Quercetin significantly decreased cyclosporin oral bioavailability in pigs and rats

Cyclosporin, an immunosuppressant with a narrow therapeutic window, is a substrate for both CYP3A4 and P-glycoprotein (Pgp). Quercetin is an inhibitor of CYP3A4 and a modulator of Pgp. This study aimed to measure the effect of quercetin on the absorption and disposition of cyclosporin in pigs and rats. Cyclosporin (Sandimmune, 10 mg/kg) was orally administered with and without a concomitant dose of quercetin (50 mg/kg) to pigs and rats. Cyclosporin concentrations in blood samples were determined by a specific monoclonal fluorescence polarization immunoassay. The pharmacokinetic parameters were calculated by noncompartmental analysis using WINNONLIN. A paired Student's t-test was conducted for statistical comparison. A study using the everted intestinal sac was carried out to evaluate the effect of quercetin on the function of intestinal Pgp. The coadministration of quercetin significantly decreased cyclosporin AUC(0-3) (area under the concentration-time curve from time zero to 3 h) by 56% and AUC(0-t) (area under the concentration-time curve from time zero to the last point) by 43% in pigs and rats, respectively, indicating that the coadministration of quercetin significantly decreased cyclosporin oral bioavailability. However, the inverted sac study showed that quercetin significantly inhibited the function of intestinal Pgp. It is suggested that concurrent use of quercetin or quercetin-containing dietary supplement or herbs with cyclosporin or other medications whose absorption and metabolism are mediated by Pgp and/or CYP3A4 should require close monitoring.     

Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract

Prolonged chemotherapy may lead to the selective proliferation of multidrug resistant (MDR) cancer cells. In MDR HepG2-DR and K562-DR cells that over-expressed P-glycoprotein (Pgp), the extract of the rhizomes of Alisma orientalis (Sam) Juzep. showed a synergistic growth inhibitory effect with cancer drugs that are Pgp substrates including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. At the same toxicity levels the herbal extract was more effective than verapamil, a standard Pgp inhibitor, in enhancing cellular doxorubicin accumulation and preventing the efflux of rhodamin-123 from the MDR cells. The extract restored the effect of vinblastine on the induction of G(2)/M arrest in MDR cells. Our data suggest that A. orientalis may contain components that are effective inhibitors of Pgp.     

Monte Carlo docking simulations of cyclomaltoheptaose and dimethyl cyclomaltoheptaose with paclitaxel

The molecular basis for the remarkable enhancement of the solubility of paclitaxel by O-dimethylcyclomaltoheptaose (DM-beta-CD) over cyclomaltoheptaose (beta-cyclodextrin, beta-CD) was investigated with Monte Carlo docking-minimization simulation. As possible guests of inclusion complexation for the host cyclic oligosaccharides, two functional moieties of the suggested solution structure of paclitaxel were used where one is the C-3'N benzoyl moiety (B-ring) and the other is a hydrophobic (HP) cluster site among the C-3' phenyl (C-ring), C-2 benzoate (A-ring), and C-4 acetoxy moieties. The energetic preference of inclusion complexation of DM-beta-CD over beta-CD was analyzed on the basis of more efficient partitioning process of DM-beta-CD into the hydrophobic cluster site of the paclitaxel.     

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.     

The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site

We examined interactions with purified tubulin of synthetic sarcodictyins A and B and eleutherobin (coral-derived antimitotic agents) and of compound 1, an analogue of sarcodictyin A methylated at the C-3 oxygen atom (i.e., the methyl ketal analogue of sarcodictyin A and thus structurally similar to eleutherobin but lacking the C-3 sugar moiety). Eleutherobin was much more active than sarcodictyins A and B, which were somewhat more active than compound 1. Effects of eleutherobin did not differ greatly from those of paclitaxel and epothilone A. Eleutherobin and epothilone A were competitive inhibitors of the binding of radiolabeled paclitaxel to tubulin polymer (apparent Ki values of 2.1 and 2.6 microM, respectively). Tubulin assembly reactions induced by all compounds were similar to the paclitaxel-driven reactions in being enhanced by the addition of microtubule-associated proteins and/or GTP to the reaction mixture and by progressively higher reaction temperatures. Antiproliferative activity was studied in six human cancer cell lines, including two paclitaxel-resistant lines with point mutations in a beta-tubulin gene. Except for compound 1, effects on cell growth were generally in accord with effects on purified tubulin. Thus, sarcodictyins A and B had IC50 values in the 200-500 nM range; paclitaxel, <10 nM (except in the resistant lines); and eleutherobin and epothilone A, 10-40 nM. The antiproliferative activity of compound 1 was more comparable to that of eleutherobin than sarcodictyin A, despite its weak interaction with tubulin. The activities of the sarcodictyins, eleutherobin, and compound 1 in the mutant ovarian lines were similar to their activities in the parental line.     

The discovery of BMS-275183: an orally efficacious novel taxane

The evolution of 2, a C-4-methylcarbonate analogue of paclitaxel with minimal oral bioavailability and oral efficacy, into its C-3'-t-butyl-3'-N-t-butyloxycarbonyl analogue (15i), a novel taxane with oral efficacy in preclinical models that is comparable to iv administered paclitaxel, is described.     

Novel C-seco-taxoids possessing high potency against paclitaxel-resistant cancer cell lines overexpressing class III β-tubulin

Novel C-seco-taxoids were synthesized from 10-deacetylbaccatin III and their potencies evaluated against drug-sensitive and drug-resistant cancer cell lines. The drug-resistant cell lines include ovarian cancer cell lines resistant to cisplatin, topotecan, adriamycin and paclitaxel overexpressing class III β-tubulin, A2780TC1 and A2780TC3. The last two cell lines were selected through chronic exposure of A2780wt to paclitaxel and Pgp blocker cyclosporine. All novel C-seco-taxoids exhibited remarkable potency against A2780TC1 and A2780TC3 cell lines, and no cross resistance to cisplatin- and topotecan-resistant cell lines, A2780CIS and A2780TOP. Four of those C-seco-taxoids exhibit much higher activities than IDN5390 against paclitaxel-resistant cell lines, A2780ADR, A2780TC1 and A2780TC3. SB-CST-10202 possesses the best all-round high potencies across different drug-resistant cell lines. Molecular modeling studies, including molecular dynamics simulations, on the drug-protein complexes of class I and III β-tubulins were performed to identify possible cause of the remarkable potency of these C-seco-taxoids against paclitaxel-resistant cell lines overexpressing class III β-tubulin.     

Functionalized chalcones as selective inhibitors of P-glycoprotein and breast cancer resistance protein

A library of chalcones with basic functionalities were screened for inhibition of P-glycoprotein (Pgp, ABCB1) by the calcein-AM accumulation assay on MDCKII/MDR1 cells. Three members that had ring A substituted with 5-(1-ethylpiperidin-4-yl) and 2,4-dimethoxy groups were found to increase calcein-AM accumulation to a greater extent than verapamil, a Pgp inhibitor. These compounds were subsequently shown to enhance the uptake of doxorubicin by MCF-7 cells that over-expressed Pgp. However, when tested for inhibition of the breast cancer resistance protein (BCRP, ABCG2) by the mitoxantrone uptake assay, the same compounds fared poorly. In comparison, a non-basic chalcone (5-14, 3-(4-chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-en-1-one) increased mitoxantrone uptake by BCRP over-expressing MCF-7 cells (MCF-7/MX) by more than 300% at 5 microM. Thus, introducing a basic group on the chalcone template enhanced Pgp inhibition at the expense of BCRP inhibition. The basic chalcones were also better Pgp inhibitors than their non-basic counterparts which may in turn be better BCRP inhibitors. Structure activity analysis showed that lipophilicity of the chalcones was not the overriding factor for Pgp inhibitory activity. Rather, good activity was associated with appropriately placed electron donor atoms, of which the meta-disubstituted dimethoxy motif on either ring A or B was of particular relevance. In spite of differing structural requirements for inhibition of Pgp and BCRP, chalcone 3-100 [3-(2,4-dimethoxyphenyl)-1-(4-(piperazin-1-yl)phenyl)prop-2-en-1-one] inhibited both Pgp and BCRP to a reasonable extent and may be a useful starting point for the design of dual inhibitors.     

A novel 2'-(N-methylpyridinium acetate) prodrug of paclitaxel induces superior antitumor responses in preclinical cancer models

The development of novel strategies for the treatment of malignancies by successful intervention in advanced stage disease is a major challenge in oncology. We tested the hypothesis that this can be achieved by the rational design of taxoid onium salts modified at C-7 and C-2' positions. The characterization of these molecules revealed a dramatically improved water solubility and prodrug behavior in plasma. Specifically, all compounds released parental paclitaxel with half-lives ranging from 0.9 to 180 min. In the absence of plasma, only the 2'-(N-methylpyridinium acetate) derivative of paclitaxel (2'-MPA-paclitaxel) revealed a complete abrogation of paclitaxel specific microtubule assembly disassembly dynamics and a 3 log reduction in cellular binding, indicating that reversible blockage of the C-2' position by methylpyridinium acetate yields a true paclitaxel prodrug. Structure/activity profiles of all compounds in tissue culture revealed cytotoxicity effective at picomolar concentrations with a panel of 16 cancer cell lines in contrast to 4 nonmalignant cell lines. Importantly, the decisive cytotoxic potential observed in vitro for all compounds correlated only with in vivo findings for 2'-MPA-paclitaxel. Specifically, the 2'-MPA-paclitaxel prodrug induced regression of primary tumors in three xenograft models of nonsmall cell lung carcinoma, ovarian carcinoma and prostate cancer, in contrast to ineffective C-7 derivatives and parental paclitaxel. At the same time, a reduced systemic toxicity of 2'-MPA-paclitaxel was observed in contrast to a far more toxic parental paclitaxel. Taken together, these findings demonstrate that the 2'-MPA-paclitaxel prodrug is a promising new candidate for cancer therapy.     

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.