Paclitaxel Conjugate Research Articles

Polyisobutylene‐paclitaxel conjugates with pendant carboxylic acids and polystyrene chains: Towards multifunctional stent coatings with slow drug release

ABSTRACTDrug‐eluting stents are used in the treatment of atherosclerosis, where the incorporation of anti‐proliferative or anti‐inflammatory drugs decreases the rate of restenosis, the recurrence of artery narrowing. However, these stents can suffer from limitations such as drug depletion and delamination of the drug‐eluting coating from the stent surface. Described here is an approach aimed at addressing these issues. Starting from a maleic anhydride adduct of polyisobutylene (PIB) prepared from butyl rubber, ring opening using paclitaxel (PTX) or a combination of PTX and polystyrene (PS) afforded covalent conjugates of PTX and PIB or PIB‐PS graft copolymers bearing pendant carboxylic acids. When coated on stainless steel, the drug release was slower than that from a control coating that ressembles a clinical formulation comprising a physical mixture of a PS‐PIB‐PS triblock copolymer (SIBS) and PTX. The PTX conjugates also exhibited enhanced adhesion to stainless steel and increased tensile strength in comparison with the starting rubber. Cytotoxicity assays indicated that the materials did not leach toxic levels of PTX into cell culture media. Nevertheless, they were capable of inhibiting the adhesion and proliferation of C2C12 cells on their surfaces. These properties are advantageous for the potential application of the materials as stent coatings. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 2209–2219

Macromolecular conjugates of paclitaxel: Synthesis, characterization, andIn Vitropaclitaxel release studies based on HPLC validated method

Biodegradable and bioresorbable macromolecular conjugates of paclitaxel were prepared. The release of drug from conjugates has been carried out in vitro at 37 °C in phosphate buffered saline solution (PBS, pH 7.4), with 0.1% (w/v) Cremophor® EL. Periodically, all the solution in the samples was removed and replaced with fresh buffer until limit of detection (LOD) of paclitaxel was released. The quantity of released drug was analyzed by means of high-performance liquid chromatography (HPLC) method. Chromatographic separations were conducted using the NUCLEODUR C18 Gravity column with a guard column at 30 °C. Mobile phase consisted of a mixture of acetonitrile, methanol, and deionized water (60:2:38). The flow rate was 1.0 mL min−1, and paclitaxel was detected at 229 nm, retention time of 3.5 min. The applied analytical method was validated according to International Conference on Harmonization (ICH) procedures or recommendations. The chromatographic separation was excellent. The linearity in the range 0.1–...

Anticancer activity of drug conjugates in head and neck cancer cells.

Sexually transmitted oral cancer/head and neck cancer is increasing rapidly. Human papilloma virus (HPV) is playing a role in the pathogenesis of a subset of squamous cell carcinoma of head and neck (SCCHN). Paclitaxel is a widely used anticancer drug for breast, ovarian, testicular, cervical, non-small cell lung, head and neck cancer. However, it is water insoluble and orally inactive. We report the synthesis of water soluble nanosize conjugates of paclitaxel, branched PEG, and EGFR-targeting peptide by employing native chemical ligation. We performed a native chemical ligation between the N-hydroxy succinimide (NHS) ester of paclitaxel succinate and cysteine at pH 6.5 to give the cysteine-conjugated paclitaxel derivative. The thiol functionality of cysteine was activated and subsequently conjugated to multiarm thiol-PEG to obtain the paclitaxel branched PEG conjugate. Finally, we conjugated an EGFR-targeting peptide to obtain conjugates of paclitaxel, branched PEG, and EGFR-targeting peptide. These conjugates show anticancer activity against squamous cell carcinoma of head and neck cells (SCCHN, Tu212).

Molecular nanofibers of paclitaxel form supramolecular hydrogel for preventing tumor growth in vivo

A conjugate of tripeptide RGD and paclitaxel can form a nanofibrous hydrogel with a high payload of 66.7%, which can serve as both carrier and cargo for delaying tumor growth in vivo.

Micellar delivery of paclitaxel using a glucosamine-based glycopolymer for the treatment of prostate cancer

Event Abstract Back to Event Micellar delivery of paclitaxel using a glucosamine-based glycopolymer for the treatment of prostate cancer Alice Du1, Hongxu Lu2 and Martina H. Stenzel2 1 University of New South Wales, Centre for Advanced Macromolecular Design, School of Chemical Engineering, Australia 2 University of New South Wales, Centre for Advanced Macromolecular Design, School of Chemistry, Australia Introduction: The use of carbohydrates in the synthesis of polymeric nanoparticles has garnered interest in recent years, especially for its potential use as a therapeutic delivery vehicle for cancer treatment. For example, recent work reported by our group utilised the natural targeting ability of fructose decorated micelles for the treatment of triple-negative breast cancer[1]. Another carbohydrate of note is chitosan, a glucosamine-based glycopolymer which has shown potential in oral delivery approaches. However, one of the major disadvantages of chitosan lies in the handling difficulty of the macrostructure and control over its molecular weight. Hence, it is the aim of this study to synthesize a glucosamine monomer which is capable of undergoing controlled polymerization. Subsequent drug loading, micelle formation and in vitro studies will be used to evaluate the efficacy of the glycopolymer micellar system as a delivery vehicle for the treatment of prostate cancer. Materials and Methods: The solubility of glucosamine in organic solvents was increased through protection of its hydroxyl and amino functional groups. 2-hydroxyethyl acrylate was used to introduce alkene functionality to afford the protected glucosamine monomer (GluHEA). The polymerisation of GluHEA was controlled using a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent chain extension was completed using 2-carboxyethyl acrylate (CEA). Paclitaxel was chemically conjugated to the CEA block and used to introduce hydrophobicity to the block. Deprotection of the glucosamine will then afford the hydrophilic block, thereby inducing micelle formation. In vitro biological studies will be conducted where the toxicity of the micelles will be evaluated against both monolayer cell culture as well as multicellular tumor spheroids. In addition, the migratory ability of the glucosamine-coated nanoparticles through Caco-2 (epithelial) cells will be investigated and compared to chitosan. Results: Synthesis of PGluHEA26-b-PCEA64 was successfully completed and controlled using 3-(benzylsulfanylthiocarbonyl sulfanyl)-propionic acid (BSPA) as the RAFT chain transfer agent (polydispersity ~1.3). Subsequent conjugation of paclitaxel to the carboxylic acid groups on the PCEA block was also successful. Conclusion: It is envisioned that the glucosamine coated micelles will offer a viable alternative to chitosan as an enhanced transcell delivery vehicle. In addition, the synthesis of a glucosamine monomer allows the structure of the block copolymer to be finely controlled, a characteristic not associated with naturally derived chitosan.

Tumor-selective peptide-carrier delivery of Paclitaxel increases in vivo activity of the drug.

Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy, and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively, and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo. NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity.

Abstract B43: ANG1005, a novel brain-penetrant drug conjugate, in CNS metastases from breast cancer: FLT-PET imaging as a predictor of response

Abstract Background: The novel drug conjugate ANG1005 consists of 3 molecules of paclitaxel covalently linked to Angiopep-2. After binding to the low-density lipoprotein receptor-related protein, ANG1005 crosses the blood brain barrier (BBB) by endocytosis. A multi-center Phase II study, with the primary endpoint of intracranial response in patients with breast cancer brain metastases is in progress. At the NCI, a biomarker substudy is evaluating 18F-FLT (3'-Fluoro-3' deoxythymidine)-PET for response assessment. Methods: Patients with measurable brain metastases from breast cancer received ANG1005 at a dose of 550mg/m2 IV once every 21 days. Before and after cycle 1, all patients on study underwent imaging with 18F-FLT, a thymidine analog and novel imaging agent; retention of 18F-FLT correlating with DNA synthesis. In order to detect brain metastases and assess response to treatment, we compared FLT PET images with MRI-gadolinium contrast scans, obtaining both dynamic and static images. We determined the percentage (%) change after treatment with ANG1005; a decrease in ≥20% was considered significant. Results: Ten patients were enrolled on study. A total of 32 target and 20 non target CNS lesions in 10 patients were imaged and analyzed by MRI and FLT-PET. At 30 minutes, SUVmax ranged from 0.8 to 6.3 at baseline (mean 2.64), and the SUV80%, (mean of top 20% SUV units) ranged from 0.7 to 5.12. The median% change from baseline to post one cycle of ANG1005 was -19.9% for SUVmax (range +85.4 to -67.7, and was median -20.2% for SUV80% (range 95.7 to -69%). Two patients had confirmed partial responses lasting 6 and 18 cycles, respectively. Six patients had stable disease and received a median of 6 cycles. Tumor reductions, as determined by MRI per CNS RECIST version 1.1 ranged from -5% to -60% in lesion size, compared to baseline. Both FLT-PET parameters correlated with the% change in MRI measurements in the target lesions (R2 = 0.64, P = 0.0002). Conclusion: The development of CNS-directed therapies designed to cross the BBB, such as the paclitaxel conjugate ANG1005, is a research priority. As contrast-enhanced MRI detection of brain metastases is representative of gadolinium leakage through the BBB, as opposed to actual tumor volume, better approaches are needed to evaluate drug efficacy. Pilot evaluations of FLT-PET imaging in this setting suggest that it is a promising tool that may serve as a complementary assessment method for breast cancer brain metastases going forward. Citation Format: Ciara C. O'Sullivan, Maria Lindenberg, Christine Bryla, Nicole Davarpanah, Cody Peer, Nicholas Patronas, Laleh Amiri-Kordestani, Sanjeeve Balasubramaniam, Tito Fojo, William D. Figg, Peter Choyke. ANG1005, a novel brain-penetrant drug conjugate, in CNS metastases from breast cancer: FLT-PET imaging as a predictor of response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B43.

PH-Sensitive Biocompatible Nanoparticles of Paclitaxel-Conjugated Poly(styrene-co-maleic acid) for Anticancer Drug Delivery in Solid Tumors of Syngeneic Mice

In the present study, we have synthesized poly(styrene-co-maleic anhydride), a biocompatible copolymer that was further conjugated with paclitaxel (PTX) via ester linkage and self-assembled to form poly(styrene-co-maleic acid)-paclitaxel (PSMAC-PTX) nanoparticles (NPs). The in vitro release of PTX from PSMAC-PTX NPs showed a higher release at lower pH than at the physiological pH of 7.4, confirming its pH-dependent release. The cell viability of PSMAC-PTX nanoparticles was evaluated using MTT assay. IC50 values of 9.05-18.43 ng/mL of PTX equivalent were observed in various cancer cell lines after 72 h of incubation. Confocal microscopy, Western blotting, and Flow cytometry results further supported that the cellular uptake and apoptosis of cancer cells with PSMAC-PTX NPs. Pharmacokinetic studies revealed that the conjugation of PTX to the PSMAC co-polymer not only increased the plasma and tumor C(max) of PTX but also prolonged its plasma half-life and retention in tumor via enhanced permeability and retention (EPR) effect. Administration of PSMAC-PTX NPs showed significant tumor growth inhibition with improved apoptosis effects in vivo on Ehrlich Ascites Tumor (EAT)-bearing BALB/c syngeneic mice in comparison with Taxol, without showing any cytotoxicity. On the basis of preliminary results, no subacute toxicity was observed in major organs, tissues and hematological system up to a dosage of 60 mg/kg body weight in mice. Therefore, PSMAC-PTX NPs may be considered as an alternative nanodrug delivery system for the delivery of PTX in solid tumors.

Synthesis and properties of arborescent polyisobutylene derivatives and a paclitaxel conjugate: Towards stent coatings with prolonged drug release

Polyisobutylene (PIB) and its copolymers are used in a wide range of commercial products owing to their high chemical stability, impermeability, elasticity, and biocompatibility. The development of arborescent PIB (arb-PIB) opens many new possibilities for tuning PIB’s properties and for introducing new functionalities. In this work, arb-PIB with short isoprene-rich terminal sequences (arb-PIB-co-IP) was functionalized to provide arborescent epoxide, allylic alcohol, and carboxylic acid derivatives of PIB. The carboxylic acid derivative was used to conjugate the antiproliferative agent paclitaxel (PTX) for investigation as a potential vascular stent coating. The thermal, tensile, and rheological properties of all of the functionalized arb-PIB materials were studied and compared to their linear analogues in order to gain insight into the effects of polymer architecture on these properties as well as to determine their suitability as potential medical device coatings. A coating using the PTX conjugate was found to release PTX much more slowly than control formulations with physically encapsulated drug, yet was still able to prevent cell adhesion and proliferation on their surfaces.

Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid

Polymer–drug conjugates have demonstrated application potentials in optimizing chemotherapeutics. In this study a new bioconjugate, HA-ss-PTX, was designed and synthesized with cooperative dual characteristics of active tumor targeting and selective intracellular drug release. Paclitaxel (PTX) was covalently attached to hyaluronic acid (HA) with various sizes (MW 9.5, 35, 770kDa); a cross-linker containing disulfide bond was also used to shield drug leakage in blood circulation and to achieve rapid drug release in tumor cells in response to glutathione. Incorporation of HA to the conjugate enhanced the capabilities of drug loading, intracellular endocytosis and tumor targeting of micelles in comparison to mPEG. HA molecular weight showed significant effect on properties and antitumor efficacy of the synthesized conjugates. Intracellular uptake of HA-ss-PTX toward MCF-7 cells was mediated by CD44-caveolae-mediated endocytosis. Compared to Taxol and mPEG-ss-PTX, HA9.5-ss-PTX demonstrated improved tumor growth inhibition in vivo with a TIR of 83.27±5.20%. It was concluded that HA9.5-ss-PTX achieved rapid intracellular release of PTX and enhanced its therapeutic efficacy, thus providing a platform for specific drug targeting and controlled intracellular release in chemotherapeutics. Statement of SignificancePolymer–drug conjugates, promising nanomedicines, still face some technical challenges including a lack of specific targeting and rapid intracellular drug release at the target site. In this manuscript we designed and constructed a novel bioconjugate HA-ss-PTX, which possessed coordinated dual characteristics of active tumor targeting and selective intracellular drug release. Redox-responsive disulfide bond was introduced to the conjugate to shield drug leakage in blood circulation and to achieve rapid drug release at tumor site in response to reductant like glutathione. Paclitaxel was selected as a model drug to be covalently attached to hyaluronic acid (HA) with various sizes to elucidate the structure–activity relationship and to address whether HA could substitute PEG as a carrier for polymeric conjugates. Based on a series of in vitro and in vivo experiments, HA-ss-PTX performed well in drug loading, cellular internalization, tumor targeting by entering tumor cells via CD44-caveolae-mediated endocytosis and rapidly release drug at target in the presence of GSH. One of the key issues in clinical oncology is to enhance drug delivery efficacy while minimizing side effects. The study indicated that this new polymeric conjugate system would be useful in delivering anticancer agents to improve therapeutic efficacy and to minimize adverse effects, thus providing a platform for specific drug targeting and controlled intracellular release in chemotherapeutics.

Self-assembled nanoparticles from hyaluronic acid–paclitaxel prodrugs for direct cytosolic delivery and enhanced antitumor activity

A prodrug-based nanosystem obtained by formulating prodrug and nanotechnology into a system is one of the most promising strategies to enhance drug delivery for disease treatment. Herein, we report a new nanosystem based on HA–PTX conjugates (HA–PTX Ns), which penetrated across cell membranes into cytosol, thus enhancing paclitaxel (PTX) delivery. HA–PTX Ns were successfully obtained based on HA–PTX, and their average particle size was approximately 200nm. Importantly, unlike other prodrug-based nanosystems, HA–PTX Ns obtained cellular entry without entrapment within the lysosomal–endosomal system by using pathways including clathrin-mediated endocytosis, microtubule-associated internalization, macropinocytosis and cholesterol-dependence. Due to significant accumulation in tumors, HA–PTX Ns had more than a 4-fold decrease in tumor volume on day 14 in contrast with PTX alone. In conclusion, HA–PTX Ns could enter cells, bypass the lysosomal–endosomal system and improve PTX delivery.

Programmed Hydrolysis in Designing Paclitaxel Prodrug for Nanocarrier Assembly.

Nanocarriers delivering prodrugs are a way of improving in vivo effectiveness and efficiency. For therapeutic efficacy, the prodrug must hydrolyze to its parent drug after administration. Based on the fact that the hydrolysis is impeded by steric hindrance and improved by sufficient polarity, in this study, we proposed the PTX-S-S-VE, the conjugation of paclitaxel (PTX) to vitamin E (VE) through a disulfide bridge. This conjugate possessed the following advantages: first, it can be encapsulated in the VE/VE2-PEG2000/water nanoemulsions because of favorable hydrophobic interactions; second, the nanoemulsions had a long blood circulation time; finally, the concentrated glutathione in the tumor microenvironment could cleave the disulfide bond to weaken the steric hindrance and increase the polarity, promoting the hydrolysis to PTX and increasing the anticancer activity. It was demonstrated in vitro that the hydrolysis of PTX-S-S-VE was enhanced and the cytotoxicity was increased. In addition, PTX-S-S-VE had greater anticancer activity against the KB-3-1 cell line tumor xenograft and the tumor size was smaller after the 4th injection. The present result suggests a new way, use of reduction, to improve the in vivo anticancer activity of a prodrug for nanocarrier delivery by unshielding the ester bond and taking off the steric block.

Covalent Polyisobutylene-Paclitaxel Conjugates for Controlled Release from Potential Vascular Stent Coatings.

The development of covalent polyisobutylene (PIB)-paclitaxel (PTX) conjugates as a potential approach to controlling drug release from vascular stent coatings is described. PIB-PTX materials containing ∼24 and ∼48 wt % PTX, conjugated via ester linkages, were prepared. The PTX release profiles were compared with those of physical mixtures of PTX with carboxylic acid-functionalized PIB and with the triblock copolymer polystyrene-b-PIB-b-polystyrene (SIBS). Covalent conjugation led to significantly slower drug release. Atomic force microscopy imaging of coatings of the materials suggested that the physical mixtures exhibited multiple domains corresponding to phase separation, whereas the materials in which PTX was covalently conjugated appeared homogeneous. Coatings of the conjugated materials on stainless steel surfaces suffered less surface erosion than the physically mixed materials, remained intact, and adhered well to the surface throughout the thirty-five day study. Tensile testing and rheological studies suggested that the incorporation of PTX into the polymer introduces similar physical changes to the PIB as the incorporation of a glassy polystyrene block does in SIBS. Cytotoxicity assays showed that the coatings did not release toxic levels of PTX or other species into a cell culture medium over a 24 h period, yet the levels of PTX in the materials were sufficient to prevent C2C12 cells from adhering to and proliferating on them. Overall, these results indicate that covalent PIB-PTX conjugates have promise as coatings for vascular stents.

Core-Cross-Linking Accelerates Antitumor Activities of Paclitaxel-Conjugate Micelles to Prostate Multicellular Tumor Spheroids: A Comparison of 2D and 3D Models.

The 2D monolayer cell culture model is often the first step in the prediction of the success or failure of a nanoparticle-based drug delivery system. However, there is often poor translation between the 2D monolayer in vitro results and the nanoparticle-drug performance in vivo. One possible way of bridging this gap is the use of multicellular tumor spheroids (MCTSs) as an intermediate in vitro model due to its 3D structure. This paper aims to quantify and compare the results obtained from traditional 2D monolayer cell cultures and 3D MCTS by studying the cytotoxic effects of free paclitaxel (PTX) and paclitaxel, which has been conjugated to a poly(ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer and self-assembled to give a micellar delivery system. The core of the micelle was cross-linked with a diamino nondegradable cross-linker to compare the effects of micelle stability on the results. Although the 2D prostate tumor cell culture results indicated that all micellar variants (IC50: 193-271 nM) were significantly less toxic than free paclitaxel (IC50: 15.2 nM), the micelles showed faster and higher cytotoxicity than free PTX in the 3D prostate MCTS. The cross-linking of micelles even showed accelerated antitumor activities to the MCTS compared with un-cross-linked micelles. The results indicate that DAO-cross-linked POEGMEA-b-PCEA-PTX conjugate micelles will be a useful nanodrug carrier for prostate cancer therapy. MCTS offers a very promising method of incorporating 3D structures into in vitro testing.

Anti-tumor activity of folate targeted biodegradable polymer–paclitaxel conjugate micelles on EMT-6 breast cancer model

Paclitaxel (PTX) is a first line chemotherapy drug for breast cancer. There have been few studies reported concerning the therapeutic efficacy of paclitaxel-conjugated polymeric micelles in breast cancer in vivo. Two kinds of PTX conjugate micelles, one of which (M(PTX)) contained 25 wt.% of PTX and the other (M(FA/PTX)) contained 22.5 wt.% of PTX and 1.4 wt.% of folate (FA), were prepared for cell apoptosis and anti-tumor activity evaluation on EMT-6 mice breast cancer models in comparison with 0.9 wt.% saline (control) and equivalent PTX. Cell apoptosis was analyzed by flow cytometry. Breast tumors were examined histologically with H&E staining and immunohistochemically by examining Bax and Bcl-2 expression. The survival status of tumor-bearing mice with different treatments was also examined. On day 5 of the drug administration, the average tumor masses were 0.49, 0.33, 0.22, and 0.18 g for the control, PTX, M(PTX) and M(FA/PTX) groups, respectively. The inhibition rates of tumor growth calculated for the three drug groups were 32.6%, 51.6% and 62.3%, respectively. The percentage of cell apoptosis based on flow cytometry was 1.0%, 36.6%, 55.9% and 66.1%, respectively, which showed statistically significant differences (p<0.05) between three drug groups and the control group. Bcl-2 expression of PTX and M(FA/PTX) groups was lower than control group (p<0.05). Bax expression of drug groups was higher than control group (p<0.05). At an equivalent paclitaxel dose of 26.7 mg/kg, the average survival time was 33 days, 31 days, 34 days and 42 days, respectively (p<0.05). The M(FA/PTX) have better anti-tumor activity and are promising in treatment of human breast cancers.

Engineering of Supramolecular Taxane Nanoparticles by Computationally Modeling Drug‐lipid Bilayer Interactions

Clinical efficacy of lipid nanoparticles (NPs) depend on high drug to lipid ratio, stability during circulation and sustained drug release after tumor accumulation. Achieving this is the holy grail of paclitaxel (PTX) lipid NPs due to its incompatible physicochemical properties that leads to low drug loading efficiency and tendency to crystallize leading to significant instability. These limitations could be overcome by understanding molecular interactions of PTX with lipid bilayer at spatiotemporal level but these remain poorly understood. Using computer simulations, we have investigated the interactions of PTX with model lipid bilayer at low and high drug contents. We found that PTX at low concentrations occupied the space in both hydrophobic and hydrophilic regions of the bilayer and did not exhibit significant destabilizing effect whereas at high concentrations it exclusively accumulated at hydrophilic tail region causing significant rifts in the bilayer. In silico dynamic simulations showed that conjugation of PTX to cholesterol using a flexible spacer (Suprataxel, STX) stabilized the lipid bilayer at high concentrations. To confirm this, we engineered supramolecular NPs of STX (STX‐NP) and characterized the physicochemical and mechanical stability and found more efficacious than PTX in vitro. We showed that STX‐NPs were more efficient than PTX in inhibiting tumor growth in 4T1 breast and K‐RasLSL/+/Ptenfl/fl ovarian cancer models (p&lt;0.01). This study shows that effective supramolecular NPs could be engineered by understanding interactions between drug and lipid layer.

Synthesis and biological evaluation of RGD peptidomimetic-paclitaxel conjugates bearing lysosomally cleavable linkers.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.

P2.01 - ANG-1005 in Patients with Brain Metastases from Breast Cancer: Correlative Imaging with 18F-FLT-PET/CT

ABSTRACT Background: ANG1005 (formerly GRN1005) is a novel drug conjugate consisting of 3 paclitaxel molecules covalently linked to Angiopep-2 designed to cross the blood brain barrier via endocytosis after binding the low-density lipoprotein (LDL) receptor-related protein (LRP). In development for taxane-sensitive brain metastases, a multi-center, single-arm study with the primary endpoint of intra-cranial overall response rate in patients with brain metastases from breast cancer is ongoing. Since MRI detection of brain metastases utilizes gadolinium leakage rather than actual tumor volume, new assessment methods are needed. A pilot study at the NCI enrolled patients to evaluate the utility of 18F-FLT (3'-fluoro-3' deoxythymidine)-PET. Methods: Patients with measurable brain metastases from breast cancer were eligible. ANG1005 was administered IV at 550 mg/m2 q 21 days. MRI imaging with gadolinium was used to determine clinical response, and compared to 18F-FLT PET/CT imaging performed before and after cycle 1. FLT incorporation reflects DNA synthesis. Dynamic scans were obtained over 30 min and a static whole body PET image at 1 hour. The % change in standard uptake value (SUV) before and after ANG1005 was determined, considering a significant change > 20%. Results: Eighteen metastatic brain lesions in eight patients were analyzed with FLT PET. The maximum (SUVmax) ranged from 0.8 to 4.0 at baseline, mean 1.8. Tumor to normal (T:N) ratios ranged from 2.9 to 22.3, mean 7.7. Twelve of the 18 lesions showed a >20% decrease post-therapy. The average % change in SUVmax was -24.8% (11 to -66.8%), and T:N ratios -7.7%. The FLT-PET response was frequently discordant with the MRI result. Two patients had confirmed partial responses with durations of response of 6 and 13 cycles. One patient had an unconfirmed PR, with progression after 6 cycles. Two patients had stable disease, receiving 6 and 8 cycles. Conclusion: Therapy for CNS metastases from breast cancer is an important unmet need, as is assessment of therapeutic outcome. ANG1005 is a paclitaxel conjugate with demonstrated activity designed to cross the blood-brain barrier. Pilot evaluations of FLT-PET imaging of brain metastases suggest it is a promising tool for detection and measurement of CNS disease.

Dendritic polyglycerol sulfate as a novel platform for paclitaxel delivery: pitfalls of ester linkage.

In this study, dendritic polyglycerol sulfate (dPGS) is evaluated as a delivery platform for the anticancer, tubulin-binding drug paclitaxel (PTX). The conjugation of PTX to dPGS is conducted via a labile ester linkage. A non-sulfated dendritic polyglycerol (dPG) is used as a control, and the labeling with an indocarbocyanine dye (ICC) renders multifunctional conjugates that can be monitored by fluorescence microscopy. The conjugates are characterized by (1)H NMR, UV-vis measurements, and RP-HPLC. In vitro cytotoxicity of PTX and dendritic conjugates is evaluated using A549 and A431 cell lines, showing a reduced cytotoxic efficacy of the conjugates compared to PTX. The study of uptake kinetics reveals a linear, non saturable uptake in tumor cells for dPGS-PTX-ICC, while dPG-PTX-ICC is hardly taken up. Despite the marginal uptake of dPG-PTX-ICC, it prompts tubulin polymerization to a comparable extent as PTX. These observations suggest a fast ester hydrolysis and premature drug release, as confirmed by HPLC measurements in the presence of plasma enzymes.

Design, synthesis and biological evaluation of novel dimeric and tetrameric cRGD-paclitaxel conjugates for integrin-assisted drug delivery.

Integrins are associated with tumour cell survival and progression, and their expression has been shown to be increased in tumours. Thus, four novel conjugates of the tripeptide integrin ligand Arg-Gly-Asp (RGD) and the cytotoxic agent paclitaxel (cRGD-PTX) were prepared to investigate the potential of the multivalent presentation of the RGD moiety in improving the antitumor efficacy of PTX by tumour targeting. PTX was conjugated to two or four integrin recognizing ligands. The influence of multivalent presentation on in vitro αvβ3-receptor affinity was confirmed. For all the conjugates compared to the previously synthesized monovalent counterparts, an enhancement of the binding strength was observed; this behaviour was more pronounced when considering the tetravalent presented RGD-conjugate. Cell growth inhibition assays on a panel of human tumour cell lines showed remarkable cytotoxic activity for all conjugates with IC50 values in a nanomolar range. Among the four conjugates, the bivalent derivative 3b was selected for in vivo studies in an ovarian carcinoma cell model xenografted in immunodeficient mice. A marked antitumor activity was observed, similar to that of PTX, but with a much more favourable toxicity profile. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted anti-tumour therapy.