Acid-sensitive Hydrazone Bond Research Articles

Acid/GSH Dual-Responsive Endosome Escape Dual Pro-drug Micelles Targeted Synergistic Treatment for A549/ADR.

The resistance of cancer cells to anticancer drugs has been recognized as one of the main reasons for chemotherapy failure. Multidrug combination therapy is one of the most effective ways to solve this problem. Therefore, in this article, we designed and synthesized a pH/GSH dual-responsive camptothecin/doxorubicin (CPT/DOX) dual pro-drug synergistic treatment system with the aim of overcoming the resistance of non-small cell lung cancer A549/ADR cells to DOX. The pro-drug cRGD-PEOz-S-S-CPT (cPzT) was obtained by linking CPT to poly(2-ethyl-2-oxazoline) (PEOz) with endosomal escape properties through a GSH-responsive disulfide bond and modifying it with the targeted peptide cRGD. The pro-drug mPEG-NH-N=C-DOX (mPX) was synthesized by attaching DOX to polyethylene glycol (PEG) through acid-sensitive hydrazone bonds. The dual pro-drug micelles cPzT/mPX configured according to the CPT/DOX mass ratio of 3:1 showed a strong synergistic therapeutic effect at IC50 with a combined therapy index CI = 0.49, far less than 1. Moreover, with the further improvement of the inhibition rate, the 3:1 ratio showed a stronger synergistic therapeutic effect than other ratios. The cPzT/mPX micelles not only had better targeted uptake ability but also showed a better therapeutic effect in both 2D and 3D tumor suppression assays relative to free CPT/DOX and significantly enhanced the penetration ability into solid tumors. In addition, the results of confocal laser scanning microscopy (CLSM) showed that cPzT/mPX could effectively overcome the resistance of A549/ADR cells to DOX by delivering DOX into the nucleus to exert its effect. Thus, this dual pro-drug synergistic therapy system combining targeting and endosomal escape ability provides a possible strategy to overcome tumor drug resistance.

A Novel Homodimer Peptide–Drug Conjugate Improves the Efficacy of HER2-Positive Breast Cancer Therapy

Tumor-targeting peptide-drug conjugates (PDCs) have become a focus of research in recent years. However, due to the instability of peptides and their short in vivo effective half-life, they have limited clinical application. Herein, we propose a new DOX PDC based on a homodimer HER-2-targeting peptide and acid-sensitive hydrazone bond, which could enhance the anti-tumor effect of DOX and reduce systemic toxicities. The PDC could accurately deliver DOX into HER2-positive SKBR-3 cells, with it showing 2.9 times higher cellular uptake than free DOX and enhanced cytotoxicity with respect to IC50 of 140 nM (vs. 410 nM for free DOX). In vitro assays showed that the PDC had high cellular internalization efficiency and cytotoxicity. In vivo anti-tumor experiments indicated that the PDC could significantly inhibit the growth of HER2-positive breast cancer xenografts in mice and reduce the side effects of DOX. In summary, we constructed a novel PDC molecule targeting HER2-positive tumors, which may overcome some deficiencies of DOX in breast cancer therapy.

An Acid-Sensitive Nanofiber Conjugate Based on a Short Aromatic Peptide for Targeted Delivery of Doxorubicin in Liver Cancer

PurposeThis study aimed to construct a DOX conjugate with liver tumor targeting and acid sensitivity based on a short aromatic peptide FFYEE, which could amplify the tumor inhibition efficacy of DOX and alleviate tissue toxicity.MethodsA novel DOX-peptide conjugate, D-gal-FFYEE-hyd-DOX, was constructed by linking DOX to the side chain of FFYEE with acid-sensitive hydrazone bond and by modifying the C-terminal of peptide with α-D-galactosamine (D-gal) as targeting ligand. The structure of D-gal-FFYEE-hyd-DOX was characterized by mass spectrometry, infrared spectroscopy (IR), and UV-Vis spectroscopy (UV-Vis). The assembly characteristics of pentapeptide FFYEE and D-gal-FFYEE-hyd-DOX were observed by transmission electron microscope (TEM). In vitro drug release, cytotoxicity, endocytosis, in vivo antitumor experiment and histopathology analysis were investigated.ResultsPeptide FFYEE endowed the D-gal-FFYEE-hyd-DOX with self-assembly performance and improved biocompatibility. D-gal-FFYEE-hyd-DOX can self-assemble into nanofibers with a diameter of ~ 40 nm in neutral aqueous solution and significantly reduced the cytotoxicity of free DOX to L02 cells. In vitro drug release results showed that D-gal-FFYEE-hyd-DOX had acid sensitivity and controlled release characteristics. The cytotoxicity and endocytosis investigations confirmed that D-gal-FFYEE-hyd-DOX enhanced the cellular uptake of DOX and inhibition effect on HepG2 cells. In vivo antitumor experiment indicated that D-gal-FFYEE-hyd-DOX could significantly inhibit the growth of liver tumor in mice and reduce the side effects of DOX.ConclusionThe conjugate D-gal-FFYEE-hyd-DOX with liver tumor targeting and acid sensitivity has the characteristics of strong tumor inhibition and low toxicity, hinting the great clinical application potential for targeted delivery of DOX in cancer treatment.

Bone targeting nano-aggregates prepared from self-assembled polyaspartamide graft copolymers for pH sensitive DOX delivery.

Nanoparticles with bone targeting ability and pH-sensitivity were prepared with polyaspartamide (PASPAM) derivatives based on polysuccinimide (PSI) grafted with octadecylamine (C18), hydrazine (HYD) and polyethylene glycol (PEG, Mw: 5000). For the bone targeting, alendronate (ALN), which has bone affinity, was grafted to PEG and doxorubicin (DOX) was conjugated with linkers of acid sensitive hydrazone bonds, which can be cleaved most effectively in an intracellular acidic environment. At pH 5.0, ∼75% of the drug was released from ALN-PEG/C18/HYD-DOX-g-PASPAM due to the effective cleavage of HYD under the acidic condition. Also, ALN-PEG/C18/HYD-DOX-g-PASPAM particles were more effectively adsorbed on the surface of bone than PEG/C18/HYD-DOX-g-PASPAM. According to an in vivo antitumor activity test, the volume of tumor treated with ALN-PEG/C18/HYD-DOX-g-PASPAM decreased (1550 mm3) when compared with the PBS control sample (3850 mm3), proving that ALN-PEG/C18/HYD-DOX-g-PASPAM is an effective drug delivery system for the treatment of bone metastasis of breast cancer.

The effect of drug position on the properties of paclitaxel-conjugated gold nanoparticles for liver tumor treatment

Structure-efficacy effect of small molecular drug attracts wide attentions, but it has always been ignored in nanomedicine research. To reveal the efficacy modulation of nanomedicine, we developed a new type of paclitaxel (PTX)-conjugated gold nanoparticles (PTX-conjugated GNPs) to investigate the influence of drug position in controlling their in vitro properties and in vivo performance. Two therapeutic ligands (TA-PEG-NH-N=PTX and TA-PTX=N-NH-PEG) were synthesized to conjugate PTX on the surface of GNPs at different positions, locating on the surface of gold conjugate and inserting between GNPs and polyethylene glycol (PEG, molecular weight 1000 Da), respectively. It was found that PEG-PTX@GNPs with PTX located between GNP and PEG exhibited higher aqueous solubility, biocompatibility, and stability. In addition, an acid sensitive hydrazone bond has been inserted between PTX and PEG in both ligands for drug release of PTX and PTX-PEG segment, respectively, at the tumor site. Further release of PTX from PTX-PEG segment is based on the esterase hydrolysis of an ester bond between PTX and PEG. This two-step drug release mechanism offers PEG-PTX@GNPs effective and sustained release behavior for desirable anticancer activity, enhanced therapeutic efficacy, and lower systematic toxicity in Heps-bearing animal models.

Poly(2-methyl-2-oxazoline) conjugates with doxorubicin: From synthesis of high drug loading water-soluble constructs to in vitro anti-cancer properties

Poly(2-oxazoline)s represent an emerging class of polymers with increasing potential in biomedical sciences. To date, most of the work on poly(2-oxazoline)-drug conjugates focused on poly(2-ethyl-2-oxazoline) (PEtOx), a biocompatible water-soluble polymer with biological properties similar to polyethylene glycol. However, the more hydrophilic poly(2-methyl-2-oxazoline) (PMeOx) shows better anti-fouling properties than PEtOx and thus indicates greater potential for the construction of polymer therapeutics. Herein, we synthesized for the first time a drug delivery system based on a linear PMeOx with a molar mass that is high enough (40 kDa) to exploit passive accumulation in the tumor by the enhanced permeation and retention effect. The anti-cancer drug doxorubicin is attached to the polymer carrier via an acid-sensitive hydrazone bond, which allows its pH-triggered release in the tumor. The in vitro study demonstrates successful cellular uptake of the PMeOx-doxorubicin conjugate via clathrin-mediated endocytosis, pH-sensitive drug release and high cytotoxicity against B16 melanoma cells. Finally, these properties were critically compared to the analogous systems based on the established PEtOx revealing that the more hydrophilic PMeOx carrier outperforms PEtOx in most of the parameters, showing higher maximal drug loading, superior cellular uptake, better anti-fouling properties, as well as improved in vitro anti-cancer efficiency. The study demonstrates the potential of PMeOx as a versatile platform for synthesis of new drug delivery systems.

Novel auristatin E-based albumin-binding prodrugs with superior anticancer efficacy in vivo compared to the parent compound

Auristatins are a class of highly cytotoxic tubulin-disrupting peptides, which have shown limited therapeutic effect as free agents in clinical trials.In our continuing effort to develop acid-sensitive albumin-binding anticancer drugs exploiting circulating serum albumin as the drug carrier, we investigated the highly toxic drug payload auristatin E to assess whether the corresponding albumin-binding prodrugs were a viable option for achieving significant and concomitant tolerable antitumor activity. To achieve our goal, we developed a new aromatic maleimide-bearing linker (Sulf07) which enhanced both water solubility and stability of the prodrugs. In this study, we describe two auristatin E-based albumin-binding drugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, which were designed to release the active compound at the tumor site in a pH-dependent manner. These prodrugs incorporate an acid-sensitive hydrazone bond, formed by the reaction of a carbonyl-containing auristatin E derivative with the hydrazide group of the water-solubilizing maleimide-bearing linker Sulf07.A panel of patient- and cell-derived human tumor xenograft models (melanoma A375, ovarian carcinoma A2780, non-small-cell lung cancer LXFA737 and LXFE937, and head and neck squamous cell carcinomas) were screened with starting tumor volumes in the range of either 130–150 mm3 (small tumors) or 270–380 mm3 (large tumors). Both albumin-binding prodrugs showed compelling anticancer efficacy compared to the parent drug auristatin E, inducing statistically significant long-term partial and/or complete tumor regressions. AE-Keto-Sulf07 displayed very good antitumor response over a wide dose range, 3.0–6.5 mg/kg (5–8 injections, biweekly). AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg (8 injections, biweekly) or at 3.8 mg/kg (4 injections, weekly), but caused cumulative skin irritation due to scratching and biting. In contrast at its MTD, auristatin E (0.3 mg/kg, 8 injections, biweekly) was only marginally active. In summary, AE-Keto-Sulf07 and AE-Ester-Sulf07 are novel acid-sensitive albumin-binding prodrugs demonstrating tumor regressions in all of the evaluated human tumor xenograft models thus supporting the stratagem that albumin can be used as an effective drug carrier for the highly potent class of auristatins.

Acid and light stimuli-responsive mesoporous silica nanoparticles for controlled release

We constructed a novel stimuli-responsive system, MSNPs 1, based on mechanized silica nanoparticles, in which mesoporous silica nanoparticles (MSNs) acted as nanocontainers to load cargo, and supramolecular switches consisting of hydrazone bond, azobenzene and α-cyclodextrin (α-CD) realized the controlled release of cargo molecules from MSNPs 1. In neutral solution and without UV light irradiation, the azobenzene component on functional stalks was in trans form, and combined with α-CD to block the mesoporous channels and prevent the cargo from escaping. Upon adjusting the solution pH to acid range, the acid-sensitive hydrazone bonds rapidly hydrolyzed, resulting in the disconnection between pseudorotaxanes and MSNs. The encapsulated cargo molecules were released simultaneously from MSNPs 1 due to the removal of supramolecular switches. On the other hand, when the neutral solution was irradiated with UV light at 365 nm, trans-azobenzene converted to cis, disassociating with α-CD to activate the switches and release the cargo. The unique acid- and light-triggered controlled release properties of MSNPs 1 have potential application in various fields. In this manuscript, doxorubicin (DOX) was selected to be stored in MSNPs 1, due to the anticancer property. MCF-7 human breast cancer cells could phagocytose DOX-loaded MSNPs 1 easily, and the released DOX showed the killing effect.

Multi-functional core-shell Fe3O4@Au nanoparticles for cancer diagnosis and therapy.

Multi-functional core-shell Fe3O4@Au nanoparticles (Fe3O4@Au-DOX-mPEG/PEG-FA NPs) conjugated with doxorubicin (DOX), methoxy poly(ethylene glycol) (mPEG), and folic acid-linked poly(ethylene glycol) (PEG-FA) were synthesized for cancer theranostic applications. In the developed NPs, the DOX was chemically conjugated at the surface of core-shell Fe3O4@Au NPs using L-cysteine methyl ester (LCME) as a linker by acid-sensitive hydrazone bond. The formation of Fe3O4@Au-DOX-mPEG/PEG-FA NPs was confirmed by 1H-NMR analysis. The TEM image and DLS studies showed that the mean diameter of the prepared NPs was about 18 and 38 nm, respectively. Due to the existence of superparamagnetic Fe3O4, the Fe3O4@Au-DOX-mPEG/PEG-FA NPs presented the saturation magnetization (Ms) value of 23 emu/g. The developed NPs displayed the maximum amount of drug release in the acidic medium than that in the mild alkaline medium because of the presence of acid-sensitive hydrazone bond. Due to the presence of FA, the Fe3O4@Au-DOX-mPEG/PEG-FA NPs displayed the increased cellular uptake through a folate-receptor-mediated endocytosis, which results in the improved cytotoxic effect on the HeLa cells. Under the laser irradiation, the cytotoxicity of Fe3O4@Au-DOX-mPEG/PEG-FA NPs was found to be improved due to the photothermal effect of Au shell existing in the NPs. These results reveal that the Fe3O4@Au-DOX-mPEG/PEG-FA NPs could be a promising tumour-targeted drug delivery system with the capabilities of combined MR/CT imaging, photothermal, and chemotherapy of tumours.

Preparation of poly(styrene-alt-maleic anhydride) grafted multi-walled carbon nanotubes for pH-responsive release of doxorubicin

ABSTRACTA pH-responsive drug release system based on doxorubicin conjugated poly(styrene-alt-maleic anhydride) grafted multi-walled carbon nanotubes (MWNT/PSMA-DOX) was prepared by the reversible addition-fragmentation chain transfer (RAFT) polymerization and acid-cleavable hydrazone linkages. Firstly, multi-walled carbon nanotubes (MWNTs) were modified with a furfuryl derivative RAFT agent by Diels-Alder reaction. The polymerization of styrene and maleic anhydride was carried out in the presence of functionalized MWNTs to afford MWNT/PSMA. The maleic anhydride ring located along the polymer chains was opened by hydrazine subsequently conjugated with doxorubicin via an acid-sensitive hydrazone bond. The composite materials were characterized by FT-IR, 1H-NMR, TGA and TEM analysis. In vitro experiments showed a burst release of doxorubicin at pH 5.0 whereas slight release at pH 7.4.

Polymer-Drug Nanoparticles Combine Doxorubicin Carrier and Heparin Bioactivity Functionalities for Primary and Metastatic Cancer Treatment.

Here, a biocompatible amphiphilic copolymer of low molecular weight heparin (LMWH) and doxorubicin (DOX) connected by an acid-sensitive hydrazone bond for enhanced tumor treatment efficacy and safety has been designed and tested. The conjugate combines DOX delivery with LMWH antimetastatic capabilities. After the nanoparticles reach the tumor site, the acidic tumor microenvironment triggers the breakage of the hydrazone bond releasing DOX from the nanoparticles, which results in an increase in the cellular uptake and enhanced in vivo antitumor efficacy. A 3.4-fold and 1.5-fold increase in tumor growth inhibition were observed compared to the saline-treated control group and free DOX treated group, respectively. The LMWH-based nanoparticles effectively inhibited interactions between tumor cells and platelets mediated by P-selectin reducing metastasis of cells in both in vitro and in vivo models. The improved safety and therapeutic effect of LMWW-DOX nanoparticles offers new potential for tumor therapy.

Abstract 2061: DK049, a novel acid-sensitive prodrug of gemcitabine: design, in vitro properties and in vivo efficacy

Abstract Gemcitabine (Gemzar®) is an anticancer nucleoside that is widely used to treat solid tumors. Unfortunately, at its recommended dose of ∼1000 mg/m2 approximately 90% are deactivated by cytidine deaminase to the inactive uridine metabolite and excreted in the urine. A further disadvantage resulting in chemo-resistance is the low expression level of the human equilibrative nucleoside transporter 1 (hENT1) on the cell surface of cancer cells thus preventing substantial uptake of gemcitabine. In order to circumvent these disadvantages, we designed an innovative gemcitabine prodrug DK049 that a.) is capped at the amino group of the cytosine moiety with acetyl benzoic acid preventing deamination, and b.) incorporates a proprietary maleimide-bearing LADR™ linker forming a stable, but acid-sensitive hydrazone bond. At its 5′-OH position a phosphate group was introduced rendering water-solubility. Following intravenous administration, DK049 binds rapidly and selectively to circulating albumin and utilizes the advantages of albumin-mediated tumor uptake as well as cellular uptake by endocytosis (1). After cleavage of the acid-sensitive benzoyl hydrazone bond, we could show that the acetyl benzoic acid is decapped by carboxylesterase 2. In a head-to-head comparison with gemcitabine (4 × 240 mg/kg on a weekly basis) in four subcutaneous human patient-derived tumor xenografts (NSCLC: LXFE397, LXFE937, ovarian cancer: OVXF899, and pancreatic cancer: Panc11159; 8-10 nude mice per group), DK049 (4 × 24 mg/kg gemcitabine equivalents or 8 × 18 mg/kg gemcitabine equivalents over 4 weeks) demonstrated distinctly superior antitumor efficacy versus gemcitabine (p < 0.05). In the LXFE937 and OVXF899 model, DK049 induced long-term significant regressions for several weeks after end of therapy without any loss in body weight or bone marrow toxicity. In summary, these results show a superior long-term antitumor efficacy of DK049 compared to gemcitabine at a significantly reduced dose. (1) Felix Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 8;132:171-83, 2008. Citation Format: David Koester, Javier Garcia, Olga Fuchs, Felix Kratz. DK049, a novel acid-sensitive prodrug of gemcitabine: design, in vitro properties and in vivo efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2061.

Bone-targeted acid-sensitive doxorubicin conjugate micelles as potential osteosarcoma therapeutics.

Osteosarcoma is a malignancy of the bone that primarily affects adolescents. Current treatments retain mortality rates, which are higher than average cancer mortality rates for the adolescent age group. We designed a micellar delivery system with the aim to increase drug accumulation in the tumor and potentially reduce side effects associated with chemotherapy. The design features are the use of the hydrophilic d-aspartic acid octapeptide as both the effective targeting agent as well as the hydrophilic micelle corona. Micelle stabilization was accomplished by binding of model drug (doxorubicin) via an acid-sensitive hydrazone bond and incorporating one to four 11-aminoundecanoic acid (AUA) moieties to manipulate the hydrophobic/hydrophilic ratio. Four micelle-forming unimers have been synthesized and their self-assembly into micelles was evaluated. Size of the micelles could be modified by changing the architecture of the unimers from linear to branched. The stability of the micelles increased with increasing content of AUA moieties. Adsorption of all micelles to hydroxyapatite occurred rapidly. Doxorubicin release occurred at pH 5.5, whereas no release was detected at pH 7.4. Cytotoxicity toward human osteosarcoma Saos-2 cells correlated with drug release data.

Lysosomal Delivery of a Lipophilic Gemcitabine Prodrug Using Novel Acid-Sensitive Micelles Improved Its Antitumor Activity

Stimulus-sensitive micelles are attractive anticancer drug delivery systems. Herein, we reported a novel strategy to engineer acid-sensitive micelles using a amphiphilic material synthesized by directly conjugating the hydrophilic poly(ethylene glycol) (PEG) with a hydrophobic stearic acid derivative (C18) using an acid-sensitive hydrazone bond (PHC). An acid-insensitive PEG-amide-C18 (PAC) compound was also synthesized as a control. 4-(N)-Stearoyl gemcitabine (GemC18), a prodrug of the nucleoside analogue gemcitabine, was loaded into the micelles, and they were found to be significantly more cytotoxic to tumor cells than GemC18 solution, likely due to the lysosomal delivery of GemC18 by micelles. Moreover, GemC18 in the acid-sensitive PHC micelles was more cytotoxic than in the acid-insensitive PAC micelles, which may be attributed to the acid-sensitive release of GemC18 from the PHC micelles in lysosomes. In B16-F10 melanoma-bearing mice, GemC18-loaded PHC or PAC micelles showed stronger antitumor activity than GemC18 or gemcitabine solution, likely because of the prolonged circulation time and increased tumor accumulation of the GemC18 by the micelles. Importantly, the in vivo antitumor activity of GemC18-loaded PHC micelles was significantly stronger than that of the PAC micelles, demonstrating the potential of the novel acid-sensitive micelles as an anticancer drug delivery system.

Abstract 779: Prodrug-mediated reversal of multidrug resistance in breast cancer cells

Abstract The P-glycoprotein (P-gp) is a 170 kDa protein that acts as an energy-dependent, transmembrane efflux pump and is encoded by the MDR1 gene. It has been shown to be responsible for multidrug resistance (MDR) in a defined subpopulation of breast cancer patients and thus represents a molecular target for circumventing MDR in this tumor indication. Third generation MDR modulators have been developed that demonstrated high selectivity for P-gp with inhibitory activities in the low nanomolar range. Although some objective responses were achieved in clinical trials, combination therapy with these MDR-modulators caused unacceptable toxicity. Targeting P-gp inhibitors to the tumor site is a means to increase their therapeutic index, and in this context binding tailor-made prodrugs to circulating albumin is an established technology to reduce the toxicity and enhance the efficacy of anticancer drugs. In this work we consequently developed an acid-sensitive albumin-binding prodrug of the P-gp inhibitor zosuquidar (LY335979) in a two-step synthesis using a maleimide hydrazone linker system established in our laboratory that first introduces acetylbenzoic acid at the OH-group of zosuquidar followed by derivatization with 6-maleimidocaproyl hydrazide to form the acid-sensitive hydrazone bond. The maleimide group enables the prodrug to bind rapidly and selectively to the cysteine-34 position of endogenous albumin after intravenous administration. HPLC analysis demonstrated rapid albumin binding of the zosuquidar prodrug as well as the quantitative release of the acetylbenzoic ester derivative of zosuquidar at pH 5.0. Subsequently, its ability to circumvent MDR was tested in two doxorubicin-resistant breast carcinoma cell lines (MCF-7/ADR and MT-3/ADR). The MDR status of these cell lines can be reversed by zosuquidar which was confirmed in a rhodamine 123 assay using fluorescence microscopy and FACS analysis. Furthermore, zosuquidar as well its acid-sensitive albumin conjugate re-sensitized cells to doxorubicin as well to an albumin-binding prodrug of doxorubicin, i.e. the 6-maleimidocaproyl hydrazone derivative of doxorubicin, achieving IC50 values in the same order of magnitude as the parental cell lines. Thus a novel formulation of zosuquidar has been developed that may be used to improve the toxicity issues and tumor targeting properties of the original compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 779. doi:1538-7445.AM2012-779

Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM.

Zosuquidar and an albumin-binding prodrug of zosuquidar reverse multidrug resistance in breast cancer cells of doxorubicin and an albumin-binding prodrug of doxorubicin

The P-glycoprotein (P-gp) is a 170-kDa protein that acts as an energy dependent, transmembrane efflux pump and is encoded by the MDR1 gene. It has been shown to be responsible for multidrug resistance (MDR) in a defined subpopulation of breast cancer patients and thus represents a molecular target for circumventing MDR in this tumor indication. MDR modulators have been developed and demonstrated high selectivity for P-gp with inhibitory activities in the low nanomolar range. Although some objective responses were achieved in clinical trials, combination therapy with these MDR modulators, such as Ca2+ antagonists caused unacceptable toxicity. Targeting P-gp inhibitors to the tumor site is a mean to increase their therapeutic index, and in this context binding of tailor-made prodrugs to circulating albumin is an established technology to reduce the toxicity and enhance the efficacy of anticancer drugs. In this study, we consequently developed an acid-sensitive albumin-binding prodrug of the P-gp inhibitor zosuquidar (LY335979) in a two-step synthesis using a maleimide hydrazone linker system established in our laboratory that first introduces acetylbenzoic acid at the HO-group of zosuquidar followed by derivatization with 6-maleimidocaproyl hydrazide to form the acid-sensitive hydrazone bond. The maleimide group enables the prodrug to bind rapidly and selectively to the cysteine-34 position of endogenous albumin after intravenous administration. HPLC analysis demonstrated rapid albumin binding of the zosuquidar prodrug as well as the quantitative release of the acetylbenzoic ester derivative of zosuquidar at pH 5.0. Subsequently, its ability to circumvent MDR was tested in two doxorubicin-resistant breast carcinoma cell lines (MCF-7/ADR and MT-3/ADR). The MDR status of these cell lines can be reversed by zosuquidar which was confirmed in a rhodamine 123 assay using fluorescence microscopy and FACS analysis. Furthermore, zosuquidar as well its acid-sensitive albumin conjugate re-sensitized cells to doxorubicin as well as to an albumin-binding prodrug of doxorubicin, i.e., the 6-maleimidocaproyl hydrazone derivative of doxorubicin, achieving IC50 values in the same order of magnitude as the parental cell lines. Thus, a novel formulation of zosuquidar has been developed that could have the potential to improve the toxicity issues and tumor targeting properties of the original compound.

Polypeptide conjugates of d-penicillamine and idarubicin for anticancer therapy

We investigated anticancer therapy with a novel combination of d-penicillamine (d-pen) and Idarubicin (Ida) in a synthetic dual drug conjugate (DDC). d-pen and Ida were covalently linked to poly(α)-l-glutamic acid (PGA) via reducible disulfide and acid-sensitive hydrazone bonds, respectively. The DDCs showed cell uptake and sustained release of the bound drugs in conditions mimicking the intracellular release media (10mM glutathione and pH 5.2). The in-vitro cytotoxicity of DDCs was comparable to unconjugated Ida in several sensitive and resistant cancer cell lines and correlated with the rate of cell uptake. In a single equivalent-dose pharmacokinetic study, DDCs enhanced the drug exposure by 7-fold and prolonged the plasma circulation half-life (t1/2) by 5-fold over unconjugated Ida. The therapeutic index of DDCs was 2–3-fold higher than unconjugated drugs. DDCs caused 89% tumor growth inhibition compared to 60% by unconjugated Ida alone and led to significant enhancement in the median survival (17%) of athymic nu/nu mice bearing NCI-H460 tumor xenografts.

Comparative evaluation of the biological properties of reducible and acid-sensitive folate prodrugs of a highly potent doxorubicin derivative

Two new water-soluble folate receptor-targeted drug conjugates that contain the highly active doxorubicin derivative N-(5,5-diacetoxybut-1-yl)doxorubicin were designed and evaluated for their biological activity against folate receptor positive tumours. The prodrugs were designed to contain an acid-sensitive hydrazone bond KO019 or in addition a disulphide bond KO013 in order to elucidate the importance of the pre-determined breaking point for their in vitro and in vivo properties. Fluorescence microscopy studies confirmed higher uptake of the prodrugs in folate receptor positive KB cells than in the folate receptor negative A549 lung cancer cells. In subsequent in vivo studies in the folate receptor positive KB xenograft model, KO019 was as active as the free drug but significantly less toxic when dosed at twice the dose of the free drug whereas KO013 showed no anticancer efficacy. As an explanation, we could show by HPLC that the prodrug KO013 that additionally contains a disulphide bond undergoes rapid disulphide exchange in murine plasma in the order of 40% after 5h at 37°C in contrast to KO019 which was essentially stable after a 5h incubation.

Combination therapy with the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin achieves complete remissions and improves tolerability in an ovarian A2780 xenograft model

We recently reported on the in superior in vivo antitumor efficacy of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206, formerly DOXO-EMCH), an albumin-binding prodrug of doxorubicin with acid-sensitive properties (see Fig. 1), in three tumor models in this journal, i.e. in three xenograft models (breast carcinoma M3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreatic carcinoma model (AsPC-1) [1] Previously, INNO-206 has shown significantly improved activity over doxorubicin in two xenograft breast cancer models and an orthotopic murine renal cell carcinoma model [2] and recently against resistant and non resistant myeloma (see http//:www.cytrx.com). INNO-206 binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site due to incorporation of an acid-sensitive hydrazone bond. The accumulation of INNO-206 in tumor tissue combined with a favorable biodistribution [3] is due to the pathophysiology of tumor tissue, characterized by a high metabolic turnover, angiogenesis, hypervasculature, a defective vascular architecture and an impaired lymphatic drainage, the so-called EPR (enhanced and retention) effect [4]. As a carrier for INNO-206, we selected albumin, which is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptideor protein-based drugs [5]. Albumin is a potential carrier for tumor targeting because it accumulates in solid tumors. In addition, we discovered that the HS-group of cysteine-34 of albumin is a unique and accessible functional group of a plasma protein for in situ prodrug conjugation considering that free thiol groups are absent from most circulating serum proteins except for albumin, which makes this group by far the most abundant thiol group in human plasma After injection directly into the blood stream, INNO-206 binds in situ to the cysteine-34 position of circulating albumin and is then transported in its albumin-bound form to the tumor site. INNO-206 is a prodrug of the anticancer agent doxorubicin in which doxorubicin is derivatized at its C-13 keto-position with a thiol-binding spacer molecule, i.e. 6-maleimidocaproic acid hydrazide. It contains an acidsensitive hydrazone linker that allows doxorubicin to be released either extracellularly in the slightly acidic environment often present in tumor tissue (pH range 6.0-6.8) [6] or intracellularly in acidic endosomal (pH 5.0-6.5) or lysosomal (pH 4.0-5.0) compartments after cellular uptake of the albumin conjugate by the tumor cell. INNO-206 was selected as an investigational product for clinical evaluation after toxicology studies in mice, rats, and dogs had shown a 2.5to 5-fold increase in the maximum tolerated dose (MTD) when compared to conventional doxoElectronic supplementary material The online version of this article (doi:10.1007/s10637-011-9686-5) contains supplementary material, which is available to authorized users.