A Cremophor-Free Formulation for Tanespimycin (17-AAG) using PEO-b-PDLLA Micelles: Characterization and Pharmacokinetics in Rats

Tanespimycin (17-allylamino-17-demethoxygeldanamycin or 17-AAG) is a promising heat shock protein 90 inhibitor currently undergoing clinical trials for the treatment of cancer. Despite its selective mechanism of action on cancer cells, 17-AAG faces challenging issues due to its poor aqueous solubility, requiring formulation with Cremophor EL (CrEL) or ethanol (EtOH). Therefore, a CrEL-free formulation of 17-AAG was prepared using amphiphilic diblock micelles of poly(ethylene oxide)-b-poly(D,L-lactide) (PEO-b-PDLLA). Dynamic light scattering revealed PEO-b-PDLLA (12:6 kDa) micelles with average sizes of 257 nm and critical micelle concentrations of 350 nM, solubilizing up to 1.5 mg/mL of 17-AAG. The area under the curve (AUC) of PEO-b-PDLLA micelles was 1.3-fold that of the standard formulation. The renal clearance (CLrenal) increased and the hepatic clearance (CLhepatic) decreased with the micelle formulation, as compared to the standard vehicle. The micellar formulation showed a 1.3-fold increase in the half-life (t1/2) of the drug in serum and 1.2-fold increase in t1/2 of urine. As expected, because it circulated longer in the blood, we also observed a 1.7-fold increase in the volume of distribution (Vd) with this micelle formulation compared to the standard formulation. Overall, the new formulation of 17-AAG in PEO-b-PDLLA (12:6 kDa) micelles resulted in a favorable 150-fold increase in solubility over 17-AAG alone, while retaining similar properties to the standard formulation. Our data indicates that the nanocarrier system can retain the pharmacokinetic disposition of 17-AAG without the need for toxic agents such as CrEL and EtOH

Toward advanced modular drug and gene delivery system

In chapter two, the development of new a nanoparticulate carrier system for gene delivery was described. The new nanocarrier consists of a blend matrix formed by a poly (lactic-eo-glycolic acid) (PLGA) and Poly(ethylene glycol) bis (3-aminopropyl) terminated (also known as JeffamineTM). Nanopartic1es were formulated based on a 50:50 weight ratio of PLGA:Jeffamine using a modified emulsification-solvent diffusion technique. The potential of these blended matrix nanoparticles for encapsulation efficiency of Calf Thymus DNA and release profile were also studied. The achieved encapsulation efficiency of Calf Thymus DNA was approximately 84% for 0.4% theoretical loading with regard to total amount of PLGA. The PLGA: Jeffamine blended nanoparticles provided continuous and controlled release of Calf Thymus DNA. The PLGA:Jeffamine nanopartic1es were also coated with PLGA-PEGMA&75and PDMAEMA-PEGMA block copolymers using a simple physical adsorption method. After surface coating of the nanoparticles, zeta potential value showed significant reduction of surface charges from -38 mV to near zero value, while TEM micrographs showed a well defined core-shell nanoparticle. In chapter three, A facile route to biocompatible poly (lactic acid-coglycolic acid)-co-poly (ethyleneglycol methacrylate) (PLGA-PEGMA) block co-polymers was described utilising a combination of ring-opening polymerisation (ROP) and Radical Addition Fragmentation Transfer (RAFT) methods. A series of PLGA-PEGMA polymers varying in comonomer content and block length were synthesised with low polydispersities. All the block co-polymers formed micelles in aqueous solution as shown by dynamic light scattering, while critical micelle concentrations were found to be in the micromolar range. The polymer micelles were able to encapsulate model drugs(carboxyfluorescein and fluorescein isothiocyanate) and selected copolymer micelles incubated with 3T3 fibroblasts as a model cell line were rapidly taken up as indicated by fluorescence microscopy assays. The combination of the polymer chemistries opens the way to highly flexible syntheses of micellar drug carrier systems. In chapter four, multifunctional and modular block co-polymers prepared from biocompatible monomers and linked by a bioreducible disulphide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block co-polymer architecture enabled well-defined nanopartic1es to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folatereceptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to non-bioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tuneable way

Toward advanced modular drug and gene delivery system

In chapter two, the development of new a nanoparticulate carrier system for gene delivery was described. The new nanocarrier consists of a blend matrix formed by a poly (lactic-eo-glycolic acid) (PLGA) and Poly(ethylene glycol) bis (3-aminopropyl) terminated (also known as JeffamineTM). Nanopartic1es were formulated based on a 50:50 weight ratio of PLGA:Jeffamine using a modified emulsification-solvent diffusion technique. The potential of these blended matrix nanoparticles for encapsulation efficiency of Calf Thymus DNA and release profile were also studied. The achieved encapsulation efficiency of Calf Thymus DNA was approximately 84% for 0.4% theoretical loading with regard to total amount of PLGA. The PLGA: Jeffamine blended nanoparticles provided continuous and controlled release of Calf Thymus DNA. The PLGA:Jeffamine nanopartic1es were also coated with PLGA-PEGMA&75and PDMAEMA-PEGMA block copolymers using a simple physical adsorption method. After surface coating of the nanoparticles, zeta potential value showed significant reduction of surface charges from -38 mV to near zero value, while TEM micrographs showed a well defined core-shell nanoparticle. In chapter three, A facile route to biocompatible poly (lactic acid-coglycolic acid)-co-poly (ethyleneglycol methacrylate) (PLGA-PEGMA) block co-polymers was described utilising a combination of ring-opening polymerisation (ROP) and Radical Addition Fragmentation Transfer (RAFT) methods. A series of PLGA-PEGMA polymers varying in comonomer content and block length were synthesised with low polydispersities. All the block co-polymers formed micelles in aqueous solution as shown by dynamic light scattering, while critical micelle concentrations were found to be in the micromolar range. The polymer micelles were able to encapsulate model drugs(carboxyfluorescein and fluorescein isothiocyanate) and selected copolymer micelles incubated with 3T3 fibroblasts as a model cell line were rapidly taken up as indicated by fluorescence microscopy assays. The combination of the polymer chemistries opens the way to highly flexible syntheses of micellar drug carrier systems. In chapter four, multifunctional and modular block co-polymers prepared from biocompatible monomers and linked by a bioreducible disulphide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block co-polymer architecture enabled well-defined nanopartic1es to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folatereceptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to non-bioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tuneable way

Nanomedicines in cancer therapy : from long-circulating drug carriers to novel therapeutic concepts

Cancer is still a leading cause of death worldwide. Despite the progress in the molecular understanding of cancer diseases, there’s an urgent need in novel therapeutics and drug delivery strategies. Many novel anti-cancer compounds in early development are characterized by unfavorable physico-chemical properties and lack in drug-like properties. As a result, many of these compounds suffer from insufficient pharmacokinetic properties and show a high accumulation in off- target tissue that can induce dose-limiting side effects. Nanomedicines depict a promising strategy to optimize the pharmacokinetics of such compounds and to deliver them to their site of action: The cancer cell. The goal of this thesis was to develop nanoparticulate drug delivery platforms for passive and active drug targeting. In addition, a novel nanoparticle-based gene therapeutic for the treatment of liver cancer was evaluated. This thesis can be summarized in two main parts as follows: In a first part, a biocompatible and biodegradable polymer was used to prepare micelles for the delivery of small molecular anticancer drugs. These micelles were tested subsequently on in vitro and in vivo models. A highly reproducible protocol for the formulation of doxorubicin-loaded micelles was developed and micelles were characterized extensively for their physico-chemical properties. Cellular uptake of micelles was analyzed and their therapeutic potential was assessed in vitro on human cancer cells. To passively accumulate in solid tumors, nanoparticles need to be long-circulating and must remain in the blood circulation for hours. Therefore, the pharmacokinetic profile and biodistribution of doxorubicin-loaded micelles in rats was analyzed and compared to the gold standard of long-circulating nanoparticles: PEGylated liposomes. In a next step, a protocol for the preparation of so-called gold-nanohybrids was developed. Such nanohybrids are valuable tools to analyze nanoparticle-cell interactions and the intracellular fate of nanoparticles in detail. Further, such nanoparticles can be used as diagnostic tools. In a last step, micelles were functionalized with an antibody for targeted drug delivery. Cellular internalization of these micelles was analyzed using an array of methods. In a second part, a novel therapeutic strategy using the main effector protein of the rat parvovirus (H-1) for the treatment of hepatocellular carcinoma (HCC) was developed. H-1 parvovirus showed promising results in the preclinical setting and was consequently tested in a clinical trial in patients suffering form glioma. Despite this development, viral therapies may be linked with several issues. Therefore, the potential of the viral effector protein NS1 for the treatment of liver cancer was analyzed after non-viral gene delivery. In a first step, the gene-delivery efficiency and the therapeutic effect were analyzed in a panel of human liver cancer-derived cell lines. Various in vitro assays were used to study the NS1-induced cell death in detail. To show that this therapeutic approach is specific for cancer cells, the treatment was furthermore tested on healthy human liver cells. To identify cells that are susceptible for this therapeutic approach, a biomarker for the sensitivity to non-viral NS1 therapy was evaluated. Finally, safety of this therapy was analyzed in mice after single and multiple dosing

Drug Delivery Technology Development in Canada

Canada continues to have a rich history of ground-breaking research in drug delivery within academic institutions, pharmaceutical industry and the biotechnology community

Advances in Nanomaterials in Biomedicine

Advances in Nanomaterials in Biomedicine” provided a platform for more than 110 researchers from different countries to present their latest investigations in various fields of nanotechnology, new methods and nanomaterials intended for medical applications. Modern achievements in the field of nanoparticle-based diagnostics, drug delivery and the use of various nanomaterials in the treatment of diseases are presented in 11 original articles. The published reviews provide a comprehensive analysis of the current information on the use of nanomedicine in the treatment and diagnosis of cancer and liver fibrosis, in the field of solid tissue engineering and in drug delivery systems