Browsing by Subject "Doxorubicin"

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

Albumin is emerging as a promising and versatile carrier to improve the pharmacokinetic and therapeutic profiles of drugs because of its unique physiological properties. The objective of this dissertation is to develop drug delivery systems that bind to and exploit the endogenous albumin in order to extend the plasma half-life of small molecule and peptide pharmaceutics for cancer and diabetes therapy. Three albumin binding drug delivery systems are developed and explored here: 1) albumin binding micelles, and 2) albumin binding peptide-drug conjugates (PDCs), both for cancer therapy, and 3) albumin binding peptide chimera for diabetes therapy. Elastin like polypeptide (ELP) is used as the recombinant expression and production platform and also as the carrier backbone for the micellar system.

For the albumin binding micelles, ELP was fused to the C-terminus of a protein-G derived albumin binding domain (ABD) and the ABD-ELP fusion was recombinantly expressed in and purified from Escherichia coli. Doxorubicin (Dox) was conjugated to the C-terminus of the ABD-ELP fusion, and conjugation of 4-5 copies of the drug to one end of the ABD-CP triggered its self-assembly into ~100 nm diameter spherical micelles. ABD-decorated micelles exhibited sub-micromolar binding affinity for albumin and also preserved their spherical morphology in the presence of albumin. In a murine model, albumin-binding micelles exhibited dose-independent pharmacokinetics, while naked micelles exhibited dose-dependent pharmacokinetics. In addition, in a canine model, albumin binding micelles resulted in a 3-fold increase in plasma half-life and 6-fold increase in plasma exposure as defined by the area under the curve (AUC) of the drug, compared with naked micelles. Furthermore, in a murine colon carcinoma model, albumin-binding nanoparticles demonstrated lower uptake by the reticuloendothelial system (RES) system organs —the liver and spleen— and higher uptake by the tumor than naked micelles. The increased uptake by s.c. C26 colon carcinoma tumors in mice translated to a wider therapeutic window of doses ranging from 20-60 mg equivalent of Dox per kg body weight (mg Dox Equiv.kg-1 BW) for albumin-binding CP-Dox micelles, as compared to naked micelles that were only effective at their maximum tolerated dose of 40 mg Dox Equiv.kg-1 BW.

For the albumin binding PDC system, 1 to 2 Dox molecules were conjugated to ABD via a pH-sensitive linker without the loss of aqueous solubility. ELP was used as a purification tag for the recombinant synthesis of ABD and was removed by an enzymatically-catalyzed reaction following drug conjugation. As with the albumin binging micells, albumin binding PDCs (ABD-Dox) showed strong nanomolar binding affinity for human and mouse serum albumin. Upon intravenous administration in mice, ABD-Dox showed an elimination half-life of about 26.5 h that is close to mouse albumin circulation time. In addition, within 2 h after administration, ABD-Dox distributed into tumor at approximately 4-fold higher concentration than free Dox and moreover while free Dox showed a quick clearance from the tumor site, ABD-Dox maintained a steady concentration in tumor for at least 72 h. The improved pharmacokinetic and pharmacodynamic profiles of ABD-Dox resulted in enhanced therapeutic efficacy in syngeneic C26 colon carcinoma and xenograft MIA-PaCa2 pancreatic tumor models compared to free Dox as well as aldoxorubicin, an albumin binding and the first-ever Dox prodrug to show superiority over Dox as a single agent in clinical trials.

Finally, for the albumin binding peptide chimera system, ABD was recombinantly fused to glucagon like peptide-1 (GLP1). In an obese and diabetic (db/db) mouse model, a single subcutaneous injection of the albumin binding GLP1 chimera provided glycemic control and effecting weight loss for over 7 days compared with PBS treated controls.

Collectively, the albumin binding technology developed here promises great potential for delivery of the traditional small molecule and peptide drugs as well as nanoparticulate therapeutics, whose clinical effectiveness is impaired by their poor pharmacokinetics and/or pharmacodynamics.