Andrew M. Rauth

BIOREDUCTIVE THERAPIES: AN OVERVIEW OF DRUGS AND THEIR MECHANISMS OF ACTION

PURPOSE Bioreductively activated drugs have been used as antimicrobials, chemotherapeutic agents, and radiation sensitizers. The present paper is an overview of their mechanism of action and application in the treatment of cancer. MATERIALS AND METHODS Drugs such as nitroimidazoles, mitomycins, and benzotriazine di-N-oxides were a focus of this research. Studies have ranged from the chemistry of the reductive process of activation to in vitro and in vivo studies in rodent and human cells, through to clinical testing. The variety of techniques and test systems brought to bear on these compounds is a strength of this field of research. RESULTS A detailed chemical understanding of the mechanism of action of a variety of bioreductives is now available. The enzymatic processes by which these drugs are activated and the cofactors involved in this activation are becoming well understood. Recent advances have been made in the design and use of dual-function bioreductives, bioreductive triggers of drug activation, and DNA-targeted bioreductives. Significant success has been demonstrated clinically with bioreductive drugs, used in combination with radiation and front-line chemotherapeutic agents. The areas of antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) are identified as new directions for bioreductive therapy. CONCLUSION The use of bioreductively-activated drugs for the treatment of cancer has made steady progress. The success obtained clinically and the new molecular approaches currently being implemented promise significant advances in the future.

A study of doxorubicin loading onto and release from sulfopropyl dextran ion-exchange microspheres

The objective of this study was to investigate various factors that influence doxorubicin (Dox) loading onto and release from sulfopropyl dextran ion-exchange microspheres (MS), and to evaluate the anticancer activity of the released drug in vitro. Dox was incorporated into the MS by incubating the MS with aqueous solutions of Dox at room temperature. The drug release was carried out at 37 degrees C in aqueous solutions containing NaCl with or without CaCl2. The kinetics of drug absorption and release, the amount of Dox released, and the stability of Dox after loading, freeze-drying, and release were determined by spectrophotometry. The cytotoxicity of Dox (the original drug or that released from MS) against murine EMT6 breast cancer cells was assessed using a clonogenic assay. An increase in the MS to drug ratio resulted in a higher absorption rate and a higher fraction of the drug extracted from the solution. The release rate and the equilibrium fraction of Dox released increased with a decrease in the initial amount of Dox loaded or an increase in the salt concentration. The addition of divalent ions (Ca2+) promoted drug release compared to NaCl alone. The percent loss of colony forming ability of the cells, a measure of cytotoxicity of the released Dox, was the same as parent Dox solutions, indicating that the drug bioactivity was fully preserved after the drug loading and release cycle. This work demonstrated that various drug release rates were achieved by varying the drug loading and that the MS-delivered Dox was effective against the cancer cells in vitro.

A novel doxorubicin-mitomycin C co-encapsulated nanoparticle formulation exhibits anti-cancer synergy in multidrug resistant human breast cancer cells.

Anthracycline-containing treatment regimens are currently the most widely employed regimens for the management of breast cancer. These drug combinations are often designed based on non-cross resistance and minimal overlapping toxicity rather than drug synergism. Moreover, aggressive doses are normally used in chemotherapy to achieve a greater therapeutic benefit at the cost of more acute and long-term toxic effects. To increase chemotherapeutic efficacy while decreasing toxic effects, rational design of drug synergy-based regimens is needed. Our previous work showed a synergistic effect of doxorubicin (DOX) and mitomycin C (MMC) on murine breast cancer cells in vitro and improved efficacy and reduced systemic toxicity of DOX-loaded solid polymer–lipid hybrid nanoparticles (PLN) in animal models of breast cancer. Herein we have demonstrated true anticancer synergy of concurrently applied DOX and MMC, and have rationally designed PLN to effectively deliver this combination to multidrug resistant (MDR) MDA435/LCC6 human breast cancer cells. DOX–MMC co-loaded PLN were effective in killing MDR cells at 20–30-fold lower doses than the free drugs. This synergistic cell killing was correlated with enhanced induction of DNA double strand breaks that preceded apoptosis. Importantly, co-encapsulation of dual agents into a nanoparticle formulation was much more effective than concurrent application of single agent-containing PLN, demonstrating the requirement of simultaneous uptake of both drugs by the same cells to enhance the drug synergy. The rationally designed combination chemotherapeutic PLN can overcome multidrug resistance at a significantly lower dose than free drugs, exhibiting the potential to enhance chemotherapy and reduce the therapeutic limitations imposed by systemic toxicity.

Pharmacology and toxicology of sensitizers: Mechanism studies

Nitroimidazoles are being studied extensively as hypoxic cell radiosensitizers. Besides their ability to selectively sensitize hypoxic cells to radiation, which depends on the parent compound, nitroimidazoles have a variety of other effects in vitro, in vivo and clinically which appear to require reductive metabolism. These other effects include direct cytotoxicity to hypoxic cells, mutagenicity and antimicrobial effects. As a first step to suggesting possible mechanisms for these other biological effects, a summary has been made of the known oxidative and reductive products of the two most widely studied radiosensitizers, metronidazole and misonidazole. Focussing on reductive products, it is clear that a great variety exists which are or may be reactive with biological molecules. Knowledge about the reduction chemistry of nitroimidazoles is new and far from complete. As a second step to suggesting possible mechanisms for these biological effects, it is important to view the problem in terms of the in vivo situation where distribution and sites of metabolism of the drug and its reduction products will be important factors. Variables such as levels of tissue oxygenation and nitroreductase activity will be important to assess. Combining basic information about the reduction chemistry of nitroimidazoles with knowledge about the pharmacology of drugs and their reduced products should allow a better assessment of mechanism of action as well as a better implementation of these drugs clinically.

A multifunctional polymeric nanotheranostic system delivers doxorubicin and imaging agents across the blood-brain barrier targeting brain metastases of breast cancer.

Metastatic brain cancers, in particular cancers with multiple lesions, are one of the most difficult malignancies to treat owing to their location and aggressiveness. Chemotherapy for brain metastases offers some hope. However, its efficacy is severely limited as most chemotherapeutic agents are incapable of crossing the blood-brain barrier (BBB) efficiently. Thus, a multifunctional nanotheranostic system based on poly(methacrylic acid)-polysorbate 80-grafted-starch was designed herein for the delivery of BBB-impermeable imaging and therapeutic agents to brain metastases of breast cancer. In vivo magnetic resonance imaging and confocal fluorescence microscopy were used to confirm extravasation of gadolinium and dye-loaded nanoparticles from intact brain microvessels in healthy mice. The targetability of doxorubicin (Dox)-loaded nanoparticles to intracranially established brain metastases of breast cancer was evaluated using whole body and ex vivo fluorescence imaging of the brain. Coexistence of nanoparticles and Dox in brain metastatic lesions was further confirmed by histological and microscopic examination of dissected brain tissue. Immuno-histochemical staining for caspase-3 and terminal-deoxynucleotidyl transferase dUTP nick end labeling for DNA fragmentation in tumor-bearing brain sections revealed that Dox-loaded nanoparticles selectively induced cancer cell apoptosis 24 h post-injection, while sparing normal brain cells from harm. Such effects were not observed in the mice treated with free Dox. Treatment with Dox-loaded nanoparticles significantly inhibited brain tumor growth compared to free Dox at the same dose as assessed by in vivo bioluminescence imaging of the brain metastases. These findings suggest that the multifunctional nanoparticles are promising for the treatment of brain metastases.

Screening of Lipid Carriers and Characterization of Drug-Polymer-Lipid Interactions for the Rational Design of Polymer-Lipid Hybrid Nanoparticles (PLN)

PurposeThe thermodynamics and solid state properties of components and their interactions in a formulation for polymer-lipid hybrid nanoparticles (PLN) were characterized for screening lead lipid carriers and rational design of PLN.MethodsVerapamil HCl (VRP) was chosen as a model drug and dextran sulfate sodium (DS) as a counter-ionic polymer. Solubility parameters of VRP, VRP-DS complex, and various lipids were calculated and partition of VRP and VRP-DS in lipids was determined. Thermodynamics of VRP binding to DS was determined by isothermal titration calorimetry (ITC). The solid state properties of individual components and their interactions were characterized using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD).ResultsDodecanoic acid (DA) was identified as the best lipid carrier among all lipids tested based on the solubility parameters and partition coefficients. VRP-DS complexation was a thermodynamically favorable process. Maximum binding capacity of DS and the highest drug loading capacity of DA were obtained at an equal ionic molar ratio of DS to VRP. In the PLN formulation, DA remained its crystal structure but had a slightly lower melting point, while VRP-DS complex was in an amorphous form.ConclusionsDrug loading efficiency and capacity of a lipid matrix depend on the VRP-DS binding and the interactions of the complex with the lipid. A combined analysis of solubility parameters and partition coefficients is useful for screening lipid candidates for PLN preparation.

Molecular interactions, internal structure and drug release kinetics of rationally developed polymer–lipid hybrid nanoparticles

This paper presents the first study of molecular interactions of ingredients and internal nanostructure in relation to drug loading and release mechanisms/kinetics of rationally designed solid polymer-lipid hybrid nanoparticles (PLN). The PLN were prepared by using a rationally selected composition that was found in our previous work to provide optimized interactions of verapamil hydrochloride (VRP) with dextran sulfate sodium (DS) and then the VRP-DS complex with dodecanoic acid (DA). The solid-state properties of the components, their molecular interactions and the morphology, particle size and internal structure of PLN were determined by use of differential scanning calorimetry, powder X-ray diffraction, (13)C nuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering. The distribution of VRP in PLN was examined by TEM imaging using a cationic gold tracer. Drug release studies were conducted in various media. Drug loading as high as 36% and loading efficiencies up to 99% were achieved in the rationally formulated PLN. Hydrogen bonding between drug, polymer and lipid and a uniform distribution of amorphous VRP within the solid lipid matrix were evident. Sustained drug release from the PLN was mainly controlled by ion exchange and diffusion processes. The results demonstrated that strong molecular interactions among the drug, the polymer and the lipid in the optimized formulation were responsible for the improved drug loading and release performance of the PLN.

Hybrid Quantum Dot-Fatty Ester Stealth Nanoparticles: Toward Clinically Relevant in Vivo Optical Imaging of Deep Tissue

Despite broad applications of quantum dots (QDs) in vitro, severe toxicity and dominant liver uptake have limited their clinical application. QDs that excite and emit in the ultraviolet and visible regions have limited in vivo applicability due to significant optical interference exerted by biological fluids and tissues. Hence we devised a new biocompatible hybrid fluorophore composed of near-infrared-emitting PbSe quantum dots encapsulated in solid fatty ester nanoparticles (QD-FEN) for in vivo imaging. The quantum yield and tissue penetration depth of the QD-FEN were characterized, and their biological fate was examined in a breast tumor-bearing animal model. It was found for the first time that chemical modification of the headgroup of QD-encapsulating organic fatty acids was a must as these groups quenched the photoluminescence of PbSe nanocrystals. The use of fatty esters enhanced aqueous quantum yields of PbSe QDs up to ∼45%, which was 50% higher than that of water-soluble PbSe nanocrystals in an aqueous medium. As a result, a greater than previously reported tissue penetration depth of fluorescence was recorded at 710 nm/840 nm excitation/emission wavelengths. The QD-FEN had much lower short-term cytotoxicity compared to nonencapsulated water-soluble QDs. More importantly, reduced liver uptake, increased tumor retention, lack of toxic response, and nearly complete clearance of QD-FEN from the tested animals was demonstrated. With a combination of near-infrared spectral properties, enhanced optical properties,and significantly improved biosafety profile, this novel hybrid nanoparticulate fluorophore system demonstrably provides real-time, deep-tissue fluorescent imaging of live animals, laying a foundation for further development toward clinical application.

5-Fluorouracil infusions and fractionated doses of radiation: studies with a murine squamous cell carcinoma

The present investigation describes the effects on a murine squamous cell tumor of combined treatment using radiation and 5-Fluorouracil (5FU), with emphasis on 5FU infusions. The tumor, SCC VII/To, was grown intramuscularly in the hind legs of C3H mice. Radiation was given locally with 100 kVp X rays either alone or in combination with 5FU by i.p. bolus injections or 4-14 day infusions using subcutaneously implanted mini-osmotic pumps. Studies with radiation alone indicated regrowth delay increased with total dose. This increase was less for fractionated than single doses. The effects of 5FU alone were compared using i.p. injections or 4, 7 or 14 day infusions. Tumor response to single i.p. bolus injections or 4 day infusions were not significantly different. Up to total drug doses of 200 mg/kg, 14 day infusions were least effective on regrowth delay, 4 day infusions were intermediate and 7 day infusions were most effective. Above total drug doses of 200 mg/kg, effects of 14 day infusions on regrowth delay increased rapidly. The LD50 for single i.p. bolus injections and 7 day infusions were similar, 230 and a total drug dose of 270 mg/kg, respectively. When a 7 day infusion of 5FU (133 mg/kg) was combined with increasing total radiation doses (1 or 5 fractions), the increase in regrowth delay was additive. Combining a fractionated dose of 5 Gy per day for 5 days (5/5 Gy) with increasing total drug doses of 5FU (single i.p. bolus injections or 4, 7 or 14 day infusions) resulted in regrowth delays that were dependent on the total dose of 5FU. Administering a 133 mg/kg dose of 5FU (via a single i.p. bolus injection or 7 day infusion) starting 2 days before, during, or immediately after 5/5 Gy gave the same regrowth delay, indicating no effect of drug sequencing. In conclusion, the above data indicate that (a) 5FU infusions (greater than 4 days) are more effective than 5FU injections on regrowth delay and (b) combinations of 5FU and radiation, produce an additive tumor response, which occurs independent of mode, schedule, and time of 5FU administration, and is dependent on 5FU total dose.

pH-Dependent doxorubicin release from terpolymer of starch, polymethacrylic acid and polysorbate 80 nanoparticles for overcoming multi-drug resistance in human breast cancer cells.

This work investigated the capability of a new nanoparticulate system, based on terpolymer of starch, polymethacrylic acid and polysorbate 80, to load and release doxorubicin (Dox) as a function of pH and to evaluate the anticancer activity of Dox-loaded nanoparticles (Dox-NPs) to overcome multidrug resistance (MDR) in human breast cancer cells in vitro. The Dox-NPs were characterized by Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The cellular uptake and cytotoxicity of the Dox-loaded nanoparticles were investigated using fluorescence microscopy, flow cytometry, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The nanoparticles were able to load up to 49.7±0.3% of Dox with a high loading efficiency of 99.9±0.1%, while maintaining good colloidal stability. The nanoparticles released Dox at a higher rate at acidic pH attributable to weaker Dox-polymer molecular interactions evidenced by ITC. The Dox-NPs were taken up by the cancer cells in vitro and significantly enhanced the cytotoxicity of Dox against human MDR1 cells with up to a 20-fold decrease in the IC50 values. The results suggest that the new terpolymeric nanoparticles are a promising vehicle for the controlled delivery of Dox for treatment of drug resistant breast cancer.