Nanotechnology has the potential to dramatically improve the drug delivery model, resulting in new classes of therapeutics as well as diagnostics. Some of the advances and opportunities were discussed earlier this year at the “NSTI BioNano” conference.
At NanoViricides, the delivery vehicle itself replaces the drug. Eugene Seymour, M.D., CEO, predicts that this new approach to destroying viruses will change the paradigm for antiviral therapeutics. The approach delivers a drug-free, “broad-structure nanoviricide that should work on up to 95 percent of all viruses.”
The technology, which was developed by Anil R. Diwan, Ph.D., president, is based upon a nanomicelle, a 20 nm biodegradable polymeric structure to which a ligand is attached as a targeting molecule. In the body, it is thought that these ligands connect to the sialic acid receptors found on most viruses.
“Although the mechanism of action has not been clearly elucidated, it has been suggested that there is fusion of the nanomicelle and viral envelope since they’re both amphiphilic. This could ultimately result in the breaching of the viral envelope,” Dr. Seymour explained. “The viral capsid containing the genome would then be released into circulation, where macrophages then could destroy the capsid and its contents.”
The technology is at the preclinical stage, and NanoViricides plans to soon file a pre-IND. The next step, according to Dr. Seymour, is to manufacture larger quantities for Phase I studies. “We’ve had success treating rabies. We were able to salvage 30 percent of the animals infected with a lethal dose,” although the treatment was administered after the animals began to show symptoms.
In tests against HIV “we’ve seen a 100 percent increase in survival times over those of the standard three-drug cocktail.” Dr. Seymour noted that tests performed by the U.S. Army involving Ebola produced positive results. He also said they have seen no toxicity in more than 1,000 animals.
According to Dr. Seymour, the implications for this technology are “pretty radical. This is a classic use for nanotechnology in medicine.” A similar approach is being used by others to develop cancer therapies using a payload of gold or doxorubicin to destroy the tumor cell.
Better than Lipids
Carigent is developing a polylactic-glycolic acid particle that can be customized to target specific tissues by attaching multiple functional groups to the particle’s surface at high densities. The particle is licensed from Yale University and is being developed for a high degree of flexibility. Seth Feuerstein, M.D., president, says the particle “has shown promise for delivering RNAi.”
Other benefits vary according to the surface of the particle and its function. It has high surface densities that Dr. Feuerstein said are “an order of magnitude better than previous attempts.” Likewise, circulating time varies substantially depending upon the surface composition. Even the delivery mechanism is flexible, allowing for surface binding or endocytosis, according to the target.
Single or multiple agents for therapeutics can be delivered in the particle, allowing for a sustained, controlled release directly into the target, “a benefit over lipids,” claimed Dr. Feuerstein. The delivery technology has applications in fields as diverse as vaccines and immune modulation.
One current version encapsulates doxorubicin for oncology. Another version, advanced through an SBIR grant, encapsulates paclitaxel. In addition, researchers are using the technology to deliver siRNA and miRNA. For example, Tarek Fahmy, Ph.D., assistant professor of biomedical engineering at Yale University, is investigating this particle for use in training the immune system, added Dr. Feuerstein.
Negatively Charged Liposomes
Novosom has developed a new class of liposomes called Smarticles® to encapsulate oligonucleotides including antisense and siRNA and sneak them through the bloodstream for tightly targeted delivery.
“Unlike the vast majority of current delivery systems, the Smarticles are negatively charged in the bloodstream,” noted Steffen Panzner, Ph.D., CSO and founder.
Once they reach the targeted cell and endocytosis begins, the pH level drops to five or four, making the vector neutral and eventually positive. That change in charge causes it to fuse with the endosome and release the oligonucleotides it carries into the cytosol.
For targeting, Novosom “takes advantage of different particle sizes but also uses the combinatorial diversity of Smarticles created through variations in lipid chemistry and mixing ratios,” said Dr. Panzner. Some versions deliver up to 90% of the injected dose to the liver, while others target tumors or inflamed tissues.
Several preclinical studies are showing efficacy. Animals display no liver toxicity, no antibody formation, no T-cell memory, and a maximum tolerated dose of 50 mg/kg in primates.
One Smarticles application targets CD40 mRNA, which is involved in inflammatory disease and B-cell cancers. The protein is present on the membranes of antigen-presenting cells that trigger an immune response and is the receptor for CD154, which is expressed on T cells.
By reducing the CD40 mRNA, the T cells cannot bind to the antigen-proliferating cells, preventing an immune response and thus inflammation. Novosom has exclusive, worldwide rights from Isis Pharmaceuticals to access antisense inhibitors that target CD40mRNA for all indications.
The company plans to move a CD40/Smarticles combination through preclinical safety trials in 2009. This would then mark the second Smarticles- based product in clinical development. The technology received a first IND in March for PNT2258, a combination of Smarticles and a bcl-2-targeting oligonucleotide from Pronai Therapeutics.
Nanoparticle Delivered siRNA
Calando Pharmaceuticals is developing a nano-based delivery system for siRNA, something that James Hamilton, M.D., CEO, said could enable siRNA as an entirely new class of therapeutics.
The company is in Phase I trials recruiting for initial compound CALAA-01. Dr. Hamilton claimed it is “the only systemically delivered siRNA in the clinic using a nanoparticle drug delivery system.” CALAA-01 targets the M2 subunit of ribonucleotide reductase.
Calando uses the same delivery system for multiple siRNA therapeutics, making it easy for the company to rapidly develop new drug candidates. A second siRNA therapeutic, CALAA-02, is in the preclinical phase and is expected to enter clinical trials in 2009. CALAA-02 targets hypoxia inducible factor-2 alpha, which is overexpressed in many cancers and is instrumental in tumorigenesis. This intracellular target has been difficult to inhibit with monoclonal antibodies or traditional therapeutics, according to Dr. Hamilton.
Calando employs a three-part delivery system called Rondel™ (RNAi Oligonucleotide Nanoparticle Delivery). The system mixes an excipient vial consisting of a linear, polycationic cyclodextin polymer backbone, a stabilizing agent, and a targeting ligand with a second vial containing anionic siRNA.
When combined, spherical particles measuring about 70 nm self-assemble, and protect the siRNA from degradation in serum. “The use of a modular excipient vial provides flexibility with regards to type of targeting ligand or type of oligonoucleotide payload,” added Dr. Hamilton.
Early studies indicate that the particles do not trigger an immune response, appear stable, and are nontoxic in animal studies. “Calando uses transferrin to target nanoparticles against transferrin receptors, which are often upregulated on the surface of a variety of tumor cell types. The binding of transferring facilitates endocytosis of the siRNA payload,” explained Dr. Hamilton. However, once the nanoparticle delivery system releases the siRNA inside the cell, the payload is highly specific for the gene knockdown target.
Preclinical mouse work showed marked tumor inhibition. For example, when athymic nude mice containing subcutaneous HT144 (human melanoma) tumors were treated with CALAA-01, tumor size was well under 500 cubic millimeters for the 31 days of treatment. In contrast, the tumors in mice treated with D5W grew only slightly slower than those of the untreated mice. Those tumors reached the 500 cubic millimeter size in about seven days, and continued growing.
“A second-generation Rondel system could include a wide variety of cellular targeting ligands,” said Dr. Hamilton. “Theoretically, using other targeting systems such as aptamers or Fab fragments would be easy to incorporate.” The therapy also has the potential to expand beyond oncology to target autoimmune inflammatory conditions and intraocular diseases.
Throughout the field, “there is considerable interest in multivalent drugs that can bind to multiple closely spaced binding sites on a target protein or protein multimer,” related Don Bergstrom, Ph.D., professor of medicinal chemistry at Purdue University’s Birck Nanotechnology Center. “The high avidity associated with such binding should result in increased drug potency. We are looking for efficient ways to construct such peptide multimers.”
His approach uses synthesized peptide-conjugated peptide nucleic acids synthesized on automated peptide synthesizers, yielding combined molecules in which the peptide portion contains a sequence that will bind a target, and the peptide nucleic acid contains a sequence that facilitates self-assembly. This work is at the early stages.
“We have constructed and characterized the complexes and have data showing their apparent uptake into MCF-7 cells, but no drug molecules have been tested.”
Dr. Bergstrom is working with Rashid Bashir, Ph.D., director of the micro and nanotechnology laboratory at the University of Illinois–Urbana-Champaign, which has constructed silicon-based field-effect nano-plate sensors for label-free electrical detection of binding. The group has covalently attached PNA to the sensors and is exploring thermally controlled hybridization of solution oligonucleotides to the sensors. Each sensor in the array can be individually heated by applying AC current.
Dr. Bergstrom said the particularly high affinity of LNA to PNA hybridization results in robust capture sequences. “The same PNA sequence is attached at every address and allowed to hybridize with the complementary inert protecting sequence. We are now working to control the heating so that only a single-heated nanoplate will undergo an exchange hybridization with a sensing sequence.”
The sensing sequence contains separate regions for binding PNA and miRNA, and allows the sequential attachment of different sensing sequences to different addresses.