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In the fight against autoimmune diseases, like HIV (pictured), researchers have turned to nanomedicine to help deliver life-saving drugs to patients.

More than a decade ago, nanotechnology became an integral part of the overall scientific research world. Governments started funding programs specifically aimed at nanotechnology, research universities opened their facilities and coursework to the “new” discipline, and journals focusing on nano research became commonplace.
And now, many researchers believe, it’s nanomedicine’s turn to do the same. Nanomedicine—which has emerged as nanotechnology’s most important sub-discipline—is the application of nanotechnology to the prevention and treatment of disease in the human body. It is already having an impact clinically among some of the deadliest diseases in the world.

“Nanomedicine is far from the stuff of science fiction. The possibilities for nanomedicine to help us diagnose, treat and image diseases are endless. Imagine a ‘smart’ nanomedicine that is able to bind to tumor cells and enhance imaging and diagnosis, at the same time as being able to deliver a gene therapy or chemotherapy agent. With the technologies available to us and our multidisciplinary teams, this will be possible in my lifetime,” said Phoebe Phillips, head of the pancreatic cancer translational research group at the University of New South Wales in Sydney.

Phillips and her team have created a nanoparticle that dramatically increases its effectiveness as an anti-cancer drug for patients with pancreatic cancers, which is one of the fastest killing cancers from time of initial detection, often leaving patients with no suitable treatment options and only weeks to live.

While nanomedicine can—and likely will—play a role in diagnostics, regenerative medicine, prosthetics and more, the effect the sub-discipline is currently having on the treatment of autoimmune diseases and cancers is significant.

Nanomedicine for HIV
Thirty years ago, a diagnosis of HIV/AIDS was essentially a guarantee of a painful, protracted death. It wasn’t until 1996 that researchers discovered antiretroviral drugs, and the potent combination therapy that leads to successful management of HIV/AIDS in most cases. However, not much has changed since that discovery. Those suffering from the autoimmune disease still require daily oral dosing of three to four pills, and chronic oral dosing has significant complications that can arise from the high pill burden experienced by patients, leading to non-adherence to therapies for a variety of reasons.

“I’ve been working in HIV for over 20 years,” Andrew Owen, professor of molecular and clinical pharmacology at the University of Liverpool (UK) told Laboratory Equipment. “I was trying to understand the variability in drug exposure that occurs between different individuals and the genetic basis for that. We were finding a lot of interesting things, but they weren’t clinically implementable. They gave us a good understanding of why drug exposure was variable, but it didn’t actually help the patients in any way.”

In an attempt to solve the problem rather than just characterize it, Owen turned to nanomedicine in 2009, eventually becoming part of the first team to conduct human trials of orally dosed nanomedicines for HIV. Since then, Owen and his interdisciplinary team at the Liverpool Nanomedicine Partnership have secured more than £20 million of research funding for a multitude of nanomedicine-based approaches to HIV, such low-dose oral delivery, long-acting injectable medications and targeted delivery of antiretrovirals.

Some of Owen’s most important research to date tackles two of the pharmaceutical industry’s biggest challenges: oral delivery of potent drugs and supply and demand.

“One of the major problems that has plagued drug discovery and drug development over the last 30 years has been compatibility with oral drug delivery,” Owen explained. “The pharmaceutical industry has wrestled with that because they can develop very potent molecules across diseases, but actually delivering those molecules orally is very challenging. As you try to design into the molecule oral bioavailabilty, you usually get further away from the potency you want.”

The Liverpool team solved this problem with the creation of Solid Drug Nanoparticles. The technology consists of combining a normal drug, in its solid form, with particles on that drug that are measurable within the nanometer scale. There are other things packed into the formulation as well, such as FDA-approved stabilizers that are proven to help disperse the drug. Owen says it is all about increasing the surface area covered by the drug.

“If you imagine you take a granulated form of the drug, you’re going to get big chunks of drugs in the intestinal tract when dissolution happens. But if you have nanometer-sized particles within the GI tract, then you are going to get a complete coating of the inside of the intestine after you take the drug,” Owen explained. “What that does is it massively increases the surface area covered by the drug, which saturates all sorts of drug influx processes within the GI tract.”

Since 80 percent of a human’s immune system is concentrated in the gut, the Solid Drug Nanoparticles are the perfect mechanism. The immune cells in the gut instinctually move toward the particles, creating a pathway for the drugs to cross the intestines, move through the lymphatic system, and finally into the systematic circulation.

In February, Owen presented the results of two trials at the Conference on Retroviruses and Opportunistic Infections (CROI) that confirmed his Solid Drug Nanoparticles can be effective at a 50 percent dose reduction. Specifically, Owen and his team applied the nanomedicine-based approach to the formulation of two drugs: efvirenz (EFV) and lopinavir (LPV). EFV is the current WHO-recommended regimen, with 70 percent of adult HIV patients in low- and middle-income countries taking the medication. At 50 percent of the dose, the patients in the trial were able to maintain plasma concentrations of the conventional dose.

Globally, the supply of drugs needed to treat every patient with HIV is outstripping manufacturing capability—meaning we, as a human species, cannot physically make enough HIV medication to treat everyone with the disease. A 50 percent reduction in dose means twice as many patients served with the existing drug supply.
Owen and his team are working with multiple global partners to move the technology forward. For the drugs already formulated, the Medicines Patent Pool and Clinical Health Access are helping to scale up and take them to market. Meanwhile, USAID’s Project OPTIMIZE is applying the nanoparticle technology to the newest HIV drugs for use in low- and middle-income countries.

For their latest collaboration with Johns Hopkins University, the Liverpool team was just awarded $3 million to examine the use of implantable technologies that can deliver drugs for weeks, or even months.

The current oral drug regimens for HIV comprises three drugs in combination—one is the major driver for efficacy, and the other two are nucleoside reverse transcriptase inhibitors that prevent resistance to the main drug. However, current injectable formulations are only available with the main drug—none include the nucleoside reverse transcriptase inhibitors.

“So, our project aims to develop the first long-acting injectable nucleoside reverse transcriptase inhibitors so that we can use them to have a fully long-acting regimen that matches the current clinical paradigm for therapy,” Owen said.

The Liverpool/Hopkins team has also thought about applying their long-acting injectable technology to other chronic diseases, such as malaria and tuberculosis, as well as some cardiovascular applications.

Nanomedicine for diabetes
When the nanoparticles he was working with as an imaging tool didn’t produce the desired results, Pere Santamaria grew frustrated—but he didn’t give up. Instead, the doctor and professor at the University of Calgary (Canada) changed his assumptions and pursued his experiment—until the data came pouring in that confirmed it wasn’t a failed experiment at all. Rather, it was a discovery.

“The discovery of Navacims was a bit serendipitous,” Santamaria told Laboratory Equipment. “Thankfully I am a little OCD and I didn’t let the failed experiment go.”
Navacims are an entirely new class of nanomedicine drugs that harness the ability to stop disease without impairing normal immunity. Santamaria has been studying Navacims for the past 17 years, ever since unintentionally developing them. He even started a spin-off company, Parvus Therapeutics, Inc., to help bring the drugs to market.

In autoimmune diseases, white blood cells, which are normally responsible for warding off foreign invaders and disease, turn on the body, attacking the good cells and causing their destruction. Each specific autoimmune disease results from an attack against thousands of individual protein fragments in the targeted organ, such as the insulin-producing pancreatic cells in the case of type 1 diabetes.

But Santamaria’s studies show that nanoparticles decorated with protein targets acting as “bait” for disease-causing white blood cells can actually be used to reprogram the cells to rightfully suppress the disease they once intended to cause.

Once the immune system recognizes the presence of a Navacim, a white blood cell is reprogrammed by epigenetic changes into a lymphocyte that no longer wants to cause tissue damage, but rather work to suppress disease. According to Santamaria, the reprogramming step is immediately followed by an expansion of that population of lymphocytes—one now-good white blood cell dividing into a million.

“Basically they turn the tables on the immune system, and then there is a very sophisticated series of downstream cellular events that arise from that reprogramming event that involves the recruitment of other lymphocytes and other cell types that completely suppress the inflammation in the organ that is being infected,” Santamaria explained. “This happens extremely efficiently and comprehensively. This is an approach that can efficiently, selectively and specifically blend a complex response without impairing basic immunity.”

In addition, the design of Navacims is modular, meaning the nanomedicine can be applied to several—if not all—autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. Navacims can be altered to target different diseases by simply changing a small portion of the “bait” molecules on the nanoparticles. Santamaria’s studies have shown this to work in about seven autoimmune diseases thus far.

In April, Santamaria’s company Parvus entered into a license and collaboration agreement with Novartis for Navacims. Under the terms of the agreement, Novartis receives exclusive worldwide rights to use Parvus’ Navacim technology to develop and commercialize products for the treatment of type 1 diabetes, and will be responsible for clinical-stage development and commercialization. Parvus will still be responsible for conducting ongoing preclinical work in the diabetes area, with some research funding from Novartis.

“We’ve had such a long time to prove ourselves, that this is not a flash in the pan, that this is something serious and robust,” Santamaria said. “We know so much about the mechanisms of our actions, and so much granularity. I think there are no other drugs that have reached the clinic with this level of understanding. That was painful in the beginning for us, but in the end it’s going to be good.”

Pere Santamaria discovered a new class of nanomedicine drugs that can halt autoimmune diseases, without impairing a person’s normal immunity. Photo: Riley Brandt, University of Calgary

Nanomedicine for solid tumors
Killing the disease without harming the patient is not just an approach for autoimmune diseases—it is also the goal for cancers, which have arguably the most egregious side effects. That’s why Penn State professor James Adair developed an investigational compound that targets and destroys cancer cells while leaving healthy cells unharmed. The compound, made from ceramide nanoliposome, is currently in a Phase I clinical trial.

The compound works by encapsulating ceramide in nanoparticles. Although ceramide is a known anticancer therapeutic agent, it has previously never been used in clinical testing to treat cancer because of its instability—it breaks down in the human body too quickly. Adair’s research and business partner, Mark Kester, resolved ceramide’s instability by outfitting the compound in a proprietary fatty lipids coating.

“Nanoliposomes have been used for decades to deliver drugs in the blood stream that otherwise would literally just float on top of the liquid,” Adair, professor of materials science and engineering, told Laboratory Equipment. “They are so hydrophobic, they wouldn’t have been deliverable. That’s where the ceramide comes in.”

Donning its protective coating, ceramide can freely flow through the body before eventually being sucked in by the tumor in question as it steals resources from its host. Once the compound infiltrates the tumor, it penetrates the cellular lining before depositing its chemotherapeutic cargo.

In dozens of animal tests, Adair and Kester found that the compound remained in the body attacking tumors for more than a day—without harming noncancerous cells. This is a far cry from current chemotherapy methods that ultimately deliver only a small percentage of the dose to a cancerous tumor, leaving the rest to harm the patient’s healthy immune system and body.

Keystone Nano, the biopharmaceutical company co-founded by Adair and Kester in 2005, recently began testing the ceramide nanoliposomes in an FDA-approved Phase I clinical trial. The patients eligible for the trial include those for whom conventional treatment, including surgery and chemotherapy, has failed, as well as those cancer patients who have no alternatives left.

As of October, three patients finished the trial, another cohort of three are presently undergoing the trial, and about 13 patients are waiting to begin.
The intent of the clinical trial is to determine the maximum tolerated dose—and thus far, there isn’t one.

“We haven’t found a maximum tolerated dose up through what should be the treatable dosing based on all the preclinical data we have,” Adair said. “So far, no adverse events or side effects have been noted.”

While phase 1 of the trial caters to patients with solid tumors, a potential phase 2 may focus on liver cancer. If all goes well, the researchers say ceramid nanoliposome could become an FDA-approved drug within a few years of trial completion.

Adair has also developed another formulation called nanojackets, which is a composite of calcium phosphate and silicate with water present in the nanoparticle. They are formulated to seek out cancer cells to deliver a chemotherapeutic agent or imaging cargo. In animal tests, that’s led to similar results as ceramide nanoliposome, where chemotherapy delivers only to the cancer, ignoring healthy cells. It has also produced another benefit: early detection. Nanojackets can be combined with fluorophores, which emit light, to detect cancer tumors using near infrared light.

“When we embed fluorophores in these particles, it will circulate for up to 96 hours in the blood stream, and gets benignly cleared through the GI tract,” Adair explained. “We find the particles in tact if they haven’t found the diseased tissue in animal models. These particles have a very long circulation time, so we can literally target the cancer cells we want to treat and diagnose.”

Adair has recently scaled up the process of formulating nanojackets in his lab—which is something most academic researchers find nearly impossible.

“We are making 20 L quantities of the formulation, and I don’t see any upper limit for the volume we can synthesize of these colloidal formulations to deliver to cancer and diagnose the disease at the earliest stage,” Adair said. “There’s a tendency to synthesize very small quantities of material, and then university technology fails at the scale up stage. I’m comfortable with the fact that we’re not going to fail at that phase.”

The researchers are hoping for a nanojacket clinical trial sometime in the next two years.

Nanomedicine for pancreatic cancer
Pancreatic cancer is the most chemo-resistant, and is therefore among the hardest cancers to treat—the best chemotherapy drug available to patients prolongs life by only 16 weeks. Pancreatic tumors have a very challenging pathology—they are surrounded by extensive scar tissue that makes up 90 percent of the tumor. It’s the scar tissue that is the problem—it blocks chemotherapy drug delivery to tumor cells, and also creates an environment that causes pancreatic cancer cell chemotherapy resistance.
It’s for these reasons the University of New South Wales’ Phillips chose to focus her nanomedicine expertise on pancreatic cancer specifically.

“I wanted to design a smart drug to overcome this obstacle,” Phillips told Laboratory Equipment. “My overall goal is to tackle both the pancreatic cancer cells and also the ‘helper cells,’ which produce the scar tissue. To truly make a difference in the treatment of pancreatic cancer patients, we cannot ignore this hard-to-treat characteristic.”

First, Phillips and her team successfully identified a key promoter of tumor growth, cancer spread and chemo-resistance in pancreatic tumors called β|||-tubulin. The researchers discovered that this gene was overexpressed in tumor samples obtained from patients. The team knew they needed to inhibit the gene, but, like many other cancer-causing genes, it is non-druggable due to its structure. That’s when Phillips turned to nanomedicine as an urgently needed alternative.

She developed a small, star-shaped nanoparticle that efficiently binds and protects a gene-silencing tool known as short interfering RNA (siRNA). Even packed with siRNA, the nanoparticle retains its small size, a key component for it to successfully penetrate the scar tissue of pancreatic tumors and specifically target β|||-tubulin. When tested in mice, the nanomedicine decreased the growth of tumors by more than 50 percent, also reducing the overall spread of the cancer.

“The significance of our nanomedicine lies in its potential to inhibit any tumor-promoting gene or cocktail of genes personalized to the genetic profile of a patient’s tumor,” Phillips said. “We believe our work will lead directly to the development of new chemotherapies that will target not just the cancer cells, but the surrounding scar tissue-producing cells, thereby improving patients’ survival prospects.”

Other cancers such as lung, ovarian and prostate also overexpress the gene β|||-tubulin, thus Phillips believes it possible to repurpose the nanomedicine to treat those cancers as well. Her lab has recently received additional support to perform further pre-clinical testing on mice, the last step required before advancing the nanomedicine to a human clinical trial.

Ceramide nanoliposome has been approved for phase one clinical human trials by the FDA. Researchers used nanotechnology to stabilize ceramide, a delicate compound that’s a known anticancer therapeutic agent. Photo: Keystone Nano via Penn State University
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