Some suggest that it takes about a decade for a new technology to really make its influence felt in any field as scientists and engineers work out the kinks. And, in fact, it was 2004 when I wrote in another publication the words: “Regardless of the drug delivery vehicle format or formulation, there is every indication that nanotech methods will continue to be an active research avenue in the pharmaceutical community.”
Time, dear readers. And the time may now be here for nanotech in life sciences.
The promise of nanotechnology as applied to pharmaceutical development (nanomedicine) has been a long one. A promise that, like any other, has had its bumps along the way as well as its successes.
“This concept of nanotechnology has been around forever, but it seems that around 2000 it started to get a bit more traction,” recounts Chris Anzalone, president and CEO of Arrowhead Research, suggesting initial interest focused on materials and silicon processing, and the idea of nanomedicine itself not really raising its profile until about 2003.
“When you shrink matter down to the nanoscale, it reacts differently than at the macroscale,” he continues. “You can control certain properties at the nanoscale that you cannot when matter is larger. So it really captured the imagination of a lot of folks, and it looked like it was going to be very interesting and really transformational.”
A decade on, however, one might ask how transformational nanotechnology has been to drug development.
“Today, there are roughly 40 products based on nanomedicine and almost all of them are drug-delivery systems,” says Laurent Levy, CEO of Nanobiotix, who adds that there are another 200 products in the clinical development pipeline globally.
“When we look at those, most of them are coming from the first-generation technology,” he continues. “A nano-something, liposome or object, that will make a drug less toxic, will give a better biodistribution or better efficacy of the drug.”
And that was largely the value proposition of nanomedicine: improving the ability to move and potentially target small-molecule drugs to diseased tissues while protecting the drug from the body and the healthy body from the drug.
It is a clarion call that is still heard in conference halls and boardrooms.
“Pharmaceutical companies do not have a ‘drug’ problem; they have a ‘drug delivery’ problem,” wrote Purdue University nanomedicine specialist James Leary in a 2013 editorial in Nanomedicine & Biotherapeutic Discovery. “It still makes little sense to deliver large amounts (perhaps 10 times or more of what should be needed) of drugs systemically when nanomedicine provides tools to decrease total patient exposure by a combination of increasing drug circulation time and providing at least partial targeting to diseased cells, while increasing local drug delivery to the cells of interest.”
In fending off the anti-hype, if you will, about the realistic merits of nanomedicine, Leary highlights a strong business reason for companies to consider the idea.
“By repackaging drugs that have already been FDA-approved, new ‘combo’ drug-devices could allow for 10-year extensions to patent lives, which can also be periodically improved for still more combo patent extensions.”
And to date, this has largely been how the industry has approached nanomedicine: as a mechanism to reformulate existing drugs, whether to improve delivery or efficacy while improving safety. Examples include pegylated interferons for hepatitis (e.g., PegIntron, Pegasys), liposomal cytarabine (DepoCyt) and daunorubicin (DaunoXome) and nanoparticle albumin-bound paclitaxel (Abraxane).
Building off this first-generation approach of repackaging approved drugs, many research groups are factoring in the reality that for many conditions, patients will receive a combination or cocktail of drugs, rather than one drug at a time. As such, they are trying to create an all-in-one pharmaceutical kit using various nanotechnologies.
In May, Zhen Gu and colleagues at North Carolina State University and the University of North Carolina–Chapel Hill described their efforts to use polyethylene glycol (PEG) to generate structures they call nanodaisies to transport a pair of anticancer drugs through the bloodstream. In this case, camptothecin is chemically linked to the PEG polymers while doxorubicin remains in the matrix solution.
“Both drugs attack the cell’s nucleus, but via different mechanisms,” explained study co-author Wanyi Tai in announcing their publication in Biomaterials.
“Combined, the drugs are more effective than either drug by itself,” added Gu, who enthused about upcoming preclinical efforts to test their platform.
But why limit yourself to two drugs, when you can do three?
In April, Massachusetts Institute of Technology (MIT) researchers did just that using the same drugs Gu tested and adding cisplatin, but rather than build a delivery vehicle and then load it with drugs, the scientists decided to incorporate the drugs during construction.
Describing their efforts in the Journal of the American Chemical Society, the particles were designed such that cisplatin is released from the complex when it interacts with cellular glutathione, camptothecin is released when its links to the complex are hydrolyzed by esterases and doxorubicin is released when the particle is hit with UV light.
When tested against ovarian cancer cells, the triplex nanoparticle more effectively killed cells than nanoparticles carrying only one or two of the drugs.
“In principle, there’s no limitation on how many drugs you can add, and the ratio of drugs carried by the particles just depends on how they are mixed together in the beginning,” said lead researcher Jeremiah Johnson in announcing the work. “If I want a particle with five drugs, I just take the five building blocks I want and have those assemble into a particle.”
Of course, in many therapeutic cases, multidrug combinations are less about hitting the body with a barrage of chemicals but rather hitting them strategically with a carefully timed regimen. And even in these situations, there is an opportunity for nanomedicine to step forward.
Mere weeks after the JACS announcement, another MIT group led by Michael Yaffe and Paula Hammond described their efforts to deliver a chemotherapeutic one-two punch that first weakened and then destroyed cancer tumors.
“We’re moving from the simplest model of the nanoparticle—just getting the drug in there and targeting it—to having smart nanoparticles that deliver drug combinations in the way that you need to really attack the tumor,” said Hammond, who had also worked with MIT’s Johnson on the triplex.
The researchers embedded anti-EGFR compound erlotinib in the surface of liposomes, as well as folate to target the liposomes, which were filled with doxorubicin. The idea was that once the nanoparticles reached the tumor cells and were ingested, erlotinib would begin to weaken the cells while doxorubicin slowly made its way into the nucleus where it would damage the DNA.
When given to mice implanted with human triple-negative breast or NSCLC tumors, the nanoparticles not only significantly shrank the tumors but they also were more effective than when the two drugs were administered more traditionally, even with dose-staggering.
“It’s like rewiring a circuit,” explained Yaffe. “When you give the first drug, the wires’ connections get switched around so that the second drug works in a much more effective way.”
“There’s a lag of somewhere between four and 24 hours between when erlotinib peaks in its effectiveness and the doxorubicin peaks in its effectiveness,” he added.
Another place where nanomedicine is starting to make significant strides is in RNA interference (RNAi), where the nanoshells and liposomes seem tailor-made to protect siRNA and microRNA molecules as they work their way through the bloodstream and into cells where they can down-regulate the aberrant gene expression associated with so many diseases.
Publishing in Pharmaceutics last year, John Rossi and colleagues at the Beckman Research Institute of the City of Hope reviewed some of the recent preclinical RNAi platforms being tested by various academic and pharmaceutical organizations, including liposomes, silica nanoparticles, protein-based systems and cyclodextrin polymers. They summarized their analysis with a list of five developments necessary to “effectively translate preclinical proof of concept to clinical efficacy,” as follows:
- RNAi gene silencing optimization with increased nuclease resistance and reduced immune activation;
- Delivery formulation for prolonged circulation time and enhanced biodistribution;
- Tissue- and cell-specific uptake;
- Efficient siRNA release from endosomes and RISC incorporation; and
- Elucidation of RISC loading and Ago2 function.
Arrowhead Research is one such company trying to leverage its nanoparticle platform, dynamic polyconjugates (DPCs), for RNAi. Arrowhead’s Anzalone sees siRNA delivery as a two-step challenge.
“One is getting stable siRNA into the right cell at the right time as opposed to cells where it’s not going to have an effect,” he says, repeating the call of small-molecule nanomedicine. “The second hurdle is once you get into a cell, that siRNA is encapsulated in endosomes.”
“The cell’s job is to degrade whatever this foreign body is and spit it out, and so the challenge is to beat the cell,” he continues, “to get that payload into the cytoplasm where it can do its job and load into RISC and knock down whatever gene product you’re trying to knock down.”
To accomplish this, Anzalone tips his hat to the scientists in Madison, who worked on Roche’s RNAi portfolio, which was acquired by and forms the base of Arrowhead’s pipeline.
“They sought to mimic what viruses do—find a way to get into the right cell and then get out of the endosome and do its business,” he explains.
In the process, he continues, they built a whole library of DPC molecules that can facilitate endocytosis and endosome lysis. The library approach—different molecules for different applications—is pivotal, he says, because it allows the company to optimize RNAi on a target-by-target basis.
Preclinical studies have shown that DPCs provide both significantly deep and sustained knockdown of targeted gene products.
“We showed a year ago, in our first HBV paper, the ability to knock down gene products three to four logs,” he enthuses. “We’re talking about 99.99 percent knockdown.”
As well, some of their experiments are showing durable knockdown of weeks or months on a single dose, which could have a significant impact on dosing schedules once the products get to patients.
The company’s initial therapeutic target was hepatitis B virus (HBV). About 400 million people globally live with chronic HBV.
As Anzalone explains, HBV seemed tailor-made for RNAi, because not only does the virus replicate new viruses when it infects hepatocytes, but it also produces tremendous quantities of specific proteins that serve to immunosuppress the host.
“The thought is, if we can silence that whole genome, we should be able to enable a functional cure, enabling the immune system to de-repress and clear the virus,” he says.
The indication was also attractive because current therapies on the market are focused on lowering the threat of viral spread between partners, which means they are lifelong prescriptions.
“And even if they do stay on that regimen, they still have an increased risk of hepatocellular carcinoma and cirrhosis arising from viral infection,” Anzalone adds.
The company also believes that interest in HBV is about to catch fire as it did with hepatitis C.
“The pharmaceutical world was focused on HIV for some time and did a great job, developed great cocktails that made that a more manageable disease,” Anzalone recounts. “It then shifted its attention, broadly speaking, to hep C and did a great job of making that probably history; we have extremely high cure rates now.”
Thus, the company believes the next shift would be into hep B, and he believes they could be at the forefront of when that shift happens.
Aside from HBV, Arrowhead is also exploring RNAi in another liver indication: alpha-1 antitrypsin deficiency (AATD), a storage disease wherein damage occurs to the liver and lungs when mutant alpha-1 antitrypsin folds improperly and cannot be secreted, so it accumulates in cells as a toxin. The hope is that by knocking down expression of the mutated enzyme, they can slow and possibly reverse the damage done to liver cells.
The company is working with patient advocacy group the Alpha-1 Foundation on this project, as Anzalone explains it, to get a better understanding of patient needs and access to patient populations as their lead product moves toward clinical trials.
But even DPCs, much like the other nanomedicine formulations described above, are largely passive complexes, simply providing conveyance and targeting for their therapeutic payloads. What if, instead, the nanotechnology itself provided the therapeutic mode of action?
According to Nanobiotix’ Levy, such innovations are starting to occur more frequently, entirely removing the concept of a small-molecule drug or biomolecule from the equation.
“When you can bring a physical tool at the subcellular level like a nanoparticle that will allow us to heat, to absorb energy, to deliver energy, then you can really change the way of seeing the medicine,” he says.
He very much sees an opportunity for principles of physics to play a role in therapeutics much as chemistry and biology have in the past and continue to do. To highlight his point, he offers a contrast between a chemical and physical approach to cancer.
“Let’s take 100 different breast cancer cell samples coming from 100 different patients,” he says. “If you take all those cells and apply a cytotoxic chemotherapy, some of them will die, some of them will not react to the chemo and some of them will start dying but then start to become resistant to the chemo.”
“If you take the exact same 100 cells coming from 100 different patients and you heat them at 80 degrees, every single cell will die,” he continues, “regardless of the genome of the patient, regardless of the genome of the cancer.
“When you bring physics back into medicine, then you can start thinking again about having some general treatments, and also you are more flexible on the price.”
As an example of a product that works purely on physical principles, he points to a therapeutic used in joint arthritis.
“We know that there is a lot of friction between bones and this causes much pain and inflammation,” he explains. “So people have developed liposomes that can act purely as a physical lubricant between the bones, restore the functions of the bones and remove inflammation and pain for the patient.”
Another example is Nanobiotix’ lead product NanoXray, a crystalline nanoparticle for local treatment of cancer. Levy describes it as a radiotherapy amplifier.
The NanoXray nanoparticles are made of hafnium oxide, an inert material capable of absorbing radiation and releasing large numbers of electrons that damage tissue near which the nanoparticles sit. The goal is to reduce the overall radiation dose required to achieve a therapeutic endpoint, thus minimizing the secondary damage associated with radiotherapy.
And because the nanoparticles are purely physical entities, they should work with almost any type of solid tumor. To further ensure this, the company is working on three formulations of the NanoXrays that can be administered either by injecting directly into a tumor, applied as a gel when used post-surgery to kill any missed cancer cells or intravenously.
Because of this diversity, Levy foresees a potentially huge market for the product.
“About six million patients a year are receiving radiotherapy, and potentially this could help many of those patients,” he says.
Researchers at Rice University, meanwhile, decided recently not to limit themselves to either physics or chemistry in their nanomedicine approach, but rather to combine the two in a platform they call quadrapeutics.
Realizing that both radiotherapy and chemotherapy can have their limitations, Dmitri Lapotko and colleagues determined how to amplify the effects of both modalities through tiny, remotely triggered nanoexplosions. These plasmonic nanobubbles occur when colloidal gold particles, targeted to tumors with cancer-specific antibodies, are hit with near-infrared laser pulses.
Thus, when a tumor is struck with the laser, the nanobubbles succeed in killing a percentage of the cells, but even in those cells that don’t die, the nanobubbles make them more susceptible to chemotherapy. As well, the gold particles serve to amplify the effect of the radiotherapy, thus hitting the cancer cells from all sides.
“What kills the most-resistant cancer cells is the intracellular synergy of these components and the events we trigger in cells,” said Lapotko in announcing the findings. “This synergy showed a 100-fold amplification of the therapeutic strength of standard chemoradiation in experiments on cancer cell cultures.”
In the study published in Nature Medicine, the researchers found that a single treatment using only 3 percent of the typical drug dose and 6 percent of the typical radiation dose was able to essentially eliminate head and neck carcinomas from mice within a week.
Back to basics
Biology isn’t about to cede the nanomedicine field completely to chemistry and physics, however, as shown by the recent work being done at Australia’s EnGeneIC.
Relying on a strange bacterial mutation that causes cells to bud off small, genome-devoid sacks of cytoplasm that company co-founder Dr. Himanshu Brahmbhatt calls nanocells, the company has discovered that these nanocells are capable of delivering a variety of chemotherapeutics and siRNAs at levels impossible with synthetic nanoparticles to date.
“Almost a million molecules of drug can be packaged in a single cell,” Brahmbhatt explains. “That is very unlike the synthetic nanoparticles, as in liposomal particles such as Doxil, where these particles will carry about 14,000 molecules of drug.”
And what’s even more promising to the company is that once the therapeutic is encapsulated, it stays there, with no leakage into the bloodstream—thus limiting off-target toxicity, he suggests.
Because the nanocells are bacterial in origin, they are coated with polysaccharides, which can be used as anchor points for bispecific antibodies. The other half of these antibodies can then be specified to the target tumor cell-surface receptor, like EGFR or HER2 receptors.
As the nanocells, also known as EDVs (or EnGeneIC delivery vehicles), move through the bloodstream, they passively accumulate at the tumor site due to the naturally leaky vasculature found around tumors and, via the cell-surface receptors, are endocytosed to deliver their payloads, whether cytotoxic drug or siRNA.
One concern early in development, says Brahmbhatt, was active clearance of the nanocells from circulation by the host immune system, which would see the bacterial baggage as a foreign invader. Remarkably, however, the immune response seemed to work in EnGeneIC’s favor.
Rather than be cleared from circulation, the nanocells appear to act as a decoy, in his words, attracting the attention of macrophages and then drawing them to the tumor site, where they find dying cancer cells.
“These macrophages then swallow up the dying tumor cells, and in doing so, they start displaying the tumor antigens in vivo,” he explains. “So you are triggering off a really big cascade of the immune response that then translates into an antitumor response rather than an antibacterial response.”
For this reason, Brahmbhatt describes EDVs as a cytoimmunotherapeutic that “not only kills tumor cells, but at the same time, in a physiologic manner, wakes up the immune system to then augment the antitumor efficacy.”
EnGeneIC is also looking at its own one-two punch scenario for EDVs, seeing them as a potential way to reverse drug resistance in cancer. The argument goes that if you know the resistance pathway that’s been activated—for example, the multidrug resistance (MDR) pump—you can first hit the tumor with an EDV carrying an siRNA to down-regulate the MDR pump.
“Then you can come in with the same EDV carrying a drug to which those same tumors were highly resistant,” Brahmbhatt enthuses. “Because you’ve turned off the pumps, these tumor cells have become exquisitely sensitive to the drug and you can eliminate the tumors.”
EnGeneIC has performed a number of Phase 1 studies in different cancers showing the EDV platform is safe and is preparing a U.S. Food and Drug Administration submission to initiate a Phase 1/2 study on several tumor types. The company is also about to commence a microRNA study in mesothelioma and expects to initiate a personalized medicine trial that will rely on drug resistance and sensitivity analysis of tissue samples from different cancer patients to tailor EDV treatment.
Small products, big challenges
One traditional caution for all of this nanomedical enthusiasm, however, is that it can be hard enough to find a viable formulation for a simple small-molecule drug and that the evolution to nanoformulations increases complexity dramatically. Even the best-designed nanomedicine may work wonderfully in the research and preclinical phases, but its ultimate manufacture may simply be commercially unfeasible.
This is a caution Nanobiotix’ Levy understands well.
“We have seen some projects under development like ultracomplex liposomes with two or three different targeting agents on the surface plus a mix of two drugs inside the liposome,” he offers. “The idea could work and it could also answer some of the unmet medical needs, but when it comes to manufacturing, that is very complicated.”
“The more complex the object, the more difficult is the manufacturing and the characterization of the product,” he continues. “If you can’t guarantee the characterization of the product, it means you can’t guarantee the quality of the product.”
Arrowhead’s Anzalone agrees.
“Bringing breakthrough technologies to the marketplace is difficult, but the science part of that is not the most difficult part,” he says. “Making good science once or twice or three times in the lab is a doable thing. But scaling that up and finding somebody who can manufacture these very new and novel materials at scale is a real challenge.”
That’s one thing he particularly likes about his company’s DPC platform, which he suggests sits in a sweet spot between being novel enough to be transformational medically, but close enough to existing technologies to be scaleable under GMP conditions.
“What’s going to kill a lot of these nanotechnologies is not the big science, it’s the block and tackling of just manufacturing,” he says. “Finding a way to make these materials at scale, make them consistently, finding capital to either make them yourself, which you almost assuredly cannot afford to do, or more appropriately find somebody who can make these, which is not straightforward at this moment.”
These exact concerns are what has EnGeneIC’s Brahmbhatt smiling, however.
The company already has in place a small-scale but modular cGMP manufacturing plant that they are currently trying to get CGA-registered, with an eye to future FDA approval. But perhaps more importantly for EnGeneIC, because EDVs originate with bacteria, the initial portion of their manufacturing is handled by microbes looking for a free meal.
“With a 10-L lab-scale bacterial culture, you can end up with 1013 nanocells—and it is very reproducible,” Brahmbhatt brags. “The human therapeutic dose is on the order of 5 x 109 EDV, so 1013 is a very large amount.”
Further enhancing the process is its relative simplicity when compared with the preparation of synthetic nanoparticles.
The harvested nanocells are co-incubated with the drug for a short period before excess drug is washed away using standard pharmaceutical filters. The drug-loaded particles are then co-incubated with the desired bispecific antibody for about an hour before excess is again filtered away. The targeted, drug-loaded therapeutic is then ready for lyophilization. No chemical engineering as is typical with synthetic nanoparticle delivery systems simplifies things dramatically.
“With just four technicians and three days, we can manufacture in excess of a thousand human doses,” he says. “The cost of goods is so low that each therapeutic that we are shipping to the various pharmacies for our clinical trials is about $30 per dose.”
Nanobiotix, meanwhile, extends its vision even further downstream, considering how the product will be used when it reaches hospitals.
“The more you request a change of the existing medical practice to develop your innovation, the higher will be the hurdle and cost for the healthcare system,” says Levy. “We’ve chosen this particular technology because X-ray equipment is present all over the world.”
NanoXray, he explains, doesn’t require the end user to use different equipment or really change their clinical protocols. The only difference from current medical practices is a single application of the product before the first radiotherapy session.
“I can guarantee that when you look at the different ongoing developments, many of those developments will require a lot of change in the existing medical practice,” he warns, “meaning that the healthcare system at the end will not only pay for the product itself but also for the change in the medical practice.”
And that might just be enough to keep a good, effective product out of the market.