Drug formulations that use nanoparticles to target and dispense therapeutic agents are making an impact in the clinic, says Mark Peplow

Source: Microspheres (DAVID MCCARTHY/SCIENCE PHOTO LIBRARY) Poly(lactic-co-glycolic acid) microspheres are being used to prepare BIND-014 — a nanoparticle used to transport docetaxel — which has shown promise in early-phase clinical trials

In the war against disease, carpet-bombing rogue cells into submission can be counterproductive. Conventional drugs often diffuse throughout the body, so large doses are sometimes needed to achieve effective concentrations at a particular site, which can cause severe side effects.

Nanomedicine offers a more tactical approach.1 Tiny particles measured in billionth of a metre — nanometres — can encapsulate and deliver drugs to dramatically enhance their effectiveness. Associating a therapeutic molecule with a nanoparticle can enhance its solubility by orders of magnitude, allowing hydrophobic drugs to be carried more easily through the bloodstream. This could help to address a serious challenge in pharmacology, since an estimated 40 per cent of new drugs are poorly soluble in biological fluids.

Even soluble drugs may circulate in the body for only a short time, necessitating frequently repeated doses. Loading an active pharmaceutical ingredient (API) within a nanoparticle can shield it from being metabolised by the body’s natural defences, and allow it to remain in circulation for hours rather than minutes. Nanoparticles can also enable the controlled release of the drug, or deliver two different drugs simultaneously to give a more powerful combination therapy.

Crucially, nanoparticles can also help in targeting specific tissues, delivering their drug cargo to just one area of the body, or around one type of cells needing treatment. This minimises systemic side effects, and may also reduce the risk of cells developing resistance to the drug.

Nanoparticles are particularly useful delivery vehicles because they can be decorated with different types of functional molecules. As well as bearing a drug, some carry targeting molecules that seek out specific receptors on the surface of a cell. They can also incorporate components that make them useful for diagnostic imaging — contrast agents for magnetic resonance imaging (MRI), or fluorescent molecules, for example — which can reveal whether the complex is hitting its mark in the body.

A wide variety of nanoparticles are now in clinical use, or making their way through trials, including liposomes, polymeric micelles and polymeric nanoparticles. Many of these featured at the 9th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, held in Lisbon, Portugal, from 31 March to 3 April, where nanomedicine was a major theme. “There was a lot of excitement and enthusiasm for it,” said Yvonne Perrie of Aston University, Birmingham, who spoke at the conference.

Tiny milestones

The most well-established nanoparticle therapies are based on liposomes, spherical structures that are most commonly built from phospholipids, such as phosphatidyl choline.2 These molecules have a hydrophilic phosphate head and a hydrophobic tail containing fatty acids. In water, the hydrophobic parts of the molecules align so that they self-assemble into lipid bilayers that surround a watery core. This core can carry hydrophilic drugs in solution, while hydrophobic drugs can be loaded into the lipid layers.

Liposomes were first investigated as drug delivery vehicles in the early 1970s, and in 1990 AmBisome became the first liposomal drug to be approved in Europe (US approval followed in 1997). AmBisome contains amphotericin B, an antifungal agent, packaged into liposomes less than 100 nanometres wide.

AmBisome takes advantage of the liposome’s tendency to accumulate in certain immune system cells found in the liver and spleen, which also contain the fungi and parasites that the drug treats. This enables the liposomes to deliver a bigger load of the API to the cells than would be possible with amphotericin B alone.

The first liposomal treatment marketed in the US was also a significant breakthrough in nanomedicine. Doxil, approved by the US Food and Drug Administration in 1995, incorporates the anticancer agent doxorubicin, and is used against Kaposi’s sarcoma, a tumour caused by a form of herpes virus, and ovarian cancer, among others. (Doxil is known outside the US as Caelyx).

Doxorubicin works by sticking between the strands of the DNA double helix, effectively shutting down replication. But it has serious side effects, particularly cardiotoxicity, which Doxil helped to reduce.3

The lipids in liposomes can be quite mobile in vivo, potentially allowing drug molecules inside to leak out before reaching their destination. Doxil addressed this problem with a surface coating of poly(ethylene glycol) (PEG), which helps to hold the liposome together and shields it from the immune system. This tactic, known as PEGylation, has been used in at least a dozen successful drugs since 1990.

Pegylation has been a successful approach to protecting liposomes, but it is not a panacea. Some patients have antibodies to PEG, which can break through the protective covering. And some pegylated drugs tend to accumulate in skin cells, where they can cause hand-foot syndrome, characterised by tingling, numbness, swelling and redness. So rather than adding a protective layer of PEG, the liposome’s properties can be changed by altering the lipids themselves. Incorporating pH-sensitive phospholipids can also make the liposomes release their drug at just the right moment — remaining stable while circulating in the blood before breaking apart when they hit the more acidic conditions inside a cell.

And instead of relying on the liposomes’ intrinsic tendency to accumulate in particular cell types — a form of passive targeting — they can also be fitted with active targeting systems, using ligands such as antibodies or peptides to lock onto specific cells. The liposome SGT-53, for instance, bears a ligand that targets transferrin receptors that are overexpressed in some tumour cells. It carries plasmids that contain the p53 tumour-suppressing gene,4 and a clinical trial is now testing whether the package can also dispense a dose of the anticancer agent docetaxel at the same time.

Hidden charge

Liposomes are also being used to deliver adjuvants, essential components of vaccines that enhance the immune systems’ response to an antigen.

Drug-makers currently have very few adjuvants to choose from — most are based on aluminium salts, a formulation developed more than 70 years ago.

The liposome CAF01 is being used as a vehicle for a new tuberculosis vaccine, for example.5 Although BCG is an effective vaccine in children, it is not good at preventing pulmonary TB in adults, and there are also safety concerns about using BCG in HIV-infected infants.

CAF01 relies on N,N’-dimethyl-N,N’-dioctadecylammonium (DDA) to form liposomes that carry the adjuvant a, a’-trehalose 6,6’-dibehenate (TDB), as well as an antigen. The package can deliver both agents to key immune cells, minimising toxicity to the rest of the body while inducing a strong immune response. The State Serum Institution in Copenhagen, Denmark, which developed CAF01, has successfully tested the liposome in a phase I clinical trial for tuberculosis.

At the Lisbon meeting, Professor Perrie presented key results from her work to understand the mechanism of CAF01 and related liposomes. Her team has found that the liposome is effective even if the antigens are on the outside of the liposome, rather than contained within its structure. That means “you can mass-produce these liposomes and then add antigens to the exterior”, Professor Perrie said. Her team has so far shown that it is possible to add antigens for tuberculosis, hepatitis B and malaria in this way.

Positively-charged liposomes such as CAF01 can aggregate at the site of injection as they combine with negatively-charged species in the body. Professor Perrie has also shown that pegylating the liposome can help prevent this and improve circulation through the body.6 “When you pegylate a liposome you avoid aggregation,” said Professor Perrie. “The PEG hides the cationic charge from the body.”

Tailor made

Researchers are also looking beyond lipids, creating similar nanocarriers from synthetic polymers.

If that polymer contains hydrophobic and hydrophilic segments it can assemble into spherical micelles. Rather than forming a liposome-like bilayer with a watery core, polymer micelles usually have a single hydrophilic outer layer enclosing a hydrophobic core. This makes them particularly suitable as carriers for hydrophobic drugs, and they are generally better at evading breakdown in the kidneys than liposomes.

Synthetic polymers can also be more easily tailored to give them specific properties — fine-tuning their solubility, their ability to carry a particular drug, or the location and timing of their breakdown in the body.

In Lisbon, Alexander Kabanov of the University of North Carolina, Chapel Hill, presented his work on a micelle called SP1049C. It relies on a block co-polymer, which contains alternating sections of PEG and polypropylene glycol, and carries doxorubicin.7

This formulation has already seen success in phase II clinical trials against advanced oesophogeal cancer.8 The block co-polymer helps to stabilise the micelle, and carries the API readily through the bloodstream. “But very soon we realised that block co-polymers had properties beyond solubilisation,” said Professor Kabanov.

Once doxorubicin is released inside cells, the polymer then blocks a transporter protein that would otherwise eject the drug. Preventing efflux of the drug increases its concentration in the cell, potentially helping to prevent drug resistance. Supratek Pharma of Montreal, Canada, a company that Professor Kabanov co-founded, now says it hopes to move to phase III trials of SP1049C.

Micelles, too, can be fitted with targeting systems, Professor Kabanov noted. Folic acid, for example, will latch on to cancer cells that tend to overexpress folate receptors, and he has conducted experiments using this approach to deliver the cancer drug cisplatin.9

The medicinal gobstopper

But some self-assembled nanoparticles run the risk of falling apart as they are diluted in the bloodstream, or if they meet molecules that can worm their way inside and disrupt their structure.

Solid polymer nanoparticles, prepared by active chemical processing rather than self-assembly, tend to be more robust.10 Drugs can be physically entrapped within the nanoparticle, or chemically attached to it; breaking down the polymer matrix then provides control over the drug’s rate of release.

BIND-014, for instance, is a nanoparticle made from poly(lactic-co-glycolic acid) and PEG that transports anticancer agent docetaxel. It also carries a chemical group on its outer shell that targets prostate-specific membrane antigens, which helps to guide it towards cancer cells; it has produced promising results in early-phase clinical trials.11

At the Lisbon meeting, Paula Hammond of the Massachusetts Institute of Technology in Cambridge, US, discussed her work on even more complex “layer-by-layer” (LbL) nanoparticles.12 “Some people tell me that it reminds them of Willy Wonka’s everlasting gobstoppers,” laughed Professor Hammond.

Some of Professor Hammond’s LbL nanoparticles have a coating of hyaluronic acid that allows the particles to move stealthily through the bloodstream, but also actively target tumour cells. Once in place, inner layers carrying the API can unload their shipment. The nanoparticles’ layers also allow different drugs to be delivered in stages, as a timed combination therapy. “Now we’re no longer relying on a solid polymer or a liposome membrane to stay together through the body and then fall apart at the tumour site,” said Professor Hammond. “This can unveil different therapies at different times.”

Professor Hammond has developed a LbL nanoparticle with one layer containing small interfering strands of RNA (siRNA) that can lower the tumour cells’ defence mechanisms; and a core that subsequently dispenses doxorubicin.

Using a mouse model of triple-negative breast cancer, Professor Hammond’s team found that the double-barrelled medicinal gobstoppers were far more effective than those containing siRNA or doxorubicin alone.13

“The more we learn about cancer, the more we realise that we need multiple approaches to tackling it,” said Professor Hammond.

Cancer crusade

Professor Hammond’s work on cancer therapeutics exemplifies a broader trend in the field. “Cancer presents a strong case for nanomedicine,” she said.

More than seven million people around the world die of cancer every year, and it is the second-largest cause of death in the US. “It affects so many people’s lives,” said Professor Hammond. Yet cancer drugs tend to be highly toxic, so targeting can keep doses relatively low, avoiding widespread side effects through the body while delivering a knockout blow to tumour cells.14

Nanoparticles also take advantage of the ‘leaky’ vasculature around tumours, escaping from blood vessels and burrowing into tumour tissue. This enhanced permeability and retention (EPR) effect means that nanoparticles in the range 20-150nm or so naturally accumulate in tumour cells.

The drug paclitaxel, for example, which is a close chemical cousin to paclitaxel, can be delivered in several of the major nanoparticle formulations. This molecule was first discovered in the late 1960’s, isolated from the bark of the Pacific yew tree. The FDA approved paclitaxel (marketed as Taxol) as a treatment for ovarian cancer in 1992, and then breast cancer two years later. It became the best-selling cancer drug of its time, reaching $1.6bn in sales in 2000.

But each treatment of Taxol contained less than 1 per cent of the API by weight. As a highly insoluble molecule, it had to be teamed with excipients such as Cremophor (polyethoxylated castor oil) in alcohol. This formulation causes unpleasant side effects, and even though they can be minimised by premedication with a steroid or antihistamine, it still limits how much of the drug a patient can receive.

One potential solution is a liposome formulation. Known as LEP-ETU (liposome entrapped paclitaxel-easy to use), it has reached phase II clinical trials against breast cancer. The drug can be administered safely in higher doses, but studies suggest that the liposome breaks down quickly once in the bloodstream.

Micelle formulations have had more success. Genexol-PM consists of a block copolymer of methoxy-PEG and poly(D,L-lactide), which encapsulates paclitaxel in a nanoparticle roughly 20-50nm across. In 2007, the formulation became the first micelle drug formulation to win regulatory approval, in South Korea. Genexol-PM can deliver more than twice the maximum tolerated dose of paclitaxel compared with Taxol, and a phase III clinical trial of this formulation (branded as Cynviloq) against breast cancer is now under way in the US.

Meanwhile, a similar micelle called NK105 uses a different block copolymer — PEG and a modified poly(aspartate) — and is also in phase III clinical trials. This polymer has enhanced stability in the bloodstream and tests in animals have shown that it leads to a 24 times greater accumulation of paclitaxel in tumour cells compared with Taxol.

Paclitaxel has also been incorporated into a solid nanoparticle by binding it to albumin, a formulation known as Abraxane that was approved by the FDA in 2005. These 130nm particles contain about 10 per cent of the API by weight and may be assisted by a targeting effect — albumin can bind to a glycoprotein secreted by the tumour cells.

Professor Kabanov presented results in Lisbon showing that an alternative micelle formulation of paclitaxel offers an even higher API loading. Using polyoxazoline polymers, his micelles reached 45 per cent of API by weight, which could deliver eight times as much paclitaxel as Taxol in tests on mice.15 This is probably due to the extremely polar micelle core, which may form hydrogen bonds with the oxygen- and nitrogen-containing chemical groups in the paclitaxel molecule. Professor Kabanov’s team is now building a cheminformatic database to help predict which other drugs might be solubilised in similar ways using polyoxazolines.

High hurdles

Nanoparticles can add complexity to a formulation, but that does not necessarily present a problem for scale-up. Professor Hammond has prepared her layer-by-layer nanoparticles using a roll-to-roll printing process, although as yet has only conducted animal tests using “blanks” containing no drugs.16

Nanoparticles can add complexity to a formulation, but that does not necessarily present a problem for scale-up.

She is now working with Liquidia, a company based in North Carolina, which has already demonstrated that this technology can make an antigen-containing nanoparticle and used it in a phase I/IIa clinical trial against influenza.

Meanwhile, Professor Perrie is making liposomes using microfluidic systems that pump mere drops of solution through miniature reaction vessels the size of a large postage stamp. The advantage is that “it would give us continuous processing of liposomes”, she said. “At the moment they’re generally produced by batches, which is not ideal.”

The chips themselves can be stacked to increase their output volume. “These things can scale up,” said Professor Perrie. “You just put more and more chips together.”

But drug formulation often poses problems for manufacturers, and nanoparticles are no exception. One of the challenges for the field lies in ensuring sufficient loading of a drug or antigen into a liposome. An excess of free API that is left over must be removed before the liposome can be administered. “If you have to remove it that’s another process step, which nobody wants,” said Professor Perrie.

Physically entrapping drugs in micelles or liposomes can cause a two-phase release, where an initial burst of API is followed by a long tail-off, which may not be the best way to deliver the drug. One way to combat this involves chemically bonding the drug to its carrier using a linker that breaks apart in certain physiological conditions: a change in pH or enzyme levels, for example.

It can also be difficult to assess how long the nanoparticles persist in the body, and to what degree they are actually delivering encapsulated drugs into cells. Those pioneering formulations, Genexol-PM and Abraxane, actually begin to break down in the bloodstream before they reach specific tumour cells. Understanding these mechanisms will be an essential factor in future developments in nanomedicine.10

Quality control is another significant issue, since nanoparticles must be manufactured in a uniform and reproducible way, and any variability in composition should not affect the overall effectiveness of the treatment. Regulatory agencies understandably want to see safety and efficacy data for the nanocarrier, as well as the API. Fulfilling these requirements can increase the costs of clinical trials, as well as making manufacturing more expensive.17

Indeed, some multifunctional nanoparticles might struggle to get into the clinic because of their high cost, said Professor Perrie. But they are still useful, she added. As the “concept cars” of the pharmaceutical world, they may never go into mass production, but they demonstrate new concepts that could be incorporated into more commercially-viable drugs.

Despite all these hurdles, there was a widespread conviction among researchers at the Lisbon conference that nanomedicine could overcome them and deliver significant improvements in drug effectiveness. “The opportunities are very strong,” said Professor Perrie.