Can creative carriers deliver the goods?

There are many reasons why conventional drug delivery technologies may not be effective and a more creative approach is required to convey the active ingredient to its destination. Dr Sarah Houlton looks at the more promising technologies in development

Red blood cells have the potential to be an ideal drug delivery vehicle

Sometimes, a simple pill just isn’t good enough to deliver a drug. The active might be destroyed by the acid or enzymes in the stomach; it might not reach the target site after absorption through the intestines; it might cause damage to healthy tissues on the way; transport across cell membranes might be poor. There are many reasons.

Some of these problems can be solved by delivering the actives via injection or infusion, or perhaps topically or via inhalation. And then there are particles comprising lipids or synthetic polymers that carry and protect the active on its journey through the bloodstream, and then promote its absorption.

There are, however, creative potential alternatives involving strategies inspired by nature. One such tactic is to use engineered red blood cells to carry drugs to the active site in the body. A group of scientists at MIT and the Whitehead Institute in Cambridge, Massachusetts, US, has managed genetically and enzymatically to modify red blood cells in a way that will enable them to carry drugs, vaccines and even imaging agents through the blood to the target site.1

A group of scientists in the US has managed genetically and enzymatically to modify red blood cells in a way that will enable them to carry drugs through the blood to the target site

Red blood cells have the potential to be an ideal drug delivery vehicle. While they are being made, their progenitor cells lose all their genetic material along with the nucleus, and thus the potential for genetically engineered red blood cells to cause tumour formation is significantly reduced. The Whitehead team started with early stage red blood cell progenitor cells, which still have the nucleus intact, and introduced genes that code for specific modified surface proteins on the cells. These proteins remain on the cell surface as the cells mature and jettison the nucleus.

The next step, deemed ‘sortagging’, involves modification of these cell surface proteins via a protein labelling technique. It uses the bacterial enzyme sortase A to form a strong chemical bond between the surface protein and another substance, which might be a small molecule drug, or an antibody that can bind a toxin. These modifications cause no damage to the cell or the surface itself.

The researchers believe there are many applications. These range from carrying clot-busting drugs to treat stroke patients or deep vein thrombosis to binding and removing bad cholesterol from the bloodstream. The cells may even be used to prime the immune system ahead of protein-based therapies, which all too often provoke an immune response on administration.

Another potential use – in which the US military is taking a keen interest and is funding via its Defense Advanced Research Projects Agency, or DARPA – is in combating the effects of biological weapons. Because red blood cells can hang around in the bloodstream for up to four months, one might envisage that antibodies designed to neutralise toxins could be attached to the cell surface. They would then circulate in the body, ready to catch any of the toxins that might be absorbed.

It is also possible to encapsulate drugs within red blood cells, rather than just attach them to the outside

It is also possible to encapsulate drugs within red blood cells, rather than just attach them to the outside. Over the years, various techniques involving electrical insertion or hypotonic red blood cell loading, followed by the cell being re-sealed, have been used to introduce both small molecule and biologic drugs into the cells. The drug can then be released either slowly by diffusion through the cell membrane, or more quickly if the cell wall undergoes lysis.

Various animal and human trials have taken place, with somewhat mixed results. One problem is that loading the cells inevitably causes damage, and this can lead the cells to break down quickly – in one study in mice, the activity of erythropoietin encapsulated within labelled red blood cells disappeared within a day, even though the labels showed the cells had a half-life in blood of about six days.2

Conversely, a study where an antisense compound was encapsulated in red blood cells showed improved hepatic delivery.3 Part of the problem is that clearly damaged red blood cells – as is the case with these cells because of the way the loading process alters the cell membrane and, sometimes, the energy transport mechanism – are naturally destroyed by macrophages and other phagocytes. For such strategies to succeed, ways of loading the cells without causing macrophage-attractive damage will be essential.

Of course, this phagocytosis process could be harnessed to enable delivery of the drug cargo to the lysosome. Lysosomal storage diseases such as Gaucher disease are an obvious target. In these conditions, enzyme deficiencies cause the accumulation of those enzymes’ substrates – in the case of Gaucher disease, the absence of the enzyme glucocerebrosidase results in the accumulation of glucocerebroside, which then builds up in the white blood cells, and organs such as the liver, kidneys and spleen. Treatment is via enzyme replacement therapy; engineered red blood cells with active lysosomal metabolism could offer the ideal route for delivery of these replacement enzymes directly to the point of activity.4

Pathogen carriers

Viruses and bacteria evolved to evade the immune system while homing in on target cells. So why not take advantage of this to deliver drugs to the cells?

Perhaps the best known application of this technique is the use of viruses to deliver gene therapy to cells. Viruses cause infection by integrating their genetic material with that of the host, enabling it to replicate, so if it were possible to replace the virus’s own genetic information with the gene therapy, could that provide a successful way of delivering new genetic material into cells?

Of course, the pathogenic virus must be rendered non-infectious so that it doesn’t cause disease, usually by deleting the part of the genome that causes viral replication. However, this must be done in such a way that viral promoters remain, and the transgene that the therapy is looking to insert is still present. A viral vector also needs to cause little or no change to the cells it is infecting. A genetic marker will often be added to facilitate the identification of cells that have taken up the transgene.

Several different viral vectors have been investigated over the years

Several different viral vectors have been investigated over the years. As the name implies, lentiviruses have slow incubation periods, and they also deliver a lot of viral RNA into the host cell’s DNA. Unusually, they can also infect non-dividing cells. However, the transgenes are often inserted at random points along the host’s chromosome, which can cause problems. A good example of this was the gene therapy trial in severe combined immunodeficiency, or SCID (perhaps better known as ‘bubble boy’ disease) in 2002, in which four of the trial subjects developed leukaemia.

Adenovirus and adeno-associated viruses (AAVs) may prove a better choice. AAVs are small viruses that infect humans but are not believed to cause disease, and thus the immune response to them is minimal. Like the lentiviruses, AAVs are able to infect non-dividing and dividing cells; however, the genetic material integrates into the host chromosomes much more specifically, with very few additional random insertions.

Adenoviruses have potential as viral vectors

One product delivered via this method has already been approved in Europe: UniQure’s Glybera (alipogene tiparvovec).5 It uses an AAV to deliver the gene that encodes for the lipoprotein lipase enzyme, and is administered via intramuscular injection. People with lipoprotein lipase deficiency are unable to synthesise this enzyme, which plays an important role in the breakdown of fatty acids. It affects maybe one or two people in a million, and leads to extremely high blood triglyceride levels. Patients also tend to experience recurrent pancreatitis, which can be fatal. Clinical trials showed a significant reduction in triglyceride levels over the long term.

Very many other clinical trials using AAV vectors to deliver gene therapy have been carried out and are ongoing, with treatments designed for a range of conditions. These include cystic fibrosis, in which the virus is delivered via aerosol to the lung, and age-related macular degeneration, with subretinal viral delivery. Several trials involving the delivery of genetic material to the brain are also in progress, albeit in the early stages, for diseases such as Alzheimer’s, and Batten’s disease in children.

Results from a Phase I trial in Parkinson’s disease, to insert the GAD gene into neurons in the brain, demonstrated that the gene transfer into the brain could be done safely, and Phase II trials showed some clinical benefit.6 However, the company developing it, Neurologix, failed to raise the necessary funding for Phase III trials, perhaps reflecting nervousness among the investment community about such an unknown technology. It went out of business in 2012.

Still in business is Celladon, a California-based biotech looking to use AAV to deliver the SERCA2a gene via infusion into the coronary arteries, where it is delivered to cardiac muscle cells in patients with advanced heart failure.7 This gene codes for the SERCA2a enzyme, which is involved in the control of calcium ion flow in and out of the cells, a process that is essential in the cardiac muscle cells’ contraction and relaxation. The enzyme is underproduced in failing heart cells, and the idea is that introducing this gene will kick off enzyme production again, restoring the cells’ function. Positive results have been achieved in Phase II trials, and maintained after the trial finished; further trials are under way.

A modified version of AAV, self-complementary adeno-associated virus, has also been engineered to improve further its delivery potential. AAV contains single-strand DNA, which must first be converted to double-strand DNA in the host cells; scAAV contains both strands, albeit shorter, speeding up the expression process. A downside is that it can provide only half the amount of genetic material. These virus products may also have increased immunogenicity.

Delivery using bacteria

DNA plasmid vectors can also be inserted into live bacteria which, as they contain RNA polymerases, are able to create the enzymes or cytokines these genes encode for at the active site. Obviously, these bacteria need to be safe and non-pathogenic, with the list of suitable bacteria including Streptococcus gordonii, which colonises mucosal membranes, and Lactococcus lactis, the lactic acid bacterium that is very commonly used in protein production. For example, L. lactis has been used to deliver interleukin-10, an anti-inflammatory kinase, directly to the intestinal mucosa with the aim of treating inflammatory bowel disease.8 A Phase I trial showed that the strategy appeared safe, and did not cause untoward adverse effects.

DNA plasmid vectors can be inserted into live bacteria; suitable candidates include Streptococcus gordonii

Meanwhile, on the drug delivery front, scientists at Aarhus University in Denmark are in the early stages of a project to engineer L. lactis to deliver drugs directly to intestinal cells in patients with Crohn’s disease. Drugs for this condition are typically given in enteric coated form so they pass through the stomach and release drugs in the intestine, where they are needed. L. lactis could provide the perfect answer – it is both non-colonising and non-invasive, and passes through the stomach unharmed. The organisms will be genetically modified to die once they have delivered the drugs they contain.

Various live attenuated pathogenic bacteria have been investigated as potential drug delivery systems, but there are drawbacks, such as a risk of their virulence returning. An alternative is to create a bacterial ghost, with no genetic material. If a Gram negative bacterium is modified such that its contents are removed but the bacterial envelope remains intact, the resulting bacterial ghost has potential as a carrier for drug molecules, or even DNA fragments. Protein E-mediated lysis can be used to create bacterial ghosts from numerous different Gram negative bacteria, including E. coli, H. pylori, Pseudomonas aeruginosa and P. putida, Klebsiella pneumonia, and salmonella species.

An early example of bacterial ghost platform technology used bacterial ghosts from Mannheimia haemolytica to deliver the cytotoxic agent doxorubicin to colorectal adenocarcinoma cells

An early example from bacterial ghost platform technology company Bird-C and the University of Vienna used bacterial ghosts from Mannheimia haemolytica to deliver the cytotoxic agent doxorubicin to colorectal adenocarcinoma cells.9 The drug was non-covalently loaded into the ghost, and given to the cells at a low concentration in an in vitro assay. Cytotoxicity was observed after incubation, in contrast to free doxorubicin at a similarly low concentration, which had a minimal effect.

Bird-C is now trying to raise funds for preclinical and clinical trials of cytotoxin-loaded ghosts in head and neck squamous cell carcinoma and peritoneal carcinomatosis, both of which have high unmet need. It also plans to start clinical trials, in collaboration with German company Pharmazentrale, next year on an E. coli ghost system in ulcerative colitis. Vaccine products are at an earlier stage of development.

The ghosts also have potential in DNA delivery and the targeting of macrophages. The large size of the ghost is a definite advantage: in one early study, up to 6000 plasmids encoding for the enhanced green fluorescent protein gene could be loaded into a single E. coli ghost, and efficiently transferred to murine macrophages – EFGP was expressed in nearly two-thirds of the macrophages.10 This could, for example, have potential in delivering gene therapy in inflammatory conditions such as rheumatoid arthritis, as macrophages tend to accumulate at injured sites.

Similar success was achieved in delivering DNA to melanoma cell lines, using ghosts derived from both E. coli and Mannheimia haemolytica.11 It has also successfully been delivered to human monocyte-derived dendritic cells.12 While it is still early days for ghosts in human therapy, the in vitro results show a good deal of promise, if they can be translated into clinical effects.

References

1. J. Shi et al. PNAS 2014, 111, 10131

2. M.I. Garin et al. Cytokine 1997, 9, 66

3. S.H. Kim et al. Biomaterials 2009, 30, 959

4. J.D. Humphreys and G. Ihler J. Lab. Clin. Med. 1080, 96, 682

5. D. Gaudet et al. Gene Therapy 2013, 20, 361

6. M.G. Kaplitt et al. Lancet, 2007, 369, 2097

7. M. Jessup et al. Circulation, 2011, 124, 304

8. H. Braat et al. Clin. Gastroenterol. Hepatol. 2006, 4, 754

9. S. Paukner et al. J Control Release 2004, 94, 63

10. S. Paukner et al. Mol. Ther. 2005, 11, 215

11. P. Kudela et al. Cancer Lett. 2008, 262, 54

12. P. Kudela et al. J. Immunother 2005; 28:136-43

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