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European Biopharmaceutical Review

Crackling the Code


A rare supply of encapsulated porcine insulin-producing cells have been shown to be a promising alternative for the treatment of type 1 diabetes, with a significant reduction of unaware hypoglycaemic events and improvement in patient quality of life

Diabetes is a chronic disease characterised by high blood glucose levels resulting from the body’s inability to produce, or respond appropriately to, sufficient insulin. Type 1 diabetes occurs when the pancreas is unable to produce sufficient insulin for the body. It affects about 34 million people worldwide and requires insulin replacement therapy to stabilise blood glucose levels. A serious and potentially fatal complication is unaware hypoglycaemia, which occurs in up to 20 per cent of type 1 diabetics. This group of patients have lost their ability to demonstrate warning symptoms (sweating, tremor and tachycardia) and during hypoglycaemic episodes they can develop dizziness, confusion and blurred vision (1,2). In severe cases, uncontrolled hypoglycaemia can lead to a coma, seizure, or even death.

Treatment Options

Type 1 diabetes mellitus is typically treated by subcutaneous injections of human insulin or analogues which, though lifesaving, need to be given for the duration of the patient’s life. Conventionally, insulin injections are generally given two or three times per day (the dosage and timing being based on the individual’s blood glucose levels and/or glycosylated haemoglobin level). However, in some cases, three or more times daily regimens are utilised in an attempt to mimic the normal insulin release pattern. Intensive insulin therapy, combined with frequent monitoring of blood glucose levels (four to seven times per day), can achieve ‘tighter’ blood glucose control throughout the day, and this substantially reduces, but does not eliminate, the development and progression of diabetic complications (3). Intensive insulin therapy is now advocated by specialist clinicians whenever feasible, but the disadvantages of this approach are that it requires at least four injections of insulin daily and is associated with a high incidence of hypoglycaemia.

Among the newer treatment strategies that have been proposed for type 1 diabetes, transplantation of pancreatic islets, sourced either from other humans or animals, has received a great deal of attention worldwide (4,5), particularly as stem cell research (for example, the use of human embryonic stem cells or induced pluripotent stem cells from patients being developed into transplantable insulin-producing cells) is still at an early stage. Islet transplantation can restore not only the insulin-secreting unit, but also the precise fine-tuning of insulin release. In some centres, good results with human islet allotransplantation have been obtained in the short-term, with some patients achieving insulin-independence over a year or two (6). However, major obstacles to the success of human islet allotransplantation include:

  • Primary non-functioning of the transplanted islets, transplantation of an inadequate number of functioning cells, or cell dysfunction and apoptosis due to host inflammatory responses mediated by macrophage activation
  • Allorejection (immune-mediated rejection responses culminating in loss or decreased function of the graft)
  • Autorejection (recurrence of the underlying disease pathogenesis that triggered diabetes resulting in destruction of the transplanted islets)
  • The requirement for continuous administration of immunosuppressive drugs which, in addition to having numerous adverse effects, do not always protect the transplanted islets, and have also been shown to have adverse effects on their function
  • The limited supply of human islet tissue (a minimum of two donor pancreata is required for each patient)

Xenotransplantation Using Porcine Islets

Due to the limitations imposed by the shortage of human islet tissue, xenotransplantation of porcine islets is a promising alternative, and some encouraging early results using this approach have been achieved (7-9). The use of porcine islets provides a practical solution to the islet supply problem since pigs are readily available, easy to breed, are not endangered species, and also exhibit morphological and physiological similarities to humans (10-12). There are other advantages to sourcing porcine islets:
  • The supply of pig cells can be readily expanded by optimising the supply of donor animals
  • Pig and human insulins have close structural and biological similarities
  • Physiological glucose levels in pigs are similar to those in humans
  • Porcine insulin has been used safely to treat diabetes for several decades, and has only been replaced by human sequence insulin relatively recently
  • The biosafety of the cells can be thoroughly determined prior to transplantation

The implanted porcine islets have the potential to mimic normal physiological insulin response in type 1 diabetics to the extent that near-normal glycaemic levels may be achievable without exogenous insulin or with a reduced requirement for it. As a result, long-term complications associated with diabetes may be prevented and patients may experience less hypoglycaemia than they currently do with recommended insulin-intensive regimens.

The porcine islets used in this study were sourced from a closed herd of designated pathogen free (DPF) pigs originating from the sub-Antarctic Auckland Islands. These animals have had essentially no contact with other pigs or humans for about 150 years, and have subsequently been bred in isolation in New Zealand. Their origin and isolation has protected them from the common porcine viruses. Any possible sources of zoonotic infection were closely monitored to ensure the pigs were free of all specified diseases. The production processes included rigorous infection surveillance procedures comprising screening for bacteria, fungi and Mycoplasma spp and bacterial endotoxin testing.

Xenotransplantation Without Immunosuppression

Cell encapsulation technology was used to provide immunoprotection for transplanted islets via the use of a semipermeable membrane which acted as a protective barrier. This procedure involves extruding a mixture of islets and an ultrapure sodium alginate solution through a droplet-generating needle into a bath of gelling cations (calcium chloride). Islets entrapped in the calcium-alginate gel are then coated with poly-L-ornithine (PLO) followed by an outer coat of alginate. The central core of alginate is then liquefied by the addition of sodium citrate and the final capsules have a diameter of 650 to 750 μm. These encapsulated islets are incubated in culture medium in cell culture flasks with media changes every two to three days as required.

The materials used in the encapsulation process are polyelectrolytes, consisting of linear or branched repeat units each carrying a negative (alginate) or positive (PLO) charge. Alginate is manufactured as a sodium salt, which is displaced during the crosslinking process. The formation of the alginate-polyornithine-alginate (APA) permselective membrane is accomplished through the sequential exposure of oppositely charged materials in a process designed to minimise exposure of stress to the islets within the capsule. This is accomplished by carefully selecting and purifying the bulk polyelectrolytes used to form the physical barrier, as well as an efficient and robust encapsulation process.

Calcium is a critical but intermediate component of the encapsulation process. Initially, crosslinking of alginate by divalent cations occurs between guluronate (G) residues, due to their 1-4 linkages, which orients the monomers such that hydroxyl and carboxylic groups can participate in ionic bonding. This crosslinking serves to solidify the sphere and although the majority of bound calcium is retained within the sphere, cations on the surface undergo dynamic and reversible binding in the presence of coating and chelating agents, poly- L-ornithine (PLO) and sodium citrate. Owing to the anisotropic nature of the crosslinking process, there is a theoretical gradient that is established, with more calcium present on the outside than within the capsule core.

The function of PLO is to impart diffusion limitations on the resulting APA membrane, as well as to provide capsule strength and robustness. As a relatively small polymer with a molecular weight of about 12 kDa and enhanced affi nity relative to calcium, PLO binds to the alginate on the surface of the capsule by displacing small amounts of calcium.

Conclusion

Clinical treatment involves introducing encapsulated porcine cells into the abdominal cavity of the patient using a simple laparoscopic procedure. A recent trial in New Zealand with 14 patients demonstrated that these cells are a safe and effective treatment, resulting in:
  • A statistically significant reduction in unaware hypoglycaemic events
  • A trend to reduction in HbA1c
  • Improvement in patient-reported quality of lif
A Phase 1/2a trial in Argentina is currently in progress and an interim report was due to be published by the end of 2012.

References

  1. Canadian Diabetes Association 2008 clinical practice guidelines for the Prevention and Management of diabetes in Canada, Canadian Journal of Diabetes 32(Suppl 1): ppS1- S201, 2008
  2. Shalitin S, Phillip M, Hypoglycemia in type 1 diabetes: a still unresolved problem in the era of insulin analogs and pump therapy, Diabetes Care 31(Suppl 2): ppS121-S124, 2008
  3. DCCT (Diabetes Control and Complications Trial) Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus, N Engl J Med 329(14): pp977-986, 1993
  4. de Kort H, de Koning EJ, Rabelink TJ, Bruijn JA, Bajema IM, Islet transplantation in type 1 diabetes, BMJ 342:d217, doi: 10.1136/bmj.d217, 2011
  5. van Belle TL, Coppieters KT, von Herrath, MG, Type 1 diabetes: etiology, immunology, and therapeutic strategies, Physiol Rev 91(1): pp79-118, 2011
  6. Ryan EA, Paty BW, Senior PA, Bigam D, Alfadhli E, Kneteman NM, et al. Five-year follow-up after clinical islet transplantation, Diabetes 54 (7): pp2,060-2,069, 2005
  7. Elliott RB, Towards xenotransplantation of pig islets in the clinic, Curr Opin Organ Transplant 16(2): pp195-200, 2011
  8. Ekser B, Cooper DK, Overcoming the barriers to xenotransplantation: prospects for the future, Expert Rev Clin Immunol 6(2): pp219-230, 2010
  9. Ekser B, Ezzelarab M, Hara H, van der Windt DJ, Wijkstrom M, Bottino R, et al, Clinical xenotransplantation: the next medical revolution? Lancet 379(9,816): pp672-683, 2012
  10. Rayat GR, Rajotte RV, Korbutt GS, Potential application of neonatal porcine islets as treatment for type 1 diabetes: a review, Ann N Y Acad Sci 875: pp175-188, 1999
  11. Korbutt GS, Ao Z, Flashner M, et al, Neonatal porcine islets as a possible source of tissue for humans and microencapsulation improves the metabolic response of islet graft post-transplantation, Ann N Y Acad Sci 831: pp294-303, 1997
  12. Daar AS, Ethics of xenotransplantation: animal issues, consent, and likely transformation of transplant ethics, World J Surg 21(9): pp975-982, 1997

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Peter Hosking is Head of Operations at Living Cell Technologies. He is responsible for the company’s manufacturing processes, quality assurance, quality control, and the Molecular Diagnostics Laboratory. Peter has extensive experience in the biotechnology sector and has held senior management positions for Nufarm Health and Sciences in New Zealand, and Pfi zer in the US. He holds a BTech from Massey University, New Zealand, an MBA from the University of North Carolina, and is a former president of the New Zealand Biotechnology Association.
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