Patent Application
20080254090
The invention relates to the use of porcine pancreatic islet cells for the treatment of diabetes.
More particularly but not exclusively it relates to the use of porcine pancreatic islet cells with associated Sertoli cells for the treatment of diabetes by xenotransplantation.
Type 1 (insulin-dependent) diabetes mellitus is a common endocrine disorder that results in substantial morbidity and mortality, and has a major financial impact on individual patients and healthcare systems. Treatment with insulin, while life-saving, often does not provide sufficient control of blood glucose to prevent the life-shortening complications of the disease, and this has given rise to intensive research into better methods of achieving and sustaining normoglycaemia. Among the newer treatment strategies that have been proposed, transplantation of pancreatic β islet cells, obtained either from other humans or animals, has received the most attention worldwide. This is because islet cell transplantation can restore not only the insulin-secreting unit, but also the precise fine-tuning of insulin release in response to multiple neural and humoral signals arising within and beyond the islets of Langerhans.
As human islet cell transplantation (allotransplantation) is limited by the shortage of human islet tissue, the use of pig islet cells is currently viewed as the most promising alternative since:
The rationale for this treatment approach (termed ‘xenotransplantation’) is that the implanted porcine islets have the potential to mimic the normal physiological insulin response in type 1 diabetics, such that near-normal blood glucose levels may be achievable without insulin or with a reduced requirement for it. As a consequence, long-term diabetes complications may be prevented and patients should experience less hypoglycaemia than they do with the currently recommended ‘intensive’ insulin regimens.
Any new treatment strategy is burdened with problems and pitfalls before it can be implemented, and xenotransplantation of porcine islet cells is no exception. There have been a number of scientific and ethical/political barriers to implementation of the procedure, but as knowledge in the field has grown, these barriers have steadily receded. The problems that have arisen include:
It is an object of the invention to provide a method of treatment of diabetes, and/or a means to aid treatment of diabetes which has improvements to, or provides an alternative from, the abovementioned methods and/or means.
According to a first aspect of the invention there is provided a method of preparing aggregates of porcine pancreatic islets and porcine Sertoli cells capable upon implantation into a recipient, of producing insulin in vivo, including or comprising the steps of:
Preferably the combination is in a predetermining ratio from 1:20,000 (islet:Sertoli cells) to 1:100; more preferably the ratio is between 1:2,000 to 1:4,000.
Preferably the culturing step is over a time period between 3 to 7 days more preferably it is for 5 days.
Preferably the isolation of the islets is followed by purification of the islets.
Preferably the isolation and purification of the islets together comprise or include the steps of:
Preferably the digestion involves Liberase H and Xylocaine.
Preferably the isolation of the Sertoli cells is followed by purification of the Sertoli cells.
Preferably the isolation and purification of the Sertoli cells together comprise or include the steps of:
Preferably the method includes the step
Preferably or alternatively the method includes a prestep (before step 1)) of virological monitoring and/or testing of one or preferably both of the islets and Sertoli cells.
Preferably the method includes additionally or alternatively a pre-step of virological monitoring and/or testing of the piglet donors.
Preferably the islets and Sertoli cells derive from the same herd, more preferably from the same donor piglet.
Preferably the piglets are one week old donors.
Preferably the piglets are monitored and/or tested for infectious agents.
Preferably the pig herd is a New Zealand pig herd.
Preferably the step of the formation of the aggregates involves: the preservations of the original characteristics and/or native structure of the islets.
According to a further aspect of the invention there is provided an aggregate of porcine islets with Sertoli cells prepared substantially according to the above method.
According to a third aspect of the invention there is provided a method of treating a patient suffering from diabetes mellitus comprising or including the steps of:
Preferably the step of implanting or administering the aggregate may be by:
According to a further aspect of the invention there is provided a device for implantation into a recipient suffering from diabetes mellitus, the device incorporating aggregates of porcine pancreatic islets and porcine Sertoli cells, the aggregates being, or possessing the characteristics of, the aggregates previously described.
Preferably the device incorporating the aggregates may be one of:
According to a further aspect of the invention there is provided a method of preparing aggregates of porcine pancreatic islets and porcine Sertoli cells prepared substantially according to
According to a further aspect of the invention there is provided an aggregate of porcine pancreatic islets and porcine Sertoli cells substantially as described herein and with reference to any one or more of
The invention disclosed herein relates to the preparation and use of an “aggregate” of Sertoli cells with porcine islets.
Prior art methods involving the use of Sertoli cells and islets (or other cells) have generally involved processing and isolation of each separately and putting together at the time of the transplant.
We have found that preparation of an aggregate, in a predetermining ratio of Sertoli to islet cells, and co-culturing allows the islets time to grow and to use the growth factors deemed from the Sertoli cells in vitro before the transplant. We have found the islets function better as they are protected by the layer of Sertoli cells.
Ideally both the islets and Sertolis are derived from the same donors. This simplifies viral screening.
By “aggregate” as used herein we specifically mean a discontinuous layer of Sertoli cells over the surface of the natural islet structure.
Rationale for Cotransplantation of Sertoli Cells with Islets
Cotransplantation of islet cells with Sertoli cells isolated from the testes of donor animals has been investigated as a means of achieving:
Sertoli cells are known to play a critical role in various physiological activities such as the synthesis of certain growth factors [e.g. insulin-like growth factors 1 and 2 (IGF-1, IGF-2) and epidermal growth factor (EGF)], immunomodulation [possibly as a result of increased secretion of transforming growth factor-beta 1 (TGF-β1)], and an anti-apoptotic (cell death inhibitory) function.
Our recent studies in experimental animal models have shown that the presence of Sertoli cells improves the in vitro functional competence of islets, and that xenotransplantation of islet-sertoli cell aggregates in diabetic rats, rabbits and NOD mice prolongs islet cell survival, leading to reversal of the diabetic state. The precise mechanism by which Sertoli cells protect islet cell grafts against immune rejection is not precisely known, but appears to be related to stimulation of the production of growth and differentiation factors by Sertoli cells.
Thus, our invention deals with cotransplantation of Sertoli cells with islets as aggregated such that the Sertoli cells can act as “nursing” cell systems for the islets, providing both efficient immunoprotection and enhancement of their functional performance and longevity.
This approach is complementary to, and synergistic with, other approaches for providing immunoprotection and functional longevity for transplanted islet cells.
In particular our invention deals with the use of islet-sertoli cell aggregates in:
We have determined, the islet cell: Sertoli cell ratio that provides optimal protection of the islets against immune rejection and maximal functional longevity may range from 1:20,000 ratio to provide maximal insulin release down to at least 1:2,000. The range is based on the findings of experimental studies with islet-Sertoli cell aggregates conducted in our laboratory, and in collaboration with the University of Perugia and National University of Singapore.
Preparation of our preferred Islet-Sertoli Cell Aggregates, in the Ratio of 1:2,000-1:4,000
The pig herd from which porcine islets and Sertoli cells for incorporation in our islet-sertoli cell aggregates are obtained comprises specific pathogen-free (SPF) NZ Large White pigs raised under strict biosecurity. Possible sources of zoonotic infections are monitored in the herd, the sows one month before farrowing, the donor piglets, and the tissue used. New Zealand is free from prion-mediated disease and many of the viral infections found in herds elsewhere in the world.
It would be envisaged by those skilled in the art that other suitable pig herds may be used if bred under suitable conditions, elsewhere in the world.
The islet cells are isolated from the pancreases of 7-day-old piglets via a major modification of the standard (Ricordi's) collagenase digestion procedure. All surgical procedures and cell processing are carried out with strict aseptic precautions. Following their isolation and purification, the islets are placed into culture tissue in RPMI medium enriched with 2% human serum albumin and 10 mmol/L nicotinamide. Culture at 37° C. in an air/5% CO2 mixture with frequent changes of medium is then performed for 48 hours.
Sertoli cells are isolated from testicular cells of male 7-day-old piglets using a standard (Rajotte's) isolation method with modifications to ensure maximal cell yield. Following a number of quality control tests of both the islets and Sertoli cells (to ensure their optimal purity, viability and freedom from microbiological contamination; see further below), both the Sertoli cells and islets are counted and the latter adjusted to islets equivalents (IEQs) of 150 μm in diameter. The Sertoli cells are then combined with the islets in a ratio of 1:2,000-1:4,000, cultured for 24 hours, and scraped to form aggregates. Following a further 24 hours in culture, the islet-Sertoli cell aggregates are then tested for viability and insulin secretory capacity before being released for transplantation.
The production process for our islet-Sertoli aggregates preferably includes rigorous infection surveillance procedures comprising virological monitoring (see further below), screening for bacterial, fungal and mycoplasmal organisms, and bacterial endotoxin testing (LAL test). The presence of either microbiological contamination or a failure of the cells to meet any of the rigid quality control criteria set by the Applicant will lead to the particular cell batch being discarded.
a) Introduction of Sertoli Cells
b) First digestion of Sertoli Cells
c) Second digestion
Are prepared according to our previously published method in WO 01/52871 (the contents of which are incorporated herein by reference).
3) Sertoli islet Aggregates:
As indicated above, testing of the transplant material for the presence of PERV (porcine endogenous retrovirus), using a highly specific and highly sensitive assay developed for this purpose, is preferably an integral part of our islet-sertoli cell aggregate production process. In addition to PERV, attention is also directed towards other potentially infectious pathogens that can cause zoonoses and xenoses, including porcine cytomegalovirus (PCMV), porcine circovirus (PCV), porcine lymphotropic herpesvirus (PLHV), encephalomyocarditis virus (EMCV), and porcine hepatitis E virus. Preferably such a multi-level virological screening strategy undertaken by us as part of our process includes:
In a study conducted at Diatranz's laboratories, the efficacy and safety of transplants of alginate-encapsulated islet-Sertoli cell aggregates (ratio 1:4000) and alginate-encapsulated islets without Sertoli cells were compared in New Zealand white rabbits with experimentally-induced diabetes (5 animals per group).
Both groups received islet cell doses of 10,000 IEQ/kg via intraperitoneal injection. The weekly average blood glucose level declined in both groups over a follow-up period of 5 weeks post-transplantation, and two rabbits in each group were considered to have responded successfully to the transplants. At subsequent postmortem examinations, no abnormal histological findings were found in abdominal organs of recipient animals in either group.
Similar results were achieved in a study of NOD (nonobese diabetic) mice that received intraperitoneal transplants of alginate-encapsulated islets in a dose of 10,000 IEQ/kg with or without Sertoli cells. Two of 5 mice that received islet Sertoli cell aggregates (ratio 1:4000) and 2 of 6 that received islets alone had a partial response, with one animal in each group exhibiting a normal blood glucose level for up to 5 weeks.
Although the ratio of 1:4,000 has been used in these studies it will be clear to those skilled in the art that other ratios may be used without departing from the scope of the invention.
Clinical Studies with Islet-Sertoli Cell Aggregates
We have conducted a number of clinical investigations for our islet-Sertoli cell aggregates. In an experiment islet-Sertoli cell aggregates were transplanted into 12 adolescent type 1 diabetics via the use of subcutaneous stainless steel implant devices that create (on surgical removal of the Teflon® rod) vascularised collagen reservoirs in which the introduced cells are mechanically protected by a steel mesh tube. Initially, two such vascularised collagen reservoirs were created on the upper abdominal wall of each patient, followed by a further two, six months later. Each patient received islet-Sertoli cell aggregates (in ratios varying from 1:30 to 1:100) corresponding to a dose of 250,000 islet equivalents (IEQs) injected into each reservoir, and this dose was repeated in each of the second two reservoirs after 6 months.
Five of the 12 patients responded favourably to this treatment. After a lag period of approximately 8 weeks, the insulin requirements of the 5 patients began to decline and usually fell further after the second transplant. Reductions in the average daily insulin dose of more than 50% were achieved after 12 months, and one patient required no insulin after this time. Improvements in mean daily blood glucose levels and in glycosylated haemoglobin (HbA1C) were also recorded. No evidence of adverse effects were detected in any of the 12 patients, and PERV monitoring tests remained negative after 12 months.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NZ03/00130 | 6/24/2004 | WO | 00 | 4/23/2007 |
