FIELD

The present application relates to strategies aimed at reducing or preventing fibrotic overgrowth on medical implant devices and thereby improve medical transplantation outcomes. In more detail, the application relates to coating of medical devices suitable for implantation with sphingolipids, in particular sulfatide to reduce or stop the growth of the fibroblasts on medical implant devices.

BACKGROUND

Pancreatic islet transplantation has been considered for many years a promising therapy for betacell replacement in patients with type 1 diabetes despite that long-term clinical results are not as satisfactory. Diabetic' s experiences increased growth of fibroblasts and adipocytes at injection sites and in islets transplantation therapy, in-growth of fibroblast impedes success.

Microencapsulation of pancreatic islets in alginate hydrogels is one of several strategies being explored as a potential cellular therapy for type 1 diabetes without the need for potentially toxic immunosuppression. However, graft retrieval from human recipients show often the presence of dense pericapsular fibrotic overgrowth with necrotic islets.

In fact, fibrotic overgrowth is problematic on several types of medical devices used for implantation. It forms a physical barrier, mainly of macrophages and fibroblasts, that prevents e.g. the transport of oxygen and other nutrients, leading to starvation, hypoxia and suboptimal function and ultimately leading to islet death in islet transplants and the other way, less amount to be host of the secretory product, ex insulin.

SUMMARY

The present invention relates to coating of medical devices suited for implantation with

sphingolipids, in particular sulfatide to reduce or stop the growth of the fibroblasts on the device.

In healthy individual’s insulin is secreted together with sulfatide and no fibroblast growth is seen despite of a high concentration of insulin in the islets. To further illustrate the problem, it can be mentioned that when insulin is injected into the femur, there is typically within a few years a certain degree of connective tissue growth, which has been stimulated by the massive amount of injected insulin.

The present application discloses that various sphingolipids especially sulfatide inhibit the growth of fibroblasts on the surface of a medical implant device. In one aspect, the present invention relates to a pharmaceutical composition comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of a medical implant device.

In another aspect, the invention relates to a medical implant device comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of said medical implant device

In a presently preferred aspect, the invention relates to a medical implant device, wherein the device is a transplanted islet device or wherein the medical implant device comprises an insulin producing cell, such as a beta cell.

In yet another aspect, the invention relates to use of a composition or device according to the invention for the treatment of diabetes.

In a further aspect, the invention relates to a method of treating, preventing or delaying insulin- producing cell rejection or islet transplantation misfunction comprising administering an effective amount of a sphingolipid or a pharmaceutically acceptable salt thereof to an islet transplantation medical implant device comprising an insulin-producing cell.

DETAILED DESCRIPTION

The present invention relates to a medical device coated with a pharmaceutically effective amount of a sphingolipid for the prevention of adhesion and/or proliferation of fibroblasts on the surface of the medical device, in particular medical implant devices.

Medical device

A medical device in the present context means any device that may be used during medical intervention. Directive 2007/47/EC defines a medical device as: Any instrument, apparatus, appliance, software, material or other article, whether used alone or in combination, together with any accessories, including the software intended by its manufacturer to be used specifically for diagnostic and/or therapeutic purposes and necessary for its proper application, intended by the manufacturer to be used for human beings for the purpose of:

Diagnosis, prevention, monitoring, treatment, or alleviation of disease

Diagnosis, monitoring, treatment, alleviation of, or compensation for an injury or handicap Investigation, replacement, or modification of the anatomy or of a physiological process Control of conception This includes devices that do not achieve their principal intended action in or on the human body by pharmacological, immunological, or metabolic means, but may be assisted in their function by such means.

Medical implant device

A medical implant device in the present context means any medical device that is used for implantation, and thus a device manufactured to replace a missing biological structure, support a damaged biological structure, or enhance an existing biological structure. In order to inhibit immunological cells to contact or interfere with the transplanted cells, the transplanted cells may preferably by inside the device. Medical implants are manmade devices, in contrast to a transplant, which is a transplanted biomedical tissue.

Macroencapsulation

Beta-02 Technologies for example has developed a medical implant device based on

macroencapsulation and active oxygen supply approach in order to facilitate the widespread adoption of islet transplantation without the associated complications. Rather than transplanting the cells directly into the human body, thus creating a need for immunosuppression, cells are transplanted into a macrocapsule that provides immunoprotection and optimal conditions for cells to thrive. The macrocapsule uses alginate as a scaffold for cells to function. Alginate is a biocompatible polymer which creates a minimal foreign-body response. As such, it is widely used as a scaffold for encapsulation of islets and cells.

In one or more exemplary embodiments, the medical implant device may be a macrocapsule. Microencapsulation

Microencapsulation is a process in which tiny particles or droplets are surrounded by a coating to give small capsules, of many useful properties. In general, it is used to incorporate food ingredients, enzymes, cells or other materials on a micro metric scale. Microencapsulation can also be used to enclose solids, liquids, or gases inside a micrometric wall made of hard or soft soluble film, in order to reduce dosing frequency and prevent the degradation of pharmaceuticals. In a relatively simple form, a microcapsule is a small sphere with a uniform wall around it.

The material inside the microcapsule is referred to as the core, internal phase, or fill, whereas the wall is sometimes called a shell, coating, or membrane. Some materials like lipids and polymers, such as alginate, may be used as a mixture to trap the material of interest inside. Most microcapsules have pores with diameters between a few micrometers and a few millimeters. The coating materials generally used for coating are: Ethyl cellulose, Polyvinyl alcohol, Gelatin or Sodium alginate. Encapsulation allows free access of oxygen and nutritional factors influent, and outgoing free secretion of insulin or other molecules in play.

The definition has been expanded, and includes most foods, where the encapsulation of flavors is the most common. The technique of microencapsulation depends on the physical and chemical properties of the material to be encapsulated.

In one or more exemplary embodiments, the medical implant device may be a microcapsule. Nanoencapsulation

As an alternative to microencapsulation, nano-thin multilayered nano-capsules have been implemented in an attempt to increase coverage of encapsulated islets and decrease islet response time for insulin release to external stimuli. Polyethylene glycol (PEG) is a hydrogel polymer, which can be used as a conformal coating on islets.

In one or more exemplary embodiments, the medical implant device may be a nanocapsule. Polymers

A number of polymers have favorable properties to serve as scaffolding material for an artificially engineered islet transplantation site. A biopolymeric scaffold allows the creation of a novel ectopic transplantation site for islets.

In one or more exemplary embodiments, the medical implant device may be a polymer. The polymer may be serving as scaffolding material for an artificially engineered islet transplantation site.

The polymer poly(DL-lactidecocaprolactone), commercially available as Neurolac, is one example. Polyactive is another candidate. This is a copolymer of poly( ethylene oxide terephtalate) (PEOT) and poly(butylene terephtalate) (PBT) and is used for orthopedic devices. Polysulfone is also a promising candidate polymer that has been shown to be compatible with the survival of islets in animal models.

Islet transplantation

In one or more exemplary embodiments, the present invention relates to a composition or device according to the present disclosure, wherein the medical implant device is an islet transplantation device.

Islet transplantation is a form of insulin replacement that matches normal physiology much more closely than exogenous insulin injections. Islet transplantation is the transplantation of isolated islets typically from a donor pancreas into another person or from stem cell derived insulin secreting cells. The result is improved glycemic control, and overall improvement in quality of life. It is an experimental treatment for type 1 diabetes. Once transplanted, the islets begin to produce insulin, actively regulating the level of glucose in the blood.

It has, however, associated with it the burden of immunosuppression.

The disadvantages of the basic approach to islet transplantation are the need for often two donor pancreases for most recipients and graft failure, which occurs within a relatively short period of time. Poor vascularization and relative hypoxia of the transplanted cells, continuing destruction by autoimmunity and allorejection, and exposure to the toxic effects of immunosuppressive drugs are all thought to contribute to early graft failure. For these reasons, islet transplantation remains an experimental treatment available only for carefully selected cases of type 1 diabetes. This will remain the case until these deficiencies are overcome, and even then, a severe shortage of donor islets will limit the number of patients that can be treated.

The problem of immune rejection remains a significant challenge not only for the transplantation of islets but also for most forms of tissue transplantation.

One approach to improve the immune rejection of islet transplantation is immunoisolation, where islets are enclosed in a barrier device that facilitates the exchange of oxygen, nutrients, and insulin but protects the islets against the host immune response.

Immunoisolation implant devices

If a sufficiently effective and safe immunoisolation device for use in islet transplantation in humans can be found, the absence of harmful immunosuppression would dramatically sway the balance of outcomes toward benefit and away from harm for many more patients with diabetes.

In one or more exemplary embodiments, the present invention relates to a composition or device according to the present disclosure, wherein the medical implant device is an immunoisolation device.

The concept of immunobarrier protection for islets was first described in the seventies, where a device was implanted in diabetic rats in which islets were placed on the outside of hollow tubes made of a semipermeable acrylic copolymer, which carried blood as an arteriovenous shunt.

This was followed by the use of a microencapsulation approach in 1980 reported successful reversal of diabetes in rat recipients of immunoisolated islets for up to 15 weeks. This was with the use of alginate microcapsules with poly-l-lysine (PLL) and polyethylenimine coatings. Macrodevices, which rely on diffusion rather than arteriovenous shunts, have also been used to provide immunoprotection to transplanted islets. In 1991 acrylic hollow fibers containing islets immobilized in alginate reversed diabetes in rats for up to 60 d.

With both micro- and macroencapsulation, inadequate oxygenation has been a significant obstacle in the way of long-term graft success.

Central necrosis is commonly observed in islets recovered after transplantation. In preclinical studies, the biocompatibility of the materials and device design issues remain ongoing problems. If these issues can be addressed, immunoisolation provides an attractive means of protecting transplanted islets from rejection. An additional benefit is that the barrier could prevent release of potentially harmful foreign donor cells, such as those derived from hESC with teratoma-forming characteristics.

3D printed vascularized device for subcutaneous transplantation

Recently, a 3D printed and functionalized encapsulation system for subcutaneous engraftment of islets or islet like cells printed with polylactic acid and the surfaces treated and patterned to increase the hydrophilicity, cell attachment, and proliferation. Surface treated encapsulation systems were implanted with growth factor enriched platelet gel, which helped to create a vascularized environment before loading human islets. The device protected the encapsulated islets from acute hypoxia and kept them functional. Also, cellular immune attack should be prevented. The adaptability of the encapsulation system was demonstrated by refilling some of the experimental groups transcutaneously with additional islets.

Insulin producing cells

The invention relates to any cell capable of producing insulin, typically beta cells. However, some of the cells e.g. in the pylorus region of the stomach are amenable to convert to beta cells, thus the invention relates to both beta cells and such immature beta cells.

In one or more particular preferred exemplary embodiments, the medical implant device comprises a cell capable of producing insulin.

Beta cell

Beta cells (b cells) are a type of cell found in the pancreatic islets of the pancreas. They make up 65-80% of the cells in the islets. The primary function of a beta cell is to produce, store and release insulin.

In one or more particular preferred exemplary embodiments, the medical implant device comprises a beta cell. Immature beta cell.

These cells can make insulin, but some don't have the receptors to detect glucose, so they can't function as a full beta cell yet but can be matured. Even alpha cells in the islet have been observed to turn into immature beta cells and then mature into real beta cells.

In one or more particular preferred exemplary embodiments, the medical implant device comprises an immature beta cell.

Islets

Pancreatic islets, also called islets of Langerhans, are tiny clusters of cells scattered throughout the pancreas. Pancreatic islets contain several types of cells, including beta cells, that produce insulin.

In one or more particular preferred exemplary embodiments, the medical implant device comprises pancreatic islets.

Sphingolipids

Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. The long-chain bases, sometimes simply known as sphingoid bases, are the first non-transient products of de novo sphingolipid synthesis in both yeast and mammals.

SphinGOMAP© at http://www.sphingomap.org is an evolving pathway map for sphingolipid biosynthesis that includes many of the known sphingolipids and glycosphingolipids arranged according to their biosynthetic origin(s).

Some compounds, specifically known as phytosphingosine and dihydrosphingosine, are mainly C18 compounds, with somewhat lower levels of C20 bases. Ceramides and glycosphingolipids are N-acyl derivatives of these compounds.

The sphingosine backbone is usually O-linked to a charged head group such as ethanolamine, serine, or choline. The backbone is also amide-linked to an acyl group, such as a fatty acid.

In one or more exemplary embodiments, the active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of a medical implant device is a sphingolipid.

The sphingolipids of the present disclosure, are fatty acids with carbon (C) chain lengths of 12-20, although very long-chain with C > 22, such as C > 20, C > 24 and C > 26 also exist.

In some embodiment, the sphingolipid may be any of the below mentioned sphingolipids. It should also be understood that any feature and/or aspect discussed above in connections with the sphingolipids as a genus according to the invention apply by analogy to any of the species disclosed herein, such as but not limited to glycosphingolipds and in particular to the explicit species of sulfatide described below.

Simple sphingolipids, which include the sphingoid bases and ceramides. Sphingoid bases are the fundamental building blocks of all sphingolipids. The main mammalian sphingoid bases are dihydrosphingosine and sphingosine, while dihydrosphingosine and phytosphingosine are the principle sphingoid bases in yeast. Sphingosine, dihydrosphingosine, and phytosphingosine may be phosphorylated. Ceramides, as a general class, are N-acylated sphingoid bases lacking additional head groups. Dihydroceramide is produced by N-acylation of dihydrosphingosine.

Ceramide is produced by desaturation of dihydroceramide by dihydroceramide desaturase 1 (DES1 ). This highly bioactive molecule may also be phosphorylated to form ceramide-1 -phosphate. Phytoceramide is produced in yeast by hydroxylation of dihydroceramide at C-4. Complex sphingolipids may be formed by addition of head groups to ceramide or phytoceramide:

Sphingomyelins have a phosphocholine or phosphoethanolamine molecule with an ester linkage to the 1 -hydroxy group of a ceramide.

Cerebrosides have a single glucose or galactose at the 1 -hydroxy position.

Sulfatides are sulfated cerebrosides.

Gangliosides have at least three sugars, one of which must be sialic acid.

Inositol-containing ceramides, which are derived from phytoceramide, are produced in yeast. These include inositol phosphorylceramide, mannose inositol phosphorylceramide, and mannose diinositol phosphorylceramide.

Glycosphingolipids are ceramides with one or more sugar residues joined in a b-glycosidic linkage at the 1-hydroxyl position.

Glycosphingolipids

Glycosphingolipids are a subtype of sphingolipids containing the amino alcohol sphingosine. They are sphingolipids with an attached carbohydrate. They consist of a hydrophobic ceramide part and a glycosidically bound carbohydrate part. In general, glycosphingolipids can be categorized into two groups: neutral glycosphingolipids (also called glycosphingolipids) and negatively charged glycosphingolipids.

In one or more exemplary embodiments, the sphingolipid of the present invention is a neutral glycosphingolipid.

In one or more exemplary embodiments, the sphingolipid of the present invention is a negatively charged glycosphingolipids.

The latter can be distinguished again by means of the charge carrier.

The structural similarity of most glycolipids is the so-called lactosylceramide, that is, a lactose disaccharide that is glycosidically bound to a ceramide.

The glycosphingolipids of the present invention also include cerebrosides, gangliosides and glo bos ides.

Ganglioside

A ganglioside is a molecule composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (e.g. n-acetylneuraminic acid, NANA) linked on the sugar chain. More than 60 gangliosides are known, which differ from each other mainly in the position and number of NANA residues.

In one or more exemplary embodiments, the glycosphingolipid of the present invention is ganglioside.

Structures of the common gangliosides:

GM2-1 = aNeu5Ac(2-3)bDGalp(1-?)bDGalNAc(1-?)bDGalNAc(1-?)bDGIcp(1-1 )Cer GM3 = aNeu5Ac(2-3)bDGalp(1-4)bDGIcp(1-1 )Cer

GM2,GM2a(?) = N-Acetyl-D-galactose-beta-1 ,4-[N-Acetylneuraminidate- alpha-2, 3-]- Galactose-beta-1 ,4-glucose-alpha-ceramide GM2b(?) = aNeu5Ac(2-8)aNeu5Ac(2- 3)bDGalp(1-4)bDGIcp(1-1 )Cer

GM 1 ,GM 1 a = bDGalp( 1 -3)bDGalNAc[aNeu5Ac(2-3)]bDGalp( 1 -4)bDGIcp( 1 -1 )Cer asialo-GM1 ,GA1 = bDGalp(1 -3)bDGalpNAc(1-4)bDGalp(1-4)bDGIcp(1 -1 )Cer

asialo-GM2,GA2 = bDGalpNAc(1-4)bDGalp(1-4)bDGIcp(1-1 )Cer

GM1 b = aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)bDGalp(1-4)bDGIcp(1-1 )Cer

GD3 = aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGIcp(1-1 )Cer

GD2 = bDGalpNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGIcp(1- 1 )Cer GD1a = aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1 -4)[aNeu5Ac(2-3)]bDGalp(1-4)bDGIcp(1- 1 )Cer

GD1 alpha = aNeu5Ac(2-3)bDGalp(1 -3)bDGalNAc(1-4)[aNeu5Ac(2-6)]bDGalp(1- 4)bDGIcp(1-1 )Cer

GD1 b = bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4 )bDGIcp(1- 1 )Cer

GT1a = aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-3 )]bDGalp(1- 4)bDGIcp(1-1 )Cer

GT1 ,GT1 b = aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1 -4)[aNeu5Ac(2-8)aNeu5Ac(2- 3)]bDGalp(1-4)bDGIcp(1 -1 )Cer

OAc-GT 1 b = aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)aXNeu5Ac9Ac(2-8)aNeu5Ac( 2-

3)]bDGalp(1 -4)bDGIcp(1 -1 )Cer

GT1 c = bDGaip(1-3)bDGalNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-8)aNeu5Ac(2-3 )]bDGalp(1-

4)bDGIcp(1-1 )Cer

GT3 = aNeu5Ac(2-8)aNeu5Ac(2-8)aNeu5Ac(2-3)bDGal(1 -4)bDGIc(1-1 )Cer

GQ1 b = aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2- 8)aNeu5Ac(2-3)]bDGalp(1-4)bDGIcp(1-1 )Cer

GGal = aNeu5Ac(2-3)bDGalp(1-1 )Cer where

aNeu5Ac = 5-acetyl-alpha-neuraminic acid

aNeu5Ac9Ac = 5,9-diacetyl-alpha-neuraminic acid

bDGalp = beta-D-galactopyranose

bDGalpNAc = N-acetyl-beta-D-galactopyranose

bDGIcp = beta-D-glucopyranose

Cer = ceramide (general N-acylated sphingoid)

In one or more exemplary embodiments, the sphingolipid of the invention is a ganglioside. Ceramides

A ceramide is composed of sphingosine and a fatty acid. Ceramides are found in high concentrations within the cell membrane of eukaryotic cells, since they are component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer.

In one or more exemplary embodiments, the sphingolipid of the invention is a ceramide.

In one or more exemplary embodiments, the glycosphingolipid of the present invention is glycosylceramide. Sulfatide

Sulfatide, also known as 3-O-sulfogalactosylceramide, SM4, or sulfated galactocerebroside, is a class of sulfolipids, specifically a class of sulfoglycolipids, which are glycolipids that contain a sulfate group.

As shown in Example 1 , the growth rate of primary human fibroblast was measured with 3H- thymidine incorporation. In this experiment the inventors shows, that several GSLs, including sulfatide inhibit growth rate of primary human fibroblasts. Data suggest that sulfatide neither induce apoptotic nor necrotic pathways in the exposed cells.

The ATP content had a tendency to be higher in the sulfatide exposed cultures, this was not significant, although it does indicate that the energy status of the fibroblasts are not adversely affected by sulfatide, but perhaps the contrary. Rather sulfatide seem to induce cell cycle arrest. In RIN cells neither sulfatide nor GalCer affected the growth rate indicating that the GSLs are not cytotoxic.

Duration experiments seem to indicate that the inhibition is reversible and that the inhibited cells display normal division behaviour four days after inhibition. The study with radio labelled sulfatide and GalCer indicate that the two GSLs are taken up by the cells, with GalCer being taken up on average twice as efficient as sulfatide, but with individual variation in GalCer uptake.

The fibroblasts were exposed to three different anabolic hormones, insulin, IGF-1 and hGH. All three increased the growth rate of the fibroblasts, and the effects of all three were effectively checked and countered by 30pg/ ml of sulfatide.

The level of growth rate increase induced by the hormones wasin the range expected and sulfatide were able to completely overrule the mitogenic effects of the hormones.

Growth hormone is acting via a different receptor than insulin and IGF-1 , and the effect of sulfatide is probably not due to an interaction between sulfatide and the IGF-1 receptor. Cells cultures without hormones displayed the same growth retardation, when exposed to sulfatide and GalCer, as hormone treated cultures (data not shown).

In one or more particular preferred exemplary embodiments, the sphingolipid of the invention is sulfatide.

Suppressing adhesion and/or proliferation of fibroblasts

A great problem when transplanting for example insulin producing cells is that the device that e.g. protects the transplanted cells against the immune cells of the host is covered by fibroblasts. Even cell cultures are jeopardized by the outgrowth of contaminating fibroblasts, since fibroblasts usually grow at much faster rates than other cell types, and the growth of fibroblasts are stimulated by the insulin.

This hurts not only the liberation of insulin to the transplanting person who typically is a patient with type I diabetes for whom more or less continued insulin secretion is crucial. Also, it hurts the oxygen supply to the insulin producing cells which is highly important for their optimal function.

Inhibition of fibroblast proliferation and adhesion on medical implant devices is in this application provided by the use of sphingolipids, such as sulfatide or a pharmaceutically acceptable salt thereof.

Thus, in one or more exemplary embodiments, the invention relates to a method of treating, preventing or delaying insulin-producing cell rejection or islet transplantation rejection comprising administering an effective amount of a sphingolipid or a pharmaceutically acceptable salt thereof to an islet transplantation medical implant device comprising an insulin-producing cell.

Oxygen transport

Oxygen transport across the immuno-isolation barrier in for example a microcapsule is crucial for encapsulated optimal islet survival and prevention of necrosis. The Beta-02 macroencapsulation device is an implantable device that furnishes islet cells with their own supply of oxygen, via a chamber that can be replenished every 24 hours.

However, by limiting the fibroblast overgrowth with the sphingolipids of the present application, in particular Sulfatide provides sufficient flow of oxygen to islets either encapsulation or otherwise supported by a medical implant device according to the present disclosure.

In one embodiment, the present invention relates to a medical implant device comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of said medical implant device provides the islet cells with an oxygen partial pressure of about 100 mm Hg from surrounding capillaries.

In one embodiment, the present invention relates to a medical implant device comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of said medical implant device provides the islet cells with an oxygen partial pressure of at least 50 mm Hg from surrounding capillaries.

In one embodiment, the present invention relates to a medical implant device comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of said medical implant device provides the islet cells with an oxygen partial pressure of at least 25 mm Hg from surrounding capillaries.

In another embodiment, the present invention relates to a medical implant device comprising a sphingolipid as an active ingredient for suppressing adhesion and/or proliferation of fibroblasts on the surface of said medical implant device provides the islet cells with an oxygen partial pressure of at least 25 mm Hg from exogenous oxygen from a replenishable gas chamber in said device.

Diabetes

Type 1 diabetes

In some embodiments, the invention relates to the use of any of the sphingolipids describe herein for prevention fibroblast overgrowth on medical implanted devices used for the treatment of type 1 diabetes in a human. In the present context pre-type 1 diabetes relates to the presence of insulitis, dysfunction and/or loss of beta cells, as well as islet autoantibody/autoantibodies.

In type 1 diabetes, the beta cells of the pancreas no longer make sufficient insulin. A person who has type 1 diabetes must take insulin daily to live. Transplanted islet cells, however, can take over the work of the destroyed cells. The beta cells in these islets will begin to make and release insulin, and thus islet transplantation will help people with type 1 diabetes live without daily insulin injections.

Type 2 diabetes

In some embodiments, the invention relates to the use of any of the sphingolipids describe herein for prevention fibroblast overgrowth on medical implanted devices used for the treatment of type 2 diabetes in a human. Diabetes type 2 is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. This is in contrast to diabetes type 1 in which there is an absolute insulin deficiency due to destruction of islet cells in the pancreas and gestational diabetes that is a new onset of high blood sugars associated with pregnancy.

Type 1 and type 2 diabetes can typically be distinguished based on the presenting circumstances. If the diagnosis is in doubt antibody testing may be useful to confirm type 1 diabetes and C-peptide levels may be useful to confirm type 2 diabetes, with C-peptide levels normal or high in type 2 diabetes, but low in type 1 diabetes.

Latent Autoimmune Diabetes of Adulthood (LADA)

In some embodiments, the invention relates to the use of any of the sphingolipids describe herein for prevention fibroblast overgrowth on medical implanted devices used for the treatment of LADA in a human. LADA is a form of type 1 diabetes that develops later into adulthood. LADA tends to develop more slowly than type 1 diabetes in childhood and, because LADA can sometimes appear similar to type 2 diabetes, doctors may mistakenly diagnose LADA as type 2 diabetes.

In one or more exemplary embodiments, the invention relates to the use of any of the sphingolipids disclosed herein, in particular sulfatide, for prevention fibroblast overgrowth on medical implanted devices used for the treatment of LADA.

In one embodiment, the LADA patients may have dramatic and recurrent fluctuations in glucose levels.

LADA is sometimes referred to as type 1.5 diabetes. This is not an official term, but it does illustrate the fact that LADA is a form of type 1 diabetes that shares some characteristics with type 2 diabetes.

As a form of type 1 diabetes, LADA is an autoimmune disease in which the body’s immune system attacks and kills off insulin producing cells.

The reasons why LADA can often be mistaken for type 2 diabetes is it develops over a longer period of time than type 1 diabetes in children or younger adults.

Whereas type 1 diabetes in children tends to develop quickly, sometimes within the space of days, LADA develops more slowly, sometimes over a period of years.

The slower onset of diabetes symptoms being presented in people over 35 years may lead a GP to initially diagnose a case of LADA as type 2 diabetes.

General

It should be understood that any feature and/or aspect discussed above in connections with the compounds according to the invention apply by analogy to the devices and methods described herein.

The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way. BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Growth rate of primary human fibroblast measured with 3H-thymidine incorporation. Cells were incubated with 0.5 pmol/ ml of human insulin and different concentrations of GSLs. Data is displayed as percentage of control cells not exposed to either insulin or GSLs. Bars indicate SEM. The data comes from four separate experiments each with two different patients, hence the slightly different responses to insulin. Sulfatide (blue line) were included in all experiments for comparison.

Figure 2

Left: Growth rate of primary human fibroblast measured with 3H-thymidine incorporation. Cells were incubated with 0.5 pmol/ml of human insulin, 10 and 100 ng/ml of human recombinant IGF-1 or 1000 and 2000 ng/ml of human recombinant growth hormone (hGH). Columns to the right displays cells coincubated with 30 pg/ml of sulfatide indicated by a+ sign. Data is presented as percentage of control cells not exposed to either hormones or sulfatide. Right: Same as left, but here the ATP content is displayed.

Figure 3

Growth rate of RIN cells measured with radio nucleoside labelling. Cells were incubated for 24 hrs with 1 - 30 pg/ml of native sulfati.de or GalCer. Data is presented as percentage of control cells not exposed toGSLs.

Figure 4

The growth rate of cells pulsed for 24 hours with radio labelled sulfatide were monitored for 5 days after sulfatide had been removed from the media. Data is the average of three different patients. Vertical bars represent s.e.m. N=3.

EXAMPLES

Example 1 Effect of sulfatide

Materials and methods

We expose human primary fibroblast to insulin in physiological (10-125 pmol/l) and hyper- physiological (200-2000 pmol/l) concentrations together with sulfatide in physiological (1 - 3 pg/ml) and hyper-physiological (30 pg/ ml) concentrations.

Cultures exposing fibroblast to either IGF-1 or human growth hormone (hGH) together with sulfatide were also conducted. hGH were included in the experiments in order to test if a mitogenic signal from a receptor different from the IGF-1 receptor could alter the outcome of GSL exposure. Cell growth was tested using 3H-thymidine incorporation. The cellular uptake of sulfatide and GalCer were determined using 3H-labelled GSLs. In order to test if sulfatide induced apoptosis or necrosis in the cell cultures the ATP content were determined in some of the cell cultures. In healthy cells the ATP levels are precisely maintained, when cells become apoptotic or necrotic, cellular ATP content is rapidly lost. If the GSL affects cellular growth generally and unspecifically, they should also be able to inhibit the growth of transformed cells. In order to test this, rat insulinoma cells (RIN) were exposed to GalCer and sulfatide and their effect on the RIN cells growth rate monitored.

Cell culture

Fibroblast cultures were derived from 6 patients undergoing eyelid reduction surgery aged 42-53 years. Cells were maintained in Dulbecco' s modified Eagle's Medium (DMEM) (Cambrex Bio Science, Verviers, Belgium) containing 10% foetal calf serum (FCS) (Kibutz industries, Halbek, Israel) at 37°C in an atmosphere of 95% air/5% C02. Cells were split 1 :4 when 80-90% confluence were attained, typically every 7-10 days. All cells used in the experiments were at the time of usage in passage 2-5. For GSL exposure experiments, cells were trypsinised and distributed into 96 well plates at a density of 25-30% (0.1 ml/well) and maintained for 24 hrs at conditions as described above. The Media was then replaced with DMEM containing 0.5 % FCS and maintained a further 24 hrs. After the 48 hrs conditioning period . GSLs from stock was added, along with insulin; each experimental well was produced in triplicate or quadruplicate. Cultures were incubated for 18 hrs upon which the media was replaced with media containing 1 pCi 3H thymidine, in addition to the glycolipids and insulin . Cells were harvested using an automated cell harvester (Dynex technologies, Chantilly VI, USA), thymidine incorporation was determined by scintillation counting.

Preparation of glycolipids and reagents

3H-labelled lyophilized GalCer and sulfatide was dissolved in demthylsulfoxide (DMSO) making stock solutions of 5 pg/ pL lyophilized GM 1-3, GD1a and glucosylceramide sulfatide was dissolved in demthylsulfoxide (DMSO) making stock solutions of 5 pg/ mI_ Insulin (Actrapid) (Novo Nordisk, Bagsvaerd, Denmark) was diluted in PBS containing 0.1 % bovine serum albumin (BSA).

Insulin like Growth Factor (VWR International.

Human Growth hormone (Phamacia AB, Stockholm, Sweden)

[methyl-3H] Thymidine (Amersham Biosciences, Little Chalfont, UK)

ATP content

Cells were cultured as for thymidine incorporation as described above. At the end of the incubation period, the ATP content in each well were determined using the ViaLight kit (Cambrex, East Rutherford, New Jersey, USA) according to the manufactures specifications.

RIN cells

Cells were initially cultured in 75-80 cm ² in RPMI 1640 supplemented with 10% foetal calf serum, at 37°C in a C02 rich atmosphere. The cells were then trypsinised and distributed to a 96 well plate. Cells were exposed to 1 , 3, 10 or 30 pg/ml of either native sulfatide or GalCer for 24 hrs. During the last 3 hrs of incubation, the cells were pulsed with 1 pCi 3H thymidine. After incubation, the cells were harvested and the 3H -labelled DNA counted as described above.

Results

Sulfatide at physiological concentrations significantly affected the 3H-thymidine incorporation of insulin stimulated fibroblasts (Figure 3). Compared to insulin stimulated cells without sulfatide, 1 pg/ ml sulfa tide on average lowered the growth rate by 20% (p<0.05), 3 pg/rnl lowered the growth rate by 28% (p<0.05). 30 pg/rnl of sulfatide decreased the growth rate by 74% (p<0.001 ). GalCer reduced the growth of the fibroblast marginally less efficient than sulfatide, 30 pg/ml of GalCer reduced the growth by 56 % compared to insulin stimulated cells. The fibroblasts were also exposed to GMI, GM2, GM3, GDIa and glucosylceramide. GDIa were the most efficient of all the tested GSLs reducing the growth rate by 81 % (p<0.0001 ). Students T-test for samples of equal variance was used.

Viability test

Duplicate microtitter plates were prepared and the energy level (ATP content) of one plate were established, the growth rate of the other plate were measured with 3Hthymidine incorporation. As can be seen in Figure 3, sulfatide decreased the growth rate considerably without significantly affecting the ATP content of the cells. There was a tendency for sulfa tide to increase the ATP content, but this was not significant.

RIN cells

Sulfatide or GalCer had no effect on the growth rate of RIN cells in culture; even at the highest concentration (30 pg/ml) the thymidine incorporation was unaffected. Growth rate recovery

The growth rate of the sulfatide pulsed cells were monitored for five days post sulfatide removal. The results, displayed in Figure 4, indicates that at day 4 after sulfatide was removed from the medium, the growth rate of the cells exposed to sulfatide were the same as cells that had not been exposed to sulfatide, the subsequent decrease in growth is probably caused by contact inhibition. Even at 40% confluence the fibroblasts displayed retarded growth as compared to lower densities (data not shown).