ISCHEMIA-REPERFUSION INJURY

The present invention concerns the use of biotinylated oligosaccharide conjugates in the field of solid organ preservation for transplantation, and in particular for the prevention and treatment of ischemia or reperfusion injury.

Solid organ transplantation is an effective treatment modality and has become the treatment of choice for end-stage diseases of the kidney, pancreas, liver, heart, lung and intestine. Transplantation is defined as a process intended to restore certain functions of the human body by transferring an organ from a donor to a recipient. The ultimate aims of solid organ transplantation are improved survival and improved quality of life. For example, patients suffering from chronic kidney disease and end-stage renal disease (ESRD) require dialysis or transplantation to sustain life. Over the past 50 years organ transplantation has become an established worldwide practice, bringing immense benefits to hundreds of thousands of patients. The use of human organs for transplantation has steadily increased during the last two decades. Organ transplantation is now the most cost-effective treatment for end-stage renal failure, while for end-stage failure of organs such as the liver, lung and heart it is the only available treatment.

The increasing use of human organs for transplantation during the past decades has led to a shortage of organs. This is widely acknowledged as the most critical factor hindering the full realization of success for solid organ transplantation. For example, to date, the number of ESRD patients waiting for a kidney graft widely exceeds the number of kidney donors. In France, 2 826 kidney transplants were performed in 2009, for 9 675 patients on the waiting list. This means that only approximately one third of patients on the waiting list each year will receive a kidney, while the others will be kept on dialysis, with dramatic impacts on their quality of life.

The lack of transplantable organs led to define new, less severe, criteria for qualifying transplantable organs. Thus, to standard donors (living donors, brain death donors) were first added expanded criteria donors (ECD, dead over 60 years-old, or dead between 50- 59 years-old and suffering from mild hypertension/kidney dysfunction, or dead from stroke), and in a further step donation after cardiac death (DCD). However, ECD or DCD organs are more susceptible to ischemia reperfusion injury (IRI) and acute or long-term deteriorations and allograft survival is lower in DCD grafts compared to donation after brain death (Vieira, 2009).

In the course of a transplant procedure organs may suffer from ischemia reperfusion injury (IRI). IRI refers to the tissue damage that occurs when blood supply returns to tissue after a period of ischemia. It is associated with all solid organ transplantation (Kosieradzki and Rowinski, 2008) and it is shown to play a major role in acute and long-term organ outcome.

IRI is directly linked to inflammation. Under hypoxia the stressed endothelial cells produce and/or release inflammatory mediators such as platelet activating factor (PAF), tumor necrosis factor (TNF) and a panel of interleukins (ILs). Cellular adhesion molecules for leukocytes are also expressed on the cell surface and together with reduction of nitric oxide (NO) production induce shrinkage of the vascular lumen and the phenomenon of no reflow (Sutton, 2009), decreasing oxygen supply to the parenchyma and increasing the injury. Invading immune cells release supplementary reactive-oxygen species (ROS), proteases and inflammatory mediators, also increasing the injury.

IRI affects all organs and its most obvious manifestation in the context of transplantation is delayed graft function (DGF) and decreased duration of graft survival (DGS). DGF is defined as a temporary divergence between the functional capacity of the engrafted organ and fulfilment of the physiologic needs of the recipient. For instance, in kidney transplantation, DGF is defined as the need for dialysis, since the engrafted kidney cannot produce urine and filter the blood, in the first one or two weeks following the procedure. Graft outcome has been shown to be correlated with the extent and intensity of IRI associated lesions: on the short-term in regards to the rate of DGF and acute rejection (Mikhalski et al, 2008), on the long-term in regards to the development of graft interstitial fibrosis and decreased survival (Pratschke et al, 2008).

A large and increasing portion of DGF organs come from DCD and ECD, which are highly predisposed to suffer IRI. Organs from DCD incur indeed a variable period of warm ischemia, between cessation of cardiopulmonary function and perfusion with cold preservation solution. This leads to warm ischemic injury, which increases the incidence of DGF.

There is currently no drug authorized for the condition of IRI associated with solid organ transplantation.

Organs waiting for transplantation are currently preserved in the cold, in a special solution in order to suppress metabolism by hypothermia. The organ is flushed with the solution and stored at 0°C to 4°C prior to implantation. Several organ preservation solutions exist and some of them have been compared in clinical studies, however there does not seem to be a trend towards superiority for any of these to date (Yuan et al, 2010). The components found in the preservation solutions are electrolytes, sugars, buffers, anti-oxidants and salts. Each solution substantially differs in their composition, but the purposes of each are similar: to prevent cellular oedema, delay cell destruction, and maximize organ function after perfusion is re-established. However none of these solutions are authorized in the treatment or prevention of ischemia reperfusion injury associated with solid organ transplantation.

Although static preservation remains the standard practice for all solid organs, there has been a recrudescence of interest in machine perfusion. Perfusion was a technique developed four decades ago that disappeared when preservation solutions improved enough to allow for static storage. Perfusion results in continuous circulation of energy substrates and washout of waste products, for organ rehabilitation and recovery, and for assessment of tissue metabolism and viability through measurement of preservation fluid parameters. In hypothermic machine perfusion, after an initial washout of blood, the kidney is connected to a perfusion device, and a solution is pumped continuously through the renal vasculature at temperatures between 1 and 10 °C.

Currently, hypothermic machine preservation for kidneys has gained popularity, particularly for organs from suboptimal donors at increased risk of DGF (Feng, 2010).

Thus, maintaining organ viability during preservation is an important prerequisite for successful outcome after transplantation. With the current practice to accept older and more injured donor organs, improvement of preservation techniques has now become a must. To date, most centers use static cold storage to preserve organs. This preservation method, however, was developed in an era with younger donors with good-quality organs. With the introduction of extended donor criteria the limitations of cold storage have probably been reached with IRI still being an issue in solid organ transplantation. Therefore there is a need to improve the preservation solution of the prior art and in particular to obtain solution that can be used in hypothermic machine perfusion and solutions that contain an agent able to prevent IRI.

Several anticoagulant compounds have been shown to prevent IRI.

According to WO94/08595, it is known to use heparin or non anticoagulant heparin in the treatment and prevention of IRI. However, heparin has never been authorized for this application.

Fondaparinux a synthetic anticoagulant oligosaccharide was shown to improve IRI organ injury, while reducing coagulation, inflammation and neutrophil accumulation (Frank et al, 2005). In this study, the effect of fondaparinux in a lethal murine model of kidney IRI was investigated. More recently, in a modified Langendorff rat heart preparation, it was shown that fondaparinux improved post IR myocardial dysfunction and vascular injury (Montaigne et al, 2008). Results suggested that mechanisms of these cardioprotective effects could involve alteration in endothelial cell to leukocyte interactions and reduced ROS production.

The short-term and long-term effects of using the direct thrombin inhibitor melagatran during cold preservation of a kidney graft were described in a porcine DCD model (Giraud et al, 2009). It was shown that melagatran was (i) directly protective against endothelial cell activation and histologic damage, (ii) lowered expression of thrombospondin-1 (Tsp-1), P-selectin, IL β-l and monocyte-chemo-attractant protein- 1 (MCP-1), (iii) protected mononuclear cells, and (iv) improved graft outcome and recovery of function.

Another direct thrombin inhibitor, hirudin, has been shown to protect against IRI in a stroke model (Karabiyikoglu et al, 2004) and ultimately protect against chronic allograft vasculopathy (Holschermann et al, 2000).

However, the anticoagulant activity of heparin, fondaparinux, melagatran and hirudin cannot be neutralized if need be which may be a problem as a certain amount of the product remains in the organ after storage in the preservation solution. Moreover, melagatran has been withdrawn from the market because of serious side-effect. It is known also that various anticoagulant display different effects on the various enzymes of the coagulation cascade and on blood coagulation as a whole. In the same way such compounds display various inhibitory effects on various other (more or less closely related) proteases. It is known also that heparin, fondaparinux and melagatran do not display the same effect in models of IRI. Thus, the global inhibitory profile of a given molecule may have a different effect on a given biological system.

Furthermore none of these compounds have been reported to be tested in an hypothermic machine perfusion. Therefore, there is a need of a synthetic anticoagulant which could be neutralized if needed and which could be used in hypothermic machine perfusion. Neutralizable anticoagulants are known. E.g. biotinylated oligosaccharides or biotinylated oligosaccharide conjugates that display an anticoagulant effect can be neutralized by injection of avidin, an egg derived protein.

The biotinyl group, which is derived from biotin (IUPAC name: 5-[(3aS,4S,6aR)-2- oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl]pentanoic acid; also known as vitamin B7) represents the following group:

Biotinyl (left) and biotin (right).

However, the presence of the biotin residue may influence the activity of the oligosaccharide and therefore the one skilled in the art would have thought that this type of compound could not be used in the treatment or prevention of reperfusion injury and in particular in the preservation of organ for solid organ transplantation.

However, the inventors have surprisingly found that the compound of formula I which contain a biotin residue has a strong activity in the prevention and/or treatment of ischemia reperfusion injury when it is added to a preservation solution used for organ preservation and in particular when such a solution is used in conjunction with a hypothermic machine perfusion:

(I)

This compound is a representative of a new class of synthetic anticoagulants with a dual mechanism of action. It combines in a single molecule an indirect factor Xa inhibitor (antithrombin-binding pentasaccharide) and a direct thrombin (factor Ila) inhibitor (peptidomimetic) (Petitou et al, 2009; WO2006/0617173). However these documents never suggest nor described that this compound could be useful in the treatment or prevention of reperfusion injury and in particular in the preservation of organ for solid organ transplantation. Because of the presence of a biotin moiety linked to a lysine inserted between the tetraethylene linker and the peptidomimetic moiety, its anticoagulant activity can be suppressed by using avidin, which is a specific antidote that binds to the biotin group and thus prevents the compound from having its effect on its targets. The anti-factor Xa (IC ₅₀ = 1 1 .68 ± 0.95 nM) and anti-factor Ila (IC ₅₀ = 1 1.26 ± 1.44 nM) activities of the compound of formula I were assessed in rat plasma by measuring the rate of hydrolysis of a chromogenic substrate (mimicking the natural substrate fibrinogen). Therefore, the present invention concerns a compound of formula (I) the salt, solvate, enantiomer, diastereoisomers and racemic mixture thereof for treating or preventing ischemia/ reperfusion injury associated disorders during surgical operations or transplantation of internal organs.

The compound of Formula I has been described in WO 2006/067173 which is incorporated herein by reference. In particular WO 2006/067173 describes its process of preparation.

Advantageously the compound of formula (I) is a 60/40 mixture o f two diastereoisomers, arising from epimerization at a lysine carbon (arrow in the formula I; 60/40 ratio of L/D lysine). Both diastereoisomers displayed similar in vitro pharmacodynamic (PD) effects on factor Xa and Ila inhibition and in vivo pharmacokinetic (PK) profiles in dogs and rats. Therefore, the configuration of the lysine motif in compound (1) structure has little influence, if any, on the PK/PD profile of the two single isomers.

In a particular embodiment, compound of formula (I) is in form of a pharmaceutical composition which is advantageously a clear, colorless and isotonic aqueous solution. It is in particular formulated in a 13 mM phosphate buffer pH 6.0. It is supplied in a vial of 1 mL at a concentration of 10 mg/mL. In a particular embodiment, ischemia/ reperfusion injury associated disorders are organ damage, delayed graft function, decreased duration of graft survival, graft rejection, graft failure, inflammation, graft interstitial fibrosis, short term or long term graft loss.

Advantageously the ischemia/reperfusion injury occurred during the transplantation process of internal organs, particularly before removal of the organ from the deceased donor (warm ischemia) or during the preservation phase before implantation of the organ in the receiver (cold ischemia) or during reperfusion of the transplanted organ.

The ischemia/reperfusion injury can also occurr during organ surgery under cardiopulmonary bypass such as for example coronary bypass surgery. In particular the internal organs are solid organs, advantageously chosen in the group consisting of kidney, pancreas, liver, heart, lung and intestine, still more advantageous it is kidney.

Advantageously, the internal organs are mammalian internal organs, in particular human internal organs.

The present invention also concerns a solution for protecting internal organs disconnected from the circulatory system from ischemic damages comprising as an active ingredient the compound of formula I as defined above or the salt, solvate, enantiomer, diastereo isomer and racemic mixture thereof.

Advantageously, the organs are as indicated above.

Advantageously the compound of formula I is present in the solution at a concentration of between 1 mg/L to 100 mg/L preferably 5 mg/L to 50 mg/L, more preferably 10-20 mg/L.

More advantageously the solution according to the present invention comprises a cardioplegia solution or an organ preservation solution. The cardioplegia solution is a water-based or blood-based solution which is used to protect the disconnected heart during surgery. The heart is bathed by this solution while it is stopped during for example coronary by pass surgery.

The organ preservation solution is used to protect the organ disconnected from the circulatory system from damage prior to transplantation.

Advantageously the preservation solution according to the present invention contains a potassium salt, such as for example potassium chloride or potassium hydroxide.

In another advantageous embodiment the preservation solution according to the present invention contains an antioxidant such as allopurinol, glutathione or mannitol.

The cardioplegia solution and organ preservation solution are advantageously chosen in the group consisting of Celsior solution, Krebs-Henseleit solution, St Thomas II solutions, Collins solution, Stanford solution University of Wisconsin (UW) solution or Histidine Tryptophan Ketoglutarate solution, more advantageously between University of Wisconsin solution or Histidine Tryptophan Ketoglutarate (HTK) solution, still more advantageously it is University o f Wisconsin so lution.

The composition of UW solution is described in Table 1 below.

Table 1 Composition of UW solution

Component name Mass of compound to add (g) Concentration (niM)

Poly (0-2-hydroxyethyl) starch 50.000 -

Lactobionic Acid (as Lactone) 35.830 105

Potassium Hydroxide 56 % 14.500 100

Sodium Hydroxide 40% 3.679 27

Adenosine 1.340 5

Allopurinol 0.136 1

Potassium Dihydrogen Phosphate 3.400 25

Magnesium Sulphate.7H ₂ 0 1.230 5

Raffmose.5H ₂ 0 17.830 30

Glutathione 0.922 3 The composition of HTK solution is described in Table 2 below.

Table 2. Composition of HTK solution

In an advantageous embodiment, the protective solution according to the present invention is maintained at a temperature of between 0 and 10 °C, advantageously between 1 and 10 °C, more advantageously between 1 and 8 °C, still more advantageously between 3 and 5°C, even still more advantageously at around 4 °C.

The present invention also consists in a method for protecting internal organs cut from the body from ischemic damages comprising contacting the organ tissues and cells with the protective solution as defined above.

Advantageously contact is carried out by a hypothermic perfusion device.

A hypothermic perfusion device is any mechanical device which can be used to infuse an organ with the protective solution according to the present invention.

In a particular embodiment, the hypothermic perfusion device is a reservoir, such as a syringe, for holding the protective solution connected to a vein and/or artery of the organ by a tube or a cannula. In another embodiment the hypothermic perfusion device is an electrically driven and controlled device, including various flow and temperature control device, and which allow the delivery of the protective solution at a controlled flow rate and temperature. Advantageously, the flow rate is of 30ml/min.

More advantageously, the hypothermic perfusion device is a Lifeport® from Organ Recovery System or RM3® from Waters Instruments/IGL group.

The protective solution according to the present invention is compatible with the hypothermic perfusion device and does not precipitate or aggregate during the perfusion through the organ. Definitions

The compound of the present invention may also be present in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compound of this invention refer to non-toxic "pharmaceutically acceptable salts." FDA approved pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts (Gould, 1984,; Berge. et ah, 1977).

Pharmaceutically acceptable salts of the acidic or basic compound of the invention can of course be made by conventional procedures, such as by reacting the free base or acid with at least a stoichiometric amount of the desired salt-forming acid or base.

Pharmaceutically acceptable salts of the acidic compound of invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc, ammonium, and salts with organic bases. Suitable organic bases include N-methyl-D- glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.

Pharmaceutically acceptable salts of the basic compound of the invention include salts derived from organic or inorganic acids. Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactiobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, subsalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide.

In the methods of treatment of the present invention, word "administering" shall encompass the treatment of the various described disorders with the specifically disclosed compound or with compound which may not be specifically disclosed, but which convert to the specified compound in vivo after administration to the subject.

In particular the compound according to the present invention can be administered to the subject in which the organ will be transplanted before the transplantation of the organ, in order to avoid the ischemia/reperfusion injury which occurs during the reperfusion of the organ and/or to treat the ischemia/reperfusion injury which has occurred before the removal of the organ and/or during the preservation phase of the organ.

In another embodiment, the compound according to the present invention can be administered to the subject after the transplantation of the organ in order to treat the ischemia/reperfusion injury which has occurred during the reperfusion of the organ and/or before the removal of the organ and/or during the preservation phase of the organ.

In another embodiment, the compound according to the present invention can be administered to the subject who will give the organ for transplantation in order to avoid the ischemia/reperfusion injury which occurs during the reperfusion of the organ and/or before the removal of the organ and/or during the preservation phase of the organ.

In another embodiment, the compound according to the present invention can be administered to the subject during surgery (before or after the removal of the organ) in order to avoid the ischemia/reperfusion injury which occurs during the reperfusion of the organ and/or before the removal of the organ and/or during the preservation phase of the organ. It is anticipated that the compound of the invention can be administered by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, rectal and topical administration. Intravenous and subcutaneous administrations of the compound of the present invention are particularly preferred.

For parenteral use, including intramuscular, intraperitoneal, subcutaneous and intravenous use, the compound of the invention will generally be provided in sterile aqueous solutions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride, sodium phosphate. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

The present compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising an agent of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Another use of the compound of the present invention requires that the compound be added to a preservation solution. It will be a solution or a powder provided with the solvent to dilute it.

In transplantation process static cold storage is generally used to preserve kidney allograft. But machine preservation is mandated for organ preservation from donors like deceased after cardiac arrest donors or marginal donors. Recent data showed that hypothermic machine perfusion was associated with a reduced risk of delayed graft function and improved graft survival after transplantation (Moers et al, 2009) (Watson CJ, et al. 2010) (Vaziri et al, 2011). Compound I could be used during conservation on perfusion machine to improve the vascular drug distribution.

Clinical investigations have demonstrated the benefits of using perfusion machines to preserve kidneys obtained from DCD donors (Maathuis, Manekeller et al. 2007; Moers, Smits et al. 2009; Pirenne, Smits et al. 2009). These machines reduce DGF incidence and therefore the necessity to dialyze the newly-transplanted patients, resulting in a decrease in long term health care costs not only for DCD donors (Wszola, Kwiatkowski et al. 2009) but also for the extended and standard criteria donors (Garfield, Poret et al. 2009). These observations have led the French health authorities to impose machine perfusion (MP) for DCD kidney preservation (http ://www.urgences- serveur . fr/prelevements-de-reins-sur-des, 141 8.html) .

The following examples and figures illustrate the invention but are not limitative.

Figure 1 represents the creatininemia level post-transplantation in function of the day after kidney transplantation in pig in an in vivo study in which pig kidney was preserved before transplantation and after warm ischemia using UW, UW + UFH and UW + Compound (I) in the form of a 60/40 mixture of L/D lysine.

Figure 2 represents the Kaplan-Meier plot of the survival of pigs after kidney transplantation in the in vivo study.

Figure 3 represents the percentage of LDH release in an in vitro study on endothelial porcine primary cells model.

Figure 4 represents the creatininemia level 14 days, 1 month and 3 months after kidney transplantation in pig in an in vivo study in which pig kidney was preserved before transplantation and after warm ischemia using UW + UFH and UW + compound (1), uninephrectomised pigs are taken as control. Figure 5 shows the presence of vimentin in kidney tissue 3 months after transplantation in unnephrectomised pigs, uninephrectomised pigs, HEP group, ENDO 10 and END016.3 groups.

EXAMPLE 1- In vivo studies I

To mimic conditions found in DCD kidneys, renal warm ischemia was induced in large white pigs by clamping the right renal pedicle for 60 min with a vascular non-traumatic clamp. Large white male pigs (INRA/GEPA, Surgeres, France) were prepared as previously described (Hauet et al, 2000) and in accordance with French guidelines of the Ethical Committee for Human and Animal Studies.

Then, the kidney was collected, cold flushed, and stored for 24 h at low temperature in University of Wisconsin (UW) preservation solution (as a control), or with UW containing either unfractionated heparin (UFH) or compound (I) in the form of 60/40 mixture of L/D lysine (referred as compound 1) prepared as indicated in WO 2006/067173 prior to implantation. Compound (1) was compared to UFH since most donors are treated with heparin as part of the intensive care they receive prior to their death. The kidney was then transplanted in the same animal, and followed for 3 months. In each experimental group, the left kidney was removed during transplantation to mimic the nephron mass in the transplanted human recipient settings. Time for anastomosis was 30±5 min.

Animals were divided into three experimental groups: UW: kidneys underwent 60 min of warm ischemia, then kidneys were preserved at 4 °C during 24 h in UW (n=5); UW+UFH: pigs were first administered by the IV route with UFH (125 IU/Kg) 10 min before the 60 min warm ischemia and kidneys were then preserved at 4 °C during 24 h in UW to which UFH was added (5 000 IU/L) (n=l l); UW+compound (1): kidneys underwent 60 min of warm ischemia, then kidneys were preserved at 4 °C during 24 h in UW to which COMPOUND 1 was added (16.3 mg/L) (n=5).

Renal function recovery was measured through serum creatinine assay (Figure 1).

For statistical analysis of groups UW+UFH vs. UW+compound 1, NCSS software (NCSS LLC, USA) was used with the Equal-Variance T-Test for Two-Sample Test comparisons in case of normality (Skewness, Kurtosis and Omnibus tests) and equality of variance (Modified-Levene Equal- Variance Test) or Aspin- Welch Unequal- Variance Test in case of normality and unequal variance. Statistical significance was accepted for p<0.05 (* p<0.05, ** p<0.01 and *** p<0.001) (p meaning the percentage of chance for the observed difference to be random occurrence).

The results from the in vivo studies were:

o There was no survival past 1 week in the UW group, due to primary non function (PNF, no resuming of urine production), which led to subsequent euthanasia of the animals. o Out of the 11 kidneys preserved with UFH-supplemented UW solution, 6 kidneys displayed PNF after one week and had to be euthanized according to ethical regulations. In this group, serum creatinine rose steadily until day 5, and then slowly decreased, without reaching pre- transplant levels.

o Replacement of UFH by compound 1 allowed a faster recovery of function (starting at day 3) followed by a significant decrease of the creatinemia level down to recovering pre-transplant levels by day 30. These results clearly demonstrated that DGF could be suppressed by the treatment with compound 1.

In addition to the 6 PNF observed after one week for kidneys preserved with UFH- supplemented UW solution, there were two more animal losses at 4 and 6 weeks, which general poor state led us to perform euthanasia. Examination of kidney grafts at these time-points revealed massive tissue invasion by immune cells and parenchyma necrosis. Substitution of UFH by compound 1 improved the survival drastically, as all kidneys recovered their function and as animal survival was allowed until the end of the experiment (Figure 2) (3 months, p<0.05, Logrank Test).

For statistical analysis of groups UW+UFH vs.UW+compound 1, to compare the survival distributions, the Logrank Test with GraphPad Prism software was used. Statistical significance was accepted for p<0.05 (* p<0.05).

In summary, in the model of DCD kidney transplantation used, preservation with the gold standard UW was unable to preserve kidney integrity during cold storage and to permit function recovery. Addition of UFH allowed for some tissue protection, slightly improving function recovery as well as allowing 27% survival at the end of the follow- up. Using compound 1 instead of UFH allowed full recovery of function for all kidneys, as well as faster recovery to pre-transplant levels.

In conclusion, these preliminary results demonstrated the superiority of compound 1 over standard UFH to improve kidney preservation and quality, optimizing long-term outcome. Considering the universal nature of IRI in organ transplantation and that the impact of IRI on endothelial cells is similar regardless of the organ, therefore compound 1 should be effective in preventing other organs from IRI.

EXAMPLE 2: In vitro studies

A study defining the benefits of compound I in the form of 60/40 mixture of L/D lysine (referred as compound 1) against IRI was conducted in an endothelial porcine primary cells model. The aim of this study was to mimic organ preservation using endothelial cells, which are the first cells affected by IRI during kidney transplantation. Cells were placed in hypoxic atmosphere for 24 h in UW solution at 4 °C with or without addition of compound 1 at 16.3 mg/L.

After hypoxia, extracellular lactate dehydrogenase (LDH) quantification was carried out to determine the extent of cell death using the in vitro toxicology assay kit from Sigma- Aldrich and including LDH. The assay is based on the reduction of NAD by LDH. The resulting reduced NAD (NADH) is utilized in the stoichiometric conversion of a tetrazolium dye. The resulting colored compound is measured spectrophotometrically. The amount of LDH activity can be used as an indicator of relative cell viability as well as cellular membrane integrity.

Results were expressed as a percentage of LDH release, i.e. [(LDH release) / (LDH release) + (LDH intracellular)]* 100. With this approach, a high amount of LDH release involves more cell death. Three groups were compared and consisted of:

-Control cells (n=8): endothelial porcine cells, cultured in adequate medium, without hypoxia and UW solution

-UW (n=8): endothelial porcine cells, incubated in hypoxia during 24 h in UW solution. -UW+compound 1 (n=8): endothelial porcine cells, incubated in hypoxia during 24 h in UW solution + compound 1 at 16.3 mg/L. (see figure 3)

Results are expressed as Mean ± SEM. For statistical analysis of groups UW vs.UW+ compound 1, NCSS software (NCSS LLC, USA) was used with the Equal- Variance T- Test for Two-Sample Test comparisons in case of normality (Skewness, Kurtosis and Omnibus tests) and equality of variance (Modified-Levene Equal-Variance Test). Statistical significance was accepted for p<0.05 (*** p<0.001).

This experiment showed that the use of compound 1 at 16.3 mg/L preserved cell integrity during IRI as demonstrated by the halving of LDH release in the extracellular compartment compared with the standard preservation method (UW alone) (p<0.001) (Figure 3).

EXEMPLE 3: In vivo studies II

A second study has been conducted to evidence the advantages to add compound (1) in UW in comparison with the addition of heparin in UW in large white pigs. This second study as the first one uses autotransplantation for assessing effects of ischemia reperfusion while avoiding any influence from any immunological phenomenon.

Large white male pigs (INRA/GEPA, Surgeres, France) were prepared as previously described (Hauet et al, 2000) and in accordance with French guidelines of the Ethical Committee for Human and Animal Studies.

The left kidney was subjected to renal warm ischemia by clamping the left renal pedicle for 60 min with a vascular non-traumatic clamp in order to mimic the conditions found in DCD kidneys. Then the left kidney was removed, washed with UW and preserved at 4°C for 24 hours in UW containing unfractionated heparin (UFH) (5000 U/L) (group HEP) or compound (1) (two concentrations have been used: 10 mg/L and 16.3 mg/L) (respectively groups ENDOIO and END016.3).

After this period of time, the right kidney is removed and replaced by the left kidney. In one group of large white pigs, one kidney is removed without any transplantation. This group (group NEP) serves as a control group for controlling the nephron mass.

Renal function recovery was then measured through serum creatinin assay (Figure 4). For statistical analysis, NCSS software (NCSS LLC, USA) was used. Newman Kulls parametric test was used for comparing samples in case of equal variances (Skewness, Kurtosis, Omnibus et Modified-Levene tests). Following ANOVA One Way, in case of unequal variances, Dunn's non parametric test was used. Number of animals per each group was 6.

Creatinin measurement enabled to evidence a rapid recovery towards normal values in ENDOIO and END016.3 groups. Concentrations of creatinin after 14 days and 1 month show that there is no statistical differences for NEP group and ENDOIO and END016.3 groups. This means that the presence of compound (1) in UW enabled the kidney to repair IR lesions to the extent that its function is equivalent to that of a kidney that suffered no injury (NEP group).

On the contrary, in HEP group the kidney function did not fully recover even 3 months after the transplantation.

Epithelial-mesenchymal transition was also evaluated through vimentin measurement (Figure 5). Epithelial cells dedifferentiation leads to fibrosis formation in renal tissue and participates in kidney malfunction. Vimentin is a dedifferentiation marker. Presence of vimentin is higher in tissues with high number of dedifferentiating cells and fibroblasts.

Statistic figures were obtained with NCSS software using Kruskal-Wallis' test (Dunn's test). Figure 5 clear shows that ENDOIO, END016.3 and NEP groups present significantly lower marking by vimentin than group HEP (*p<0.05). Number of animals for the unnephrectomised group was 4 and 5 for the others.

Example 3 clear by demonstrates that use of compound (1) in UW is more beneficial than use of heparin in UW during kidney transplantation for the preservation thereof. Best results were found with a concentration of 16.3 mg/L of compound (1) in UW.

BIBLIOGRAPHIC REFERENCES

Berge, S.M. et al. , J. Pharm. ScL, 1977, 66 (1), 1-19

Feng, S. (2010) Donor intervention and organ preservation: where is the science and what are the obstacles? Am J Transplant, 10, 1155-1162.

Frank, R.D., Schabbauer, G., Holscher, T., Sato, Y., Tencati, M., Pawlinski, R. & Mackman, N. (2005) The synthetic pentasaccharide Fondaparinux reduces coagulation, inflammation and neutrophil accumulation in kidney ischemia-reperfusion injury. J Thromb Haemost, 3, 531-540.

Giraud, S., Thuillier, R., Belliard, A., Hebrard, W., Nadeau, C, Milin, S., Goujon, J.M., Manguy, E., Mauco, G., Hauet, T. & Macchi, L. (2009) Direct thrombin inhibitor prevents delayed graft function in a porcine model of renal transplantation. Transplantation, 87, 1636-1644.

Gould, P.L., International ! Pharm., 1984, 33, 201-271.

Hauet, T., Goujon, J.M., Vandewalle, A., Baumert, H., Lacoste, L., Tillement, J. P., Eugene, M. & Carretier, M. (2000) Trimetazidine reduces renal dysfunction by limiting the cold ischemia/reperfusion injury in autotransplanted pig kidneys. J Am Soc Nephrol, 11, 138-148.

Holschermann, H., Bohle, R.M., Schmidt, H., Zeller, H., Fink, L., Stahl, U., Grimm, H., Tillmanns, H. & Haberbosch, W. (2000) Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts. Circulation, 102, 357-363. Karabiyikoglu, M., Hua, Y., Keep, R.F., Ennis, S.R. & Xi, G. (2004) Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab, 24, 159-166.

Kosieradzki, M. & Rowinski, W. (2008) Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant Proc, 40, 3279-3288.

Mikhalski, D., Wissing, K.M., Ghisdal, L., Breeders, N., Touly, M., Hoang, A.D., Loi, P., Mboti, F., Donckier, V., Vereerstraeten, P. & Abramowicz, D. (2008) Cold ischemia is a major determinant of acute rejection and renal graft survival in the modern era of immunosuppression. Transplantation, 85, S3-9.

Moers, C, Smits, J.M., Maathuis, M.H., Treckmann, J., van Gelder, F., Napieralski, B.P., van Kasterop-Kutz, M., van der Heide, J.J., Squifflet, J. P., van Heurn, E., Kirste, G.R., Rahmel, A., Leuvenink, H.G., Paul, A., Pirenne, J. & Ploeg, R.J. (2009) Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med, 360, 7-19.

Montaigne, D., Marechal, X., Lancel, S., Decoster, B., Asseman, P. & Neviere, R. (2008) The synthetic pentasaccharide Fondaparinux prevents coronary microvascular injury and myocardial dysfunction in the ischemic heart. Thromb Haemost, 100, 912- 919.

Petitou, M., Nancy-Portebois, V., Dubreucq, G., Motte, V., Meuleman, D., de Kort, M., van Boeckel, C.A., Vogel, G.M. & Wisse, J.A. (2009) From heparin to COMPOUND 1 : the long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile. Thromb Haemost, 102, 804-810.

Pratschke , J . , Weiss , S . , Neuhaus , P . & P ascher, A . (2008) Review of nonimmunological causes for deteriorated graft function and graft loss after transplantation. Transpl Int, 21, 512-522.

Sutton, T.A. (2009) Alteration of microvascular permeability in acute kidney injury. Microvasc Res, 77, 4-7. Vaziri N, ThuiUier R, Favreau FD, Eugene M, Milin S, Chatauret NP, Hauet T, Barrou B. Analysis of machine perfusion benefits in kidney grafts: a preclinical study. J Transl Med. 2011 Jan 25;9: 15.

Vieira, L. (2009) Expanded criteria donors offer hope for patients needing kidney transplant. Jaapa, 22, 33-36.

Watson CJ, Wells AC, Roberts RJ, Akoh JA, Friend PJ, Akyol M, Calder FR, Allen JE, Jones MN, Collett D, Bradley JA. Cold machine perfusion versus static cold storage of kidneys donated after cardiac death: a UK multicenter randomized controlled trial. Am J Transplant. 2010 Sep; 10(9): 1991-9.

Yuan, X., Theruvath, A.J., Ge, X., Floerchinger, B., Jurisch, A., Garcia-Cardena, G. & Tullius, S.G. (2010) Machine perfusion or cold storage in organ transplantation: indication, mechanisms, and future perspectives. Transpl Int, 23, 561-570.