WO2012164321A1

WO2012164321A1 - Organ and tissue transplantation solution containing bmp-7

Info

Publication number: WO2012164321A1
Application number: PCT/HR2011/000025
Authority: WO
Inventors: Dragica BOBINAC
Original assignee: Medicinski Fakultet U Rijeci
Priority date: 2011-05-30
Filing date: 2011-05-30
Publication date: 2012-12-06

Abstract

The present invention relates to a method for preserving of organs and/or tissue and/or bioengineered tissue products. This method comprises obtaining an organ and/or organ parts and/or tissue from dead or living bodies and/or the tissue is a bioengineered tissue product and bringing it into contact with the basic cold storage solution containing bone morphogenetic protein-7 (BMP-7 protein, also known as osteogenic protein 1; OP-1). Further, this invention relates to the basic storage solution containing BMP-7 in concentrations 1-50 μg/kg as a primary active substance responsible for efficient prevention of the damage to organ and/or tissue and/or cell during cold preservation awaiting transplantation.

Description

ORGAN AND TISSUE TRANSPLANTATION SOLUTION CONTAINING BMP-7

FIELD OF THE INVENTION

The present invention relates to a method for preserving of organs and/or tissue and/or bioengineered tissue products. This method comprises obtaining an organ and/or organ parts and/or tissue from dead or living bodies and/or the tissue is a bioengineered tissue product and bringing it into contact with the basic cold storage solution containing bone morphogenetic protein-7 (BMP-7 protein, also known as osteogenic protein 1 ; OP-1). Further, this invention relates to the basic storage solution containing BMP-7 in concentrations 1-50 pg/kg as a primary active substance responsible for efficient prevention of the damage to organ and/or tissue and/or cell during cold preservation awaiting transplantation.

BACKGROUND OF THE INVENTION

Transplantation is now the last-resort treatment for the end stage of both acute and chronic organ diseases; however this therapy is hampered by the extreme sensitivity of organs to ischemia-reperfusion injury. This type of damage occurs during the period starting at the recovery of organs from the donor until their reperfusion in the recipient. The reintroduction of blood flow to the ischemic organ is necessary to rescue the organ from necrosis and permanent loss of function. However, it may cause acute cellular and organ dysfunction. Depending on the severity of acute organ injury from ischemia and reperfusion, in a large proportion of organ transplantations (20-80%), delayed graft functions is observed. Furthermore, damage to the organs during ischemia/reperfusion is also associated with increased risk of long-term graft failure.

Due to the lack of suitable donors, organs from donors that would be in the past considered as unsuitable (non-heart beating donors) are presently transplanted into recipients. Organs from these donors suffer further warm ischemic injury from circulatory arrest until organ preservation. As a result, most of these organs experience a delayed graft function and do not even show functional recovery at all. The key step to minimize the incidence of these negative outcomes of transplantation is the improvement of organ preservation solutions.

The main goal of organ preservation is the maintenance of the functional, biochemical and morphological integrity of the graft in transit from donor to recipient: from harvest to revascularization.

Organs can survive for only very brief period (30 to 60 minutes) after disconnecting from the circulatory system of the donor. The resultant oxygen depletion and loss of substrates, energy and other substances, as well as accumulation of metabolic end products impairs organ tissue and causes cellular death.

Two main strategies are presently used to preserve organs and improve the results of transplantation. One is to stimulate the physiological environment of the organ while it is outside of the body and, most likely, being transported to the recipient. The other strategy is to use cooling or hypothermia as a way to slow down metabolic processes and inhibit cell death.

Using these two strategies can significantly improve the viability of the organ and enhance the success of organ function after reperfusion in the recipient.

Ischemia-reperfusion after cold preservation is considered as major non-immunologic antigen- independent factor that influences graft outcome (Chatauret, N et al. Preservation strategies to reduce ischemic injury in kidney transplantation: pharmacological and genetic approaches. Curr Opinion in Org Transplant 2011, 16:180-187; Lutz et al. Anti-inflammatory treatment strategies for ischemia reperfusion injury in transplantation. Journal of Inflammation 2010, 7: 27-35) Ischemia-reperfusion injury is the result of a prolonged oxygen deprivation in a tissue leading to hypoxia and is initiated by the cascade of molecular and cellular events, including release of proinfammatory mediators and the attraction of various cell types that infiltrate the tissue. Hypoxia results in an ATP-depletion of the cells leading to swelling of mitochondria eventually causing a release of cytochrome c from the mitochondria. Cytochrome c activates an apoptotic signaling cascade involving caspases 1 and 9.These events participate in the induction of an inflammatory response via generation of IL-1 β as well as programmed cell death (apoptosis by activation of different caspases). Moreover, ATP depletion induces a cellular edema that occurs particulary during cold ischemia when Na/K ATPase is inhibited (Kosieradzki M, Rowinski W: Ishemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transpl. Proc. 2008, 40:3279-3288). A crucial mediator or ischemia-reperfusion injury are oxygen derived free radicals (Ma A et al.. Antioxidant therapy for prevention of inflammation, ischemic reperfusion injuries and allograft rejection. Cardiovasc. Hematol. Agents Med. Chem. 2008, 6:20-43) Particularly important is hydrogen peroxide, which can induce TNFa, a pleiotropic inflammatory cytokine.

Additionally, a number of intracellular adaptive metabolic responses occur among them an increase in the intracellular C²⁺ concentration with generation of calcium pyrophosphate complexes and the formation of uric acid. Both of them act as "danger signals" which through myriad of reactions stimulate secretion of further proinflammatory cytokines/chemokines (Meldrum DR et al. Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by P38 mitogen activated protein kinase-dependent mechanism. Surgery 1998, 124:291- 296; Martinon F et al. the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing prolL-beta. Mol. Cell 2002, 10:417-426; Moynagh, PN: Toll-like receptor signaling pathways as key targets for mediating the anti-inflammatory and immunosuppressive effects of glucocorticoids. J Endoc nolol 2003, 179:139-144)

This is followed by an infiltration of lymphocytes, mononuclear cells/macrophages and granulocytes into the injured tissue. The increase in intracellular Ca²⁺ concentration enhances activation of phospholipases as well as proteases. These include calpains (cleaving protein kinase c, fodrin, components of the cytoskeleton) and caspases which execute programmed cell death (apoptosis).

An important effect of hypoxia on a tissue is the development of metabolic acidosis. It occurs as a result of hypoxia when anaerobic glycolysis is the only way to generate the energy. However, it can induce and inflammatory response when perfusion of the respective tissue is restores after hypoxia as well as hypothermia (Polderman KH: Mechanisms of action, physiological effects, and complications of hypothermia. Crit Care Med 2009, 37:5186-202)

Due to the chronic inflammation, tissue will be remodeled which can eventually lead to organ fibrosis with consecutive loss of function (Gueler F. et al. Long-term effects of acute ischemia and reperfusion injury. Kidney Int 2004, 66.523-527)

These subsequent short- and long term changes induced by hypoxia influence the structure and function of the organ and may compromise graft survival. A number of solutions have been developed to minimize the pathophysiological changes that occur in the graft tissue as a result of hypothermic ischemia. The ingredients of the solutions simulate an optimal environment to protect and preserve cellular functioning both during extracorporeal phase, and upon reperfusion.

Each solution substantially differs in their composition, but the purposes of each are similar: to prevent cellular edema, to delay cell destruction and to maximize organ function after perfusion is established

Examples of these solutions are: Euro-Collins solution (high concentrations of potassium (110 mM), phosphate (60 mM), and glucose (180 mM)); University of Wisconsin solution (complex solution, golden standard for renal and hepatic preservation, has osmolality of 320 mmol/kg and pH 7.4 at room temperature, composed of: potassium (135 mmol/l), sodium (35 mmol/L), magnesium (5 mmol/l), lactobionate (100 mmol/l), phosphate (25 mmol/l), sulphate (5 mmol/l), raffinose (30 mmol/l), adenosine (5 mmol/l), allopurinol (1 mmol/l), glutathione (3 mmol/l), insulin (100 U/l), dexamethasone (8 mg/l), hydroxyethil starch (HES, 50 g/l), bactrim 0.5 ml/l); Celsior solution (sodium 100 mmol/l, potassium 15 mmol/l, magnesium 13 mmol/l, calcium 0.25 mmol/l, lactobion, te 80 mmol/l, glutathione 3 mmol/l, glutamate 20 mmol/l, mannitol 60 mmol/l, histidine 30 mmol/l and ET-Kyoto solution (sodium 100 mmol/l, potassium 44 mmol/l, phosphate 25 mmol/l, trehalose mmol/l, HES 30 g/l, gluconate 100 mmol/l).

Despite the fact that those solutions have made extended cold preservation feasible, their protective effect on organ and/or tissue is far from optimal. It has been demonstrated for example that prolonged cold ischemia causes vascular complications (Mor et al. Prolonged preservation in University of Winsconsin solution associated with hepatic arthery thrombosis after orthotopic liver transplantation. Transplantation 1993: 56(6): 1399-402.) and blood vessel constriction when the organ is cold flushed (Ramella-Virieuex SG et al. Nifedipine improves recovery function of kidneys preserved in a high-sodium, low-potassium cold storage solution: study with the isolated perfused rat kidney technique. Nephrol. Dial. Transplant 1997; 12(39:449-55). Moreover it has been shown that hydroxilethil starch induces red blood cell aggregation which causes blood stasis in the organ and triggers further inflammatory events (Morariu AM et. al, Hyperaggregating effect of hydroxiethyl starch components and University of Wisconsin solution on human red blood cell: a risk of impaired graft perfusion in organ procurement? Transplantation 2003:76(1 ):37-43; Monariu AM et al. Acute isovolemic hemodilution triggers proinflammatory and procoagulatory endothelial activation in vital organs: role of erythrocyte aggregation. Microcirculation 2006; 13(5):397-409)

Although cold ischemia times for deceased donor kidney transplants have been reduced in the past 20 years from an average of 24 hours during 1988-1992 to 18 hours in 2003-2007, the incidence of delayed graft function has remained at 25% each year. The effect of prolonged cold ischemia times (> 30 h ) on graft survival was a 1% reduction over 10 years for deceased donor kidneys under age 35 and a 4% reduction for deceased donor kidney over age 55 compared with the graft survival results for deceased donor kidneys in either age group transplanted within 18 hours. (Cecka JM . Kidney transplantation in the United States. Clin Transpl. 2008:1-18)

Therefore there is a clear unmet clinical need for improved organ preservation solutions that would have significantly higher protective effect on organs and/or tissues. One of the approaches would be to apply a pharmacological approach and introduce biologically active compounds to the preservation solutions. Although the mechanism of ischemia-reperfusion injury is a multifactor process and there are many target sites that are affected by it, it might be possible to find additives that would interfere with the process of damage in a way that damage to the organ and/or tissue would be significantly reduced and/or prolonged.

Several groups of compounds are regarded as candidates for such a pharmacological approach. These are Ca²⁺ channel blockers (nisoldipine or verapamil), antioxidants (deferoxamine, melatonin), protein kinase inhibitors (FR167653) and trophic factors (insulin-like growth factor-1 , hepatocyte growth factor) (Zaouali MA et al. Pharmacological strategies against cold ischemia reperfusion injury. Current Opin Pharmacother, 2010 11(4)537-555).

Many of these candidates have failed to be developed for a variety of reasons: low efficacy in vitro, low preclinical efficacy, preclinical data were not translated into clinical efficacy, safety issues etc.

Prior art where organ preservation solution containing pharmacological additives are disclosed can be exemplified by:

US 2004/0053205 A1 (Flush preservation solution) describes addition of Ca²⁺ channel blockers; WO 01/20982 A2 (Solution for the preservation of hearts) describes addition of cyclosporine

US 2005/0233299 (Method for preserving organs for transplantation with a HGF- containing solution) where addition of human recombinant hepatocyte growth factor to organ preservation solution is described

Bone morphogenetic protein-7 is a member of the TGF-β superfamiliy of ligands that is required during normal skeletal, kidney and eye development (Dudley AT et al. A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev 1995, 9:2795-2807; Godin RE et al. Regulation of BMP-7 expression during kidney development Development 1998, 125:3473-3482; Luo G et al. BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning, Genes Dev 1995, 9:2808-2820) In the kidney BMP-7 is thought to be required for continued epithelial tubule development in the embryonic kidney. The expression of BMP-7 continues in the adult kidney and this expression has been suggested to decrease in response to injury (Almanzar MM et al. Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury. J Am Soc Nephrol 1998, 9:1456-1463; Simon M et al. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am J Physiol 1999; 276, F382-9) Administration of BMP-7 to animals in a model of renal ischemia has been reported to increase their survival which correlates with decrease in resulting infarct size, necrosis and infiltration and activity of neutrophils following reperfusion (Vukicevic S et al. Osteogenic protein-1 (bone morphogenetic protein-7) reduces seventy of injury after ischemic acute renal failure in rat. J Clin Invest 1998, 102:202-214).

The present invention discloses for the first time novel improved organ and/or tissue and/or bioengineered tissue preservation solution that uses an addition of bone morphogenetic protein-7 as a primary active ingredient.

DISCLOSURE OF THE INVENTION

The present invention provides a solution for use in organs and/or tissue and/or cells and /or bioengineered tissue products perfusion, preservation or reperfusion. The present invention provides solution that consists of basic cold storage solution and an effective amount BMP-7. The solution of the present invention consists of basic cold storage that could be but it is not limited to standard (normal) saline solution and/or any other solutions or procedures designed to preserve viability of organs, tissues, cells and bioengineered tissues where these designed preservation solutions may include, but are not limited to University of Wisconsin solution(Viaspan™), Euro-Collins solution, Celsior solution, Krebs-Henseleit solution, St. II Thomas solution, Stanford solution, Polysol® solution, Custodiol® HTK solution, Hypothermasol® solution, IGL-1 solution, Lifor solution, Trans Send solution, ET-Kyoto solution, siRNA solution or similar.

One embodiment according to the present invention is a solution where a basic storage solution or procedure is designed for treating organs, tissues, cells or bioengineered tissue prior and/or after cryopreservation and/or vitrification regardless of the indented use of (transplantation medicine, regenerative medicine, assisted reproductive medicine, bio- pharmaceutical research, forensic sciences, or other).

BMP-7 protein is a known protein with primary structure of SEQ 1 : (Human BMP-7 protein - Oezkaynak E. et al. OP-1 cDNA encodes an osteogenic protein in the TGF-beta family EMBO J. 1990; 9:2085-2093)

A solution of the present invention contains BMP-7 protein that has an amino acid sequence with more than 90 % of homology to sequence SEQ No1.

In a further embodiment, a solution of the present invention preferably contains BMP-7 protein that has a an amino acid sequence with more than 95 % of homology to sequence SEQ No1

In a further embodiment, a solution of the present invention more preferably contains BMP-7 protein that has a an amino acid sequence with 100 % of homology to sequence SEQ No1

The solution of the present invention contains BMP-7 protein in concentrations of about 1-50

Mg/kg-

In an embodiment, the present invention solution contains BMP-7 protein preferably in concentrations of about 5-20 pg/kg.

In an embodiment, the present invention solution contains BMP-7 protein more preferably in concentrations of about 10 pg/kg.

In one embodiment present invention uses the solution of the invention in virtually all adult or embryonic human or animal organs and/or tissue and/or cells and /or bioengineered tissue products perfusion, preservation or reperfusion.

A method of preserving an organ and/or tissue may comprise the steps of:

a) obtaining the organ and/or tissue and/or cells from a suitable source; and

b) maintaining the organ and/or tissue and/or cells in a solution of the present invention

EXPERIMENTAL RESULTS

The inventors have found that the solution of present invention provides significantly better results in protecting the rat kidneys from cold ischemia injury during 24 hours in direct comparison with normal saline and University of Wisconsin solutions.

Methods

Animals and surgical preparation

Male Wistar rats (3 months old; weight 250 - 300g) were supplied by the Institute for Medical Research and Occupational Health (Zagreb, Croatia). Animals were housed in a climate controlled facility with a constant 12h light - dark cycle, had free access to water and were fasted 12h before operation. The experimental protocol of this study was approved by the Faculty ethical committee, and all experimental procedures were performed in accordance with Croatian legislation on Laboratory Animal Experiments.

Animals were anesthesized with ketaminhydrochloride (0,1 mg/g body weight i.p) and xylazinhydrochloride (0,02 mg/g body weight i.p). During surgical procedure animals were placed on a heating pad to maintain body temperature at 36.5-37°C. A midline incision was performed in the supine position. Viscera were exteriorized and wrapped in wet gauze to expose both kidneys, supra- and infrarenal portion of aorta and vena cava inferior. The suprarenal part of aorta and vena cava inferior were cannulated with venous cannula. The supra- and infrarenal portion of aorta and vena cava inferior were clamped. Both kidneys were flushed via the intraaortic cannula in situ with respective solution (temperature 4°C) at a pressure of 100 mm H₂0 for 3 min. Perirenal fat tissue was than dissected and both kidneys (en bloc) with their vascular pedicles were removed, additionally flushed with 5ml of respective solution (4°C), placed in small container with respective preservation solution and stored at 4°C for 6, 12 and 24h.

Preservation solutions and study design

Both kidneys of five animals (N=10) in control group were flushed with cold saline, immediately frozen in liquid nitrogen and stored at -80°C. The kidneys exposed to cold ischemia were perfused with three different preservation solutions, each applied to 15 animals. From each animal both kidneys were perfused. Kidneys in the first group were perfused with saline (5ml), in the second group with University of Wisconsin solution (5 ml) and in the third group with rhBMP-7. RhBMP-7 protein was administered in 10[ig/kg dosage with saline as vehicle and applied in total volume of 5ml. After perfusion the kidneys were exposed to cold ischemia for 6, 12 or 24h. So, each of the examined group was divided depending on time of cold ischemia and for each time point 10 kidneys were analyzed. After determined period of cold ischemia kidneys were frozen in liquid nitrogen and stored at -80°C. Altogether 50 animals were involved in the experimental procedures and both kidneys from each animal were analyzed.

Chemicals and reagents

Chemicals were purchased from Sigma - Aldrich, Inc. (St. Louis, MO, USA), Panreac Quimica S.A.U. (Barcelona, Spain), and Kemika (Zagreb, Croatia). RANSOD kit for SOD and RANSEL kit for GSH-Px determinations were obtained from Randox Laboratories Ltd. (Crumlin, UK). Recombinant human BMP-7 protein (concentration 0.1 mg/ml) was purchased from MyBioSource (San Diego, CA, USA) and UW - Viaspan from DuPont (Wilmington, DE, USA).

Sample preparation

Kidney tissue were weighted and homogenized in ice cold phosphate buffered saline, pH 7, 4. The homogenates were aliquoted (100μΙ) and used for lipid peroxidation and protein carbonyl content assays. Remaining homogenate was centrifuged at 13000rpm for 30 min and the obtained supernatant was used as the source for enzyme assay. Supernatant was aliquoted and stored at -20°C in order to measure the activities of superoxide dismutase (SOD) and gluthation peroxidase (GPH-Px). All preparation procedures were performed at +4°C.

Lipid peroxidation assay

Lipid peroxidation was determined spectrophotometrically by measuring the thiobarbituric acid reactive substances (TBARS) level, using a modification of method previously described by Ohkawa et al. (1979). Briefly, 200 μΙ of homogenate was mixed with 100 μΙ of 8.1 % sodium duodecyl sulphate and incubated for 10 min at room temperature. Subsequently, 900 μΙ of 20% acetic acid was added to samples and centrifuged at 10,600 rpm for 15 min. Supernatants (1 ml) were heated with 0.8% thiobarbituric acid solution (1 ml) in water bath at 95°C for 60 min. Samples were cooled on ice for 5 min and 2ml of n-butanol:pyridine (15:1 , v/v) was added. From pink-stained solution TBARS were extracted by centrifugation at 4800rpm for 10 min. The absorbance of upper organic layer was read at 532nm. The concentration of lipid peroxidation products was calculated as MDA (malondyaldehide) equivalent using a molar extinction coefficient for the MDA-thiobarbituric acid complex of thiobarbituric acid reactive substances (TBARS). TBARS were expressed as micromole MDA per miligram of protein.

Protein carbonyl assay

Protein carbonyl content (PCC) was determined spectrophotometrically after labeling with 2,4-dinitrophenylhydrazine (DNPH) following a modification of the procedure of Levine et al.²⁴ Briefly, 450 μΙ of homogenate was mixed with 100 μΙ of 20% SDS and incubated for 10 min at RT. Samples were centrifuged at 10600rpm for 10min at 4°C, supernatant (200μΙ) was aliquoted in two tubes and either 200μΙ of 2 M HCI (control) or 0.5 ml of 2 M HCI containing 10 mM DNPH (experiment) was added. Samples were incubated for 1 h on RT and vortexed every 15min, following the addition of 500 μΙ 20%TCA and 10 min incubation on ice. The samples were centrifuged at 14000rpm for 10min at 4°C, and supernatants were discarded. The protein pellet was washed three times with 1 ml ethanol: ethyl acetate (1:1 ,v:v). The pellet was then broken up with sonication and dissolved in 8M urea at 37°C for 30min. The absorbance of samples was read with a spectrophotometer (375 nm). Protein concentrations were determined at 280 nm using an absorption coefficient of 1 mg/ml. The carbonyl contents were calculated from the 375/280 absorbance ratio of each sample minus the respective control using a molar absorption coefficient of 22,000 M-1 cm-1 at 370 nm. Results are reported as the nmol carbonyl/mg protein (mean ± standard error mean).

Enzyme assay

SOD activity was determined in samples using method that employs xanthine and xanthine oxidase to generate superoxide radicals, which react with 2 iodophenyl-3-(4- nitrophenyl)-5-phenyltetrazolium (INT) chloride, to form a red formazan dye. One unit of enzyme activity was defined as the quantity of SOD required to cause a 50% inhibition of the absorbance change per min of the blank reaction. The final color was measured at 505 nm

GSH-Px activity was measured according to the Paglia and Valentina method. GSH-Px catalyses the reduction of cumene hydroperoxide using glutathione as a reducing agent. Oxidased glutathione is converted to reduced state in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP+. Oxidation of NADPH is monitored by decrease in absorbance at 340nm, whose rate is directly proportional to the GSH-Px activity in sample.

Protein content quantification

Protein content was measured by the spectrophotometric method of Bradford (1976) using bovine serum albumin as standard.

Statistical analysis

Data from each animal (N=5 animals/time point) were averaged and expressed as the mean ± SEM for each time point. Statistical significance was calculated according to the oneway analysis of variance (ANOVA), followed by Duncan^'s multiple range post-hoc test and defined as P<0.05.

Results Lipid peroxidation levels

TBARS levels, a measure of lipid peroxidation and cellular damage, increased in the kidney tissue exposed to cold ischemia. A time dependant increase in TBARS levels was especially observed in the kidneys preserved by saline or UW solution. (Figure 1A).

Figure 1. Lipid peroxidation, protein carbonyl content (PCC) and antioxidant enzyme activities (SOD and GSH-Px) in the groups treated with saline, Viaspan (UW) and Bone Morphogenetic Protein -7 (BMP-7) as well as in the control group (kidneys without cold ischemia) at several time points (6, 12 and 24 h). (A) The levels of thiobarbituric acid reactive substances (TBARS) as measure of lipid peroxidation, (B) the levels of PCC, (C) the activity of superoxide dismutase (SOD), (D) the activity of gluthatione peroxidase (GSH-Px). The bars represent mean ± S.E.M. (N=10). * P<0.05 significantly different from the control group; # P<0.05 significantly different from the UW and BMP-7 group; & P<0.05 significantly different from the UW and saline group.

TBARS levels in the kidneys preserved by rhBMP-7 were not statistically altered in relation to the control values after 6 and 12 h of cold ischemia. However, after 24 h of cold ischemia significant elevation of TBARS levels in rhBMP-7 treated group was found. Contrary, in the kidneys preserved by UW solution or saline, TBARS levels were significantly higher at all time points compared to the control group (P = 0.001).

Furthermore, the kidneys preserved by rhBMP-7 had statistically significant lower TBARS levels towards saline or UW groups at all time points (P=0.028). Moreover, the kidneys preserved by UW solution during first six hours of cold ischemia produced higher TBARS levels compared to the TBARS levels found in rhBMP-7 treated kidneys after 24 h of cold ischemia.

Preservation by UW solution did not decrease significantly TBARS levels compared to the saline treated group.

Protein carbonyl content

Protein carbonyl content (PCC) is an indicator of ROS induced protein oxidative damage. PCC increased in the kidney tissue exposed to cold ischemia and within all groups, but especially in the groups treated with saline or UW solution, that increase was time dependant (Figure 1 B).

In the kidneys preserved by rhBMP-7 solution PCC slowly increased after 24 h of cold storage but there was no significant elevation of protein carbonyl content in respect to the control group at all time points. The isolated kidneys preserved with UW solution or saline showed significantly elevated the protein carbonyl content in the comparison to the control group at all time points (P=0.001 ).

PCC was significantly lower in the rhBMP-7 treated kidneys towards saline or UW solution treated groups at all time points (P=0.021).

In the kidneys preserved by UW solution time dependant increase of PCC was not observed following 6 and 12 h time points, but was expressed after 24 h of prolonged cold ischemia. Antioxidant enzymes^* activities

Figure 1C and 1 D show the SOD and GSH-Px activities in the rat kidneys preserved by certain preservation solution after 6, 12 or 24 h of cold ischemia.

In comparison to the control group, it could be seen that after 6 h of cold ischemia SOD activity significantly increases in all three examined groups. But, after 6 h of cold ischemia the increase of SOD activity was significantly higher (P=0.038) in the kidneys perfused with rhBMP- 7 than in the kidneys perfused with saline or UW solution. The increase of SOD activity in the kidneys preserved by rhBMP-7 was four folds higher compared to the control group after 6 h of cold ischemia. After 12 or 24 h SOD activity decreased in all kidneys exposed to the cold ischemia.

After 6 and 12 h of cold ischemia all three preservation solutions maintained the GSH-Px activity at the similar level as it was in the control group. After 24 h of cold ischemia GSH-Px activity decreased, and the decrease was significantly lower for all three examined groups in the comparison to the control group (P=0.024). However, the kidneys preserved by rhBMP-7 showed still significantly higher of enzyme activity in comparison to the kidneys preserved by UW solution after 24 h of cold ischemia (P=0.041).

SOD and GSH-Px activity levels time-dependently decreased in the kidney tissue exposed to the cold ischemia.

Claims

PATENT CLAIMS

1. A solution for use in organs and/or tissue and/or cells and /or bioengineered tissue products perfusion, preservation or reperfusion wherein the solution consists of basic cold storage solution and effective amount BMP-7.

2. A solution according to claim 1 where the basic storage solution is standard (normal) saline solution and/or any other solutions or procedures designed to preserve viability of organs, tissues, cells and bioengineered tissues. The designed preservation solutions may include, but are not limited to University of Wisconsin solution(Viaspan™), Euro-Collins solution,

®

Celsior solution, Krebs-Henseleit solution, St. II Thomas solution, Stanford solution, Polysol solution, Custodiol® HTK solution, Hypothermasol® solution, IGL-1 solution, Lifor solution, Trans Send solution, ET-Kyoto solution, siRNA solution or similar.

3. A solution according to claim 1 where a basic storage solution or procedure is designed for treating organs, tissues, cells or bioengineered tissue prior and/or after cryopreservation and/or vitrification regardless of the indented use of (transplantation medicine, regenerative medicine, assisted reproductive medicine, bio-pharmaceutical research, forensic sciences, or other).

4. A solution according to claim 1 where BMP-7 has a an amino acid sequence with more than 90 % of homology to sequence SEQ No1 :

5. A solution according to claim 1 where BMP-7 has an amino acid sequence preferably with more than 95% of homology to sequence SEQ No1 :

6. A solution according to claim 1 where amino acid sequence of BMP-7 is more preferably 100% homologous to the sequence SEQ No1 :

7. The solution according to claim 1 , where BMP-7 is at concentration of about 1-50 pg/kg.

8. The solution according to claim 1 , where BMP-7 is at concentration of about 5-20 pg/kg.

9. The solution according to claim 1 , where BMP-7 is at concentration of about 10 pg/kg.

10. The solution according to claim 1 for use in virtually all adult or embryonic human or animal organs and/or tissue and/or cells and /or bioengineered tissue products perfusion, preservation or reperfusion.

11. A method of preserving an organ and/or tissue, comprising the steps of:

a) obtaining the organ and/or tissue and/or cells from a suitable source; and

b) maintaining the organ and/or tissue and/or cells in a solution according to claim 1