AU2008201676A1 - Flush preservation solution - Google Patents

Description

17/04/2008 10:51 61-3-99901337 PATENT ATTORNEY SERV PAGE 04/54

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD

PATENT

ORIGINAL

Applicant(s): UNIVERSITY OF LEEDS Actual Inventor(s): David Potts and Jeremy P A Lodge Address for Service: PATENT ATTORNEY SERVICES 26 Ellingworth Parade Box Hill Victoria 3128 Australia Title: FLUSH PRESERVATION SOLUTION Divisional of Australian Patent Application No. 2002215131 The following statement is a full description of this invention, including the best method of performing it known to me/us:- 1 COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 05/54 00 0 0 TTFLUSTf P SEVATION SQ LUION ci The present invention relates to a flush preservation solution to keep cells 7O without a blood supply alive, the use thereof to prevent damage to cells in r---r transplantation, surgery, experimentally and in vitro, a method for O preservation, flush or flush preservation, a method for treatment and a kit of 00 parts comprising the solution.

N

I Organ transplantation is now available for kidney, liver, heart, lung, pancreas and intestine. At retrieval a transplant organ is flushed through its vasculature with a preservation solution. This solution is designed to facilitate the reduction of temperature of the organ, prevent cell swelling, remove oxygen free radicals, control pH, reduce ischacrnic damage, extend the safe time for which organs can be kept out of the body and facilitate recovery of the organ upon reperfusion.

Important flush solutions were introduced, by Belzer in 1967 and Collins in 1969, subsequently modified to Euro-Collins Marshall (1976), Bretschneider (see Isemer et al 1988), and others. University of Wisconsin solution the most successful of all solutions, was introduced in 1988 by Belzer and his colleagues. There remains a need for improved flush preservation. Recent evidence indicates that a high quality graft provides both better immediate function and a longer functional graft lifetime.

This area of the literature has been reviewed by: Belzer, 1993; Belzer Southard, 1988; Bonventre and Weinburg, 1992; Changani et al, 1999; Churchill Kneteman, 1998; Collins, 1997; Collins Wicomb, 1992; D'Allesandro et al, 1994; Muhlbacker et al, 1999 and Southard Belzer, 1995.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2l00 10:51 c 1- -o 9 -l 7 .PATE..NI ATTORNEY SERV PAGE 06/54 00 2 1 A simple flush solution containing only sodium phosphate and sucrose was shown by Andrews and Coffey in 1982, and by Coffey and Andrews in 1983 to protect the morphology of kidney tubules from ischaemic damage. We \D carried out functional tests and found a similar solution (PBS 140) to be highly 5 successful in the preservation of kidney tubules as judged by microperfusion (Pirie and Potts, 1986) when compared to established preservation solutions NC (EC and Collins C2 solutions). PBS140 was found to provide better 00 Spreservation of morphology and function of proximal tubules than other 0 solutions. PBS140 was also found to be effective in the preservation of whole kidney function by Ferwana, Pirie and Potts in 1988 and 1989. In experimental transplantation Lam, Mavor, Potts and Giles (1989) found PBS140 to provide highly effective preservation which compared favourably with Hyperosmolar Citrate and UW solution (Lodge, Perry, Sldnner, Potts and Giles 1991). PBS140 was tested clinically at Leeds and found to work well for human kidney transplantation (Abmad, Kashi, Helmy, Hadingham, Potts and Lodge, 1997).

With the advent of improved immunosuppression came the need for a high performance multi-organ flush solution.

Numerous workers have investigated the development of improved solutions, and in particular of simpler solutions incorporating a number of additives.

Efforts have been made to rationalise the results obtained with different formulations. Nevertheless it is apparent that apparently interchangeable additive types may in fact have different effects from those anticipated, this makes formulations extremely specific both in terms of a given type of additive, and in terms of other components present with a given additive since the function is dependent on the formulation as a whole and not simply on an individual constituent,- It is moreover apparent that a universal flush solution is a misleading objective, since different organs have quite widely differing requirements. Individual workers have however attempted to focus on COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 07/54 00 3 essential components which form the basis for flush solutions which may be adapted for specific rgans A number of reviews of the subject are of relevance, in particular "Evaluation of preservation of the intra abdominal O organs, F 0 Blzer, Transplantation Proceedings, vol 25, No 4 (August) 1993, P2527-2530, "Organ Preservation" D'Alessandro et al, Horizons in Organ Transplantation, Vol 74, No. 5, 1994 page 1083-1093, "A comparison of OO Flushing Solutions for Liver procurement using an isolated perfused Porcine 0 Model", Bell et al, Aust. N.Z.J.Surg. (1994) 64, 565-568, "New Organ preservation Solutions", Collins et al, Kidney Interational, vol 42, Suppl. 38 (1992) PS197-S202, "Improved Preservation Solutions for Organ Storage", Changani et al, Transplantation, vol. 68, 345-355, No. 3, 1999 and "Investigation of a Primary Requirement of Organ Preservation Solutions", Churchill et al, Transplantation, vol, 65, 551-559, No. 4, 1998. Although in part instructive, the individual work and the reviews present so many variables that any meaningful deductions cannot as such be drawn. Design concepts are proposed, such as designing a formulation around essential cellular constituents which are lost or broken down during preservation, and a contrasting concept of designing formulations around constituents which interact with the natural cellular functions and constituents to prevent breakdown, maintain certain functioning or arrest other functioning and the like. The result is that any effective flush solution is likely to be derived by diligent experiment, rather than by deduction or analysis from existing work.

Accordingly there remains a need for a commercially viable and effective flush solution which enables extended preservation of cells in particular of organs and living tissues, including engineered organs and tissues, which provides improved versatility, effectiveness and reperfusion in transplantation, in surgery, including any situation of warm or cold ischaemia, cardioplegia and open heart surgery, whole limb, whole body, or in experimentation.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 08/54 4 00 We have carried out experiments to develop an improved solution for liver C' transplantation. It emerged tlat sucrose-phosphate buffer solution provided good basic ~preservation. The incorporation of a number of additives produced a solution that provided better preservation than UW solution as judged by greater bile flow, bile acid exlraction, and reduced liver weight gain and release of liver enzymes. This approach provided a new flush solution for use on kidney and liver.

\O hThe heart has different requirements. The new solutions developed for kidney and liver o were not found to be suitable for heart. However, a basic sucrose-phosphate buffer 00 10 solution, with some modifications was found to be effective in the preservation of heart o tissue. These different requirements are almost certainly related to the muscular structure ¢C of the heart.

The above references to and descriptions of prior proposals or products are not intended to be, and are not to be construed as, statements or admissions of common general knowledge in the art in Australia.

According to a first aspect of the invention there is provided a flush preservation solution for the preservation of cells in the absence of a blood supply comprising: i) water for injection; and ii) at least one saccharide; and iii) at least one component with pH buffer properties; and iv) at least one component with calcium transport blocking properties or an anticalcium action activity; vi) together with a thromboxane inhibitor.

The flush solution may consist only of these components, in which case it is suited for preservation of universal cell types and functioning, in particular for preservation of simple cell systems, alternatively it may be provided together with one or more further substituents specifically suited to the preservation of a desired type or function of cell, in particular in the preservation of complex cell systems such as organs or living tissue, COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 18:51 61-3-99901337 PATENT ATTORNEY SERV PAGE 09/54 more particularly for small or lage animals, most particularly human organs and living tissue.

By means of the present invention we have surprisingly found that a highly effective universal basic solution may be supplemented to equal effect with a number of different constituents for different application. This has a number of advantages in terms of Simproving existing solutions, with reduced damage during preservation and the possibility to extend preservation periods, in addition to the provision of a formulation kit o from which to create a particular desired formulation, with the associated convenience 0 10 and cost implications which will render such formulation commercially viable.

N The prior art has attempted to teach the basis for a universal preservation solution, however, teaching is widely divergent, and even the identification of a combination of saccharide and pH buffer is not universally accepted. The present invention has found the is hereinbefore defined flush preservation solution to be universally acceptable, based on experiments and without attempting to rationalise the underlying preservation mechanism.

All components of the flush preservation solution of the invention satisfy National or International Pharmacopoeial Standards of purity where applicable. Water for injection is typically purified and de-ionized prior to sterilization.

A saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide is selected from sucrose, raffinose, mannitol, and is preferably sucrose.

A pH buffer is selected from a sodium phosphate buffer, a potassium phosphate buffer and the like, preferably NazHP0 4 NaH.PO4, KzHPO 4 KH, P0 4 and the like.

A calcium transport blocker or anti-calcium activity agent is selected from any known calcium transport or channel blocker such as nicardipine, diltiazem, verapamil, nisoldipine, chlorpromazine or trifluorperazine, preferably nicardipine and/or diltiazem.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 10/54 6 00 m by Without b g lmitd to ry reference is made hereinbelow to components by function based on commonly accepted physiological activity, however for the avoidance Sof doubt, components listed may contribute additional r different function to attibuted, and this should not be seen as a limitation thereof. Additionally functional equivalents t those listed may be considered within the scope of this invention.

Sreferably the flush preservation solution comprises one or more additional components selected from: 0 0i ly impermeable into ceDs, preferably is an v) at least one anion that is largely impermeable into c is 0 impermeant sequestering anion; vi) at least one component with colloid osmotic properties.

An impermeant sequestering anion preferably comprises lactobionate or lactobionic acid.

thromboxane inhibitor prevents blood clotting and preferbly comprises aspirin.

A colloidal osmotic preferably comprises polyethylene glycol (peg), succinylated gelatin (as in Gelofusine), Ficoll (a polysaccharide) or a starch product.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 11/54 0 0 <7 Alternativly or additionally, the flush presrvaton solution of the invntion may comprise one or more components selected from: N viii) inorganic or organic solutes; i) a component or components with calci chelating propertes; and i x) a component or compnents with iron chelating propertis.

00 o preferably an inorganic or organic solute comprises an inorganic solute and is an electrolyte including cations and/or anions, foI example selected from Na K CI-, 0 Mg" and the like Preferably a calcium chelator comprises citrate or EGTA and an iron chelater comprises

EDTA.

Alternatively or additionally the flush preservation solution of the invention may comprise one or more components selected from: xi) one or more amino acids xii) at least one component that is effective against oxygen free radicals or the production of oxygen free radicals; xiii) and at least one component of the energy supply system or which influences the energy supply system or a ketone body.

Preferably an amino acid is glutaimine, glycine or n-acetyleysteine.

Preferably oxygen free radical inhibitors are selected from allopurinol and reduced glutathione, more preferably a combination thereof.

Preferably an energy supply system component comprises adenosine.

Preferably a ketone body comprises beta-hydroxy butyrate.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 12/54 Alternatively or additionally the flush preservaton solution of the invention may comprise additional components for a specific function selected from: IO xiv) at least one component that acts reversibly upon crosabridge function in rmuscle, preferably butane-dione-rnonoxine; o xv) ,at least one component that influences the insertion and removal of 00 proteins into and from cell membranes, preferably Taxol; xvi) at least one component of the intracellular signal transduction system or which modifies this system, preferably a protein kinase inhibitor or a calmodulin inhibitor, xvii) at least one component that has a membrane stabilising action, preferably ranolazine, and the like.

The flush preservation solution of the invention is preferably formulated to comply with a desired range of the pharmacopoeially acceptable physical properties. Preferably the solution has a pH in the range 6.5-7.8, more preferably 6.5-7.0, most preferably 6.8-7.0.

Preferably the solution has osmolality in the range 300-450 mosmol/1, more preferably 350-400 mosmol/1.

In a further aspect of the invention there is provided a process for the preparation of a flush preservation solution as hereinbefore defined. The solutions are suitably made up by methods as known in the art by simple admixture under pharmacopoeially acceptable conditions. Preferably components are determined and incorporated in a desired molar concentration.

It will be appreciated that variation may be specific or non-specific to the effectiveness of the solution and that an amount of variation which has no COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 6 1 -3-98901337 PATENT ATTORNEY SERV PAGE 13/54 00 9 effet on the performance of the fluid is considered within the scop of this effect on the performance ofh ountd ofvrfcainb ouont of vorification by inentiOll. Selection of omaponent type, requiring an am routin expeimfefation, is considerd within t e scope of this invention.

aont in the range 50-150 Preferably a component (ii) is present in an amount i te rge mmol/l, for example approximate ly 100 mxnolll; each component (iii), 0 (viii) is present in a total amount in the range 15-75 mmoV for eample o approximatelY 15-20 or 40-70 mmcl/l; component (xi) is present in an amount in the range 15-30 mmol/l, for example approximately 20 mmol1b; component (xii) is present in a total amount in the range up to 5 mmoll, for example approximately 3-5 mxnoll; component xiii (ketone body component) is present in an amount in the range of up to 40mmoIll, for example component (vii) is present in an amount in the range 0.5-3.0 mmol/l, for example approximately 0.75 1.33 mmol/1I, such as 1.0 mmol, and 20,000 mw; component (vi) is present in an amount in the range 0.3-1.0 nmmrnol/l1, for example 0.5 mmol/1; component (iv) is present in an amount in the range 0.0005-0.-i mmol/l, for example approximately 0.0005-0.5 mmol1.

Other components will typically be present in minor amounts for example in the range up to 1 mmol/l.

In the case of certain components present as 2 or more types, the relative amounts may be critical or non-citical. Preference xii) is for approximately 3 mmcl/1 reduced glutathione and 0.35 to 0.4, more preferably 0.4 mmol/ 1 allopurinol.

Preference viii) is for electrolytes as follows: Na 50-150 mmoIl/1 K* 0-25 mmol/l CL- 0-100 rmnol/i COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 14/54 00 O OH-0-75mmol/1 Ca 2 0-2 mmol/1 O- Mg" 0 10mmol/1 Preferably a solution according to the invention is prepared and stored under anoxic condition in the absence of UV light. It is a particular advantage that the solution as Sdefined comprising components may be stored for extended periods, and Sadditional components added immediately prior to use.

00 10 The flush preservation solution of the invention as hereinbefore defined preferably Scomprises the basis components (iv) together with additional components for specific C" function. The solution for use in preserving organs is particularly of greater complexity than that for preserving simple cell systems, however we have found that the solution may nevertheless be relatively straightforward.

Preferably a flush preservation solution for liver, kidney and pancreas preservation comprises comombination of component classes viii), xii), iii), xi), ii), vi), iv), vii) as previously defined.

Preferably a flush preservation solution for intra abdominal organs such as intestine, bowel and the like comprises components (iv) as hereinbefore defined together with at least one component selected from (vi) and (vii), and more preferably additionally (viii), (xii) and (xiii).

Preferably an organ used to preserve muscular organs such as the heart comprises components (iv) as hereinbefore defined together with components selected from (viii), and more preferably additionally at least one component (xiv) as hereinbefore defined.

Preferably a flush preservation solution for liver, kidney and pancreas preservation comprises a combination of component classes given below, more preferably of the specific type listed, most preferably substantially in the amount listed: COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-99901337 PATENT ATTORNEY SERV PAGE 15/54 00 0 0 SCompoent Type Amount mmol/L v) Lactobionic acid I viii) K.OH viii) NaOH o xii) Glutathione 3 00 iii) NazHPO 4 26.45 o iii) NaH 2 PO4 16.66 C xi) Glutamine ii) Sucrose 100 vi) Aspirin xii) Allopurinol 0.4 iv) Nicardipine 0.005 vii) Peg (20,000 MW) 1 Preferably a flush preservation solution for heart preservation comprises a combination of component classes given below, nmore preferably of the specific type listed, most preferably substantially in the amount listed: Component Type Amount mmol/L: iii) Na 2

HPO

4 42.3 iii) NaHPO 4 .2H 2 0 10.67 iii) KH 2 P04 16 ii) Sucrose 100 viii) MgC 2 viii) CaC1 2 0.6 viii) NaC1 12 iv) Diltiazem We have moreover surprisingly found that the effectiveness of constituents of a flush preservation solution according to the invention is effected by the COMS ID No: ARCS-187164 Received by IPAustralia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 16/54 00 12 presnco of other constituents. Without being limited to this theory it seems that the effect of certain constituents is either negated or simply not transmitted to a target site, in the absence of constituents ensuring the maintenance of other cell functions. For example it is believed that the presence of an impermeant sequestering anion and the presence of a c colloid osmotic agent (vii) is more effective in cobination than alternative, o0 and the presece of both allopurinol and reduced glutathione as oxygen free Sradical quench (xii) is more effective than the individual compounds. We C, have found that this is particularly usefil in selecting specific solutions for preservation of specific cell, tissue or organ types, whereby certain combinations are useful for one type and may simply be substituted by those combinations suited for another type. As hereinbefore described, the substitution may be related to the presence of a substituent in the normally functioning cells or may interact with cell functioning in some manner. In a preferred embodiment of the invention we found that the impermeant sequestering anion together with the colloid osmotic agent (vii) are suited for preservation of intra abdominal organs, and are substituted by a muscle cross bridge function agent (xiv) in tailoring a fluid for use in preservation of muscular organs such as the heart.

Accordingly the finding according to the present invention is that certain substituents are essential for the preservation of the principle cell functions essential to all cell types and these have been identified as the component (iv) as hereinbefore defined. Whilst this formulation may be highly effective or satisfactory in preserving simple tissue or cellular systems, if it is desired to preserve organs or cell systems requiring or providing unusual or more complex cell function it is necessary to incorporate substituents specifically directed to preserve the requisite or provided function, whether this be muscular, electrical, specific membrane activity, energy supply and the like.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 17/54 13 00 0 In a further aspect of the invention there is provided a method for the preparation of a flush preservation solution comprising adding components under pharmacopoeially <1 acceptable conditions in sequence to water, with the exception of component vii) and 5 unstable components if any, and dissolving, adding component vii) if any and making the solution nearly up to volume and finally making up to volume to regulate pHI, sterilising IN) and cooling. The solution may be stored if desired with subsequent addition of any unstable components immediately prior to use.

10 In a further aspect of the invention there is provided the use of a flush preservation 00 Ssolution as hereinbefore defined as a flush solution, preservation solution or flush C' preservation solution for the preservation of cells in the absence of a blood supply, in particular to prevent damage to organs, living tissues and cells. The solution is suited for use with small or large animal or mammalian, in particular human organs, living tissues and cells.

The use of the solution may be in transplantation including organs from heart beating or non. heart beating donors, in surgery including any situation of warm or cold ischaemia, cardioplegia or open heart surgery, whole limb or whole body preservation, in experimentation on living tissues, in culturing and preserving engineered cells, tissues and organs and the like. Preferably the solution is used as a flush solution brought into contact with cells, living tissues, organs, limbs or the whole body via the vascular system, and optionally additionally serves as a preservation solution for storage of flushed cells, tissues and organs. In an embodiment of the invention a first solution. as hereinbefore defined is used to flush the cells, tissue, organ, limb or body and a second solution is used for the preservation thereof, or part thereof. For example the flush solution comprises components (iv) as hereinbefore defined and the preservation solution comprises additional components for continued functioning or prevention of damage to certain cellular or organ functions specific to the cell, tissue or organ in question.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 18/54 00 14 In a further aspect of the invention there is provided a method for flushing, preserving or flush preservation of cells, in. particular living cells, tissues or organs whereby the cells, tissue or organs are brought into contact with a s solution as hereinbefore defined. The method may be for simple hypothermic storage, whereby the cells, tissue or organ are flushed with solution, removed o from the normal locus, cooled preferably to temperatures normally in the range between zero and 4 C and stored. We have found that cells, tissues or 0 0 organs can be stored for extended periods exceeding those currently practised, C1 for example, heart has been stored for periods of the order of 6-12 hours, and kidney and liver for periods of the order of 48 hours or more. Additionally or alternatively the method is for the preservation of cells, particularly tissue or organs, whereby the cells, tissue or organs have been flushed and brought into a hypothennic state and are contacted with the preservation solution by immersion or perfusion.

Preferably the method of the invention comprises administering to the cells, tissue, organ or to a patient a biologically effective amount of the solution of the invention, at an effective rate or in an effective concentration to maintain or enhance function thereof. Preferably the method is a method for preserving certain cell, tissue or organ function, for example cell metabolism, and/or for temporarily arresting certain functions, for example muscular activity, breakdown or excretion of essential cell components and the like, and/or excretory products for example in the form of bile or urine and the like.

Ischaemia is the situation that results from the stopping of blood flow through an organ. The effects are due to lack of oxygen and nutrients, and accumulation of carbon dioxide and other waste products. It is more damaging at body temperature than in the cold which is why transplant organs are flushed and cooled. Organ donors have frequently suffered trauma and the donor organ may therefore have been subjected to a period of warm ischaemia as a result of the trauma. Adding a period of warm ischaemia experimentally COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 19/54 00 O prior to flush copies this situation. It is an advantage that our solution provides protection ,1 from such warm ischaemia.

Preferably flush perfusion is carried out at a pressure of up to 300 mmHg, more preferably in the range atmospheric to 200 mmHlg, more preferably in the range up to 160 mmHg, more preferably up to 100 mmHg, most preferably up to 50 mmHg.

\0S In a further aspect of the invention there is provided a kit of parts comprising a 'flush Spreservation solution having components (iv) as hereinbefore defined, together with 00 10 individual components selected from one or more of (xvii) as hereinbefore defined, O for use in the preparation of one or more flush solutions for specific purpose, and serving C] as a universal flush, preservation or flush preservation solution.

The invention is now illustrated in non-limiting manner with reference to the examples.

When used in this specification and claims, the terms "comprises" and "comprising" and variations thereof mean that the specified features, steps or integers are included. The terms are not to be interpreted to exclude the presence of other features, steps or components.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 20/54 lip: tfp. a lpn*nl.nc!U 00 O16

O

FIsh Areservadon Solutions Tsed in the Invention As Comparison 5 The preservation solutions involved in this study arc shown in the Tables 1 2 and 3, amounts are given in mmol/L. SOLS were made up from a flask half C' filled with water, to which any or all of lactobionate, KOH, sodium phosphate, 00 glutamine, sucrose, aspirin, nllopurinol and nicardipine were added in 0 sequence and dissolved. The colloid (PEG) was then added and the solution made nearly up to volume. NaOH was added to set the pH. The solution was then made up to volume. All solutions were sterilized by filtration and stored in glass bottles at 4°C and used within 3 days of preparation. Reduced glutathione was added during preparation or immediately before use.

Comparatiye UW is a standard original conmmercial solution (viaSpan/BELZER UW, DU PONT PHARMA). PBS140 (not shown) is composed of phosphate and sucrose (140mmol/L).

Chemicals Lactobionic acid (Sigma, L-2398), Allopurinol (Sigma. A-8003), Glutamine (Sigma, G-7029), Reduced Glutathione (Sigma, G-4251), Aspirin (Sigmam.

A-5376), Diltiazem (Sigma. D-2521), Taurocholic Acids (Sigma, T-4009), Polyethylene Glycol (Sigma, P-2263), Nicardipine (Sigma, N-7510), Adenosine (Sigma. A-9251), Glycine (Sigma, 007126), N-Acetyl-Cysteine (Sigma, A-8199), Albumin, Bovine (Sigma, A-7906), Alanine (Sigma, A- 5824), Glucose (BDH, 10117 Gelofusine Braun Medical Ltd., Lot: 8274D14 KOH (BDH, 102104V), NaOH (BDH, 102524X), NazHPO, COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 21/54 hnlui~ww\uBnlnQ*ra.nall 17 (BDH, 1.02494C), NaH 2

PO

4 21-120 (BDII, 310324Q), Sucrose (DI, 102745C), MgCl 6H20D (DH, 101494 V).

Tabe I Comnosition of Commercially Available Preservatigrn Solutions University of Wisconsin Solution Contents in mrnol/I

KHPO

4 KCI Adenosine Olutathione 3 MgS0 4 Raffinose A.11opurinol 0.01 K-Lactobionate 100 Pentastarch Osmolality 320 n.osmol/L PH 7.4 COMS ID No: ARCS-i87164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 22/54 tytpiEntnwto nUrw' Table 2 Composition of the cold reservation solutions of the invention used in the study MmoI/L SOL 4 SOL 5 SOL 6 SOL 7 SOL 8 SOL 12 SOL 13 50 Lactobionate 50 50 50 50 KOH 5 5 5 5 5 NaOH 40 40 40 5 5 55 Glutathione 3 3 3 3 3 3 3 Na 2

HPO

4 26.45 26.45 26.45 26.45 26.45 26.45 26.45 G]utarnine 20 20 20 20 20 20 NaH 2 FO4 2120 16.66 16.66 16.66 16.66 16.66 16.66 16.66 Sucrose 100 100 100 100 100 100 100 Aspiin 0.5549 0.5549 0.5549 0.5549 0.5549 0.$5P 05549 Allopurinol 0.3673 0.3673 0.3673 0.3673 0.3673 0.3673 0.3673 Diltiazm 0.0221 0.0221 0.0221 0.0221 0.0221 0.0221 Adenosine Gelofusine 100 300 100 300 (l/L) Nicardipine 0.005 Osmolality 300 320 350 280 300 300 300 pH 7.0 7.0 7.0 7.0 7.0 7.0 COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2002 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 23/54 00 Tabl 3 Cmnustiox of he cld peseratio johtions of the lvnin(ol) NAObint 50 50 50 50 5050 6.5 2.5 26.45 26.45 26.45 -26,45 126.45 26.45 42.3 Cl] Gmnc 20 20 262 20 20 10.67 00 NaHPR0 4 1666 16,6 16.66 1.6 1-6 1.6 1,6 66 06 CiScoe 100 100 100 100 100 10 10 10 100 Aspirin o-5549 0-5549 0.5549 0.5549 0.5549 0,5549 0,5549 Alnwnl 0.3673 0,3673 -0.3673 0.367 0.3673 0.36-73-60374 Diltiazer, 0.0221 0.0221 0,0221- 0.0221 0.0221 0-0221_ Nicardipina 0.005 0.005 AdWxoRTine Glycine N-Acetyl- MgCI 1 611,0 CaCE, 2}I,0 r 133 .0 ,4 Polyethylene 0.133 1.29 1.33 1.3 13 1.

Oamollt 300 30 220 340 380 290 380 380 PH E 7.0 7-0 170 7. 07.0 7.0 17.0 Molecular weight of polyethylene Glycol *8000 daltcnD #20000 dalton SQL 4, SOL 5, SQL 6, SOL 7, SQL 8, SOL 12, SOL 13, SOt 14, SQL SOL 16, SQL 17, SQL 18, SOL 19, SQL 20 were all based on SOL 4 but with different additives. The effects of individual additives were studied, and the more significant are mentionled as follows. Specific results are given below in respect of SQL When glutatbione was replaced with glycine and ri-acetylcysteine an advantage was seen in 24 hour cold rait liver preservation, as evidence by greater bile production than in SOL 4 group during the reperfusio-n. When nicardipine was added in place of diltiazern in SQL 4, the new solution was COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 24/54 00 2 0 found to be more effective than SOL 4 in 24 hour cold rat liver preservation, as indicated by greater bile production and less enzyme release than that for the SOL 4 group during the reperfusion In a comparison of different PEGs, I PEG 20K appears slightly more effective than PEG 8K, as evidenced by IN 5 numerically less AST and LDH release during the reperfusion. The o concentration of PEG 20K in SOL 17 was raised from 0.133mmol/L to 1.33 00 mmol/L to yield SOL 18 which was found to be more effective in 24 hour rat Sliver cold preservation than SOL 17, SOL 4 and UW solution, as indicated by C- greater bile production and less enzyme release and higher bile acid extraction.

The final solution SOL 20 was more effective than SOL 4 or UW solution in 24 hour and 48 hour rat liver cold preservation, as evidenced by greater bile production and less enzyme release and higher bile acid extraction. SOL has advantages for protection of the rat liver from ischemia damage.

LIVER PRESERVATION: MATERIALS AND METHODS 1. ISOLATED PERFUSED RAT LIVERS (IPRL) The method for this model described by Gores and colleagues (Gores ct al, 1986) was adopted and modified for this study. Inbred male Wistar rats weighing 230-280 gram were obtained from the Biomedical Service of the University of Leeds.

1.1 Donor Liver Harvesting The rat was anesthetized by intraperitoneal injection of pentobarbital (60mg/kg) without prior fasting. The abdomen was opened through a long midline incision. The liver was checked to be without tumor or any evidence of inflammation. The common bile duct was located, cut in its anterior wall and cannulated with a polyethylene catheter of 1 mm in diameter which was secured in place by a silk ligature, the bile duct was then divided distally. ml of normal saline solution containing 500 units of heparin was injected via the penile vein. The abdominal aorta was isolated and cannulated with a 2 mm COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 25/54 hUptt*ww.pfltflpl gl-r& 21 diameter polythylene catheter, through which the liver was flushed in situ with 30 ml of one of the ice-cold experimental preservation solutions. In the meantime, the thoracic cavity was opened, the thoracic aorta was clamped, and the inferior vana cava (IVC) was cut open to allow the perfusate to escape.

Ice-cold normal saline was put around the liver to cool it. Another 2 mm diameter polyethylene catheter was inserted into the portal vein and secured by a silk ligature, 15 ml of the same preservation solution was perfused through the liver via this catheter. The donor liver was then removed and stored at 4 OC for 24-48 hours in the same preservation solution. In the preflush warm ischaemia group, the rat heart was stopped before the livers were flushed in situ with the PBS140 or UW solution. In the pre-reperfusion warm ischaemia group, 30 minutes warm ischemia in a chamber of 37 'C was imposed just before the reperfusion.

1.2 Reperfusion of stored livers.

The basic solution for the isolated liver reperfusion in the present study was physiological saline (Table 4).

Table 4: The Composition of Physiological Saline.

Components mM/L NaCI 114.0 IKaHP04 31H20 MgC1 2 61-120 1.2 Na-Lactate (CHO 5 0 3 Na) NaFHC0 25.0 CaCla- 2H 2 0 L-Alanine Ostnolality (mOsm/L) 297 PH COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 26/54 l, it f :L EImph'W.atnlmtii Washed bovine red blood cells were added to increase the oxygen capacity.

2% of the bovine serum albumin (fraction 5, Sigma) was added to maintain the oncotic pressure. The composition of the perfusate is outlined in Table Table 5: The composition of the perfusate Composition volume Physiological Saline 160ml Fresh Bovine Red Blood'Cells Bovine Serum Albumin 5g in solution) Taurocholic Acid 60-80mg (about Fresh bovine blood was obtained from the abattoir. The blood was centrifuged for 10 minutes at 3000 rpm, the red blood cell were separated and washed with normal saline twice and then decanted into the final perfusate. The haernatocrit of the perfusate was around 30-35%. The pH was measured and titrated to 7.4 by the addition of 8.4% sodium bicarbonate, this usually needed 1.3 Apparatus.

The perfusion apparatus consisted of a perspex cabinet in which the temperature was maintained at 37 OC by two fan heaters, a roller pump (Watson-Marlow a membrane oxygenator (COBE*), two syringe pumps, a magnetic stirrer and a perfusion circuit connecting the liver (in a perspex container) via the portal vein eannulation at one end to a glass reservoir at the other. All components were inside the cabinet except for the pump. The roller pump drove the circulation, two syringe pumps were for the separate injection oftaurocholic acid and sodium bicarbonate (Fig la).

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 27/54 lvpm:'www.painflhIlI'R* ntn 00 0 23 1.4 Reperfusion The perfusion circuit including the oxygenator was filled in advance with the perfusate which had been oxygenated with a 95 O0/C0 2 mixture at a IN flow rate of 100-200 ml/min. Following cold storage, the livers were r- I\ 5 transferred to the liver container, placed in the perspex cabinet and the portal cannula connected to the inflow tubing. Extreme care was taken to ensure that cO no air bubbles entered the cannula, in order to avoid embolism to the liver.

00 0 Both the circulation pump and the syringe pump for the taurocholic acid were C1 started simultaneously. Taurocholate (sodium salt, Sigma) was pumped into the perfusion solution at a rate of 60-80 tnol/br. 8.4% of sodium bicarbonate solution was added into the reservoir as required to maintain the pH of perftisate to about which was measured by blood gas analyser (CIBA- CORNING). The reperfusion flow rate was 15 m/min. Bile volume was recorded in consecutive 15 minute periods. Samples were obtained from inflow (portal vein), and outflow (hepatic vein) at each 30-minute intervals for the measurement of pH, pO2 and pCO 2 and the samples from the hepatic vein were also used for the determination of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotran.sferase (ALT). At the end of reperfision, samples fiom the perfusate were taken for the m easurement of taurocholic acid concentration to calculate the bile acid extraction percentage. The bile was collected, recorded and analysed for its concentration of bile acids at the end of the 2-hour of reperfusion. The bile volume was expressed as 1.L (g liver)"'. (15 min.)' or l1. (g liver)'. (2hr)'.

Liver weights were recorded before and after storage to yield the net liver weight and the liver weight change. The liver weight was also recorded at the end of the reperfusion period to calculate the change in weight.

Presentation of Results In order to facilitate both presentation of statistical analysis and also visualization of results observations are presented in both table and graphical form in most instances.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 28/54 00 0 0 S24 1.6. Statistical Analysis Data are expressed as mean± SEM throughout. In each experimental group, the animal number is 6 unless otherwise stated. The statistical tests used in this thesis are Students t'test or onc way analysis of variance with Dunnets Stest for multiple comparison with a control, or Tukey test for multiple C'0 comparisons. Significance was assumed at P< 0.05.

00 0.1 SOL 20 was developed from results obtained with SOL4-19. The results compare the effect of the new solution with solution 4 and UW solution at 24 hours cold rat liver preservation.

1.5.1 Bile Flow.

In these three groups, the bile production in SOL 20 was the best seen in this model. Bile flow commenced within 5 minutes of the livers being placed in the circuit and continued throughout the whole 2 hours of the reperfusion period. The appearance of the bile in each group was excellent: the colour was deeply yellow, and very clear. The bile flow rates per gram of liver for the 2 hours were: 93.4±5.3, 103.4±6.7 and 198.3±18.0 1l for the SOL 4, UW and SOL 20 groups respectively. The best result was with SOL 20, with significant differences compared either with SOL 4 or with LTW groups (Fig 80). The results of bile flow rates [l1. (g liver)"'. (15min)"] in consecutive 15 minute periods during the rcpcrffision for the SOL 4, UW and SOL 20 groups are displayed in Table 6 and cumulative results in Figure 2.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 29/54 http*~JJwpatA#t:ArcJM Table 6: Bile flow rates [pl. (g liver)"'. (5lmin)f] in consecutive minute periods during the reperfusion for the SOL 4, UW and SOL groups following 24-hour cold preservation.

Timo (min.) 0-15 15-30 30-45 45-60 60-75 75-90 90-105 105-120 SOL 4 5.72±1.3 10.14±1.6S10.72±0.98*14.45±11.3* 13.091.0*14,45±1,J 1 J.38±0.871 1.09±0.62* UW 4.9910.99 972±1.3K14.01±2.2* 19.01±1.7* 16.90±1.2*16.96+1.4*11.92±0.63* 9.78t1.1* SOL 20 9.94±23 1942±3A 24.10±4.4 35.15±3.3 2833±3.8 30.7641.9 26.84*2.4 23.782.3 P<0.05 (vs SQL 1.5.2. Enzyme release.

1.5.2.1 ALT release: During the reperfusion period, livers stored in SOL group for 24-hour released significantly Icss ALT than that for the UW or SOL 4 groups (Table otherwise the ALT released from the livers stored in the UW were no difference compared from that in SOL 4.

Table 7: ALT release during the reperfuson for the SOL 4, UW and SOL 20 groups following 24-hour cold preservation.

Time (min.) Rinse 30 60 90 120 SOL4 64.& UW 74.5± SOL 20 38.6± P<0.05 (vs SOL t6.8 30.3±4.3* 9.8" 24.8-*2.9 2.9 16.22±1.1 32,0±2.9* 44.9±3.2* 80.7-8.1* 37.6±3.7 50.3±3.5" 73.0±6.7* 20.6±1.2 28.6±2.2 35.411.7 1.5.2.2 AST release: The AST released from the livers stored in the SOL 20 were less than that either in the UW or SOL 4, with significant differences at the Rinse and the 30, 120 minute points compared with the UW group and at the 30, 90, 120 minute points compared with the SOL 4 group during the reperfasion (Table There were no differences between the SOL 4 and UNW groups.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 30/54 .l r4 I. rtt~ p m iT*' dl0 11 1 Table 8: AST release during the reperfusion for the SOL 4, UW and SOL 20 groups following 24-hour cold preservation.

Time (min.) Rinse 30 60 90 120 SOL 4 38.7±6.8 25.2±3.0* 32.9±6.2 49.0±8.0* 86.6±14* UW 55.3±6.4* 23.812.0* 33.6±1.8 46.2±2.7 69,2t3.6* SOL 20 27.911.3 13.3±0.92 21.6±1.2 26.6±1.4 30.2±1.5 P<0.05 (vs SOL 1.5.2.3 LDH release: The livers stored in the SOL 20 group for 24-hour released significantly loss LDH than that either in the UW or SOL 4 groups (Table Compared with the UW group, whilst the livers stored in the SOL 4 also released less LDH, the difference was not statistically significant.

Table 9: LDH release during the reperfusion for the SOL 4, UW and SOL 20 groups following 24-hour cold preservation Time (min.) Rinse 30 60 90 120 SOL 4 544±36* 204+20* 241±31* 317±57* 461±52* UW 565±56* 183±18* 216*18* 295:24* 390±21* SOL 20 358±12 92±18 106±16 130±16 172±25 P<0.05 (vs SOL 1.5.3. The bile acid concentrations in bile during the reperfusion for the SOL 4, UW and SOL 20 groups were 6700±417, 6725±371 and 6529±317 )gmol/L respectively, there was no significant differences between these groups (Fig The bile acid extraction during the reperfusion for the SOL 20 group was higher than that for the SOL 4 or UW groups (Fig Compared with the COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 31/54 00 0 27 SUW group, the bile acid extraction for the SOL 4 group was not significantly l[different.

\D 1.5.4. The weight of all the livers stored in SOL 4, UW or SOL 20 decreased rafter 24-hour cold preservation. There was a significant difference between o SOL 4 and UW or SOL 20 groups (Table 10). The liver weights for the SOL N 20 group was also decreased after the 2 hours of reperfusion, in the other two 00 O groups, the liver weights were increased after the reperfusion, there were significant differences between SOL 20 and SOL 4 or UW.

Table 10: Liver weights change after 24-hour cold preservation and after 2-hour reperfusion for the SOL 4, UW and SOL 20 groups.

After Preservation After Reperfusion SOL 4 -0-78:0.82 3.97±1.0" UW -2.39±0.62** 5.27±1-1 SOL 20 -2.13±0.72** -0.18±1.8 P<0.05 (vs SOL 20); P<0.05 (vs SOL 4) 1.5.5. The oxygen consumptions of the livers stored in SOL 4, UW and SOL during the reperfusion period were: 0.360±0.007, 0.417±0.016 and 0,646±0.04 pmol. (g liver)", (min)' respectively, there were significant differences for the SOL 20 group compared with the SOL 4 or UW groups, also significant difference between the UW and SOL 4 groups (Fig A completely independent fra-ther set of observations was made on PBSH, the results are shown in Figs 6-10 inclusive. These results confirm that PBSL causes less swelling of the liver than UW (Fig Bile flow after preservation in PBSL was approaching twice that following preservation in UW (Fig 7).

AST release was lower with PBSL (Fig 8) as were ALT (Fig 9) and LDH (Fig COMS ID No: ARCS-187164 Received byIP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 32/54 00 0 0 28 All of these results were highly significant confirming the advantages of PBSL over UW preservation solution.

S2. Heart Preservation: materials and methods.

I\ o Experiments were carried out under a Home Office Licence in compliance Swith Home Office guidelines for animal care.

0 Cl 2.1 Perfusion apparatus.

An isolated working rat heart model was used as described by Nccly JR This apparatus consisted of the following parts: 1) Langendorff reservoir with water jacket and glass filter at the bottom outlet.

2) Heart Chamber and cannula assembly. A Teflon bung held two stainless steel cannulas grooved to accommodate ligatures. One cannula was straight to fit the aorta and was fitted with four side arm connectors for Langendorf perfusion, cardioplegia perfusion, pressure and heart rate measurement, and for aortic outflow measurement. The other cannula was angular to facilitate cannulation of the left atrium. The perfused heart, supported by the two cannulae fitted loosely into a water jacketed heart chamber.

3. Aortic and atrial bubble traps and compliance chamber.

4. Oxygenation chamber consisting of three water jacketed condenser units with taper connections which provided a large surface area to facilitate oxygenation.

5. Cardioplegia reservoir, situated 60cm above the heart and through which preservation solution was infused into the heart COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 33/54 hti/IWWW, teintlelenp.n a 00 0 0 29 6. Peristaltic pump and filter used to recycle perfision solution through the oxygenation chamber.

\o 7. Pressure gauge and pen recorder to record heart rate and aortic pressure.

IO

Cl A simplified diagram of the heart reperfusion apparatus is shown in Fig lb.

00 0 2.2 Perfusion medium.

The perfusion solution was modified Krebs-henseleit buffer (KHB) equilibriated with 95% oxygen and 5% CO 2 at 37 0 C. The final concentration of salt of this buffer (mmol/1) was NaC1 118, ICCI 4.7, MgSO 4 1.2, CaCl 2

KH

2 P0 4 1.2 and NaHICO 3 2.3 Study Protocol.

Male Wistar rats (250-320g) were anaesthetised with intraperitoneal injection of sodium pentobarbitone (0.26-0.28ml). Heparin (250IU) was injected intravenously immediately before surgery. The abdomen was opened to facilitate exposure to the chest, thoracotomy was performed and the heart excised and immediately placed in ice cold saline. Cannulation of the aorta was then carried out and the heart attached to the perfusion apparatus followed by perfusion of KIB solution at 37"C for an initial wash out and equilibration period during which the heart regained the beating state. During this period the left atrium was cannulated in order to allow the heart to be switched to a pumping mode. In this configuration KHB drains to the left ventricle which in turn pumped the solution to the aorta, from which it passed to the compliance chamber. This chamber was partly filled with air to provide some elasticity to the otherwise rigid system. Pressure development in this chamber forced the COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 34/54 httJf i. pi*teiItlUn.nTWV t 00 3 e, solution through a flow meter to a bubble trap situated 100cm above the heart from which it returned via the oxygenation chamber to be recycled.

k\ Control measurements of cardiac functions included heart rate systolic D 5 pressure aortic flow coronary flow (CF) and cardiac output (CO) every five minutes for a duration of 15 minutes. Hearts that achieved aortic Ci flow of less than 45 ml/min, coronary flow of less than 16ml/min or heart rate 00 0 of less than 240 beats/min were excluded from the study. The hearts were then 0. flushed with one of the four preservation solutions (25ml) from the cardioplegia reservoir situated above the heart, and stored for 6 hours at 4 0

C.

The solution tested were St Thomas' Hospital Solution no 2 (STH2), University of Wisconsin Solution, Celsior Solution (CS) and Phosphate Buffered Sucrose Solution (PBS-I). The minimum no of hearts included in each group was 6.

After storage for the desired period, reperfusion in a Langendorff mode for a min equilibration period was carried out, followed by conversion to a working heart mode by stopping the Langendorff pe-fusion and commencing perfusion through the left atrium. The working heart mode lasts for 30 min during which measurement of the cardiac function was made every 5 minutes for comparison to their individual pre-ischacmic control values. Recovery of cardiac functions was expressed as a percentage of the preischaemic control values.

In summary the protocol was as follows:- Langendorff mode (2-3 min) Working mode (15 min) Cardioplegia (25ml of flush solution).

Preservation. (6 hour) Langendorffreperfusion (15 min) COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 35/54 I. :r t4 nn:~m~irwMlimllnn Working reperfusion (30 mmin) Statistical analysis.

Results in each group were expressed as the meant SEM. Means of groups were compared using one-way analysis of variance (ANOVA). Statistical significance was accepted at a P value of less than 0.05.

2.4 Results.

Table 11. Results (mnean±SEM) for the four experimental groups are shown in the table, expressed as the recovery of pre-ischaemic function for each of the haemodynamic parameters shown.

COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51_ 61-3-98901337 PATENT ATTORNEY SERV PAGE 36/54 00 0 S32

REFERENCES

Andrews PM and Coffey AIC (1982). Laboratory Investigation 46, 100-120.

0 Belzer FO, Ashby BS and Dunphy JE (1967). Lancet 2, 536-539.

Belzer FO and Southard JH (1988). Transplantation 45, 673-676.

Coffey AK and Andrews PM (1983). Transplantation 35, 136-143.

o Collins GM, Bravo-Shugarman M and. Trasald PI (1969). Lancet 2, 1219-1222.

00 Ferwana O, Pirie SC and Potts DJ (1988). Clinical Science 74,261-268.

SFerwana O, Piric SC and Potts DJ (1989). Clinical Science 77, 77-84 10 Isemer FE, Ludwig A, Schuck O, Bretschneider HJ and Peiper HJ (1988) Transplantation Proceedings 20, 885-886.

Lam FT, Mavor A, Potts DJ and Giles G (1989). Transplantation 47, 767-771.

Ross I, Marshall VC and Escott MO (1976). Transplantation 21,498-501.

Pirie SC and Potts DJ (1986). Clinical Science 70, 443-452 COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17

Claims (12)

1. Flush preservation solution for the preservation of cells in the absence of a blood supply comprising: N i) water for injection; and D ii at least one saccharide; and Siii) at least one component with pH buffer properties; and Cl 00 iv) at least one component with calcium transport blocking properties or an 0 Santi-calcium action activity together with vi) a thromboxane inhibitor.

2. Flush preservation solution as claimed in Claim I wherein a thromboxane inhibitor comprises aspirin.

3. Flush preservation solution as claimed in any of Claims I and 2 which comprises one or more additional components selected from: v) at least one impermeant sequestering anion; and vii) at least one component with colloid osmotic properties.

4. Flush preservation solution as claimed in any of Claims 1 to 3 wherein a saccharide is selected from sucrose, raffinose and mannitol. Flush preservation solution as claimed in any of Claims 1 to 4 wherein a pH buffer is selected from a sodium phosphate buffer and a potassium phosphate buffer and mixtures thereof.

6. Flush preservation solution as claimed in any of Claims 1 to 5 wherein a calcium transport blocker is selected from nicardipine, diltiazem, verapamil, nisoldipine, chlorpromazine and trifluorperazine. COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 38/54 00 3.4 O O

7. Flush preservation solution as claimed in any of Claims 1 to 5 wherein a component with an anti-calcium action activity, comprises a calcium chclator, preferably citrate. S. Flush preservation solution as claimed in any of Claims 3 to 7 wherein ,D an impermeant sequestering anion comprises lactobionate or lactobionic acid. 00 O 9. Flush preservation solution as claimed in any of Claims 3 to 8 wherein a colloidal osmotic comprises polyethylene glycol (peg), succinylated gelatin, a polysaccharide such as Ficoll or a starch product. Flush preservation solution as claimed in any of Claims I to 9 which additionally comprises one or more components selected from: viii) inorganic or organic solutes; ix) a component or components with calcium chelating properties; and x) a component or components with iron chelating properties.

11. Flush preservation solution as claimed in Claim 10 wherein an inorganic solute is an electrolyte including cations and/or anions, selected from Na, Cl', OH', Ca 2 and Mg'.

12. Flush preservation solution as claimed in any of Claims I to 11 which additionally comprises one or more components selected from: xi) one or more amino acids xii) at least one component that is effective against oxygen free radicals or the production of oxygen free radicals; xiii) and at least one component of the energy supply system or which influences the energy supply system or a ketone body. COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 39/54 00 O p

13. Flush preservation solution as claimed in Claim 12 wherein an amino acid is selected from glutamine, glycine and n-acetylcysteine.

14. Flush preservation solution as claimed in Claim 13 or 14 wherein an oxygen free radical inhibitor is selected from allopurinol and reduced glutathione and combinations thereof. 0 o

15. Flush preservation solution as claimed in any of Claims 1 to 14 which 0 comprises additional components selected from: xiv) at least one component that acts reversibly upon crossbridge function in muscle, preferably butane-dione-monoxime; xv) at least one component that influences the insertion and femoval of proteins into and from cell membranes, preferable Taxol; xvi) at least one protein kinase inhibitor or a calmodulin inhibitor; xvii) at least one component that has a membrane stabilising action, preferably ranolazinc. .16. Flush preservation solution as claimed in any of Claims I to 15 which has a pH in the range 6.8-7.0 and/or which has osmolality in the range

300-450 mosmol/1, preferably 350-400 mosmol/L. 17, Flush preservation solution as claimed in any of Claims 1 to 16 wherein component (ii) is present in an amount in the range 50-150 rinol/L each component (iii), (viii) is present in a total amount in the range 15-75 mmol/1; component (xi) is present in an amount in the range 15-30 mmol/I; component (xii) is present in a total amount in the range up to mmol/l; component (vii) is present in an amount in the range 0.5-3.0 mmal/1; component (vi) is present in an amount in the range 0.3-1.0 mmol/l; component (iv) is present in an amount in the range 0.0005-0.1 COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 40/54 00 36 O O C mmol/l; and other components are present in minor amounts in the range OD up to 1 mmol/1. 18. Flush preservation solution for liver, kidney and pancreas preservation which, comprises a combination of component classes given below sO Component: I\ viii), xii), iii), xi), ii), vi), iv), vii) as defined in any of Claims 1, 3, O& 12. O 00 S19. Flush preservation solution for liver, kidney and pancreas preservation which comprises a combination of component classes given below of the specific type listed Component Type v) Lactobionic acid viii) KOH viii) NaOH xii) Glutathione iii) Na 2 HP04 iii) NaH2P04 xi) Glutamine ii) Sucrose vi) Aspirin xii) Allopurinol iv) Nicardipine vii) Peg (20,000 MW) Flush preservation solution for liver, kidney and pancreas preservation which comprises a combination of component classes given below of the specific type listed and substantially in the amount listed when made up to volume in water COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 41/54 00 37 O O CI Component Type -Amount mmol/L v) Lactobionic acid viii) KOH viii) NaOH xii) Glutathione 3 iii) Na 2 HP04 26.45 iii) NaH 2 PO 4 16.66 0 xi) Glutanine o ii) Sucrose 100 0 vi) Aspirin xii) AIlopurino! 0.4 iv) Nicardipine 0.005 vii) Peg (20,000 MW) 1 21. Method for the preparation of a flush preservation solution as claimed in any of Claims 1 to 20 comprising adding components under pharmacoeially acceptable conditions in sequence to water, with the exception of component vii) if present and unstable components if any, and dissolving, adding component vii) and any unstable components and making the solution nearly up to volume and finally making up to volume to regulate pH, sterilising and cooling. 22. A flush preservation solution as claimed in any of Claims 1 to 20 for use as a flush solution, preservation solution or flush preservation solution for the preservation of cells in the absence of a blood supply, in particular to prevent damage to organs, living tissues and cells, 23. Flush preservation solution for use as claimed in Claim 22 in transplantation including organs from heart beating or non heart beating donors, in surgery including any situation of warm or cold ischaemia, COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 42/54 00 38 O cardioplegia or open heart surgery, whole limb or whole body preservation, in experimentation on living tissues or in culturing and Spreserving engineered cells, tissues and organs, limbs or the whole body. r- 24. Flush preservation solution for use as claimed in Claims 22 or 23 which is brought into contact with cells, living tissues, organs, limbs or the Swhole body via the vascular system, and optionally additionally serves 00 as a preservation solution for storage of flushed cells, tissues and organs. 0 Flush preservation solution for use as claimed in any of Claims 22 to 24 wherein a first solution as hereinbcfore defined is used to flush the cells, tissue, organ, limb or body and a second -solution is used for the preservation thereof or part thereof 26. Flush preservation solution for use in flushing, preserving or flush preservation of cells, in particular living cells, tissues or organs whereby the cells, tissue or organs are brought into contact with the solution as claimed, in any of Claims 1 to 2 for simple bypothermic storage, whereby the cells, tissue or organ are flushed with solution, removed from the normal locus, cooled preferably to temperatures normally in the range between zero and 4°C and stored. 27, Flush preservation solution for use as claimed in Claim 2 wherein the solution is for the preservation of cells, particularly tissue or organs, whereby the cells, tissue or organs have been flushed and brought into a hypothermic state and are contacted with the preservation solution by immersion or perfusion. COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17 17/04/2008 10:51 61-3-98901337 PATENT ATTORNEY SERV PAGE 43/54 00 39 O 0C 28. Flush preservation solution for use as claimed in any of Claims 2Qn. e 27 which comprises administering to the cells, tissue, organ or to a patient a biologically effective amount of the solution of any of Claims 1 i_ to 20, at an effective rate or in an effective concentration to maintain or enhance function thereof, wherein the method is a method for preserving ID certain cell, tissue or organ function, and/or for temporarily arresting ID certain fiuntions. 00 29. Flush preservation solution for use in flushing, preserving or flush Spreservation of cells, wherein the cells are flushed and brought into a. hypothermic state and contacted with a preservation solution as hereinbefore defined in any of Claims 1 to 20 by immersion or perfusion. Use as claimed in any of Claims 22 to 29 wherein flush perfusion is carried out at a pressure of up to 300 mmHg. 31. Use as claimed in any of Claims 22 to 29 wherein flush perfusion is carried out at a pressure of up to 200 mmHg, 32. Use as claimed in any of Claims 22 to 29 wherein flush perfusion is carried out at a pressure of up to 100 mmHg. 33. Kit of parts comprising a flush preservation solution as claimed in any of Claims I to 20 having components (iii) and together with individual components as hereinbefore defined in any of Claims I to for use in the preparation of one or more flush solutions for specific purpose, and serving as a universal flush, preservation or flush preservation solution. COMS ID No: ARCS-187164 Received by IP Australia: Time 10:58 Date 2008-04-17