KVD solution for transplantable organs

Details KVD solution for transplantable organs KVD solution for transplantable organs

2000-05-05

2003-04-08

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Differentiated tissue or organ other than blood, per se, or...

Including perfusion; composition therefor

Reexamination Certificate

active

06544726

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of preservation of biological materials for transplantation, and more particularly to compositions and methods for the resurrection and preservation of organs, tissues and cells from mammals.
2. Background and Description of the Related Art
Organ transplantation has become a relatively common procedure. Major solid organs that are routinely transplanted include the liver, kidney, heart, pancreas, lung and small bowel. The success of organ transplantation is due, in part, to the development of preservation solutions which allow for the shipment of cadaveric organs to transplant centers around the country. Cadavers are the main source of organs for transplantation. To optimally utilize all available organs it is necessary to be able to preserve viability while the organ is being shipped to the most suitable recipient. Suitability is generally based upon histocompatability matching of six-antigen matches between the donor and recipient.
When transplant organs are removed from the donor's body, the blood supply is interrupted. This action also interrupts the source of the organ's supply of oxygen, carbon dioxide, nitric oxide and nutrition, as well as the liquids that contain the necessary salts to create the correct osmotic pressure for a healthy osmotic environment for the tissue. Organ preservation methods are directed at minimizing the effects of interrupting the blood supply.
There are two main methods for clinical organ preservation. Simple cold storage is the most common and involves flushing the blood out of the organ and infusing it with a cold preservation solution. The second method is machine perfusion and involves continuous perfusion of the organ with a perfusate maintained at a temperature of 4° C. to 8° C. Perfusion is done at low pressure and usually with the pulsatile flow of about 0.6 to 10 ml/min/g of tissue. An advantage of perfusion is that end products from metabolism can be removed and that oxygen and other substrates can be delivered to the organ. Thus, energy requiring reactions that continue, even at hypothermia, can be supplied with a constant source of ATP derived from mitochondrial oxidative phosphorylation. Perfusion, therefore, provides longer preservation of organs than cold storage, and in general does a better job of preserving the quality of organs for short periods of time.
In 1967, Belzer et al. (“Belzer”) developed the method of continuous perfusion for preserving dog kidney for three days. The initial attempts at perfusion used plasma as the perfusate, but vascular injury resulted from the cold-induced precipitation of lipoproteins in the glomerular vessels. Belzer was able to overcome this limitation by first freezing the plasma and then filtering the precipitated lipoproteins. This led to the development of cryoprecipitated plasma (CPP) as the first successful perfusion fluid for hypothermic storage of kidneys. The solution worked well for a three day preservation of dog kidney but was unsuccessful for longer periods. Additionally, the CPP solution was unstable and needed to be prepared fresh for each use. Subsequently, a plasma based perfusion solution was developed that removed the lipid components by mixing silica gel with the plasma. The silica gel fraction of this plasma-based perfusion solution was as effective as CPP and was also shelf stable. Others attempted to develop perfusates based on saline mixed with human serum albumin as a colloid. All of the perfusates were about equally effective for kidney preservation and have been used clinically.
Belzer's interest in improving machine perfusion of kidneys to obtain longer term perfusion (5-7 days), led to the development of the composition commonly known as the University of Wisconsin Solution (Table 1). The original Wisconsin Organ Preservation Solution has allowed preservation of a variety of organs for transplantation including heart, liver, kidney and lungs, (Transplant Proceedings, 20-Supplement 1, p. 945, 1988, incorporated herein by reference as if set forth in its entirety herein).
TABLE 1
5%
hydroxyethyl starch having a molecular weight of from about
200,000 to about 300,000 and a degree of substitution of from
0.4 to 0.7.
25
mM KH
2
PO
4
3
mM glutathione
5
mM adenosine
A0
mM glucose
10
mM HEPES Buffer (Sigma Chemical Company)
5
mM magnesium gluconate
1.5
mM CaCl
2
105
mM sodium gluconate
200,000
units of penicillin
40
units insulin
16
mg Dexamethasone
12
mg Phenol Red
pH 7.4-7.5
This solution has found widespread clinical application for the preservation of the major intra-abdominal organs, and is the subject of three issued U.S. Patents (U.S. Pat. No. 4,798,824; U.S. Pat. No. 4,873,230; U.S. Pat. No. 4,879,283), all of which are incorporated herein by reference as if set forth in their entirety herein.
Even with the advent of improved techniques and organ preservation solutions, over the years, reperfusion injury (RI) still occurs. This injury is most commonly recognized as oxidative damage to the organ. For example, Starzl et al. reported substantial deterioration of liver after 20 hours (Transplantation, 51, pp. 1000-04, 1991). Yamaguchi et al. has observed that highly toxic oxidizing substances, such as peroxynitrite and hypochlorite, can be formed as part of RI (Hepatology, 29, pp. 777-84, 1999). Furthermore, Bahr et al. has reported that damage to the liver can cause net accumulation of extra cellular matrix and matrix degradation by a family of zinc-dependent metalloproteinases, including collagenases, gelatinases, stromolysins, MT-MMP's (Hepatology 29, pp. 839-45, 1999).
Modified forms of the University of Wisconsin Solution have been shown to have certain benefits for prolonged cardiac preservation. Nutt et al. compared the effects of twenty-four hour cold storage with perfusion preservation using a modified University of Wisconsin Solution. They found that perfusion with the modified solution provided function that was comparable of that of the cold storage control. (Circulation 1992; 86 [Suppl II]: II-333-II-338, herein incorporated by reference).
It was widely thought that one mechanism of RI was xanthine oxidase activation and its reaction with hypoxanthine, xanthine, or other substrates which can produce a superoxide anion from molecular oxygen. However, the toxicity of superoxide anion itself was never clear. It is known that superoxide anion reacting with nitric oxide can produce a highly toxic substance known as peroxynitrite anion. If the peroxynitrite anion is in the presence of a proton (H+), peroxynitrous acid is produced. Peroxynitrous acid can attack almost any biochemical entity. Peroxynitrite itself can cause DNA to be nicked or split resulting in a major insult to the cell and can eventually lead to apoptosis.
Churchill et al. have recently described chemical solutions that are suitable for flushing the blood from a donor organ prior to transplantation in U.S. Pat. No. 5,834,178 herein incorporated by reference in its entirety.
In addition to interrupting the blood supply in the organ prior to transplant into the recipient, cells of the donor are trapped in situ and provide challenges of their own. Regardless of how many loose cells can be washed from the organ prior to transplant with perfusion, it is not possible to clear all of the host cells, i.e., lymphocytes, monocytes, macrophages or neutrophils, of the donor from the organ to be transplanted. The host cells represent an antigenic challenge to the recipient in addition to the antigenic load the new organ itself represents. These cells that remain in the donor organ can be activated to produce substances toxic to the recipient.
A crucial mechanism which is activated during the transplantation of an organ is known as nuclear factor (Nf) kappa b, which is known to be manifested as a gene stimulation mechanism in the cells that remain in the donor organ. Once this mechanism is initiated, many genes of the immuno-inflammatory system that have a Nf kappa b gene control mech

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