WO2008125884A2

WO2008125884A2 - Preservation composition

Info

Publication number: WO2008125884A2
Application number: PCT/GB2008/050262
Authority: WO
Inventors: Richard Robbins; Jeff Budd
Original assignee: Safemed Limited
Priority date: 2007-04-16
Filing date: 2008-04-16
Publication date: 2008-10-23
Also published as: GB0707275D0; WO2008125884A3

Abstract

The present invention provides a preservation composition which comprises a disinfectant, an alcohol and micro-organism free water. The compositionmay further comprise an alginate, polyethylene glycol,an essential oil and a secondary preservative. The disinfectant may be an iodine based disinfectant such as PVP iodine. The alcohol may be a short chain branched or un-branched C1 to C8 alcohol suchas isopropyl alcohol. The secondary preservative may be ascorbic acid, sodium nitrite, sodium sulphite, sodium iodate, sodium chloride and mixtures thereof. The present invention also provides a kit comprising containers together containing separately, or as mixtures, all the constituents of such a composition. The present invention further provides a method for the preservation of animal tissue or organs which comprises placing the tissue or organ in such compositions.

Description

Preservation Composition

The present invention relates to a composition and method for preserving tissues and organs.

Preservation and storage of tissues and organs is normally carried out by immersion in a preserving liquid which is an aldehyde, predominately formaldehyde. Formaldehyde confers excellent preservative properties on the tissue and is an effective disinfectant and anti-microbial agent. Formaldehyde acts as a chemical fixing agent reacting with the soluble albumins (proteins) in the cells and converting them to albuminoids, which are gels. This process results in the sterilisation of the tissue or organ which is then preserved. However the process results in a change in the appearance of the tissue or organ and also in a reduction in its size and change in shape due to the "pickling"' effect of the aldehyde. In addition this process can also result in the destruction of the entire DNA which reduces the use which can be made of the preserved tissue or organ. Although formaldehyde is very effective: in preserving tissues, it is nevertheless a very hazardous material and subjects the user to significant health risks. Studies have indicated that formaldehyde is a potential human carcinogen and airborne concentrations above 0.1 parts per million (ppm) can cause irritation of the eyes, nose and throat. The severity of the irritation increases as concentrations increase; at 100 ppm it is immediately dangerous to life and health. It has been linked to many different forms of cancer: e.g. nasal, lung, testicular and brain cancer as well as leukaemia. Within the last twenty-five years the use of formaldehyde in the workplace has become subject to regulation. The US Department of Labour,

Occupational Safety and Health Administration (OSHA) has placed increasingly stringent limits upon formaldehyde vapour exposure in the workplace. The permissible exposure limit (PEL) is 0.75 ppm measured as an

8 hour time weighted average; it also specifies a 2 ppm short term exposure limit - i.e. the maximum exposure allowed during a 15 minute period. Even with careful practice, users are often subject to high doses of formaldehyde.

In addition, the disposal of formaldehyde can create problems, as it should not enter sewerage systems or water courses. Its effect on soil and soil organisms is not known but formaldehyde has been shown to be toxic to fish and related species when introduced into water systems. Recommended disposal is via incineration.

US Patent No. 5,670,333 discloses formaldehyde-free tissue preservative compositions useful in the field of mortuary science and histology. Disclosed in this patent are compositions of an aqueous solution of ethanol, ethanedial, a long polymer and polar aprotic solvents as an arterial injection fluid for use in preserving animal bodies. Also disclosed is a formaldehyde-free composition of aqueous solutions of ethanedial, a polar aprotic solvent, a proteolytic enzyme, a surfactant, an anti-microbial agent and optionally a chelating agent as a pre-injection cleansing composition for the circulatory system in preparation for the administration of the inventive tissue preservative composition. In addition, this patent describes a formaldehyde- free body cavity fluid for use in the embalming process which comprises an aqueous solution of ethanol, an organic compound, a polar aprotic solvent ethanedial and Bisphenol A.

We have now devised a composition and a method for preserving human and animal tissue and organs which does not require the use of aldehydes.

According to the invention there is provided a preservation composition which comprises a disinfectant, an alcohol and micro-organism free water.

Preferably the composition also includes an alginate. The alginate may be present in the composition in the range of 0 - 50 g/l. Preferably the minimum amount of alginate is at least 2 g/l or 5 g/l, and the maximum amount is no more than 10 g/l or 5 g/l.

Alginates are normally seaweed based compounds and are preferably used in the form of a salt, e.g. sodium alginate. The alginates preferably should have an average chain length corresponding to a polymer molecular weight of from about: 10 to 200 kilodaltons and preferably from about 12 to 80 kilodaltons. Molecular weights and average chain lengths below these ranges yield a calcium alginate gel with a physical strength which is insufficient to provide the integrity required for implantation and long term exclusion of immune system agents.

The mannuronate to guluronate molar ratio of the alginate is preferably from about 1.2 to 6 and is preferably from about 1.5 to 2.5. Average molecular weight and overall mannuronate to guluronate molar ratios are initially determined substantially by material origin but can be adjusted somewhat by physical and chemical methods. Molecular weights can be - A - reduced, for example, by partial acid hydrolysis, thermal degradation or sonication. High molecular weights can be obtained by controlled precipitation methods with concomitant alteration of alginate composition or by dialysis, molecular filtration, or gel exclusion chromatography. The mannuronate to guluronate ratio can be increased or decreased by selective precipitation or solubilisation.

The preferred disinfectant is an iodine based disinfectant. Suitable iodine based disinfectants include polyvinylpyrrolidone-iodine (known as Povidone-lodine and/or PVP-lodine) which is preferred. Polyvinylpyrrolidone is a non-ionic, non-detergent water soluble organic polymer that is characterised by unusual complexing ability, by its colloidal properties and by its physiological inertness. Its iodine complex is a well known iodophor that is a highly effective germicide providing a broad spectrum of microbiocidal action against virtually all microbes. Iodine is the active disinfectant. The disinfectant may be present in the composition in the range of 1 - 120 g/l. Preferably the minimum amount of disinfectant is at least 5 g/l or 10 g/l, and the maximum amount is no more than 100 g/l or 25 g/l.

Preferably the composition further comprises polyethylene glycol, and more preferably polyethylene glycol 400. This is added to maintain the suppleness of the tissue/organ being preserved. The polyethylene glycol may be present in the composition in the range of 10 - 150 ml/I. Preferably the minimum amount of polyethylene glycol is at least 50 ml/I, and the maximum amount is no more than 120 ml/I.

Preferably the composition further comprises an essential oil or mixtures of essential oils. Suitable essential oils include rosemary oil and tea tree oil. The essential oil can act as a fragrance and as an additional preservative/disinfectant. Tea tree oil is oil from the leaves of the tea tree (Maleuca alterifolia), e.g. obtained by crushing the leaves and distilling. The active ingredient is terpinen-4-ol (typically 30-40%) which can be used in a synthetic form. Rosemary oil is oil from the Rosemary plant (Rosmarinus officinalis) that can be similarly obtained.

The alcohol is preferably a short chain Ci to Cs alcohol or polyol that may be branched or un-branched. The preferred alcohol is isopropyl alcohol.

The alcohol may be present in the composition in the range of 10 - 350 ml/I. Preferably the minimum amount of alcohol is at least 25 ml/I, 50 ml/l or 100 ml/I, and the maximum amount is no more than 300 ml/l, 120 ml/l or 100 ml/l.

In order to achieve micro-organism free water, preferably the water is distilled or deionised water and more preferably of pharmaceutical quality.

Optionally there is further provided a secondary preservative. The secondary preservative may be selected from ascorbic acid, sodium nitrite, sodium sulphite, sodium iodate, sodium chloride and mixtures thereof. Sodium iodate can assist in the prevention of autolysis.

Preferably the composition of the invention is formed by mixing the disinfectant, alcohol and, if present, one or more of the optional components in micro-organism free water.

Preferably the composition comprises:

- 0 to 50 g/l of alginate;

- 1 to 120 g/l of disinfectant;

- 10 to 350 ml/l of alcohol; - 10 to 150 ml/l of polyethylene glycol; - 0 to 5 ml/1 of essential oil; and

- 0 to 20 g/l of secondary preservative; with the balance being micro-organism free water. More preferably the composition comprises: - 0 to 5 g/l of alginate;

- 1 to 25 g/l of disinfectant; - 100 to 300 ml/l of alcohol;

- 50 to 120 ml/l of polyethylene glycol;

- 0 to 1 ml/l of essential oil; and - 0 to 10 g/l of secondary preservative; with the balance being micro-organism free water. In use the components, such as the alginate and disinfectant may be kept in separate containers and a mixture formed with water when it is to be used. Therefore according to the present invention there is also provided a kit comprising a plurality of containers together containing all the constituents of a composition as previously described. There can be from two containers to as many as there are components, although it is preferred that at least some of the components are already mixed.

According to the present invention there is also provided a method for the preservation of animal tissue or organs which method comprises forming a solution of an alcohol and a disinfectant in micro-organism free water, and placing the tissue or organ in the solution or in a gel formed by that solution.

Such a method could use a composition as previously described.

It is important that the alginate and disinfectant are free of micro- organisms; normally the disinfectant will kill any micro-organisms it contacts, but the alginate should be kept out of contact with air, e.g. in a sterile air-tight container.

When carrying out the method of the present invention, preferably a sterile air sealable container is used to store the tissue or organ; this can be made of glass or a sterile plastics material, e.g. made of a high density plastic which can be sterilised by irradiation, exposure to ultra violet light, steam sterilised etc. It can also be sterilised by the action of the composition of the present invention.

In use a container is typically filled to about 60% of its volume with a mixture of composition according to the present invention, and the tissue or organ placed in the liquid. Further composition is made up and poured over the tissue or organ to fill the container; the liquid composition penetrates all the free space in the organ and displaces the air; the composition then forms around the tissue or organ and the container sealed with an air tight lid. The tissue or organ can then be preserved for long periods in excess of 6 weeks.

Any suitable container may be used to hold the composition of the present invention and tissue. It may be in the form of a flexible or semi-rigid bag and may be shaped to facilitate stacking. The container should be made of an inert material unaffected by the composition or any body part to be stored in the container, e.g. a plastics material, which is flexible or semi rigid. Materials which can be used are polyolefins and polyvinyls such as polyvinyl chloride e.g. of thickness of 0.15 to 0.25mm. Depending on the nature and purpose of the container there is preferably an entrance in a surface of the container, e.g. on the top, preferably the entrance is closed by a means which can only be used once, e.g. a suitable single or double Zip (RTM). The size of the container depends on the size of the organ, tissue or body to be preserved. In the case of whole bodies, the container is suitably sized e.g. 6' 6" X 4' X 3'.

In use the composition, according to the invention, is made up, the container is partly filled and the composition allowed to at least partly gel and the body part or organ placed in the container and the container filled.

Normally there is mild agitation to completely cover/fill the organ or body part with the preserving composition. The container is filled to the top and sealed from the air. The organ or body part is preferably stored at a cool temperature, e.g. less than 5°C.

The length of time which the organ can be preserved has not been ascertained, but work has shown very little deterioration of a heart after considerable time.

If it is needed to have access to the tissue: or organ, the container can be opened and the tissue or organ removed. To preserve the tissue or organ again the process can be repeated.

Unlike previous methods, the DNA of the tissue or organ is virtually undamaged by the composition and this can be of great value in forensic or diagnostic applications. The invention can be used, with any animal tissue or organ and can be applied to human or any other mammals, birds etc.

It is a feature of the present invention that the composition is dermatologically inert, biodegradable and non carcinogenic. Example

Various solutions according to the present invention were made by adding to one litre of pharmaceutical quality distilled water, appropriate quantities of the other components to achieve concentrations as laid out in 5 Table 1 below.

Table 1

These components were mixed together to form solutions. The solutions were then introduced into containers in which (or subsequently to which) tissue samples were located. The solutions, except 12 and 14, formed 10 gels suspending the tissue and preserving it. Samples 12 and 14 remain liquid in nature, but were also effective. As discussed above it is also possible to partially fill a container with a solution prior to introduction of a tissue sample, so that the sample may be placed on a formed gel rather than rest at the bottom of the container.

Claims

1. A preservation composition which comprises a disinfectant, an alcohol and micro-organism free water.

2. A composition as claimed in claim 1 , which further comprises an alginate.

3. A composition as claimed in claim 1 or claim 2, which further comprises polyethylene glycol.

4. A composition as claimed in any of the preceding claims in which the disinfectant is an iodine based disinfectant.

5. A composition as claimed in claim4, wherein the iodine based disinfectant is PVP iodine.

6. A composition as claimed in any of the preceding claims which further comprises an essential oil.

7. A composition as claimed in claim 6, in which the essential oil is selected from rosemary oil and tea tree oil.

8. A composition as claimed in any of the preceding claims, in which the water is distilled water or deionised water.

9. A composition as claimed in any of the preceding claims, in which the alcohol is a short chain branched or un-branched Ci to Cs alcohol.

10. A composition as claimed in claim 9, in which the alcohol is isopropyl alcohol.

1 1 . A composition as claimed in any of the preceding claims, in which there is further provided a secondary preservative.

12. A composition as claimed in claim 1 1 , in which the secondary preservative is selected from ascorbic acid, sodium nitrite, sodium sulphite, sodium iodate, sodium chloride and mixtures thereof.

13. A composition as claimed in any of the preceding claims, which comprises: 0 to 50 g/l of alginate;

5 to 120 g/l of disinfectant;

10 to 350 ml/l of alcohol;

0 to 5 ml/l of essential oil;

10 to 150 ml/l of polyethylene glycol; 0 to 20 g/l of secondary preservative; and the balance being micro-organism free water.

14. A composition as claimed in any of the preceding claims, which comprises:

0 to 5 g/l of alginate; 1 to 25 g/l of disinfectant;

100 to 300 ml/l of alcohol; 0 to 1 ml/l of essential oil; 50 to 120 ml/l of polyethylene glycol; 0 to 10 g/l of secondary preservative; and the balance being micro-organism free water.

15. A kit comprising a plurality of containers together containing separately, or as mixtures, all the constituents of a composition as claimed in any of claims 1 to 14.

16. A method for the preservation of animal tissue or organs which method comprises forming a solution of an alcohol and a disinfectant in microorganism free water as claimed in any of claims 1 to 14, and placing the tissue or organ in the solution or in a gel formed by that solution.