JPH0733601A - Composition for tissue preservation - Google Patents

Abstract

PURPOSE: To obtain a composition capable of providing an energy source for an isolated tissue at the time of surgery and simultaneously suppressing the production and accumulation of lactic acid. CONSTITUTION: This composition comprises a ketonic substance and/or its precursor, glucose and a phosphate buffered balanced salt aqueous solution. The composition is especially useful as a rich energy source for preservation of an isolated tissue and a peripheral tissue at the time of surgery and simultaneously capable of suppressing the intracellular production and accumulation of lactic acid. The composition can usually be utilized for ophthalmic or usual surgery; however, other methods for utilization (e.g. local administration or preservation and washing of a donor tissue before transplantation) can be carried out.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION

1. TECHNICAL FIELD The present invention relates to a novel composition which is particularly useful as a tissue-rich energy source during the preservation of isolated tissues and during surgery, and at the same time suppresses the production and accumulation of intracellular lactate. It can be used for eye surgery and surgery in general, but for other uses (eg, local administration and preservation and washing of donor tissue prior to transplantation)
Is also possible.

2. Background information Generally, irrigating solutions
n) is used for topical administration to the outer surface of the eye and to the skin, as well as for cleaning tissue during surgery and for maintaining the wet tissue of surgery. However, in eye surgery, replacement of aqueous humor and / or vitreous with perfusate occurs as a result of surgery (corneal transplantation (penetrating corneoplasty), cataract extraction, intraocular lens implantation and vitrectomy) . In these cases, the perfusate solution remains in the eye until the components of the perfusate are absorbed by the surrounding tissue or the solution eventually reaches parallel with body fluids and is cleared into the blood circulation. Therefore, the perfusate used is physiological (tonicity, pH, etc.)
Is not only compatible with at least the anterior chamber (an
terrior chamber and vitreous body (vitre)
It is also necessary to include components that allow the cells to maintain viability and the ability to perform physiological functions until they reach a sufficient equilibrium with physiological fluids.

In eye surgery, the components of the perfusate are of particular importance to the cornea and lens. Neither tissue has blood vessels. The cornea derives its nutrition mainly from the anterior chamber of the eye, and to a lesser extent from tears and limbal vessels. The lens obtains its nutrition from the anterior chamber and vitreous humor. The retina, ciliary body, and iris are vascularized tissues; they derive their nutrition from circulating plasma in the vascular network. Thus, the components of the perfusion solution do not have as significant an effect on these tissues as on the cornea and lens.

In the cornea, a monolayer of endothelium that covers the posterior surface contains liquid and ion transport sites that help maintain the cleanability and thus transparency of the cornea. In the lens, the ion transport site is located in the epidermis under the capsule. Generally, Na + -K + ATPase is present in the cytoplasmic membrane and has intracellular Na + and K + contents of 10 mM and 100, respectively.
Helps maintain at mM, about 120 mM Na +
And promotes transport against a 10 mM K + extracellular salt gradient. Sufficient energy is required for cells (and tissues) to perform this liquid and / or ion transport activity.

Glucose is a major energy source in mammals. The cornea, lens, and retina are highly active glycolytic tissues that utilize glucose to produce lactic acid even under aerobic conditions. When the isolated cornea is stored in a medium containing glucose, excess lactic acid is produced and accumulated and becomes acidic, and the metabolic activity of the tissue is inhibited. In vivo, lactic acid produced in vascular tissues is removed via the blood circulation, and lactic acid produced in non-vascular tissues is released into the anterior chamber of the eye and removed by the clearance mechanism of blood circulation. In humans, anterior chamber lactate is maintained at a constant low level of approximately 4.0-4.5 mM.

Glucose is an important and useful energy source for tissues when present in solution for in vivo administration.
The utilization of glucose by the glycolytic system produces ATP at a high rate, which is independent of O ₂ . However, this is not an efficient energy production pathway, only 2 moles of ATP are produced when 1 mole of glucose is utilized. At this time, 2 mol of lactic acid is also produced. When the tissue clearance mechanism of the operated eye is not sufficiently effective, lactic acid accumulates and the pH drops, resulting in inhibition of tissue metabolic activity. In contrast, in tissues with high mitochondria, complete oxidation of glucose to CO ₂ and H ₂ O occurs via mitochondria, and 38 mol of ATP is produced for consumption of 1 mol of glucose (glycolysis). Including the oxidation of 2 moles of NADH produced in the system). It is therefore clear that utilization of substrates by mitochondrial oxidation is an efficient energy production pathway when oxygen is available. Lactic acid is not produced under these conditions. Furthermore, when anaerobic glycolysis is inhibited via the Pasteur effect due to hyperventilation, lactic acid accumulation is reduced or minimized (Krebs, HA, Essays. Essays Biochem. No. 8
Vol. 1, p. (1972)).

Glucose is not directly utilized as a substrate in mitochondria, but must first be converted to pyruvate by glycolysis. Ketone bodies and their precursors (eg short chain fatty acids and ketogenic amino acids) are immediately oxidized in mitochondria, producing 32 moles of ATP for every mole of acetyl molecule utilized. Thus, ketone bodies and their precursors are energy-rich or energy-efficient molecules. In addition, oxidation of ketone bodies and their precursors enhances respiration, which inhibits anaerobic glycolysis via the Pasteur effect.

The ketone body is acetone (CH3COCH
3), acetoacetate (CH3COCH2COO-)
And β-hydroxybutyrate (CH3CHOHCH
2COO-) is a general term. The pathway for the formation of ketone bodies from fatty acids and ketogenic amino acids is described in FIG. Ketone bodies are metabolites of fatty acids and ketogenic amino acids in tissues, including leucine, lysine, phenylalanine, tyrosine and tryptophan.
In nature, fatty acids have the general formula CH3 (CH2) nCOOH
(N is an even integer). Acetic acid is the smallest fatty acid molecule with n = 0. Ketone bodies are stable forms of high-energy metabolites of acetyl-CoA for storage and release in the body. That is, ketone bodies are immediately produced in vivo from fatty acids and ketogenic amino acids. Furthermore, β-hydroxybutyrate and acetoacetate are N
Mutual conversion via the catalysis of AD + -linked β-hydroxybutyrate dehydrogenase:

[Chemical 1]

These indicate that ketone bodies, fatty acids, and ketogenic amino acids are interrelated molecules. When energy is needed, the ketone bodies and their precursors are utilized in the tissue to produce acetyl CoA,
It is further oxidized in the mitochondria via the Krebs cycle to CO ₂ and H ₂ O, producing high energy in the form of ATP.

When the precursors of ketone bodies (fatty acids and ketogenic amino acids) are administered (in humans and animals), acetyl CoA is produced, which is further oxidized to produce high levels of ATP. When acetyl-CoA is overproduced, ketone bodies are produced, the main metabolic site of which is the liver. Of these ketone bodies produced, acetone is not utilized and is excreted from breath, sweat and urine. On the other hand, acetoacetate and β-hydroxybutyrate are released to peripheral tissues where they are converted to acetyl CoA and further oxidized in the mitochondria to CO ₂ and H ₂ O through acetyl CoA and the citric acid cycle, and are abundant in peripheral tissues. Supply the perfect energy.

In peripheral tissues, acetoacetate is also produced from ketone-forming amino acids. When acetoacetate is present in excess, it is converted to β-hydroxybutyrate for storage, or converted to acetyl-CoA and further oxidized in the mitochondria to CO ₂ and H ₂ O, resulting in high levels. Produces levels of ATP. In peripheral tissues, fatty acids are decomposed into acetyl-CoA via β-oxidation (see Figure 1), which is further oxidized to CO ₂ and H ₂ O in mitochondria to produce high levels of A.
Produces TP.

The cornea, lens and retina have high glycolytic activity and metabolize glucose to produce a large amount of lactic acid. Under normal conditions glucose is the energy source of eye tissue in vivo. The lactic acid produced is removed by a clearance mechanism via the blood circulation. During or immediately after eye surgery, the mechanism of lactic acid removal by blood circulation is ineffective. Excess lactic acid accumulates in the anterior chamber of the eye and the vitreous when glucose is included in the perfusion solution as an energy source for eye tissue during eye surgery. Utilization of glucose lowers extracellular pH, which causes accumulation of lactic acid in cells (and tissues) and inhibits metabolic activity (Chen and CH and Chen, S. et al.
C. ), Arch Biochem Biofiz (Arch.B)
iochem. Biophys. ) Volume 276, page 70 (1990)). This problem is solved by adding ketone bodies and their precursors into the perfusion solution as an alternative energy source for eye tissue. In the tissues of the eye, ketone bodies and their precursors are immediately utilized to form acetyl-CoA, which in the mitochondria passes through the Krebs cycle to produce CO ₂
And it is oxidized in _{H 2} O to produce high levels of ATP. This process inhibits glycolysis by Pasteur effect and suppresses lactic acid production.

Further, ketone bodies are known to be a suitable energy source for the starved brain, muscle and kidney (Olaon RE, Nature (N.
195: 597, 1962; Bassenge, E., et al., American Journal of Physiology (Am. J. Phus).
iol. ) 208, 162, 1965; Owen, OE, et al., Journal of Clinical Investigation (J. Clin. In.
vest. ) 46, 1589, 1967; and Hawkins, RA, et al., Biochemistry Journal (Biochem. J.) 1st.
22:13, 1971, and 125: 54
P. 1, 1971).

The compositions of the present invention are not solely intended to function in eye surgery as artificial fluids that replace eye fluids or body fluids in general surgery, or as perfusion solutions themselves. Rather, it is an object of the present invention to provide ketone bodies and precursors thereof during storage of isolated tissue and as a temporary high energy source of tissue during and after surgery, at least the anterior chamber of the eye. And allowing the cells to perform viability and physiological and biological functions until the vitreous is in sufficient equilibrium with physiological fluids.

That is, the ketone body and its precursor and glucose in the balanced preparation are particularly useful for isolated tissues during storage and peripheral tissues during surgery. Glucose is
It serves as an energy source for tissues with little or no mitochondria (eg, corneal stroma and lens). Ketone bodies and their precursors are rich in energy. These are CO ₂ and H ₂ O in mitochondria through the Krebs cycle.
It is immediately oxidised to produce no other metabolic waste. The generated carbon dioxide is hydrated to carbonic acid,
It is further oxidized to bicarbonate at pH 7.4.
It has been suggested that bicarbonate is required for the process of ion transport across the corneal endothelium (Hodosi (Hodosi
n, S. ) Et al., Journal of Physiology (J. Physiol.) 263, 563, 19
1976). In addition, the oxidation of ketone bodies and their precursors not only produces high levels of ATP, but also suppresses glycolysis via the Pasteur effect and consequently inhibits lactic acid production.

In the present invention, the balanced salt (bala)
nced salts) are included in the components to produce a physiologically compatible solution for in vivo administration. Phosphate in the component is a component of oxidative phosphorylation of ATP production process when the ketone body and its precursor are oxidized. Furthermore, phosphoric acid acts as a buffer having a high buffering capacity in the physiological pH range of pH 7.2-7.4.

The theory and intended application of the invention is based on the beach (Veec) described in US Pat. No. 4,663,289.
It is different from the theory of h). The invention of Beech teaches that the metabolic processes of living animal cells are one or more fixed ratios [HCO ₃ −] / [CO ₂ ], [L-lactate-] / [pyruvate- ], And the [β-hydroxybutyrate] / [acetoacetate] pair, which is based on the theory that p
The effect is similar to a "weak acid / conjugated base" paired buffer system for adjusting H.

Vech's theory is based on several hypotheses: Both a fixed ratio of both substrates are taken up by cells or tissues. 2. A proportion of the substrate (in the tissue) that accumulates then lodges in living cells to control metabolic activity. 3. Substrate uptake and metabolic regulation by these substrate pairs is similar in all tissues.

Many of the experiments that underlie the invention of Vech were carried out in rat liver (Vech et al., Biochemistry Journal (B).
iochem. J. ) Volume 115, p. 609, 1969
Year; and Vech et al., Journal of Biological Chemistry (J. Biol. Che.
m. ) 254, 6538, 1979). The liver is the center of metabolism in humans and animals. The metabolic processes in the liver are different from those in peripheral tissues. Thus, the mechanisms that control metabolic processes may work in the liver, but not necessarily in peripheral tissues. For example, in the liver, ketone bodies are produced through β-oxidation of fatty acids.
The ketone bodies produced are not used in the liver, but are transported to the peripheral tissues via the blood circulation. Here they are coupled to the production of high levels of ATP via the Krebs cycle
₂ and H ₂ O (the principle of biochemistry (Phncip
less Biochemistry, Lehninger, Worth Publishers, In
c. )). That is, β-hydroxybutyrate is an efficient alternative energy source for peripheral tissues, but not a liver alternative energy source. The present invention extracts β-hydroxybutyrate without conjugating it with acetoacetate and metabolizes it to produce ATP, and at the same time suppresses the production and accumulation of lactic acid in cells. Utilize activity.

The lactic acid produced is released into the blood by normal cells actively degrading sugars (eg erythrocytes, skeletal muscle, cornea and retina) and then extracted from the blood by the liver and heart and metabolized. To be done. Lactate is translocated through the cytoplasmic membrane with pyruvate as a competitive inhibitor (Spencer and Lenninger (Spence).
rand Lehninger), Biochemistry Journal (Biochem. J.) 154, 40.
P. 5, 1976). Ketone bodies are produced in the liver and released into the blood, then extracted and metabolized from the blood by peripheral tissues (eg cornea and lens) (Principles of biochemistry (Pr
inciples of Biochemistr
y), Leninger, Worth Publishers
s, Inc. )). The transport mechanism of ketone bodies through the cytoplasmic membrane has not been established yet.

The metabolism of pyruvic acid, lactic acid, acetoacetate, and β-hydroxybutyrate varies depending on cells or tissues. Heart and liver lactate dehydrogenase (LDH)
Isozymes promote the oxidation of low concentrations of lactate to pyruvate; corneal, lens, and retinal LDH isozymes help reduce low concentrations of pyruvate to lactate. Β-hydroxybutyrate dehydrogenase (BDH) in liver
Isozymes promote the reduction of low concentrations of acetoacetate to β-hydroxybutyrate; BDH isozymes in peripheral tissues (eg corneas) promote the oxidation of low concentrations of β-hydroxybutyrate to acetoacetate.
Different LDH and BDH isozymes in different tissues, substrate-pairs (eg [lactate] / [pyruvate] or [β-hydroxybutyrate] / [pyruvate] and [β-hydroxybutyrate] ] / [Acetoacetate]) makes it difficult to establish a general ratio for all tissues. Food intake and other metabolic reactions Substrate uptake due to similarities, continuous influx of these chemicals, and efflux of these chemicals and removal by other metabolic reactions make it more difficult to establish a general ratio of substrate-pair It has become.

In the application of culture media for tissue preservation, the theory of Lindstrom et al. (US Pat. No. 4,695,536) was basically used by scientists in the field of tissue culture for many years. It is a tissue culture technology. Specifically, Lindstrom et al. Teach a method of corneal preservation consisting of the following methods:

(A) Earls salt
s) containing 50 ml of basal medium Gibco '
s) minimal basic medium, 25 m without L-glutamine
M HEPES buffer, final concentration of medium is 1
% Of L-glutamine (200 mM) and 50-100 μg of garamyci.
Corneal preservation medium containing antibiotics containing n); serum from the group of bovine serum, fetal bovine serum or human serum; and chondroitin sulfate, sodium hyaluronate, keratan sulfate, polyvinyl-pyrrolidone, methylcellulose, hydroxypropylmethylcellulose, cellulose gum. And an effective amount of high molecular weight molecules selected from the group consisting of dextran; (b) filling a corneal storage container with mixed medium; and (c) a cornea-sclear ring in a container containing medium.
1-scleralrim) and maintained the medium at 4-3
Maintaining a temperature of 4 ° C allows for medium and long term storage, and the system allows for changes in temperature ready for tissue transport.

This theory is based on the fact that the isolated cornea is
Kaufman (MacCarey-Kaufmann) medium (tissue culture medium 199 plus 5% dextran; McCray and Kaufmann.
Kaufman, H .; ), Invest. Ophthalmol. Volume 13,
165, 1974) with or without bovine serum supplementation (Lindstrom).
, American Journal of Off Salmology (A
m. J. Ophthalmol. ) Volume 95, 869
(1977, 1977) do not describe any mechanism by which the production and accumulation of lactic acid is inhibited when cultured or stored. MacCarey et al.
Ofsalmol (Invest. Ophthalmo
l. ) Volume 13, p. 165, 1974)
Dextran is added to tissue culture medium (TC199) as a dehydrating agent, which makes the active osmolality of the medium slightly hypertonic (about 305-340 mOsM). Dextran in this slightly hypertonic solution exerts a colloid osmotic pressure on the stored cornea, giving an artificial dehydration effect.
However, the presence of dextran in the medium has no inhibitory effect on lactate production and accumulation.

In stark contrast to this, the Che
n) et al. (US Pat. No. 4,873,186)
Isolated corneas are incubated with Dulbecco's phosphate buffered saline (PBS) containing β-hydroxybutyrate or in TC199 containing β-hydroxybutyrate (also known as medium 199). Describe the mechanism by which high levels of ATP are simultaneously produced in tissues and the production and accumulation of lactic acid are inhibited when stored in. Specifically, Chen et al. Disclose a method of prolonging the time that an isolated cornea of surgical nature is stored, which method is used in a corneal storage medium containing the stored cornea. , (1) short chain fatty acids, (2) ketone bodies, and (3) a ketogenic amino acid capable of inhibiting lactic acid production by the isolated cornea in an amount sufficient to inhibit lactic acid production, or Containing an amount of at least one compound selected from the group consisting of ketone body precursors.

The corneal preservation medium is TC1 containing Hank's salt.
Formulated by replacing the bicarbonate balanced salt solution portion of a tissue preservation medium such as 99 with the composition of the present invention. In this case, the NaCl concentration must be reduced in order to keep the total concentration of anions and cations physiologically compatible. As a result, the components in the tissue culture medium other than NaCl are not completely decomposed, and the active osmolality is slightly hypotonic. The active osmolality of the solution is isotonic at 290 mOsM, or 285-300 m
Must be in the OsM range. Sugars permeable to the plasma membrane (eg mannitol, sucrose and dextran) are used for adjusting the osmolality. The use of these does not exert a colloid osmotic pressure on the stored cornea because the solution is isotonic. Therefore, MacCare
y) et al. (MacCarey et al., Invest. Ophtha.
1 mol. ) Vol. 13, 165, 1974) is completely different from the intended application of dextran.

Β-hydroxybutyrate is a reducing agent. In solution, it is oxidized by O ₂ especially at elevated temperatures. Therefore, it is necessary to use degassed water to make the compositions of the present invention. The resulting solution is stored under vacuum and β
- prevents hydroxybutyrate is oxidized by O _2, can be extended resulting life. If the solution is allowed to equilibrate with air when opening the bottle for clinical applications, O ₂ will be available for the biological function of the tissue (or cells).

Alternatively, β-hydroxybutyrate is O ₂
Antioxidants of the group consisting of citric acid, phenylalanine and vitamin E may be used to prevent oxidization by the. The preferred antioxidant is citric acid, and the concentration of citric acid in the solution is in the range of 8-12 mM. However, upon long-term storage, O ₂ in solution also oxidizes the antioxidant, reducing the effectiveness of the antioxidant in preventing β-hydroxybutyrate from being oxidized by O ₂ . Therefore, the use of degassed water is the preferred method for making the compositions of the present invention.

[0029]

SUMMARY OF THE INVENTION The present invention relates to a novel composition comprising a ketone body and / or its precursor, glucose, a phosphate buffered balanced salt aqueous solution. The present invention also provides a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution in water to preserve the isolated tissue described above and to efficiently and physiologically reproduce the tissue during surgery. It relates to a process for preparing a composition, which comprises mixing in an amount sufficient to form a composition which effectively meets the requirements for its chemical function.

FIG. 1 shows a pathway of ketone body formation from a fatty acid and a ketone-forming amino acid.

FIG. 2 shows B as described in Example 1.
FIG. 7 is a reproduction of a photograph of the feline endothelium after perfusion of the anterior chamber with SS by spectroscopic microscopy.

FIG. 3 shows B as described in Example 1.
FIG. 6 is a reflection microscopy photocopy of the cat endothelium after perfusion of the anterior chamber with SS-plus.

FIG. 4 is a reflection microscopy photocopy of the cat endothelium after perfusion of the anterior chamber with the composition of the present invention as described in Example 1.

FIG. 5 shows the “corneal thickness vs. time” of in vitro deswelling of isolated corneas in the presence of the composition of the invention, as described in Example 2. It is a plot.

FIG. 6 shows B as described in Example 2.
FIG. 4 is a plot of “corneal thickness versus time” of in vitro deswelling of isolated corneas in the presence of SS-plus.

FIG. 7: In vivo contraction of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. (Deswelling) is a plot of "corneal thickness versus time". FIG. 8 shows in vivo deswelling of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. "Corneal thickness versus time" is plotted.

Broadly stated, the composition of the present invention comprises a ketone body and / or its precursor, glucose,
And a phosphate buffered balanced salt solution.
The composition of the present invention comprises its components (particularly the above-mentioned ketone bodies and / or the like) so that it can effectively fulfill the requirements for efficient physiological and biochemical functions of the eye and other peripheral tissues. Utilizes some of the special properties of precursors, glucose, and phosphate buffered balanced salt solutions.

Accordingly, the composition of the present invention comprising a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution in water, with or without a high density of mitochondria (or no mitochondria at all). Case), or generally with or without other tissues, to meet the minimum essential nutritional requirements of eye tissues. For example, eyes and other peripheral tissues perfused with the compositions of the present invention after surgery have energy source-dependent metabolic functions (eg, fluid and ion transport and protein synthesis) and physiological functions such as maintaining a thin, transparent cornea. Can be executed.

Therefore, the composition of the present invention can be used, for example, for cornea 3
It provides a well-balanced and abundant source of energy for eradicated tissue represented by three different organizational layers. That is, the composition of the present invention has a need for high respiratory activity of endothelium, a need for high glycolytic activity of endothelium and stroma, and a need for achieving basic intracellular energy level and ion transport of corneal fluid. Can meet.

Representative compositions of the present invention suitable for use as a perfusate solution, along with their component ranges, are set forth in Table 1 below.
Described in.

[0041]

[Table 1]

Representative anions and cations included in the compositions of the present invention are set forth in Table 2 below. Table 2 also shows the concentration of these ions in the composition of the invention.
The calculated osmolarity of the composition of the present invention is about 300 to about 320 mO when all salts are completely dissolved.
The range is sM. The actual osmolality of the prepared solution of the composition of the present invention is about 285 to about 300 mOsM.
Is the range.

[0043]

[Table 2]

According to one embodiment of the present invention, the ketone bodies of the composition of the present invention are β-hydroxybutyrate ion and acetoacetate ion. β-hydroxybutyrate ion is the D-isomer of β-hydroxybutyrate, β
Selected from the group consisting of a mixture of D-racemic and L-racemic hydroxybutyrate, and a mixture of D- and L-isomers of β-hydroxybutyrate, most preferably β-hydroxybutyrate Is the D-isomer. The concentration of β-hydroxybutyrate is preferably 5-
Preferred ketone body precursors of the composition of the present invention in the range of 30 mM are fatty acids selected from the group consisting of acetic acid and butyric acid, and preferred ketogenic amino acids are leucine, lysine, phenylalanine, tyrosine and tryptophan. Is selected from the group consisting of:

The concentration of the precursor of the ketone body is preferably 0.
It is in the range of 1-25 mM. The concentration of the ketogenic amino acid is preferably in the range of 0.1-5.0 mM,
The most preferred range is 1-2 mM. The ketogenic amino acid is preferably at a total concentration of 7.5-12.5.
mM, most preferably the total concentration is 10 mM.

The preferred fatty acid is acetic acid, preferably 1
A concentration in the range 5-25 mM, or butyric acid,
The concentration is preferably in the range of 5-15 mM. The concentration of acetic acid is most preferably 20 mM and the concentration of butyric acid is most preferably 10 mM.

The pH of the composition of the present invention is preferably 7.3.
Adjusted to -7.5.

To prevent the oxidation of β-hydroxybutyrate by O ₂ and prolong the life of the solution at room temperature, it is preferred to use degassed water to make the compositions of this invention.

A portion of NaCl, preferably in the amount of 15-35 mM, or most preferably 25-30 mM, is
Glucuronic acid, D-galacturonic acid, D-mannuronic acid,
One or more sugar acids selected from the group consisting of D-gluconic acid and D-glucaric acid (sugar)
acid) equivalent Na + salt.

A sufficient amount of 40-to form a composition that simultaneously prevents adhesions that occur during eye surgery and effectively meets the requirements for efficient physiological and biochemical function of the eye tissue. The addition of neutral polysaccharide dextran in an amount in the range of 500 kilodaltons increases the viscosity of the composition according to the invention. Suitable concentrations of neutral high molecular weight dextran are in the range 2% -20%.

In another aspect, the invention provides a composition of the invention, Eagle's medium, Dulbecco's medium and Daniel.
iel's) consisting of a member of the minimum essential amino acids and the minimum of essential vitamins selected from the group consisting of
It relates to a corneal preservation medium. This solution is commercially available and, as will be apparent to those skilled in the art, the amount added will vary depending on the particular conditions.

In another aspect, the invention relates to a corneal preservation medium in which a balanced salt solution of tissue culture medium has been replaced with a composition of the invention; the total concentration of cations and anions in the solution is balanced. To maintain the same concentration as the saline solution, the NaCl concentration is preferably reduced to within the range of 70-100 mM, or most preferably 85 mM; the active osmolarity of the solution.
Is adjusted to isotonicity of 285-300 mOsM or most preferably 290 mOsM; mannitol, sucrose and dextran are added if necessary. The tissue culture medium is preferably TC199 with Hank's salt or Eagle's minimal minimal medium with Hank's salt.

The pH of the corneal preservation medium is preferably 7.10.
-7.50 range, most preferably 7.25-
The range is 7.40.

15-35 mM, or most preferably 2
A portion of NaCl in the amount of 5-30 mM is equivalent to one or more sugar acids consisting of D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid. It is replaced with Na + salt.

In another aspect, the invention provides that the phosphate buffered balanced salt solution of the composition of the invention comprises a tissue culture medium suitable for corneal preservation but in which unfavorable lactic acid production normally occurs. It relates to a corneal preservation medium replacing the bicarbonate buffered balanced salt solution of the corneal preservation composition. Furthermore, the corneal preservation medium contains at least one compound selected from the group consisting of short-chain fatty acids and ketone bodies capable of inhibiting the production of lactic acid by the cornea and present in an amount sufficient to inhibit the production of lactic acid. Including.

In yet another aspect, the invention comprises a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor.
It relates to a corneal preservation composition.

In yet another aspect, the present invention provides for the preparation of tissue for organ transplantation (eg corneal transplantation), which comprises the composition of the present invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor. For formulations. In yet another aspect, the invention relates to a perfusate solution, eg, for topical administration, comprising a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum derived factor.

In yet another aspect, the invention comprises a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor.
For example, it relates to an organ medium for administration.

The method of preparing the solution of the present invention provides a sufficient amount of the composition to form a composition that effectively meets the requirements for efficient physiological and biochemical function of the eye and other peripheral tissues described above. Mixing glucose and ketone bodies and / or their precursors in a degassed phosphate buffered saline solution. This solution is bottled under vacuum to completely remove O ₂ to prevent β-hydroxybutyrate from being oxidized by O ₂ when stored at room temperature for extended periods. If this solution is used immediately or for a short period of time, bottling under vacuum is useful,
Not necessarily required.

Since high concentrations of Ca ²⁺ and Mg ²⁺ produce phosphate precipitates, all components except CaCl ₂ and MgCl ₂ must first be thoroughly degassed with water (preferably 90-95% of total volume). ) It is preferable to dissolve it. Mix the solution thoroughly and add P with 1N D-glucuronic acid or NaOH.
Adjust H to be in the range 7.3-7.5. Then 0.5 M stock solution (or 0.2-1.0 M range) of CaCl ₂
And MgCl ₂ are added. Check the pH and adjust if necessary to 7.3-7.5, preferably 7.4. Finally, add water to adjust the volume. Deionized doubly distilled water is preferably used to prepare this solution. All ingredients used are of the reagent grade.
e).

Several variations are possible in the preparation of the perfusion solution of the present invention. Some of them are described below.

1. The phosphate buffer is prepared using dipotassium phosphate and monopotassium phosphate. In this case, the total potassium ion concentration increases to 15 mM.

2. Phosphate buffer is prepared using disodium phosphate and monosodium phosphate. In this case 8-
12mM KCl must be added, NaCl is 5
mM subtracted.

3. Phosphate buffer is prepared with any other phosphate and phosphoric acid and the pH is adjusted appropriately to 7.4. In each case, the final concentration of K + should be 8-12 mM. The pH can vary in the range 7.3-7.5.

4. Substituting some of the NaCl with Na + salts of one or more sugar acids including D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid to give chlorine. Concentrations should be maintained within the physiological range of 90-105 mM.

5.0.1-5.0 mM, preferably 1-
One or more ketogenic amino acids consisting of 2 mM leucine, lysine, phenylalanine, tryptophan and tyrosine can be added as a component of the perfusion solution. Subtract equal amounts of NaCl to adjust Na ⁺ concentration.

6. 2 part of β-hydroxybutyrate
It can be replaced with double the amount of acetic acid (the smallest molecule of the ketone body precursor short chain fatty acid).

It is also possible to use the sodium salts of other short chain fatty acids in the range of 7.10 mM, or 5-15 mM, such as butyric acid and caproic acid. However, the use of these aqueous solutions is limited due to their low solubility.

8. The β-hydroxybutyrate can be replaced with acetate or acetoacetate, with β-hydroxybutyrate being preferred. β-hydroxybutyrate is stable and inexpensive. It is also readily utilized by cells and is more energy efficient than acetoacetate. One mole of β-hydroxybutyrate produces 64 moles of ATP and one mole of NADH, which produces an additional 3 moles of ATP.

9. Since only D-isomers are used in cells, the D-isomer of β-hydroxybutyrate is preferred as the composition of the invention. However, the D-, L-racemate or the D-, L-isomer mixture is also low in price, and commercial products are readily available, and these can also be used.

10. If desired, sugars such as mannitol and sucrose or neutral polysaccharides such as dextran can be used to adjust the osmolality of the solution to the range of 290 mOsM, or about 285-300 mOsM. In such adjustment, Na ⁺ concentration and Cl ⁻
The concentration does not change. If both Na ⁺ and Cl ⁻ concentrations are too high, an equal amount of sugar should be used instead of NaCl.

11. 5 to increase the viscosity of the solution
% Or 2-20% neutral high molecular weight dextran (preferably 70-500 kilodaltons) can be used. Very high viscosity compositions are used to prevent adhesions resulting from surgical wounds in eye surgery, such as the lens or iris adsorbing to the corneal endothelium or the iris adsorbing to the lens. It is useful. in this case,
NaCl is 2.5m per 1% of dextran added
Only M is subtracted.

The compositions of the present invention are primarily used during the interim period after surgery in the eye, such as corneal transplants, intraocular lens transplants, cataract extractions, and vitrectomy, to remove eye and other eradication. It is formulated with the minimum of essential ingredients used to enable the tissue (or cells) to maintain their viability and ability to perform physiological functions. By interim period is meant the time after surgery until the solution in the anterior chamber and / or vitreous reaches sufficient equilibrium with the body's physiological solution. As described above, the composition of the present invention can be used for topical administration to, for example, the skin and eyes, preservation and lavage of donor tissue before transplantation, and general surgery.

The composition of the present invention contains a physiologically compatible salt solution whose concentration range is similar to that found in plasma. In the present invention, phosphate (typically 10 mM) can be used both as a buffer and as a substrate for ADP phosphorylation for the production of ATP in energy metabolism. Phosphate has a pH of 7.2-
It has a strong buffering capacity at 7.4. Bicarbonate is used in other perfusion solutions currently on the market (e.g. BSS-Plus from Alcon, Fort Worth, Tex.). However, when a bicarbonate buffer is used, bicarbonate is not used as a component of the composition of the present invention because the CO ₂ partial pressure (P _co2 ) changes the pH of the solution. Further, without the addition of bicarbonate from the outside, bicarbonate is generated by the respiration (that is, oxidative phosphorylation) of the tissue itself. In this case, this substrate is oxidized (in mitochondria) to C
O ₂ and H ₂ O are produced. Next, the generated CO ₂ is hydrated to H ₂ CO ₃ , which is further decomposed into H ₂ CO ₃ .
CO ₃ ⁻ is produced. About 1.3μ in the rabbit cornea
CO ₂ is produced at a rate of mol / cm ² / hour. In the human cornea, this rate is 30-50% higher.

In mitochondria, β-hydroxybutyrate is immediately oxidized to acetoacetate and NADH by the catalytic action of β-hydroxybutyrate dehydrogenase. Acetoacetate is also succinyl CoA
React with to produce acetoacetyl CoA, which is further degraded by thiophorase to produce acetyl CoA. In cells, fatty acids are utilized by β-oxidation to produce acetyl CoA.
Ketogenic amino acids are also assimilated in cells to produce acetoacetate or acetoacetyl CoA, which in turn produces acetyl CoA. Acetyl CoA from these reactions is then oxidized to CO ₂ and H ₂ O in the mitochondria via the citric acid cycle. The pathway for the formation of ketone bodies from fatty acids and ketogenic amino acids is briefly described in FIG. Oxidation of acetyl-CoA is an energy efficient process, which yields 30 moles ATP and 2 moles GTP (ie 32 moles ATP total) for the use of moles acetyl CoAl. Inorganic phosphate is the source of oxidative phosphorylation, which is coupled to the electron transport chain and reacts with ADP to produce ATP.

As is clear from the table below, about 47% of the glucose consumed in the cornea is glycolytic and 53% is respiratory (Chen and Chen (Ch
End and Chen), Ark Biochem. Biophys. 276, 70, 1990).

[0077]

[Table 3]

The% glucose consumption in the corneal tissue layer is shown in brackets in Table 3 above. The interstitium consumes glucose by the glycolytic system at a rate twice as fast as respiration. In the lens, mitochondria are present only in the epidermis. Therefore, glucose is an important source of energy in the stroma and lens (especially cells within the nucleus of the lens). There is little or no mitochondria utilizing acetyl CoA in these cells. In other eye tissues, such as the mitochondrial dense corneal endothelium and photoreceptors, ketone bodies and their precursors are energy efficient substrates. Moreover, in these tissues, the oxidation of acetyl-CoA causes an increase in respiration, which inhibits the anaerobic glycolysis system by the Pasteur effect.

Thus, the composition of the present invention comprising a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution may or may not have a high density of mitochondria, or no mitochondria. Meet the minimum essential nutritional requirements of absent eye tissue, or other peripheral tissue in general.

The composition of the present invention is used as a simple corneal preservation composition suitable for short-term administration as disclosed by Chen et al. (US Pat. No. 4,873,186, cited above). can do. According to Chen's theory, at least one compound selected from the group consisting of short-chain fatty acids and ketone bodies is mixed with a tissue culture medium to simultaneously inhibit the lactate production caused by the cornea and increase high energy. Used for storage of the isolated cornea to produce.

According to one aspect of the present invention, the composition of the present invention, essential amino acids, and Eagle's medium (Science, Vol. 130, p. 432, 1959).
Year; Proc. Sok Expe Biol
c. Exp. Biol. ) Vol. 89, 362, 195
5 years, Dulbecco's medium (virology
gy) Vol. 8, 396, 1959 and Vol. 12,
185, 1960) or Daniel's medium (Proc.
Sok Expe Biol (Proc. Soc. Exp.
Biol. ) Vol. 127, p. 919, 1968), a corneal preservation medium comprising the essential vitamin is prepared. Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer solution 20-30 mM
Is effective in increasing the buffering power. Pre-prepared minimal essential amino acids and essential vitamins are commercially available. Essential amino acids and vitamins in solution are generally relatively short-lived and stored in cold places for short periods (usually 6-1
Since it has to be used within 2 months), the use of degassed water is effective but not always necessary for the preparation of the composition of the present invention used as a corneal preservation medium.

In a second aspect of the invention, the corneal membrane is replaced by replacing the balanced salt solution of a tissue culture medium such as TC199 containing Hank's salt or Eagle's minimal basal medium containing Hank's salt with the composition of the present invention. The storage medium is formulated. To maintain the total concentration of cations and anions in the medium at physiologically compatible levels, NaCl
The concentration is reduced within the range of 70-100 mM. The solution should be maintained isotonic at 290 mOsM, or within an active osmolality range of about 285-300 mOsM. The active osmolality is adjusted with sugars such as mannitol and sucrose, or neutral high molecular weight polysaccharides such as 40 and 70 kilodaltons dextran.

In a third aspect, Chen
Replacing the bicarbonate balanced salt solution of the corneal preservation composition of the theory of et al. (US Pat. No. 4,873,186, supra) with the phosphate buffered balanced salt solution of the composition of the present invention. The corneal preservation medium is prepared by. 15-35m
M, preferably a portion of NaCl in an amount of 25-30 mM, and one or more sugar acids including D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid. It is preferred to maintain the Cl ^- concentration in the physiological range of 90-105 mM by substituting it with the sodium salt of. In the theory of Chen et al., At least one compound selected from the group consisting of short chain fatty acids and ketone bodies is mixed with tissue culture medium and used for corneal preservation. The bicarbonate buffered balanced salt solution is part of the tissue culture medium component.

In a fourth embodiment, a suitable corneal preservation composition is described by Chen et al. (US Pat. No. 5,073,
No. 492) in combination with a synergistic composition as described above, consisting of the formulation of the above-mentioned first, second or third aspect. In the theory of Chen et al., A synergistic composition is basically
It consists of a synergistically effective mixture of vascular endothelial growth factor (VEGF), uridine, thymidine and serum derived factor (SDF). The stoichiometric ratio of uridine to thymidine is 10 μM uridine to 0.5 μM thymidine. Typically, dialyzed fetal bovine retina extract (0.1 mg / ml) and fetal bovine serum (3 mg / ml) were added to VEGF and SD, respectively.
Used as a source of F.

In a fifth aspect, the composition of the present invention is combined with the synergistic composition of Chen et al. (US Pat. No. 5,073,492) described in the fourth aspect to produce organs. Formulations are prepared for the preparation of tissues for transplants (including corneal transplants).

In a sixth aspect, the corneal preservation medium of the first or second aspect described above is the same as that described in the fourth aspect by Chen et al. (US Pat. No. 5,073,492).
No.) synergistic composition to produce a perfusion solution for topical administration to stimulate endothelial cell regeneration.

In the seventh embodiment, the corneal preservation medium of the first or second embodiment described above is the same as that described in the fourth embodiment by Chen et al. (US Pat. No. 5,073,492).
No.) synergistic composition to produce a tissue culture medium for administration to accelerate the corneal endothelial cell regeneration process prior to corneal transplantation.

The solutions of the present invention were prepared in a degassed double distilled water volume equal to about 95% of the total volume, with the exception of the Ca ²⁺ and Mg ²⁺ salts, in the exact amounts indicated in the preparation method. It is prepared by dissolving in. If necessary, the pH of the solution is adjusted to 7.4 with D-gluconic acid or NaOH. Then Ca ²⁺ and Mg ²⁺ salts are added and the volume is adjusted with degassed double distilled water. Recheck pH and adjust if necessary. The solution is then sterilized by any suitable means, such as autoclaving or using a 0.22 μm filter (Nalge Co., Rochester, NY). Next O ₂ from the solution to prevent the β- hydroxybutyrate is oxidized by O ₂
Bottles under vacuum for complete removal.

The composition of the present invention (for example, the above-mentioned first,
When used as part of a formulation (as in aspects 2, 4, 5 and 7) it is preferred to first prepare a concentrated ^stock solution free of Ca ²⁺ and Mg ²⁺ . The exact calculated amount is then weighed, combined with another portion of the ingredients and double distilled water is added to bring the volume to about 95%. Add Ca ²⁺ and Mg ²⁺ , recheck pH and readjust if necessary.
The stock solution is preferably a 2- to 10-fold concentrated solution, more preferably a 2- to 5-fold concentrated solution. If not used immediately, store the stock solution with Nalgen.
e) 0.22 or 0.45 such as (Nalge Co., Rochester, NY)
Sterilize by passing through a μm filter. Sterilized solution at 4 ℃
Store in the refrigerator. Because the solution contains relatively short-lived essential amino acids, vitamins and other components, it must be stored at 4 ° C and used for a short period of time (usually about 6-12 months). Therefore, the use of degassed water for the preparation of these solutions is useful, but not necessary.

[0090] For synergistic composition comprising a Mg ²⁺ sufficient amount, they shall not Mg ²⁺ if combining a composition of the present invention the synergistic compositions.

The invention is illustrated in detail in the examples below.
The examples described herein are for purposes of illustration and are not intended to limit the invention in any way.

Example 1 The effectiveness of the composition of the present invention as a balanced energy source is demonstrated by the two most commonly used perfusion solutions at present: BSS and BSS-Plus (Alcon Laboratories, Inc.). In
c. ), Fort Worth, Texas) (B)
SS-plus is reported to be more effective than BSS). BSS-Plus is a tripeptide,
Oxidized glutathione exists. Dixstein (Di
Kstein et al. (J. Phys)
iol. ), 221, 29, 1972), glutathione is effective for corneal fluid transport.

The compositions of the present invention are shown in Table 1, but in this example 20 mM acetate was used instead of 10 mM β-hydroxybutyrate and citric acid was used as the antioxidant. Changes in the concentrations of these components within the ranges shown in Table 1 are similarly effective. However, the active osmolality of the solution is in the range of about 285-300 mOsM, preferably 2
Must be maintained at 90 mOsM. Since the purity of the salt varies from lot to lot, the active osmolality of the solution varies from preparation to preparation. If the osmolality is too low, adjust by adding mannitol, or if it is too high, N
Instead of aCl, sodium D-glucuronate or the Na ⁺ salt of sugar acid is used.

In this experiment, two nicks each having a length of about 2 mm were made on the corneas of six cat eyes.
A 25 gauge cannula was inserted into the anterior chamber through one of the cuts in each cornea. The solution of the present invention shown in Table 1 above was used at a pressure of about 15 mmHg and a rate of about 7 ml / min, except that 20 mM acetate was used instead of 10 mM β-hydroxybutyrate and citric acid was used as an antioxidant. Thus, a total of 1 liter was injected per cornea.

For comparison, the same procedure was carried out using BSS in some cases and BSS-plus in others instead of this solution.

All corneas were examined 1, 2 and 6 days after injection. The morphology of the endothelium was examined using a reflection microscope and corneal thickness was measured using a digital caliper.

As shown in FIG. 2, in the cornea injected with BSS, endothelial cells were extensively lost, endothelium polymorphism was observed, and corneal swelling was observed 1 day after injection. It was As shown in FIGS. 3 and 4, on the first day after the injection of BSS-plus or the perfusion solution of the present invention, the shape of the endothelial cells remained polygonal (normal shape) and the corneal thickness was normal. there were. When observed 6 days after the injection, the cornea injected with the solution of the present invention (FIG. 4) was identified as BSS-plus injected cornea (FIG. 5) in terms of endothelium polymorphism and corneal thickness. Somewhat good.

The above results show that the perfusion solution of the present invention is superior to BSS-Plus in that it maintains the endothelium and the thin and transparent cornea.

In this animal study, injection of the test solution into the anterior chamber of the eye was performed for a much longer period than would be done with normal eye surgery. Therefore, it was concluded that the composition of the present invention has an effective action on the eye tissue even in clinical application.

Example 2 It is well known that the endothelium, which includes an ion pump and a liquid pump, helps to maintain the cornea in a highly hydrated state and thus to maintain clarity. Endothelial fluid and ion transport are energy-dependent processes. If the donor dies (or the cornea is isolated) and is maintained at 4 ° C for a period of time, the cornea swells due to insufficient energy production at 4 ° C to clean the cornea. Increasing the temperature of the solution increases energy production and causes the cornea to contract. This process is called temperature reversal. Thus, the cleansing activity of this isolated cornea, which is based on temperature reversal, is a measure of the corneal barrier, liquid pump and metabolic function, and the efficiency of exogenous metabolites for the cornea to perform these functions. Is a measure of.

According to the method of the present invention, the weight is 2.5-3.0.
kg Male New Zealand Albino (New Zea)
Land albino) rabbits were killed by overdosing with pentobarbital intracardiacally and kept in a cold room at 4 ° C, in some cases eyes closed with tape for 24 hours, in other cases 4
After being closed for 8 hours, swelling of the cornea was induced and a decrease in metabolic activity was induced. 1.5mm scleral ri
m) was excised, lightly washed in Dulbecco's PBS and coo β-vision viewing chamber (Coope) containing 22 ml of the solution of the invention shown in Table 1.
r VisionViewing Chamber). The corneal thickness was measured with a digital thickness gauge connected to a reflection microscope. "Corneal thickness vs. time" was plotted to determine the corneal lavage activity according to the tissue contraction rate.

For comparison, instead of this solution, BSS-
The same method was performed using a plus.

It is noted that this experimental method is similar to the in vivo wash process after the donor cornea has been transplanted into the recipient.

FIG. 5 shows a typical “corneal thickness vs. time” plot of rabbits swollen in vitro at room temperature (22-23 ° C.) in the solutions of the invention shown in Table 5 above. Tissues were isolated from rabbits 24 or 48 hours after their death. The cleaning activity according to the contraction rate of the cornea (6 each) measured 24 and 48 hours after death was 137.3 ± 5.4 μm / hour and 63.3 ± 6.7 μm, respectively.
/ Hour.

As shown in FIG. 6, in the presence of BSS-plus, the rate of contraction of the isolated corneas (6 each) was 89.7 ± 24% at 24 and 48 hours after death, respectively.
The values were 6.4 μm / hour and 30.3 ± 3.8 μm / hour. This rate was 35% and 50% lower, respectively, compared to that observed in the presence of the solution of the invention (FIG. 5).

The above results indicate that the compositions of the present invention are superior to BSS-Plus in terms of efficiency as an energy source for fulfilling the physiological and biochemical functions of tissues (and cells). ing. This result further supports the results of the experiment described in Example 1.

Example 3 Chen et al. (US Pat. No. 4,873,186)
No., supra), the theory is that when mixed with tissue culture medium, β-hydroxybutyrate is used to preserve isolated cornea, peripheral tissue, the production and accumulation of lactic acid in the preserved tissue is suppressed. At the same time, it was revealed that a high level of energy as ATP was produced by inhibition. To preserve donor corneal viability and endothelial pump function in vivo,
The efficacy of the corneal preservation medium containing the composition of the invention was compared to Optizol (manufactured by Optimir, Irvine, Calif., Manufactured by Cmiron), which is currently the best commercially available product. It is one of the corneal preservation media). Chondroitin sulfate and dextran are contained as a dehydrating agent in optisol in a sufficient amount to exert a colloid osmotic pressure on tissues.

For this experiment, a corneal preservation medium was prepared in which the bicarbonate and balanced salt solutions of TC199 were replaced with the composition of the present invention. A typical one, shown in Table 1, excludes glucose and reduces NaCl by 15 mM. Since TC199 already contains glucose, it is not necessary to add extra glucose. The reduction of 15 mM NaCl is to ensure that the total concentration of cations and anions is the same after replacing the TC199 bicarbonate and balanced salt solutions with the composition of the present invention. Even if the concentrations of these components are changed within the ranges shown in Table 1, it is similarly effective. However, the active osmolality of the solution is 290 mM isotonic, or about 285-300 mOsM.
Should be maintained in the range of 10 to provide the solution with an osmotic effect on the tissue. The active osmolality is adjusted with sugars such as mannitol and sucrose, or neutral polysaccharides such as dextran.

The solution was applied to a 0.22 μm Nalgene filter membrane.
25 m by filtration (Nalge Co., Rochester, NY).
Place in a 1 scintillation vial and store refrigerated at 4 ° C.

According to the method of this experiment, the body weight was 3.0-3.5.
kg Male New Zealand Albino (New Zea)
Land albino) rabbits were sacrificed by intracardiac administration of excess pentobarbital, and corneas with 1.5 m scleral rings were isolated. For comparison, one of a pair of corneas from the same donor was stored in corneal storage medium containing the composition of the invention and another was stored in Optizol. After storage for 7 days in some cases and 11 days in other cases, the donor corneas were removed from the storage medium, and PB from Dulbecco's was removed.
Lightly washed with S. A 7.5 mm button of the donor cornea was cut with a trephine and transplanted into a recipient rabbit. About 30-4 for transplant operation
It took 5 minutes. Measure the corneal thickness of the donor immediately after transplantation, then every 30-60 minutes until 7 hours later, once or twice daily for up to 1 week, and once a week for up to 4 weeks. It was measured.

[0111] In general, isolated corneas swell during storage at 4 ° C, which is reversible if the preserved corneal viability, cellular metabolic activity and endothelial liquid pump function are retained. is there. When transplanted into the recipient, the donor's cornea, which is well preserved in biological function, is rapidly washed and the clarity of the implant is immediately achieved. If not well retained, the donor's cornea is washed slowly and even swells after implantation.

As shown in FIG. 7, the donor corneas stored in the medium containing the composition of the present invention for 7 days after transplantation rapidly (about 16.2 μm / hour, average of 4 transplanted corneas).
The corneas that have been washed and stored for 11 days are washed at a slower rate (approximately 1.4 μm / hr, average of 4 transplanted corneas). The half-life (t _1/2 ) for the cornea stored for 7 days and 11 days after transplantation to return to the normal thickness of about 340 to 370 μm was 2.4 and 33.6 hours (4 times each).
Individual implants) (Table 4).

In contrast to this, as shown in FIG. 8, donor corneas stored in Optizol for 7 days were gently swollen for about 3 hours after transplantation and then about 0.5 μm / hour (4 transplants). Swelling at an estimated rate of (corneal mean). Donor corneas stored for 11 days in Optizol were
It swelled significantly for hours and then at an estimated rate of about 0.4 μm / hour (average of 4 transplanted corneas). The half-life (t _1/2 ) for the cornea stored for 7 days and 11 days after transplantation to return to the normal thickness of about 340-370 μm was 133 and 311 hours (4 grafts each), respectively. Presumed to be present (Table 4).

[0114]

[Table 4]

The results obtained in these in vivo experiments showed that the corneal preservation medium containing the composition of the present invention was significantly more significant than optizole in terms of survival activity of corneal tissue and retention of the liquid pump function of corneal endothelium. It clearly shows that it is excellent.

Thus, the experiments described in Examples 1, 2 and 3 show that the present invention is a unique composition. It is clear that the compositions of the invention are particularly useful for in vitro and in vivo applications as an energy source for peripheral tissues to perform their physiological and biochemical functions and to maintain tissue viability. .

In addition to its clinical properties, the compositions of the present invention have the following properties not possessed by other perfusion solutions such as BSS-Plus:

1) The composition of the present invention serves as an abundant energy source for cells and has a mechanism of suppressing the production and accumulation of lactic acid. 2) The composition of the present invention is chemically more stable than BSS-plus and has a longer life. 3) The composition of the present invention has a lower manufacturing cost than BSS-Plus. 4) The buffer solution (phosphate buffer solution) of the composition of the present invention is stable and has a large capacity. On the other hand, BSS-Plus uses a buffer system of sodium bicarbonate, and the pH changes depending on the CO ₂ partial pressure. 5) The composition of the present invention does not require a mixing step prior to administration and is therefore easy to use.

Although the invention has been described with respect to several specific embodiments, it will be apparent to those skilled in the art that many modifications can be made without departing from the scope of the invention. Therefore, it should be understood that those modifications are also included in the scope of the claims.

[Brief description of drawings]

FIG. 1 shows a pathway of ketone body formation from a fatty acid and a ketogenic amino acid.

FIG. 2 is a BSS as described in Example 1.
Reflection microscopy (s) of cat endothelium after perfusion of the anterior chamber with
It is a photograph of a specific microscope.

FIG. 3 shows the BSS as described in Example 1.
-A reflection micrograph of the cat's endothelium after positive perfusion of the anterior chamber.

FIG. 4 is a reflex micrograph of feline endothelium after perfusion of the anterior chamber with the composition of the invention as described in Example 1.

FIG. 5: “Corneal thickness vs. time” of in vitro deswelling of isolated corneas in the presence of a composition of the invention, as described in Example 2.
Is a plot of.

FIG. 6 shows the BSS as described in Example 2.
-"Corneal thickness vs. time" of in vitro deswelling of isolated corneas in the presence of plus.
Is a plot of.

FIG. 7 shows in vivo transplanted donor corneas pre-stored at 4 ° C. in medium containing a composition of the invention for 7 and 11 days as described in Example 3. 1 is a plot of "corneal thickness versus time" of contraction (deswelling).

FIG. 8: In vivo contraction of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. (Deswelling) is a plot of "corneal thickness versus time".

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 2, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title of Invention Composition for preserving tissue

[Claims]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. TECHNICAL FIELD The present invention relates to a novel composition which is particularly useful as a tissue-rich energy source during the preservation of isolated tissues and during surgery, and at the same time suppresses the production and accumulation of intracellular lactate. It can be used for eye surgery and surgery in general, but for other uses (eg, local administration and preservation and washing of donor tissue prior to transplantation)
Is also possible.

2. Background information Generally, irrigating solutions
n) is used for topical administration to the outer surface of the eye and to the skin, as well as for cleaning tissue during surgery and for maintaining the wet tissue of surgery. However, in eye surgery, replacement of aqueous humor and / or vitreous with perfusate occurs as a result of surgery (corneal transplantation (penetrating corneoplasty), cataract extraction, intraocular lens implantation and vitrectomy) . In these cases, the perfusate solution remains in the eye until the components of the perfusate are absorbed by the surrounding tissue or the solution eventually reaches parallel with body fluids and is cleared into the blood circulation. Therefore, the perfusate used is physiological (tonicity, pH, etc.)
Is not only compatible with at least the anterior chamber (an
terrior chamber and vitreous body (vitre)
It is also necessary to include components that allow the cells to maintain viability and the ability to perform physiological functions until they reach a sufficient equilibrium with physiological fluids.

In eye surgery, the components of the perfusate are of particular importance to the cornea and lens. Neither tissue has blood vessels. The cornea derives its nutrition mainly from the anterior chamber of the eye, and to a lesser extent from tears and limbal vessels. The lens obtains its nutrition from the anterior chamber and vitreous humor. The retina, ciliary body, and iris are vascularized tissues; they derive their nutrition from circulating plasma in the vascular network. Thus, the components of the perfusion solution do not have as significant an effect on these tissues as on the cornea and lens.

In the cornea, a monolayer of endothelium that covers the posterior surface contains liquid and ion transport sites that help maintain the cleanability and thus transparency of the cornea. In the lens, the ion transport site is located in the epidermis under the capsule. Generally, Na + -K + ATPase is present in the cytoplasmic membrane and has intracellular Na + and K + contents of 10 mM and 100, respectively.
Helps maintain at mM, about 120 mM Na +
And promotes transport against a 10 mM K + extracellular salt gradient. Sufficient energy is required for cells (and tissues) to perform this liquid and / or ion transport activity.

Glucose is a major energy source in mammals. The cornea, lens, and retina are highly active glycolytic tissues that utilize glucose to produce lactic acid even under aerobic conditions. When the isolated cornea is stored in a medium containing glucose, excess lactic acid is produced and accumulated and becomes acidic, and the metabolic activity of the tissue is inhibited. In vivo, lactic acid produced in vascular tissues is removed via the blood circulation, and lactic acid produced in non-vascular tissues is released into the anterior chamber of the eye and removed by the clearance mechanism of blood circulation. In humans, anterior chamber lactate is maintained at a constant low level of approximately 4.0-4.5 mM.

Glucose is an important and useful energy source for tissues when present in solution for in vivo administration.
The utilization of glucose by the glycolytic system produces ATP at a high rate, which is independent of O ₂ . However, this is not an efficient energy production pathway, only 2 moles of ATP are produced when 1 mole of glucose is utilized. At this time, 2 mol of lactic acid is also produced. When the tissue clearance mechanism of the operated eye is not sufficiently effective, lactic acid accumulates and the pH drops, resulting in inhibition of tissue metabolic activity. In contrast, in tissues with high mitochondria, complete oxidation of glucose to CO ₂ and H ₂ O occurs via mitochondria, and 38 mol of ATP is produced for consumption of 1 mol of glucose (glycolysis). Including the oxidation of 2 moles of NADH produced in the system). It is therefore clear that utilization of substrates by mitochondrial oxidation is an efficient energy production pathway when oxygen is available. Lactic acid is not produced under these conditions. Furthermore, when anaerobic glycolysis is inhibited via the Pasteur effect due to hyperventilation, lactic acid accumulation is reduced or minimized (Krebs, HA, Essays. Essays Biochem. No. 8
Vol. 1, p. (1972)).

Glucose is not directly utilized as a substrate in mitochondria, but must first be converted to pyruvate by glycolysis. Ketone bodies and their precursors (eg short chain fatty acids and ketogenic amino acids) are immediately oxidized in mitochondria, producing 32 moles of ATP for every mole of acetyl molecule utilized. Thus, ketone bodies and their precursors are energy-rich or energy-efficient molecules. In addition, oxidation of ketone bodies and their precursors enhances respiration, which inhibits anaerobic glycolysis via the Pasteur effect.

The ketone body is acetone (CH3 COCH
3), acetoacetate (CH3 COCH2 COO
-) And β-hydroxybutyrate (CH3CHO
HCH2 COO-) is a general term. The pathway for the formation of ketone bodies from fatty acids and ketogenic amino acids is described in FIG. Ketone bodies are metabolites of fatty acids and ketogenic amino acids in tissues, including leucine, lysine, phenylalanine, tyrosine and tryptophan. In nature, fatty acids have the general formula CH3 (CH2) n
It is represented by COOH (n is an even integer). Acetic acid is the smallest fatty acid molecule with n = 0. Ketone bodies are stable forms of high-energy metabolites of acetyl-CoA for storage and release in the body. That is, ketone bodies are immediately produced in vivo from fatty acids and ketogenic amino acids. Furthermore, β-hydroxybutyrate and acetoacetate are converted to each other via the catalysis of NAD + -linked β-hydroxybutyrate dehydrogenase:

Embedded image Acetoacetate + NADH + H + = D-β-hydroxybutyrate + NAD +

These indicate that ketone bodies, fatty acids, and ketogenic amino acids are interrelated molecules. When energy is needed, the ketone bodies and their precursors are utilized in the tissue to produce acetyl CoA,
It is further oxidized in the mitochondria via the Krebs cycle to CO ₂ and H ₂ O, producing high energy in the form of ATP.

When the precursors of ketone bodies (fatty acids and ketogenic amino acids) are administered (in humans and animals), acetyl CoA is produced, which is further oxidized to produce high levels of ATP. When acetyl-CoA is overproduced, ketone bodies are produced, the main metabolic site of which is the liver. Of these ketone bodies produced, acetone is not utilized and is excreted from breath, sweat and urine. On the other hand, acetoacetate and β-hydroxybutyrate are released to peripheral tissues where they are converted to acetyl CoA and further oxidized in the mitochondria to CO ₂ and H ₂ O through acetyl CoA and the citric acid cycle, and are abundant in peripheral tissues. Supply the perfect energy.

In peripheral tissues, acetoacetate is also produced from ketone-forming amino acids. When acetoacetate is present in excess, it is converted to β-hydroxybutyrate for storage, or converted to acetyl-CoA and further oxidized in the mitochondria to CO ₂ and H ₂ O, resulting in high levels. Produces levels of ATP. In peripheral tissues, fatty acids are decomposed into acetyl-CoA via β-oxidation (see Figure 1), which is further oxidized to CO ₂ and H ₂ O in mitochondria to produce high levels of A.
Produces TP.

The cornea, lens and retina have high glycolytic activity and metabolize glucose to produce a large amount of lactic acid. Under normal conditions glucose is the energy source of eye tissue in vivo. The lactic acid produced is removed by a clearance mechanism via the blood circulation. During or immediately after eye surgery, the mechanism of lactic acid removal by blood circulation is ineffective. Excess lactic acid accumulates in the anterior chamber of the eye and the vitreous when glucose is included in the perfusion solution as an energy source for eye tissue during eye surgery. Utilization of glucose lowers extracellular pH, which causes accumulation of lactic acid in cells (and tissues) and inhibits metabolic activity (Chen and CH and Chen, S. et al.
C. ), Arch Biochem Biofiz (Arch.B)
iochem. Biophys. ) Volume 276, page 70 (1990)). This problem is solved by adding ketone bodies and their precursors into the perfusion solution as an alternative energy source for eye tissue. Ketone bodies and their precursors are immediately utilized in the tissues of the eye to form acetyl CoA, which in the mitochondria is CO ₂ via the Krepp cycle.
And it is oxidized in _{H 2} O to produce high levels of ATP. This process inhibits glycolysis by Pasteur effect and suppresses lactic acid production.

Furthermore, ketone bodies are known to be a suitable energy source for the starved brain, muscles and kidneys (Olamn RE, Nature.
195: 597, 1962; Bassenge, E., et al., American Journal of Physiology (Am. J. Phus).
iol. ) 208, 162, 1965; Owen, OE, et al., Journal of Clinical Investigation (J. Clin. In.
vest. ) 46, 1589, 1967; and Hawkins, RA, et al., Biochemistry Journal (Biochem. J.) 1st.
22:13, 1971, and 125: 54
P. 1, 1971).

The compositions of the present invention are not solely intended to function in eye surgery as artificial fluids that replace eye fluids or body fluids in general surgery, or as perfusion solutions themselves. Rather, it is an object of the present invention to provide ketone bodies and precursors thereof during storage of isolated tissue and as a temporary high energy source of tissue during and after surgery, at least the anterior chamber of the eye. And allowing the cells to perform viability and physiological and biological functions until the vitreous is in sufficient equilibrium with physiological fluids.

That is, the ketone body and its precursor and glucose in the balanced preparation are particularly useful for isolated tissues during storage and peripheral tissues during surgery. Glucose is
It serves as an energy source for tissues with little or no mitochondria (eg, corneal stroma and lens). Ketone bodies and their precursors are rich in energy. These are CO ₂ and H ₂ O in mitochondria through the Krebs cycle.
It is immediately oxidised to produce no other metabolic waste. The generated carbon dioxide is hydrated to carbonic acid,
It is further oxidized to bicarbonate at pH 7.4.
It has been suggested that bicarbonate is required for the process of ion transport across the corneal endothelium (Hodosi (Hodosi
n, S. ) Et al., Journal of Physiology (J. Physiol.) 263, 563, 19
1976). In addition, the oxidation of ketone bodies and their precursors not only produces high levels of ATP, but also suppresses glycolysis via the Pasteur effect and consequently inhibits lactic acid production.

In the present invention, the balanced salt (bala)
nced salts) are included in the components to produce a physiologically compatible solution for in vivo administration. Phosphate in the component is a component of oxidative phosphorylation of ATP production process when the ketone body and its precursor are oxidized. Furthermore, phosphoric acid acts as a buffer having a high buffering capacity in the physiological pH range of pH 7.2-7.4.

The theory and intended application of the invention is based on the beach (Veec) described in US Pat. No. 4,663,289.
It is different from the theory of h). The invention of Beech teaches that the metabolic processes of living animal cells are one or more fixed ratios [HCO ₃ −] / [CO ₂ ], [L-lactate-] / [pyruvate- ], And the [β-hydroxybutyrate] / [acetoacetate] pair, which is based on the theory that p
The effect is similar to a "weak acid / conjugated base" paired buffer system for adjusting H.

Vech's theory is based on several hypotheses: Both a fixed ratio of both substrates are taken up by cells or tissues. 2. A proportion of the substrate (in the tissue) that accumulates then lodges in living cells to control metabolic activity. 3. Substrate uptake and metabolic regulation by these substrate pairs is similar in all tissues.

Many of the experiments that underlie the invention of Vech were carried out in rat liver (Vech et al., Biochemistry Journal (B).
iochem. J. ) Volume 115, p. 609, 1969
Year; and Vech et al., Journal of Biological Chemistry (J. Biol Che.
m. ) 254, 6538, 1979). The liver is the center of metabolism in humans and animals. The metabolic processes in the liver are different from those in peripheral tissues. Thus, the mechanisms that control metabolic processes may work in the liver, but not necessarily in peripheral tissues. For example, in the liver, ketone bodies are produced through β-oxidation of fatty acids.
The ketone bodies produced are not used in the liver, but are transported to the peripheral tissues via the blood circulation. Here they are coupled to the production of high levels of ATP via the Krebs cycle
₂ and H ₂ O (the principle of biochemistry (Princi
Ples of Biochemistry, Lehninger, Worth Publishers, In
c. )). That is, β-hydroxybutyrate is an efficient alternative energy source for peripheral tissues, but not a liver alternative energy source. The present invention extracts β-hydroxybutyrate without conjugating it with acetoacetate and metabolizes it to produce ATP, and at the same time suppresses the production and accumulation of lactic acid in cells. Utilize activity.

The lactic acid produced is released into the blood by normal cells actively degrading sugars (eg erythrocytes, skeletal muscle, cornea and retina) and then extracted from the blood by the liver and heart and metabolized. To be done. Lactate is translocated through the cytoplasmic membrane with pyruvate as a competitive inhibitor (Spencer and Lenninger (Spence).
rand Lehninger), Biochemistry Journal (Biochem. J.) 154, 40.
P. 5, 1976). Ketone bodies are produced in the liver and released into the blood, then extracted and metabolized from the blood by peripheral tissues (eg cornea and lens) (Principles of biochemistry (Pr
inciples of Biochemistr
y), Leninger, Worth Publishers
s, Inc. )). The transport mechanism of ketone bodies through the cytoplasmic membrane has not been established yet.

The metabolism of pyruvic acid, lactic acid, acetoacetate, and β-hydroxybutyrate varies depending on cells or tissues. Heart and liver lactate dehydrogenase (LDH)
Isozymes promote the oxidation of low concentrations of lactate to pyruvate; corneal, lens, and retinal LDH isozymes help reduce low concentrations of pyruvate to lactate. Β-hydroxybutyrate dehydrogenase (BDH) in liver
Isozymes promote the reduction of low concentrations of acetoacetate to β-hydroxybutyrate; BDH isozymes in peripheral tissues (eg corneas) promote the oxidation of low concentrations of β-hydroxybutyrate to acetoacetate.
Different LDH and BDH isozymes in different tissues, substrate-pairs (eg [lactate] / [pyruvate] or [β-hydroxybutyrate] / [pyruvate] and [β-hydroxybutyrate] ] / [Acetoacetate]) makes it difficult to establish a general ratio for all tissues. Food intake and other metabolic reactions Substrate uptake by mimicking, continuous influx of these chemicals, and removal of these chemicals by efflux and other metabolic reactions further establish a general substrate-to-pair ratio. It's getting harder.

In the application of culture media for tissue preservation, the theory of Lindstrom et al. (US Pat. No. 4,695,536) was basically used by scientists in the field of tissue culture for many years. It is a tissue culture technology. Specifically, Lindstrom et al. Teach a method of corneal preservation consisting of the following methods:

(A) Earls salt
s) containing 50 ml of basal medium Gibco '
s) minimal basic medium, 25 m without L-glutamine
M HEPES buffer, final concentration of medium is 1
% Of L-glutamine (200 mM) and 50-100 μg of garamyci.
Corneal preservation medium containing antibiotics containing n); serum from the group of bovine serum, fetal bovine serum or human serum; and chondroitin sulfate, sodium hyaluronate, keratan sulfate, polyvinyl-pyrrolidone, methylcellulose, hydroxypropylmethylcellulose, cellulose gum. And an effective amount of high molecular weight molecules selected from the group consisting of dextran; (b) filling a corneal storage container with mixed medium; and (c) a cornea-sclear ring in a container containing medium.
1-scleralrim) and maintained the medium at 4-3
Maintaining a temperature of 4 ° C allows for medium and long term storage, and the system allows for changes in temperature ready for tissue transport.

This theory is based on the fact that the isolated cornea is
Kaufman (MacCarey-Kaufmann) medium (tissue culture medium 199 plus 5% dextran; McCray and Kaufmann.
Kaufman, H .; ), Invest. Ophthalmol. Volume 13,
165, 1974) with or without bovine serum supplementation (Lindstrom).
, American Journal of Off Salmology (A
m. J. Ophthalmol. ) Volume 95, 869
(1977, 1977) do not describe any mechanism by which the production and accumulation of lactic acid is inhibited when cultured or stored. MacCarey et al.
Ofsalmol (Invest. Ophthalmo
l. ) Volume 13, p. 165, 1974)
Dextran is added to tissue culture medium (TC199) as a dehydrating agent, which makes the active osmolality of the medium slightly hypertonic (about 305-340 mOsM). Dextran in this slightly hypertonic solution exerts a colloid osmotic pressure on the stored cornea, giving an artificial dehydration effect.
However, the presence of dextran in the medium has no inhibitory effect on lactate production and accumulation.

In stark contrast to this, the Che
n) et al. (US Pat. No. 4,873,186)
Isolated corneas are incubated with Dulbecco's phosphate buffered saline (PBS) containing β-hydroxybutyrate or in TC199 containing β-hydroxybutyrate (also known as medium 199). Describe the mechanism by which high levels of ATP are simultaneously produced in tissues and the production and accumulation of lactic acid are inhibited when stored in. Specifically, Chen et al. Disclose a method of prolonging the time that an isolated cornea of surgical nature is stored, which method is used in a corneal storage medium containing the stored cornea. , (1) short chain fatty acids, (2) ketone bodies, and (3) a ketogenic amino acid capable of inhibiting lactic acid production by the isolated cornea in an amount sufficient to inhibit lactic acid production, or Containing an amount of at least one compound selected from the group consisting of ketone body precursors.

The corneal preservation medium is TC1 containing Hank's salt.
Formulated by replacing the bicarbonate balanced salt solution portion of a tissue preservation medium such as 99 with the composition of the present invention. In this case, the NaCl concentration must be reduced in order to keep the total concentration of anions and cations physiologically compatible. As a result, the components in the tissue culture medium other than NaCl are not completely decomposed, and the active osmolality is slightly hypotonic. The active osmolality of the solution is isotonic at 290 mOsM, or 285-300 m
Must be in the OsM range. Sugars permeable to the plasma membrane (eg mannitol, sucrose and dextran) are used for adjusting the osmolality. The use of these does not exert a colloid osmotic pressure on the stored cornea because the solution is isotonic. Therefore, MacCare
y) et al. (MacCarey et al., Invest. Ophtha.
1 mol. ) Vol. 13, 165, 1974) is completely different from the intended application of dextran.

Β-hydroxybutyrate is a reducing agent. In solution, it is oxidized by O ₂ especially at elevated temperatures. Therefore, it is necessary to use degassed water to make the compositions of the present invention. The resulting solution is stored under vacuum and β
- prevents hydroxybutyrate is oxidized by O _2, can be extended resulting life. If the solution is allowed to equilibrate with air when opening the bottle for clinical applications, O ₂ will be available for the biological function of the tissue (or cells).

Alternatively, β-hydroxybutyrate is O ₂
Antioxidants of the group consisting of citric acid, phenylalanine and vitamin E may be used to prevent oxidization by the. The preferred antioxidant is citric acid, and the concentration of citric acid in the solution is in the range of 8-12 mM. However, upon long-term storage, O ₂ in solution also oxidizes the antioxidant, reducing the effectiveness of the antioxidant in preventing β-hydroxybutyrate from being oxidized by O ₂ . Therefore, the use of degassed water is the preferred method for making the compositions of the present invention.

[0029]

SUMMARY OF THE INVENTION The present invention relates to a novel composition comprising a ketone body and / or its precursor, glucose, a phosphate buffered balanced salt aqueous solution. The present invention also provides a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution in water to preserve the isolated tissue described above and to efficiently and physiologically reproduce the tissue during surgery. It relates to a process for preparing a composition, which comprises mixing in an amount sufficient to form a composition which effectively meets the requirements for its chemical function.

FIG. 1 shows a pathway of ketone body formation from a fatty acid and a ketone-forming amino acid.

FIG. 2 shows B as described in Example 1.
FIG. 7 is a reproduction of a photograph of the feline endothelium after perfusion of the anterior chamber with SS by spectroscopic microscopy.

FIG. 3 shows B as described in Example 1.
FIG. 6 is a reflection microscopy photocopy of the cat endothelium after perfusion of the anterior chamber with SS-plus.

FIG. 4 is a reflection microscopy photocopy of the cat endothelium after perfusion of the anterior chamber with the composition of the present invention as described in Example 1.

FIG. 5 shows the “corneal thickness vs. time” of in vitro deswelling of isolated corneas in the presence of the composition of the invention, as described in Example 2. It is a plot.

FIG. 6 shows B as described in Example 2.
FIG. 4 is a plot of “corneal thickness versus time” of in vitro deswelling of isolated corneas in the presence of SS-plus.

FIG. 7: In vivo contraction of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. (Deswelling) is a plot of "corneal thickness versus time". FIG. 8 shows in vivo deswelling of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. "Corneal thickness versus time" is plotted.

Broadly stated, the composition of the present invention comprises a ketone body and / or its precursor, glucose,
And a phosphate buffered balanced salt solution.
The composition of the present invention comprises its components (particularly the above-mentioned ketone bodies and / or its ketone bodies) so that the requirements for efficient physiological and biochemical functions of peripheral tissues of the eye and pond can be effectively met. Utilizes some of the special properties of precursors, glucose, and phosphate buffered balanced salt solutions.

Accordingly, the composition of the present invention comprising a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution in water, with or without a high density of mitochondria (or no mitochondria at all). Case), or generally with or without other tissues, to meet the minimum essential nutritional requirements of eye tissues. For example, eyes and other peripheral tissues perfused with the compositions of the present invention after surgery have energy source-dependent metabolic functions (eg, fluid and ion transport and protein synthesis) and physiological functions such as maintaining a thin, transparent cornea. Can be executed.

Therefore, the composition of the present invention can be used, for example, for cornea 3
It provides a well-balanced and abundant source of energy for eradicated tissue represented by three different organizational layers. That is, the composition of the present invention has a need for high respiratory activity of endothelium, a need for high glycolytic activity of endothelium and stroma, and a need for achieving basic intracellular energy level and ion transport of corneal fluid. Can meet.

Representative compositions of the present invention suitable for use as a perfusate solution, along with their component ranges, are set forth in Table 1 below.
Described in.

[0041]

[Table 1]

Representative anions and cations included in the compositions of the present invention are set forth in Table 2 below. Table 2 also shows the concentration of these ions in the composition of the invention.
The calculated osmolarity of the composition of the present invention is about 300 to about 320 mO when all salts are completely dissolved.
The range is sM. The actual osmolality of the prepared solution of the composition of the present invention is about 285 to about 300 mOsM.
Is the range.

[0043]

[Table 2]

According to one embodiment of the present invention, the ketone bodies of the composition of the present invention are β-hydroxybutyrate ion and acetoacetate ion. β-hydroxybutyrate ion is the D-isomer of β-hydroxybutyrate, β
Selected from the group consisting of a mixture of D-racemic and L-racemic hydroxybutyrate, and a mixture of D- and L-isomers of β-hydroxybutyrate, most preferably β-hydroxybutyrate Is the D-isomer. The concentration of β-hydroxybutyrate is preferably 5-
Preferred ketone body precursors of the composition of the present invention in the range of 30 mM are fatty acids selected from the group consisting of acetic acid and butyric acid, and preferred ketogenic amino acids are leucine, lysine, phenylalanine, tyrosine and tryptophan. Is selected from the group consisting of:

The concentration of the precursor of the ketone body is preferably 0.
It is in the range of 1-25 mM. The concentration of the ketogenic amino acid is preferably in the range of 0.1-5.0 mM,
The most preferred range is 1-2 mM. The ketogenic amino acid is preferably at a total concentration of 7.5-12.5.
mM, most preferably the total concentration is 10 mM.

The preferred fatty acid is acetic acid, preferably 1
A concentration in the range 5-25 mM, or butyric acid,
The concentration is preferably in the range of 5-15 mM. The concentration of acetic acid is most preferably 20 mM and the concentration of butyric acid is most preferably 10 mM.

The pH of the composition of the present invention is preferably 7.3.
Adjusted to -7.5.

To prevent the oxidation of β-hydroxybutyrate by O ₂ and prolong the life of the solution at room temperature, it is preferred to use degassed water to make the compositions of this invention.

A portion of NaCl, preferably in the amount of 15-35 mM, or most preferably 25-30 mM, is
Glucuronic acid, D-galacturonic acid, D-mannuronic acid,
One or more sugar acids selected from the group consisting of D-gluconic acid and D-glucaric acid (sugar)
acid) equivalent Na + salt.

A sufficient amount of 40-to form a composition that simultaneously prevents adhesions that occur during eye surgery and effectively meets the requirements for efficient physiological and biochemical function of the eye tissue. The addition of neutral polysaccharide dextran in an amount in the range of 500 kilodaltons increases the viscosity of the composition according to the invention. Suitable concentrations of neutral high molecular weight dextran are in the range 2% -20%.

In another aspect, the invention provides a composition of the invention, Eagle's medium, Dulbecco's medium and Daniel.
iel's) consisting of a member of the minimum essential amino acids and the minimum of essential vitamins selected from the group consisting of
It relates to a corneal preservation medium. This solution is commercially available and, as will be apparent to those skilled in the art, the amount added will vary depending on the particular conditions.

In another aspect, the invention relates to a corneal preservation medium in which a balanced salt solution of tissue culture medium has been replaced with a composition of the invention; the total concentration of cations and anions in the solution is balanced. To maintain the same concentration as the saline solution, the NaCl concentration is preferably reduced to within the range of 70-100 mM, or most preferably 85 mM; the active osmolarity of the solution.
Is adjusted to isotonicity of 285-300 mOsM or most preferably 290 mOsM; mannitol, sucrose and dextran are added if necessary. The tissue culture medium is preferably TC199 with Hank's salt or Eagle's minimal minimal medium with Hank's salt.

The pH of the corneal preservation medium is preferably 7.10.
-7.50 range, most preferably 7.25-
The range is 7.40.

15-35 mM, or most preferably 2
A portion of NaCl in the amount of 5-30 mM is equivalent to one or more sugar acids consisting of D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid. It is replaced with Na + salt.

In another aspect, the invention provides that the phosphate buffered balanced salt solution of the composition of the invention comprises a tissue culture medium suitable for corneal preservation but in which unfavorable lactic acid production normally occurs. It relates to a corneal preservation medium replacing the bicarbonate buffered balanced salt solution of the corneal preservation composition. Furthermore, the corneal preservation medium contains at least one compound selected from the group consisting of short-chain fatty acids and ketone bodies capable of inhibiting the production of lactic acid by the cornea and present in an amount sufficient to inhibit the production of lactic acid. Including.

In yet another aspect, the invention comprises a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor.
It relates to a corneal preservation composition.

In yet another aspect, the present invention provides for the preparation of tissue for organ transplantation (eg corneal transplantation), which comprises the composition of the present invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor. For formulations. In yet another aspect, the invention relates to a perfusate solution, eg, for topical administration, comprising a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum derived factor.

In yet another aspect, the invention comprises a corneal preservation medium of the invention and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor.
For example, it relates to an organ medium for administration.

The method of preparing the solution of the present invention provides a sufficient amount of the composition to form a composition that effectively meets the requirements for efficient physiological and biochemical function of the eye and other peripheral tissues described above. Mixing glucose and ketone bodies and / or their precursors in a degassed phosphate buffered saline solution. This solution is bottled under vacuum to completely remove O ₂ to prevent β-hydroxybutyrate from being oxidized by O ₂ when stored at room temperature for extended periods. If this solution is used immediately or for a short period of time, bottling under vacuum is useful,
Not necessarily required.

Since high concentrations of Ca ²⁺ and Mg ²⁺ produce phosphate precipitates, all components except CaCl ₂ and MgCl ₂ must first be thoroughly degassed with water (preferably 90-95% of total volume). ) It is preferable to dissolve it. Mix the solution thoroughly and p. With 1N D-glucuronic acid or NaOH.
Adjust H to be in the range 7.3-7.5. Then 0.5 M stock solution (or 0.2-1.0 M range) of CaCl ₂
And MgCl ₂ are added. Check the pH and adjust if necessary to 7.3-7.5, preferably 7.4. Finally, add water to adjust the volume. Deionized doubly distilled water is preferably used to prepare this solution. All ingredients used are of the reagent grade.
e).

Several variations are possible in the preparation of the perfusion solution of the present invention. Some of them are described below.

1. The phosphate buffer is prepared using dipotassium phosphate and monopotassium phosphate. In this case, the total potassium ion concentration increases to 15 mM.

2. Phosphate buffer is prepared using disodium phosphate and monosodium phosphate. In this case 8-
12mM KCl must be added, NaCl is 5
mM subtracted.

3. Phosphate buffer is prepared with any other phosphate and phosphoric acid and the pH is adjusted appropriately to 7.4. In each case, the final concentration of K + should be 8-12 mM. The pH can vary in the range 7.3-7.5.

4. Substituting some of the NaCl with Na + salts of one or more sugar acids including D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid to give chlorine. Concentrations should be maintained within the physiological range of 90-105 mM.

5.0.1-5.0 mM, preferably 1-
One or more ketogenic amino acids consisting of 2 mM leucine, lysine, phenylalanine, tryptophan and tyrosine can be added as a component of the perfusion solution. Subtract equal amounts of NaCl to adjust Na ⁺ concentration.

6. 2 part of β-hydroxybutyrate
It can be replaced with double the amount of acetic acid (the smallest molecule of the ketone body precursor short chain fatty acid).

It is also possible to use the sodium salts of other short chain fatty acids in the range of 7.10 mM, or 5-15 mM, such as butyric acid and caproic acid. However, the use of these aqueous solutions is limited due to their low solubility.

8. The β-hydroxybutyrate can be replaced with acetate or acetoacetate, with β-hydroxybutyrate being preferred. β-hydroxybutyrate is stable and inexpensive. It is also readily utilized by cells and is more energy efficient than acetoacetate. One mole of β-hydroxybutyrate produces 64 moles of ATP and one mole of NADH, which produces an additional 3 moles of ATP.

9. Since only D-isomers are used in cells, the D-isomer of β-hydroxybutyrate is preferred as the composition of the invention. However, the D-, L-racemate or the D-, L-isomer mixture is also low in price, and commercial products are readily available, and these can also be used.

10. If desired, sugars such as mannitol and sucrose or neutral polysaccharides such as dextran can be used to adjust the osmolality of the solution to the range of 290 mOsM, or about 285-300 mOsM. In such adjustment, Na ⁺ concentration and Cl ⁻
The concentration does not change. If both Na ⁺ and Cl ⁻ concentrations are too high, an equal amount of sugar should be used instead of NaCl.

11. 5 to increase the viscosity of the solution
% Or 2-20% neutral high molecular weight dextran (preferably 70-500 kilodaltons) can be used. Very high viscosity compositions are used to prevent adhesions resulting from surgical wounds in eye surgery, such as the lens or iris adsorbing to the corneal endothelium or the iris adsorbing to the lens. It is useful. in this case,
NaCl is 2.5m per 1% of dextran added
Only M is subtracted.

The compositions of the present invention are primarily used during the interim period after surgery in the eye, such as corneal transplants, intraocular lens transplants, cataract extractions, and vitrectomy, to remove eye and other eradication. It is formulated with the minimum of essential ingredients used to enable the tissue (or cells) to maintain their viability and ability to perform physiological functions. By interim period is meant the time after surgery until the solution in the anterior chamber and / or vitreous reaches sufficient equilibrium with the body's physiological solution. As described above, the composition of the present invention can be used for topical administration to, for example, the skin and eyes, preservation and lavage of donor tissue before transplantation, and general surgery.

The composition of the present invention contains a physiologically compatible salt solution whose concentration range is similar to that found in plasma. In the present invention, phosphate (typically 10 mM) can be used both as a buffer and as a substrate for ADP phosphorylation for the production of ATP in energy metabolism. Phosphate has a pH of 7.2-
It has a strong buffering capacity at 7.4. Bicarbonate is used in other perfusion solutions currently on the market (e.g. BSS-Plus from Alcon, Fort Worth, Tex.). However, when a bicarbonate buffer is used, bicarbonate is not used as a component of the composition of the present invention because the CO ₂ partial pressure (P _co2 ) changes the pH of the solution. Further, without the addition of bicarbonate from the outside, bicarbonate is generated by the respiration (that is, oxidative phosphorylation) of the tissue itself. In this case, this substrate is oxidized (in mitochondria) to C
O ₂ and H ₂ O are produced. Next, the generated CO ₂ is hydrated to H ₂ CO ₃ , which is further decomposed into H ₂ CO ₃ .
CO ₃ ⁻ is produced. Rabbit corneas produce CO ₂ at a rate of about 1.3 μmol / cm ² / hour. In the human cornea, this rate is 30-50% higher.

In mitochondria, β-hydroxybutyrate is immediately oxidized to acetoacetate and NADH by the catalytic action of β-hydroxybutyrate dehydrogenase. Acetoacetate is also succinyl CoA
React with to produce acetoacetyl CoA, which is further degraded by thiophorase to produce acetyl CoA. In cells, fatty acids are utilized by β-oxidation to produce acetyl CoA.
Ketogenic amino acids are also assimilated in cells to produce acetoacetate or acetoacetyl CoA, which in turn produces acetyl CoA. Acetyl CoA from these reactions is then oxidized to CO ₂ and H ₂ O in the mitochondria via the citric acid cycle. The pathway for the formation of ketone bodies from fatty acids and ketogenic amino acids is briefly described in FIG. Oxidation of acetyl-CoA is an energy-efficient process, and 30 moles of ATP and 2 moles of GTP (ie 32 moles of ATP in total) are produced for the use of 1 mole of acetyl CoA. Inorganic phosphate is the source of oxidative phosphorylation, which is coupled to the electron transport chain and reacts with ADP to produce ATP.

As is clear from the table below, about 47% of the glucose consumed in the cornea is glycolytic and 53% is respiratory (Chen and Chen (Ch
End and Chen), Ark Biochem. Biophys. 276, 70, 1990).

[0077]

[Table 3]

The% glucose consumption in the corneal tissue layer is shown in brackets in Table 3 above. The interstitium consumes glucose by the glycolytic system at a rate twice as fast as respiration. In the lens, mitochondria are present only in the epidermis. Therefore, glucose is an important source of energy in the stroma and lens (especially cells within the nucleus of the lens). There is little or no mitochondria utilizing acetyl CoA in these cells. In other eye tissues, such as the mitochondrial dense corneal endothelium and photoreceptors, ketone bodies and their precursors are energy efficient substrates. Moreover, in these tissues, the oxidation of acetyl-CoA causes an increase in respiration, which inhibits the anaerobic glycolysis system by the Pasteur effect.

Thus, the composition of the present invention comprising a ketone body and / or its precursor, glucose, and a phosphate buffered balanced salt solution may or may not have a high density of mitochondria, or no mitochondria. Meet the minimum essential nutritional requirements of absent eye tissue, or other peripheral tissue in general.

The composition of the present invention is used as a simple corneal preservation composition suitable for short-term administration as disclosed by Chen et al. (US Pat. No. 4,873,186, cited above). can do. According to Chen's theory, at least one compound selected from the group consisting of short-chain fatty acids and ketone bodies is mixed with a tissue culture medium to simultaneously inhibit the lactate production caused by the cornea and increase high energy. Used for storage of the isolated cornea to produce.

According to one aspect of the present invention, the composition of the present invention, essential amino acids, and Eagle's medium (Science, Vol. 130, p. 432, 1959).
Year; Proc. Sok Expe Biol
c. Exp. Biol. ) Vol. 89, 362, 195
5 years, Dulbecco's medium (virology
gy) Vol. 8, 396, 1959 and Vol. 12,
185, 1960) or Daniel's medium (Proc.
Sok Expe Biol (Proc. Soc. Exp.
Biol. ) Vol. 127, p. 919, 1968), a corneal preservation medium comprising the essential vitamin is prepared. Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer solution 20-30 mM
Is effective in increasing the buffering power. Pre-prepared minimal essential amino acids and essential vitamins are commercially available. Essential amino acids and vitamins in solution are generally relatively short-lived and stored in cold places for short periods (usually 6-1
Since it has to be used within 2 months), the use of degassed water is effective but not always necessary for the preparation of the composition of the present invention used as a corneal preservation medium.

In a second aspect of the invention, the corneal membrane is replaced by replacing the balanced salt solution of a tissue culture medium such as TC199 containing Hank's salt or Eagle's minimal basal medium containing Hank's salt with the composition of the present invention. The storage medium is formulated. To maintain the total concentration of cations and anions in the medium at physiologically compatible levels, NaCl
The concentration is reduced within the range of 70-100 mM. The solution should be maintained isotonic at 290 mOsM, or within an active osmolality range of about 285-300 mOsM. The active osmolality is adjusted with sugars such as mannitol and sucrose, or neutral high molecular weight polysaccharides such as 40 and 70 kilodaltons dextran.

In a third aspect, Chen
Replacing the bicarbonate balanced salt solution of the corneal preservation composition of the theory of et al. (US Pat. No. 4,873,186, supra) with the phosphate buffered balanced salt solution of the composition of the present invention. The corneal preservation medium is prepared by. 15-35m
M, preferably a portion of NaCl in an amount of 25-30 mM, and one or more sugar acids including D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid. It is preferred to maintain the Cl ^- concentration in the physiological range of 90-105 mM by substituting it with the sodium salt of. In the theory of Chen et al., At least one compound selected from the group consisting of short chain fatty acids and ketone bodies is mixed with tissue culture medium and used for corneal preservation. The bicarbonate buffered balanced salt solution is part of the tissue culture medium component.

In a fourth embodiment, a suitable corneal preservation composition is described by Chen et al. (US Pat. No. 5,073,
No. 492) in combination with a synergistic composition as described above, consisting of the formulation of the above-mentioned first, second or third aspect. In the theory of Chen et al., A synergistic composition is basically
It consists of a synergistically effective mixture of vascular endothelial growth factor (VEGF), uridine, thymidine and serum derived factor (SDF). The stoichiometric ratio of uridine to thymidine is 10 μM uridine to 0.5 μM thymidine. Typically, dialyzed fetal bovine retina extract (0.1 mg / ml) and fetal bovine serum (3 mg / ml) were added to VEGF and SD, respectively.
Used as a source of F.

In a fifth aspect, the composition of the present invention is combined with the synergistic composition of Chen et al. (US Pat. No. 5,073,492) described in the fourth aspect to produce organs. Formulations are prepared for the preparation of tissues for transplants (including corneal transplants).

In a sixth aspect, the corneal preservation medium of the first or second aspect described above is the same as that described in the fourth aspect by Chen et al. (US Pat. No. 5,073,492).
No.) synergistic composition to produce a perfusion solution for topical administration to stimulate endothelial cell regeneration.

In the seventh embodiment, the corneal preservation medium of the first or second embodiment described above is the same as that described in the fourth embodiment by Chen et al. (US Pat. No. 5,073,492).
No.) synergistic composition to produce a tissue culture medium for administration to accelerate the corneal endothelial cell regeneration process prior to corneal transplantation.

The solutions of the present invention were prepared in a degassed double distilled water volume equal to about 95% of the total volume, with the exception of the Ca ²⁺ and Mg ²⁺ salts, in the exact amounts indicated in the preparation method. It is prepared by dissolving in. If necessary, the pH of the solution is adjusted to 7.4 with D-gluconic acid or NaOH. Then Ca ²⁺ and Mg ²⁺ salts are added and the volume is adjusted with degassed double distilled water. Recheck pH and adjust if necessary. The solution is then sterilized by any suitable means, such as autoclaving or using a 0.22 μm filter (Nalge Co., Rochester, NY). Next O ₂ from the solution to prevent the β- hydroxybutyrate is oxidized by O ₂
Bottles under vacuum for complete removal.

The composition of the present invention (for example, the above-mentioned first,
When used as part of a formulation (as in aspects 2, 4, 5 and 7) it is preferred to first prepare a concentrated ^stock solution free of Ca ²⁺ and Mg ²⁺ . The exact calculated amount is then weighed, combined with another portion of the ingredients and double distilled water is added to bring the volume to about 95%. Add Ca ²⁺ and Mg ²⁺ , recheck pH and readjust if necessary.
The stock solution is preferably a 2- to 10-fold concentrated solution, more preferably a 2- to 5-fold concentrated solution. If not used immediately, store the stock solution with Nalgen.
e) 0.22 or 0.45 such as (Nalge Co., Rochester, NY)
Sterilize by passing through a μm filter. Sterilized solution at 4 ℃
Store in the refrigerator. Because the solution contains relatively short-lived essential amino acids, vitamins and other components, it must be stored at 4 ° C and used for a short period of time (usually about 6-12 months). Therefore, the use of degassed water for the preparation of these solutions is useful, but not necessary.

[0090] For synergistic composition comprising a Mg ²⁺ sufficient amount, they shall not Mg ²⁺ if combining a composition of the present invention the synergistic compositions.

The invention is illustrated in detail in the examples below.
The examples described herein are for purposes of illustration and are not intended to limit the invention in any way.

Example 1 The effectiveness of the composition of the present invention as a balanced energy source is demonstrated by the two most commonly used perfusion solutions at present: BSS and BSS-Plus (Alcon Laboratories, Inc.). In
c. ), Fort Worth, Texas) (B)
SS-plus is reported to be more effective than BSS). BSS-Plus is a tripeptide,
Oxidized glutathione exists. Dixstein (Di
Kstein et al. (J. Phys)
iol. ), 221, 29, 1972), glutathione is effective for corneal fluid transport.

The compositions of the present invention are shown in Table 1, but in this example 20 mM acetate was used instead of 10 mM β-hydroxybutyrate and citric acid was used as the antioxidant. Changes in the concentrations of these components within the ranges shown in Table 1 are similarly effective. However, the active osmolality of the solution is in the range of about 285-300 mOsM, preferably 2
Must be maintained at 90 mOsM. Since the purity of the salt varies from lot to lot, the active osmolality of the solution varies from preparation to preparation. If the osmolality is too low, adjust by adding mannitol, or if it is too high, N
Instead of aCl, sodium D-glucuronate or the Na ⁺ salt of sugar acid is used.

In this experiment, two nicks each having a length of about 2 mm were made on the corneas of six cat eyes.
A 25 gauge cannula was inserted into the anterior chamber through one of the cuts in each cornea. The solution of the present invention shown in Table 1 above was used at a pressure of about 15 mmHg and a rate of about 7 ml / min, except that 20 mM acetate was used instead of 10 mM β-hydroxybutyrate and citric acid was used as an antioxidant. Thus, a total of 1 liter was injected per cornea.

For comparison, the same procedure was carried out using BSS in some cases and BSS-plus in others instead of this solution.

All corneas were examined 1, 2 and 6 days after injection. The morphology of the endothelium was examined using a reflection microscope and corneal thickness was measured using a digital caliper.

As shown in FIG. 2, in the cornea injected with BSS, endothelial cells were extensively lost, endothelium polymorphism was observed, and corneal swelling was observed 1 day after injection. It was As shown in FIGS. 3 and 4, on the first day after the injection of BSS-plus or the perfusion solution of the present invention, the shape of the endothelial cells remained polygonal (normal shape) and the corneal thickness was normal. there were. When observed 6 days after the injection, the cornea injected with the solution of the present invention (FIG. 4) was identified as BSS-plus injected cornea (FIG. 5) in terms of endothelium polymorphism and corneal thickness. Somewhat good.

The above results indicate that the perfusion solution of the present invention is superior to BSS-Plus in that it maintains the endothelium and the thin and transparent cornea.

In this animal study, injection of the test solution into the anterior chamber of the eye was performed for a much longer period than would be done with normal eye surgery. Therefore, it was concluded that the composition of the present invention has an effective action on the eye tissue even in clinical application.

Example 2 It is well known that the endothelium, which includes an ion pump and a liquid pump, helps to maintain the cornea in a highly hydrated state and thus to maintain clarity. Endothelial fluid and ion transport are energy-dependent processes. If the donor dies (or the cornea is isolated) and is maintained at 4 ° C for a period of time, the cornea swells due to insufficient energy production at 4 ° C to clean the cornea. Increasing the temperature of the solution increases energy production and causes the cornea to contract. This process is called temperature reversal. Thus, the cleansing activity of this isolated cornea, which is based on temperature reversal, is a measure of corneal brier, fluid pump and metabolic function, and the efficiency of exogenous metabolites for the cornea to perform these functions. Is a measure of.

According to the method of the present invention, the weight is 2.5-3.0.
kg Male New Zealand Albino (New Zea)
Land albino) rabbits were killed by overdosing with pentobarbital intracardiacally and kept in a cold room at 4 ° C, in some cases eyes closed with tape for 24 hours, in other cases 4
After being closed for 8 hours, swelling of the cornea was induced and a decrease in metabolic activity was induced. 1.5mm scleral ring
m) was excised, lightly washed in Dulbecco's PBS and the Cooper Vision Viewing Chamber (Coope) containing 22 ml of the solution according to the invention as shown in Table 1.
r VisionViewing Chamber). The corneal thickness was measured with a digital thickness gauge connected to a reflection microscope. "Corneal thickness vs. time" was plotted to determine the corneal lavage activity according to the tissue contraction rate.

For comparison, instead of this solution, BSS-
The same method was performed using a plus.

It is noted that this experimental method is similar to the in vivo wash process after the donor cornea has been transplanted into the recipient.

FIG. 5 shows a typical “corneal thickness vs. time” plot of rabbits swollen in vitro at room temperature (22-23 ° C.) in the solutions of the invention shown in Table 5 above. Tissues were isolated from rabbits 24 or 48 hours after their death. The cleaning activity according to the contraction rate of the cornea (6 each) measured 24 and 48 hours after death was 137.3 ± 5.4 μm / hour and 63.3 ± 6.7 μm, respectively.
/ Hour.

As shown in FIG. 6, in the presence of BSS-plus, the rate of contraction of the isolated corneas (6 each) was 89.7 ± 24% at 24 and 48 hours after death, respectively.
The values were 6.4 μm / hour and 30.3 ± 3.8 μm / hour. This rate was 35% and 50% lower, respectively, compared to that observed in the presence of the solution of the invention (FIG. 5).

The above results indicate that the compositions of the present invention are superior to BSS-Plus in terms of efficiency as an energy source for fulfilling the physiological and biochemical functions of tissues (and cells). ing. This result further supports the results of the experiment described in Example 1.

Example 3 Chen et al. (US Pat. No. 4,873,186)
No., supra), the theory is that when mixed with tissue culture medium, β-hydroxybutyrate is used to preserve isolated cornea, peripheral tissue, the production and accumulation of lactic acid in the preserved tissue is suppressed. At the same time, it was revealed that a high level of energy as ATP was produced by inhibition. To preserve donor corneal viability and endothelial pump function in vivo,
The efficacy of the corneal preservation medium containing the composition of the present invention was compared to Optizol (manufactured by Optiron, Irvine, Calif., Manufactured by Chiron), which is currently the best commercially available product. It is one of the corneal preservation media). Chondroitin sulfate and dextran are contained as a dehydrating agent in optisol in a sufficient amount to exert a colloid osmotic pressure on tissues.

For this experiment, a corneal preservation medium was prepared in which the bicarbonate and balanced salt solutions of TC199 were replaced with the composition of the present invention. A typical one, shown in Table 1, excludes glucose and reduces NaCl by 15 mM. Since TC199 already contains glucose, it is not necessary to add extra glucose. The reduction of 15 mM NaCl is to ensure that the total concentration of cations and anions is the same after replacing the TC199 bicarbonate and balanced salt solutions with the composition of the present invention. Even if the concentrations of these components are changed within the ranges shown in Table 1, it is similarly effective. However, the active osmolality of the solution is 290 mM isotonic, or about 285-300 mOsM.
Should be maintained in the range of 10 to provide the solution with an osmotic effect on the tissue. The active osmolality is adjusted with sugars such as mannitol and sucrose, or neutral polysaccharides such as dextran.

The solution was applied to a 0.22 μm Nalgene filter membrane.
25 m by filtration (Nalge Co., Rochester, NY).
Place in a 1 scintillation vial and store refrigerated at 4 ° C.

According to the method of this experiment, the body weight was 3.0-3.5.
kg Male New Zealand Albino (New Zea)
Land albino) rabbits were sacrificed by intracardiac administration of excess pentobarbital, and corneas with 1.5 m scleral rings were isolated. For comparison, one of a pair of corneas from the same donor was stored in corneal storage medium containing the composition of the invention and another was stored in Optizol. After storage for 7 days in some cases and 11 days in other cases, the donor corneas were removed from the storage medium, and PB from Dulbecco's was removed.
Lightly washed with S. The 7.5 m button of the donor cornea was cut with a trephine and transplanted into a recipient rabbit. About 30-4 for transplant operation
It took 5 minutes. Measure the corneal thickness of the donor immediately after transplantation, then every 30-60 minutes until 7 hours later, once or twice daily for up to 1 week, and once a week for up to 4 weeks. It was measured.

[0111] In general, isolated corneas swell during storage at 4 ° C, which is reversible if the preserved corneal viability, cellular metabolic activity and endothelial liquid pump function are retained. is there. When transplanted into the recipient, the donor's cornea, which is well preserved in biological function, is rapidly washed and the clarity of the implant is immediately achieved. If not well retained, the donor's cornea is washed slowly and even swells after implantation.

As shown in FIG. 7, the donor corneas stored in the medium containing the composition of the present invention for 7 days after transplantation rapidly (about 16.2 μm / hour, average of 4 transplanted corneas).
The corneas that have been washed and stored for 11 days are washed at a slower rate (approximately 1.4 μm / hr, average of 4 transplanted corneas). The half-life (t _1/2 ) for the cornea stored for 7 days and 11 days after transplantation to return to the normal thickness of about 340 to 370 μm was 2.4 and 33.6 hours (4 times each).
Individual implants) (Table 4).

In contrast to this, as shown in FIG. 8, donor corneas stored in Optizol for 7 days were gently swollen for about 3 hours after transplantation and then about 0.5 μm / hour (4 transplants). Swelling at an estimated rate of (corneal mean). Donor corneas stored for 11 days in Optizol were
It swelled significantly for hours and then at an estimated rate of about 0.4 μm / hour (average of 4 transplanted corneas). The half-life (t _1/2 ) for the cornea stored for 7 days and 11 days after transplantation to return to the normal thickness of about 340-370 μm was 133 and 311 hours (4 grafts each), respectively. Presumed to be present (Table 4).

[0114]

[Table 4]

The results obtained in these in vivo experiments showed that the corneal preservation medium containing the composition of the present invention was significantly more significant than optizole in terms of survival activity of corneal tissue and retention of the liquid pump function of corneal endothelium. It clearly shows that it is excellent.

Thus, the experiments described in Examples 1, 2 and 3 show that the present invention is a unique composition. It is clear that the compositions of the invention are particularly useful for in vitro and in vivo applications as an energy source for peripheral tissues to perform their physiological and biochemical functions and to maintain tissue viability. .

In addition to its clinical properties, the compositions of the present invention have the following properties not possessed by other perfusion solutions such as BSS-Plus:

1) The composition of the present invention serves as an abundant energy source for cells and has a mechanism of suppressing the production and accumulation of lactic acid. 2) The composition of the present invention is chemically more stable than BSS-plus and has a longer life. 3) The composition of the present invention has a lower manufacturing cost than BSS-Plus. 4) The buffer solution (phosphate buffer solution) of the composition of the present invention is stable and has a large capacity. On the other hand, BSS-Plus uses a buffer system of sodium bicarbonate, and the pH changes depending on the CO ₂ partial pressure. 5) The composition of the present invention does not require a mixing step prior to administration and is therefore easy to use.

Although the invention has been described with respect to several specific embodiments, it will be apparent to those skilled in the art that many modifications can be made without departing from the scope of the invention. Therefore, it should be understood that those modifications are also included in the scope of the claims.

[Brief description of drawings]

FIG. 1 shows a pathway of ketone body formation from a fatty acid and a ketogenic amino acid.

FIG. 2 is a BSS as described in Example 1.
Reflection microscopy (s) of cat endothelium after perfusion of the anterior chamber with
It is a reproduction of a photograph by a specific microscope.

FIG. 3 shows the BSS as described in Example 1.
-Reproduction of a photomicrograph by reflection microscopy of the feline endothelium after positive perfusion of the anterior chamber.

FIG. 4 is a reflection microscopy photocopy of cat endothelium after perfusion of the anterior chamber with a composition of the invention as described in Example 1.

FIG. 5: “Corneal thickness vs. time” of in vitro deswelling of isolated corneas in the presence of a composition of the invention, as described in Example 2.
Is a plot of.

FIG. 6 shows the BSS as described in Example 2.
-"Corneal thickness vs. time" of in vitro deswelling of isolated corneas in the presence of plus.
Is a plot of.

FIG. 7 shows in vivo transplanted donor corneas pre-stored at 4 ° C. in medium containing a composition of the invention for 7 and 11 days as described in Example 3. 1 is a plot of "corneal thickness versus time" of contraction (deswelling).

FIG. 8: In vivo contraction of transplanted donor corneas pre-stored for 7 and 11 days at 4 ° C. in medium containing the composition of the invention as described in Example 3. (Deswelling) is a plot of "corneal thickness versus time".

Continuation of front page (72) Inventor Smithy. Chen Killarney Court 13704, Phenix, Maryland, United States

Claims (28)

[Claims] 1. A phosphate buffered balanced salt in an amount sufficient to form a composition that effectively meets the needs of the eye and other peripheral tissues for efficient physiological and biochemical functions. A composition comprising an aqueous solution, glucose, and at least one ketone body and its precursor. 2. The composition according to claim 1, wherein the ketone body is β-hydroxybutyrate ion and acetoacetate ion. 3. The β-hydroxybutyrate ion is β
From the group consisting of D-isomers of hydroxybutyrate, mixtures of D-racemic and L-racemic forms of β-hydroxybutyrate, and mixtures of D- and L-isomers of β-hydroxybutyrate The composition of claim 2, which is selected. 4. The concentration of β-hydroxybutyrate is 5-
The composition of claim 2, which is in the range of 30 mM. 5. The precursor of the ketone body is a short chain fatty acid selected from the group consisting of acetic acid and butyric acid, and leucine,
The composition of claim 1 which is a ketogenic amino acid selected from the group consisting of lysine, phenylalanine, tyrosine and tryptophan. 6. The concentration of the precursor of the ketone body is 0.1-25.
The composition of claim 5, which is in the mM range. 7. A composition according to claim 5, consisting of one or more ketogenic amino acids, each at a concentration in the range of 0.1-5.0 mM. 8. The composition according to claim 5, wherein the total concentration of ketogenic amino acids is 7.5-12.5 mM. 9. The composition of claim 5, wherein the short chain fatty acid is acetic acid at a concentration in the range of 15-25 mM or butyric acid at a concentration in the range of 5-15 mM. 10. The composition according to claim 1, wherein the pH is adjusted to 7.3-7.5. 11. A portion of NaCl in an amount of 15-35 mM is D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D
-Gluconic acid and D-glucaric acid (D-gluc
A composition according to claim 1, which is substituted with an equal amount of Na ⁺ salt of one or more sugar acids selected from the group consisting of aric acid). 12. A sufficient amount of 40 to form a composition that simultaneously prevents adhesions during eye surgery and effectively meets the requirements for efficient physiological and biochemical functions of the eye tissue. The composition according to claim 1, wherein the viscosity of the composition is increased by the addition of the neutral polysaccharide dextran in an amount in the range of -500 kilodaltons. 13. The composition according to claim 12, wherein the concentration of neutral high molecular weight dextran is in the range of 2% -20%. 14. The composition of claim 1, Eagle's medium, Dulbecc.
A corneal preservation medium consisting of a minimum member essential amino acid selected from the group consisting of co's medium and Daniel's medium, and a minimum essential vitamin. 15. The balanced salt aqueous solution in the tissue culture medium is replaced by the composition according to claim 1, and the NaCl concentration is 70 so that the total concentration of cations and anions is the same as that of the balanced salt aqueous solution. It has been reduced to the range of -100 mM and the active osmolality of the solution (osmolarit
y) isotonicity (is) within the range of 285-300 mOsM.
corneal preservation medium adjusted to osteonicity). 16. The tissue culture medium is TC containing Hank's salt.
199 (TC 199 with Hank's sa
16. The corneal preservation medium according to claim 15, which is Its). 17. The tissue culture medium is a Eagle's minimum base containing Hank's salt.
ssential medium withHank '
16. The corneal preservation medium according to claim 15, which is s salts). 18. The corneal preservation medium according to claim 15, wherein the NaCl solution is reduced to 85 mM. 19. The osmolality of the solution is 290 mOsM.
The corneal preservation medium according to claim 15, which is adjusted to have isotonicity. 20. The corneal preservation medium of claim 15, further comprising at least one member of the group consisting of mannitol, sucrose and dextran. 21. The corneal preservation medium according to claim 15, wherein the pH is in the range of 7.10-7.50. 22. A portion of NaCl in an amount of 15-35 mM is one selected from the group consisting of D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, D-gluconic acid and D-glucaric acid. The corneal preservation medium according to claim 15, which is substituted with an equal amount of Na ⁺ salt of sugar acid or more. 23. (1) A bicarbonate-buffered balanced salt solution of a corneal preservation composition comprising a tissue culture medium, which is suitable for preservation of the cornea but which normally causes undesirable production of lactic acid. A composition capable of inhibiting the production of lactic acid by the cornea, which is substituted with a phosphate buffered balanced salt solution of the composition according to (2) and is present in an amount sufficient to inhibit lactic acid production (2). A corneal preservation medium comprising at least one compound selected from the group consisting of chain fatty acids and ketone bodies. 24. A corneal preservation composition comprising a corneal preservation medium according to claim 14, 15 or 23 and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor. 25. The composition of claim 1, and VE.
A formulation for the preparation of tissue for organ transplantation, which comprises a synergistically effective mixture of GF, uridine, thymidine and a serum-derived factor. 26. A formulation according to claim 25 for the preparation of corneal transplant tissue. 27. An irrigation solution comprising a corneal preservation medium according to claim 14 or 15 and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor. 28. An organ medium comprising the corneal preservation medium according to claim 14 or 15 and a synergistically effective mixture of VEGF, uridine, thymidine and a serum-derived factor.