CA2042152A1 - Additive for irrigation solution or surgical solution - Google Patents

Abstract

ADDITIVE FOR IRRIGATION SOLUTION OR SURGICAL SOLUTION
ABSTRACT OF THE DISCLOSURE
An additive for intraocular irrigation solution or surgical solution which provides the anterior chamber and posterior chamber of the eye with protection during surgical procedures that require irrigation. The additive for the solutions is composed of a balanced salt solution containing dextrose supplemented with chondroitin sulfate. Other chemical compositions can supplement the additive.

Description

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CR05S REFERE~CE~ TO CO-PENDING APPLICATIONS

This application is a continuation-in-part of Serial No. 7~1~407, filed August l, 1985, entitled Irrigation Solution, now U.S. Serial No. 4,696,917, which is a continuation-in-part of ~e~i~ No. 836,156, filed March 4, 1986, entitled Surgical Solution, ~.
BAC~GROUND OF THE INVENTION

1. Field of the Invention: The present inven-tion pertains to an additive for an irrigation solution or a surgical solution for the anterior and posterior chamber of the eye. The additive can also be used in other solutions, such as corneal preservation medium, for other surgical or medical applications.
2. Desaription of the Prior Art: There are two intraocular irrigation solutions commonly being used in ophthalmic surgeries. These two irrigation solutions are balanced salt solution and BSS Plus. BSS is a balanced salt solution that incorporates a sodium citrate and sodium acetate buffering system. BSS Plus consists of a balanced salt solution with a bicarbonate buffering system, with glucose added as an additional osmotic agent and energy source. An additional compo-nent, oxidized glutathione, is reduced by the ocular cells and serves as an anti-oxidant. In addition, Lactated Ringers Solution is used when BSS and BSS Plus are not available.

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E~UMM~RY OF THE INVENTION
The general purpose of the present invention is an additive for an irrigation solution or surgical solution which provides the anterior and posterior chamber of the eye protection during surgical procedures that require irrigation. This additive specifically protects the corneal endothelium in anterior segment surgery. The corneal endothelium and other anterior and posterior chamber structures are in direct contact with the additive in the solution.
According to one embodiment of the present invention, an additive includes: 1. A protective coating agent of a highly negatively charged glycosaminoglycan such as chondroitin sulfate which is a naturally occurring, biodegradable material normally found in the human cornea; In some cases, 2. An effective antioxidant such as 2-mercaptoethanol, that can be utilized in both the oxidized and reduced forms by human corneal endothelial cells. 2-mercaptoethanol i5 used as a carrier of cystine into the cells.
Cystine may well be the limiting amino acid for the synthesis of protein as well a glutathione. The addition of 2-mercaptoethanol increases the intracellular le~el of glutathione, and aids in membrane and maintenance of cell junctional complexes:

3. An energy source, such as dextrose, i5 the starting molecule for glycolysis, a central pathway for recovery of chemical energy as ATP (adenosine triphosphate). 4.
An additional energy source such as a pyruvate, is provided for additional biosynthetic synthesis that may be required by the ocular cells after surgical trauma.

5. An amino acid cystine in the presence oE 2-15~:

mercaptoethanol, is provided so that corneal endothe-lial cells can constantly utilize cystine in the additive, and maintain intracellular cystine and glutathione levels during intraocular perfusion~ 6. A
bicarbonate buffer system, important in intracellular fluid, helps maintain intercellular pH, fluid movement, corneal deturgesence, and membrane potential difference across the corneal endotheli.um.
The base medium of the irrigation solution or surgical solution with the additive consists o~ a Balanced Salt Solution or Lactated Ringers Solution containing five essential ions.
One significant aspect and feature of the additive of the present invention is to protect the anterior and posterior segments of the cornea during surgical procedures, to maintain homeostasis after surgical trauma, and to provide necessary metabolic substrates that may be needed for wound repair.
Another significant aspect and feature of this additive is use in other applications when added to the irrigation solution, such as: 1. An ophthalmic irrigating and lubricating eye drop; 2. In the solution for burn wounds; 3. As a general surgical solution for use in surgeries where a solution is required; 4. As a vehicle for ophthalmological drugs, such as drops; 5. As an anticataract drug: 6. As an eye drop; 7. As a corneal preservation medium additive; 8.
As a general irrigation solution including orthopedics, such as athroscopy; urology, such as cystoscopy;
neurosurgery: as well as in artificial insemination, obstectrics, and gynecology (o~GYN); 9. As a wound-healing solution, or 10. As an anti-ulcer drug.

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DESCRIPTION OF THE_PREFE R D EMBODIMENTB
The solution additive such as to an irrigation solution or a surgical solution is used at a concentration of 1-100 parts for each 500 parts o~ a commercially available balanced salt solution (BSS) base such as that sold by Iolab or Alcon.
Typically, the additive concentrate contains one or more of the components in Table A.
TABLE A
Preferred component Ran~e. Component glycosaminoglycan0.001-550mg/ml chondroitin sulfate an antioxidant O-lOmM2-mercaptoethanol energy source 0-20n~ dextrose 0-20mM pyruvate amino acid O-lOmM cystine buffer O-lOOmMsodium bicarbonate The formulation is a representative concentration oE
the additive made up in a total volume of 1-50ml balanced salt solution or water.
The additive can then be added to 500ml of balanced salt solution as in Table B. This irrigation solution additive is therefore defined at proportionate concentrations at all other volumes ~o~s~

TAB~E B

Additive vf 50ml made up in Balancè Sal-t Solution (BSS) Base Medium:
chondroitin sulfate 44.00mg/ml 2-mercaptoethanol 5.50mM
dextrose 55.00mM
pyruvate ll.OOmM
cystine 33.00mM
sodium bicarbonate 275.00mM

Balance Salt Solution ~BSS) Base Medium~
sodium chloride .64%
potassium chloride .075%
calcium chloride .048~
magnesium chloride .030%
sodium acetate .39 sodium citrate .17~
or made up in distilled injection grade water. The final concentration of the additive after dilution with an appropriate amount of Balance Salt Solution is described in Table C.

TABLE C
Final Concentration Final Approximate Ranges of Component Concentration final concentration chondroitin sulfate 4.00mg/ml (O-lOOmg/ml 2-mercaptoethanol 0.50mM (0-2mM) dextrose 0.92mg/ml (0-2OmM) pyruvate 1.OOmM (O-2OmM) cystine 3.00mM (O-lOmM) sodium bicarbonate 25.00mM (O-lOOmM) This additive is therefore defined at proportionate concentrations at all other volumes.

2~S2 GROUPINGS
An add~tive concentrate comprising one or more of the following components in BalancPd Salt or like solution. (lyophilized) a. a glycosarninoylycan : l.Omg/ml to 550mg/ml.
1. chondroitin sulfate 2. dermatan sulfate 3. heparan sulfate 4. heparin sulfate 5. keratan sulfate 6. hyaluronic acid b. an antioxidant 1. ascorbic acid : lmM - 20mM
2. glutathione in : lOug/ml - 500mg/ml 3. Dl - ~- tocopheral : .Olmg/ml - lmg/ml 4. 2-mercaptoethanol : .lmM to 8mM
c. Glycoproteins that promote cellular adhesion and migration:
1. Laminin, a large glycoprotein having a molecular weight of approximately 1,000,000 daltons. The laminin molecule has the shape of an ~symmetric cross, comprised of 3 B chains of 200,000 daltons each, and one A chain o~ 400,000 daltons. The chains are held toyether by disulfide bonds. The single A chaln contains a binding site for heparin sulfate. The B chains contain type IV
collagen binding sites. The intersection of the three B chains is the locus for cell binding. Laminin provides cells with physiological ;~0~ 5~:

compatible extracellular matrix that will foster attachment cytoplasmic spreading and proliferation.
Laminin : lug/ml - 300mg/ml 2. Fibronectin is an extracellular matrix-associated glycoprotein composed of two disulfide bonded subunits of 22~,000 daltons each. Fibronectin has the potential to interact with several cell surface associated macromulecules including collagen, glycosaminoglycans and cell surface receptors. Fibronectin promotes cell adhesion and migration of human corneal endothelial cells, epithelial cells and fibroblasts.
Fibronectin : lOOng/ml - lOOmg/ml d. Cell growth factors or growth supplements:
1. Fibroblastic growth factor (FGF), a single chain polypeptide, isolated and purified from the pituitary, human (hFGF) fibronectin or bovine fibronectin (bFGF), acidic or basic forms.
Molecular weight ranye of 14K to 16K~
This factor has been demonstrated mitogenic in vitro to a wide variety of cells comprising mesoderm and neuroectoderm tissue.
This also includes synthetic formulated FGF basic peptides consisting of~ 24) Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly-l~is-Phe-Lys-~sp-Pro-Lys-Arg-Leu-Try 2~2~5;2 and synthetic formulat~d FGF acidic peptides consisting of: (1-11) Phen-Asn-Leu-Pro-Leu-Gly-Asn-Tyr~Lys-Lys-Pro Fibroblastic growth factor : lOOng/ml lOOmg/ml 2. Endothelial cell growth supplement (ECGS), prepared ~rom the hypothalamus as a lyophilized extract. This growth supplement has demonstrated mitogenic in vitro to a wide variety of endothelial cells: i.e., human corneal endothelial cells, human umbilical vein endothelial cells, and mouse Balb/c fibroblasts.
Endothelial Cell Growth supplement : 200ug/ml - 500mg/ml 3. Urogastrone or Epidermal growth factor ~EGFj, a single chained polypeptide, is composed of 53 amino acids, containing 3 disulfide bonds and has been isolated from mouse submaxillary ylands (mEGF) and human urine (hEGF). This growth factor has been demonstrated to be mitogenic in vitro for a wlde variety of cells of ectodermal and mesodermal origin.
This also consists of syllthetic mouse EGF:
Asn-Ser-Tyr-Pro-Gly-Cys-Pro-Ser-Ser-Tyr-Asp-Gly-Tyr Cys-Leu-Asn-Gly~Gly-Val-Cys-Met-His-Ile-Glu-Ser-Leu-Asp-Ser--Tyr-Thr-Cys-` . J ~ ~

~ Asn-Cys-Val-Ile-Gly-Tyr-Ser-Gly-j Asp-Arg-Cys-Gln-Thr-~rg-Asp-¦ Leu-Arg-Trp-Trp-Glu-Leu-Arg And synthetic EGF [Cys(Acm) 20'31] (20-31) Cys-(Acm)-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys(Acm) And human EGF synthetic receptor peptide:
Asp-Val-Val-Asp-Ala-Asp-Glu Tyr-Leu-Ile-Pro-51n Epidermal Growth Factor : .lug/ml - lOOmg/ml 4. Bovine pituitary extract (BPE) an aqueous extract of bovine or human pituitary glands. This growth supplement has demonstrated mitogenic in vitro to a wide variety of eplthelial cells: i.e., human corneal epithel~um, human epidermal keratinocytes Bovine Pituitary Extract : lOOng - 500mg/ml 5. Insulin a polypeptide hormone that functions in the regulation of cellular carbohydrate metabolism and the synthesis of cellular protein, RNA and neutral lipids~
Insulin : .lug/ml - lOmg/ml 6. Transferrin : .lug/ml - lOmg/ml 7. Sodium Selenite : .lng/ml - lOOug/ml e. Extracellular Matrix Proteins 1. IV collagen: the main collagen in basal laminae.

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IV collagen in a range of about 5mg/ml -lOg/ml.
2. Enactin in a range of about lug/ml -lOOmg/ml.
f. Buffering Agents:
1. HEPES buffer (N'-2-hydroxyethylpiperazine-N'-Ethanesulfonic Acid) HEPES buffer : lOmM - 250mM
2. Sodium bicarbonate : .OlmM to 250mM
g. Energy Sources:
1. Pyruvate : 5mM - lOOmM
2. dextrose : .5mM - lOOmM
h. Amino Acids: .OlmM - 50mM
1. Cystine 2. Inosine 3. Adenine 4. Adenosine ~ i. Precursors of cell membrane lipids:
1. ethanolamine : .OlmM - 20mM
2. phosphoethanolamine : .OlmM - 20mM
3. sialic acid : .OOlmM - lOmM

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EFFECT OF TWO HOUR_IRRIGATION
WITH B~L~NCED ~ALT BOL TIOII WITH ADDITIVE WITHOUT
CYSTINE ON THE: ~LUTATHION13 CONTENT OF CAT L~NSE~:
To determine that the in vitro effects of 2-mercaptoethanol could be duplicated in vivo, the following studies were done. ~n two hour anterior chamber perfusion studies, using a balanced salt solution with additive (chondroitin sulfate 4.0mg/ml;
2-mercaptoethanol .5mM dextrose .92mg/ml: pyruvate 1.OmM; sodium bicarbonate 25mM) (without cystine) in cats, the glutathione content of the lens was compared to the non-irrigated control 24 hours after perfusion.

Irrigation with Additive Control Total Glutathione -1530.27 ~ 26.03 ug/lens 1435.39 + 46.39 ug/lens 6.20% Difference A 6.2% difference in total glutathiona was seen between the irrigated lens and the control lens. This increase in glutathione content is due to de novo syn-thesis.

~0~; :152 E:FFECT OF TWO HOUR IRRIGATION WITH BALl~NCED ~ALT
80LUT ON WITH ADDITIVE CONT~INING 3mM CYE~TINE ON THE
GLUTATHIONE CONTENT OF CAT LEN~:E~
To determine if the in vivo effect of the irrigation additive, containing 2~mercaptoethanol, could be enhanced by the addition of 3mM cystine, the following studies were done. In two hour anterior chamber perfusion studies, using a balanced salt solution with the additive containing 3mM cystine, in cats, the glutathione content of the perfused lens was compared to the non-irrigated control 24 hours after perfusion.

BSS with Additive with 3mM Cystine Control Total Glutathione -1495.73 ~ 35.75 ug/lens 1378.25 ~ 31.25 ug/lens 7.85% Difference A 7.85% increase in total glutathione was seen be-tween the irrigated lens and the control lens. This increase in glutathione content is due to de novo syn-thesis. Balanced salt solution with additive contain-ing 3mM cystine appears to have enhanced the effect of 2-mercaptoethanol, in vivo by increasing the lens concentration of glutathione.

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THB EFFECT OF_2-MERCAPTOETIII~NOI-ON HVMAN AND RABBIT LENE~ IN VITRO
In the investigational work with human corneal en-dothelial cells/ it is recognized the limited capacity to synthesize cystine via the methionine pathway. In addition, corneal endothelial cells are deficient in capacity to uptake cystine, and that intracellular cystine and glutathione contents decrease considerably in n~rmal culture medium containing cystine. Then examined was the qlutathione content of human and rabbit lenses. Reduction of glutathione content in human lens has been associated with the formation of certain types of cataracts. Gluathione is made up of three amino acids, cystine, glutamic acid, and glycin~.
Cystine has been implicated as the limiting amino acid for the synthesis o~ protein as well as glutathione.
An increase in the intracellular cystine pool by the addition of 2 mercaptoethanol may cause the increase in cellular glutathione contentJ because the cellular pool of cystine is very low as compared with that of glu-tamic acid and glycine.
In three days, in vitro rabbit lens incubation studies utilizing 2-mercaptoethanol, the glutathione content of the lens was compared to the control lens incubated without mercaptoethanol.

Medium with 2-mercaptoethanol Control Total Glutathione -657.61 + 24.70 ug/lens 615.51 ~ 24.70 ug/lens Percent Oxidized - 3.94 8.54 6.4% Dif~erence ln Total Glutathione Only a small difference in total glutathione was found in these studies. The rabbit lens is capable of 2~2~

producing enough cystine to maintain intracellular glutathione levels during lens incubation in vitro..
The 2-mercaptoethanol was able to ~naintain the glu-tathione in a reduced form greater than the control.
In three day, in vitro human lens incubation stud-ies utilizing 2-mercaptoethanol, the glutathione con-tent of the lens was compared to the control lens incu-bated ~-ithout 2 ~mercaptoethanol.

Medium with 2-mercaptoethanol Control Total Glutathione -230.91 + 15.49 ug/lens 176.16 ~ 2-.26 ug/lens Percent Oxidized - 13.02 - 17.B7 23.70% Difference in Total Glutathione _._ _ _ _ _ _ _ _ With the media containing 2-mercaptoethanol, there was a 23.7~ increase in the total glutathione content mainly in the reduced form.

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MODE OF OP~ATION
The base of this additlve is a compl~tely de~ined balance salt solu~ion supplemented with dextrose and with the addition of one to five corneal and retinal enhancing agents. These are chondroitin sulfate, 2-mercaptoethanol, pyruvate, cystine, and sodium biGarbonate. The exposure time to the additive solution in th~ normal ophthalmic surgical procedure, such as an intraocular lens implant, is normally 3-8 minutes, a very limited tlme period. But upon special occasions, the anterior chamber ls filled with the solution and allowed to remain there until the aqueous humor is remade, which may take up to 2~ hours. During this time the anterior chamber cells are deprived of necessary nutrients, normally supplied by the aqueous humor. Although the anterior segment cells, most significantly the corneal endothelium, are supplied with nutrients from their basal side, most of the metabolic uptake is from the anterior surface. The corneal endothelium maintains the clarity of the-cornea by actively pumping salts and water out of the connective tissue stroma into the anterior chamber of the eye. The Na~/K+ ATPase pump of these endothelial cells requires ATP and reduced pump sites to keep this pump working. When the pumping action of these corneal endoth~lial cells is reduced, the cornea imbibes fluids and becomes thickened and loses optical clarity.
Therefore, an irrigation solution with an additive with a reducing agent is of considerable advantage.
one of the major disadvantages of BSS Plus, with the reducing agent glutathione and the blcarbonate buffering system, is the lack of stability of the 2~4L21S2 solution once prepared. The glutathione component of the solukion is added separately, and the solution is stable for only a 24 hour period. The buffering abil-ity of the bicarbonate in BSS Plus is greatly reduced onca the solution is exposed to the atmosphere.
The present additive provides the reducing agent 2-mercaptoethanol, that can be utilized in both the reduced and oxidized forms eliminating the instability of a glutathione containing irrigation solution. This effective reduclng agent can be utilized by human corneal endothelial cells.
Five additional components have been added to the solution to increase its effectiveness in protecting and repairing the anterior segment o~ the cornea during and after surgical trauma. ~1) Chondroitin sulfate, a highly negative charged glycosaminoglycan is added to replace any glycosaminoglycans that may be removed from the surface of the corneal endothelial cells from the disruption of aqueous flow or surgical trauma.
Glycosamimoglycans are necessary for membrane stabil;ity and the maintenance of the three-dimensional structUre of receptor proteins. These receptor proteins are re-quired for the metabolic processes of the cell.
Chondroitin sulfate act.s as a protective coating for the anterior segment cells.
An additional substrate, (2) pyruvate, is provided for additional biosynthetic synthesis that may be required by thesa anterior segment cells after surgical trauma. There are 20 standard amino acids in proteins, all having different carbon skeletons.
Correspondingly, there are 20 different catabolic path-ways for their degradation. Carbon skeletons of 10 of 2~4~:~5Z
i the amino acids are ultimately broken down to yield acetyl-CoA which enters the citric acid cycle directly.
Five of the ten are degraded to acetyl-CoA via pyru-vate. The five amino acids entering vla pyruvate are alanine, cystine, glycine, serine, and threonine.
(3) 2-mercaptoethanol, an anti-oxidant, used in the proposed irrigation additive has been shown to be effective at concentrations of 10 to 1000uM, and its action resembles those of macrophages or feeder layèr cells. It is proposed that human corneal endothelial cells have a low capacity to synthesize cystine via the methionine pathway. Methionine furnishes the sulfur atom and serine furnishes the carbon chain in the biosynthesis of cystine. The end result of a complex series of reactions is the replacement of the -OH group of serine with an -SH group, derived from methionine, to form cystine.
In addition, corneal endothelial cells are defi-cient in their capacity to take up cystine and that intracellular cystine and glutathione contents decrease considerably in normal culture medium containing cys-tine. In the presence of 2-mercaptoethanol; corneal endothelial cells constantly utilize (4) cystine in the irrigation solution and the cellular cystine and glu-tathione levels are maintained during intraocular per-fusion. This effect seems to be the most crucial func-tion of 2-mercaptoethanol in stimulating the growth of the cells and ~s not explained by mere reduction of cystine to cystine in the irrigation solution. It is the mixed disul~ide of 2-mercaptoethanol and cystine, which is formed by the reaction of 2-mercaptoethanol /
. . .J ....................... , 2~ 52 with cystine in the irrigation solution, that plays an important rola in the action of 2-mercaptoethanol.
The 2-mercaptoethanol acts repeatedly as a carrier of cystine. The 2-mercaptoethanol does not accumulate in the cells, and it escapes to the irrigation solution and reacts with the cystine. Thus, 2-mercaptoethanol is continuously taken up by the cells in the form of the mixed disulfid2 with cystine and is returned to the irrigation solution in is reduced form. With the aid of this cyclic action of 2-mercaptoethanol, the cells are able to utilize cystine constantly.
(5) A sodium bicarbonate buffer system is important in intracellular fluid to help maintain intercellular pH, fluid movement, corneal deturgesence, and membrane potential difference across mammalian cells, such as the corneal endothelium.

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Claims

1. An additive concentrate comprising one or more of the following components in a Balanced Salt or like solution:
a. glycosaminoglycan;
b. an antioxidant;
c. at least one energy source;
d. cystine; and, e. a buffer, the concentration of the components in the concentrate whereby combining 1-100 parts by volume of concentrate with 500 parts by volume of Balanced Salt Solution.