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WO1998032839A1 - Agent de deshydratation de cornees en culture d'organes - Google Patents

Agent de deshydratation de cornees en culture d'organes Download PDF

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Publication number
WO1998032839A1
WO1998032839A1 PCT/EP1998/000324 EP9800324W WO9832839A1 WO 1998032839 A1 WO1998032839 A1 WO 1998032839A1 EP 9800324 W EP9800324 W EP 9800324W WO 9832839 A1 WO9832839 A1 WO 9832839A1
Authority
WO
WIPO (PCT)
Prior art keywords
substitution
corneas
swelling
hydroxyethyl starch
dextran
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1998/000324
Other languages
German (de)
English (en)
Inventor
Claudia Redbrake-Adams
Martin Reim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fresenius SE and Co KGaA
Original Assignee
Fresenius SE and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fresenius SE and Co KGaA filed Critical Fresenius SE and Co KGaA
Priority to JP53157298A priority Critical patent/JP2001510996A/ja
Priority to EP98904113A priority patent/EP1007635A1/fr
Publication of WO1998032839A1 publication Critical patent/WO1998032839A1/fr
Priority to US09/359,374 priority patent/US6162642A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts

Definitions

  • the present invention relates to an agent for swelling of skins, especially corneas in organ culture, both human and animal, preferably human corneas, in particular corneas from eyes, containing the culture medium and a swelling substance, and the use of hydroxyethyl starch as swelling substance in the organ culture of hides, especially corneas and in particular corneas of eyes or for the production of an agent for the swelling of corneas, especially corneas of eyes, in the organ culture.
  • Cornneas are understood here to mean corneas in general, but especially the corneas of the eyes.
  • the hides can be of both human and animal origin (for example from pigs).
  • the invention on corneas of the eyes is described below by way of illustration, without being restricted thereto.
  • the object of the invention is to provide agents which are suitable for keeping the cornea swollen throughout storage or for swelling the gelatinic cornea before the transplantation, but which do not have the disadvantageous effects of the agents previously used.
  • hydroxyethyl starch with an average molecular weight M w of 70,000 to 200,000, a degree of substitution MS from 0.15 to 0.5, a degree of substitution DS from 0.15 to 0.5 and a ratio of substitution at C2 for substitution at C6 of the anhydroglucose units of> _ 8 as swelling substance instead of dextran there is a better swelling of the corneas without simultaneously causing greater damage to the endothelium and furthermore a significant improvement in the metabolic state of the cornea .
  • hydroxyethyl starch with an average molecular weight M w of 70,000 to 200,000, a degree of substitution MS of 0.15 to 0.5, a degree of substitution DS of 0.15 to 0.5 and a ratio of the substitution at C2 to the substitution at C6
  • Anhydroglucose units of _> _ 8 preferably a hydroxyethyl starch with an average molecular weight of 100,000 to 160,000, a degree of substitution MS from 0.2 to 0.45, a degree of substitution DS from 0.2 to 0.4 and a ratio of the substitution at C2 to Substitution at C6 of the anhydroglucose units from 8 to 20.
  • hydroxyethyl starch with an average molecular weight of 130,000 ⁇ 20,000 (HES 130), a degree of substitution MS from 0.38 to 0.45, a degree of substitution DS from 0.32 to 0, 40 and a ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units from 8 to 20, such as, for example, hydroxyethyl starch an average molecular weight of 117,000, a degree of substitution MS of 0.39, a degree of substitution DS of 0.34, a ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 11, hydroxyethyl starch with an average molecular weight M ⁇ 133,800, a degree of substitution MS of 0.44, a degree of substitution DS of 0.38, a ratio of substitution at C2 to substitution at C6 of the anhydroglucose units of 12.4, hydroxyethyl starch with an average molecular weight Mw 148.700,
  • the hydroxyethyl starch used according to the invention can be produced according to the process described in EP 0 402 724 B1 and DE 39 19 729.
  • EP 0 402 724 B1 and DE 39 19 729 are hereby expressly referred to for the purposes of disclosure.
  • the agent according to the invention or the hydroxyethyl starch used according to the invention can be used according to the invention both to keep the cornea from swelling during the entire storage (long-term culture), that is to prevent the swelling of the cornea from the outset, and also to prevent the swollen cornea from being stored To transplant transplant.
  • swelling substance or "agent for swelling” used in the present invention refers both to the use for swelling of the cornea after storage and to the use as a permanent additive to the culture medium in order to keep the cornea in the swollen state during the entire storage hold.
  • the agent according to the invention comprises the respective culture medium in which the hydroxyethyl starch is contained.
  • the hydroxyethyl starch is suitably used in the culture medium in concentrations of 1 to 20% (w / v), concentrations in the range from 2 to 15% (w / v) being preferred, and in particular the hydroxyethyl starch in a concentration of 5 to 10% (w / v), for example 7.5% (w / v), is contained in the culture medium.
  • the hydroxyethyl starch used according to the invention is suitably added to the culture medium in a concentration of 1 to 10% (w / v), preferably 1 to 5% (w / v) and in particular from 2 to 7.5% (w / v) added.
  • the culture medium is used according to the invention Hydroxyethyl starch in a concentration of 1 to 20% (w / v), preferably 2 to 15% (w / v), in particular 5 to 10% (w / v), for example 7, 5% (w / v) added.
  • the corneas are stored in the culture medium under commonly used corneal bank conditions, e.g. at refrigerator temperatures (around 4 ° C) or at temperatures in the range of 30-37 ° C, in open or closed culture systems.
  • Suitable culture media are all customary known cell and tissue culture media for organ cultures of human and animal corneas.
  • suitable culture media for corneas are the TC 199 (Müller, MC et al. In Ophthalmic Res. 20 (1988), pages 44-53), modified TC 199 medium (Reim, M. Klin. Mbl. Walesicoheilk. 196 ( 1990), pages 76-80), MEM (Minimal Essential Medium) (Invest Ophthalmol Vis sei 12 (1973), 176-180), modified MEM (see e.g. Redbrake, C, Habilitationsschrift Aachen, 1996, p. 15; modification from MEM, for example with Earle's salts, with Hank's salts and the like.).
  • MEM or modified MEM or TC 199 is preferably used as the culture medium.
  • the degree of swelling of the corneas is dependent on the concentration of the hydroxyethyl starch used according to the invention, as shown by FIG. 5, which uses hydroxyethyl starch with an average molecular weight of 117,000, an MS of 0.39, a DS of 0.34 and a ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 11 in 10%, 7.5% and 5% concentration in the culture medium is made clear (cf. Examples 1-3).
  • the corneas have been stored for different lengths of time, there is a clear dependence on the hydroxyethyl starch Source of concentration. This suggests that hydroxyethyl starch leads to uniform de-swelling even with different swellings.
  • the hydroxyethyl starch used according to the invention leads to a significantly better swelling of the corneas, which is expressed in the lower pachymetry values and the lower water content, and no increased endothelial damage.
  • the hydroxyethyl starch used according to the invention provides significantly better metabolic values and the recovery in the organ culture is not lost.
  • a toxic effect as postulated for dextran is not found.
  • a corneal material is available for the transplant which is in an energetically better condition than after the swelling with dextran and which can therefore survive the transplant better.
  • the swelling with the hydroxyethyl starch used according to the invention permits the possibility of extending the storage period in a swelling medium beyond the previous time of 4 days and also the possibility of a permanent addition as a swelling substance for the entire duration of the organ culture.
  • Figure 1 relates to Examples 1 and 2 and shows the corneal thickness (HH thickness) after one-day de-soiling with HES (w 117,000, MS 0.39, DS 0.34 and the ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 11) in 5% or 10% concentration or with dextran 500 in 5% concentration in the culture medium.
  • HES corneal thickness
  • FIG. 2 relates to example 2 and illustrates the endothelial cell values of the corneas after one day of swelling with HES (Mw 117,000, MS 0.39, DS 0.34 and the ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 11) or with dextran 500 in 5% concentration in the culture medium.
  • Figures 3 and 4 relate to Example 3 and show the corneal thickness and the endothelial cell values after one day of swelling with HES (M w 117,000, MS 0.39, DS 0.34 and the ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units from 11) in 7.5% concentration or with dextran 500 in 5% concentration in the culture medium.
  • HES HES
  • Figure 5 illustrates the dependence of the degree of de-swelling on the
  • Ratio of substitution at C2 to substitution at C6 of the anhydroglucose units of 11) in the culture medium (cf. Examples 1, 2 and 3).
  • FIGS. 6 to 11 relate to example 4 and show the results of comparative tests with HES (M w 133,800, MS 0.44, DS 0.38 and the ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 12.4 ) in 7.5% concentration or with dextran 500 in 5% concentration in the culture medium with regard to their influence on the metabolic state of the cornea by one-day swelling by means of the swelling substances used.
  • HES M w 133,800, MS 0.44, DS 0.38 and the ratio of the substitution at C2 to the substitution at C6 of the anhydroglucose units of 12.4
  • dextran 500 in 5% concentration in the culture medium with regard to their influence on the metabolic state of the cornea by one-day swelling by means of the swelling substances used.
  • FIG. 6 the different hydration of the cornea
  • FIG. 7 the glucose content of the cornea
  • FIG. 8 the lactate content of the cornea measured in each case
  • Figure 9 shows the measured level of ATP in the cornea. ADP or to AMP,
  • Figure 10 shows the sum of the measured adenosine phosphates in the cornea
  • Figure 11 shows the respective energy status of the cornea.
  • FIGS. 7 to 11 also show the values obtained after 15 days of storage of the cornea in the culture medium (MEM).
  • the cornea of the eye was always used as the cornea.
  • HES hydroxyethyl starch
  • Modified MEM with the following composition was used as culture medium:
  • the MEM powder medium with Earle's salts used contained the following components (each in mg / 1): amino acids
  • EGF Extracellular Growth Factor
  • Vitamin A alcohol can. 1
  • the endothelial cell values were 2,231 ⁇ 296 endothelial cells per mm 2 for the hydroxyethyl starch-containing culm medium and 2,102 + 283 endothelial cells per mm for the culm medium containing dextran.
  • the corneas were pachymetrized 5 times each on the apex corneae using the pach-pen (Meuter company) before freezing. The endothelium was then documented photographically. 5 photographs were taken (1 photograph centrally, top, bottom, right and left) and the number of endothelial cells per mm 2 was determined twice on each picture.
  • Kulmr medium The same medium as described in Example 1 was used as the culm medium, except that 50 g of hydroxyethyl starch was added to the modified MEM to produce the hydroxyethyl starch-containing medium and 50 g of dextran 500 to the modified MEM to produce the modified medium were.
  • FIGS. 3 and 4 show a significantly better swelling with the Kulmr medium containing 7.5% hydroxyethyl starch (corneal thicknesses: Kulmr medium containing hydroxyethyl starch 0.634 ⁇ 0.072 mm; dextran-containing Kulmr medium 0.805 ⁇ 0.085 mm) without resulting in a correspondingly greater damage to the endothelium.
  • the endothelial cell values showed for the culture medium containing hydroxyethyl starch 9
  • the modified MEM described in Example 1 was used as the cooling medium, the modified MEM used in the case of the medium containing hydroxyethyl starch 75 g of the hydroxyethyl starch (7.5% concentration) and the modified MEM in the case of the medium containing dextran 50 g of dextran 500 (5% concentration) contained.
  • the conditions used corresponded to the conditions given in the examples mentioned above.
  • the statistical evaluation of the results was carried out in the Apple Stat Work Program.
  • the Man-Whitney U test was used.
  • the content of glucose, lactate, ATP, ADP and AMP and the adenylate energy batch were determined for the assessment.
  • the human cornea is mainly fed from the aqueous humor, from which glucose is absorbed, which is converted into pyruvate in the cornea by glycolysis and to lactate under anaerobic conditions. If there is a lot of oxygen, the aerobic route can also be followed via the citrate cycle and the respiratory chain.
  • the ratio of anaerobic to aerobic glycolysis is 65% to 35% and is thus far on the side of anaerobic glycolysis. Therefore, glucose as a substrate for energy production and lactate as an end product of the main metabolic pathway are important parameters for the metabolic performance of the human cornea.
  • the cells When generating energy through glycolysis, the cells recruit the high-energy adenosine phosphates: adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). These high-energy phosphates are used for all important cell functions, such as Cell migration, cell division, active transport mechanisms etc. are required. The more adenosine phosphates, the more functions the cell can perform. In the cornea - as with all other tissues - the sum of the adenosine phosphates also reflects the amount of cells. In contrast, the adenylate energy charge is a relative parameter that describes the condition of the individual cell and its energy balance.
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • AMP increased in swelling using HES.
  • the difference is statistically significantly different both from the swelling using dextran and from the values before the swelling.
  • the value using HES was 58.8233 + 46.5900 nmol per g dry weight and that using dextran 32.0286 ⁇ 47.4505 nmol per g dry weight (see FIG. 9).
  • the energy status is not significantly different in the three groups.
  • the value using HES was 0.55 + 0.16 and that using dextran 0.65 + 0.16 (see FIG. 11).

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  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Cet agent de déshydratation de cornées, notamment de la cornée des yeux en culture d'organes, contient le milieu de culture et comme substance déshydratante de l'amidon hydroxyéthylique avec un poids moléculaire moyen MW de 70.000 à 200.000, un degré de substitution MS de 0,15 à 0,5, un degré de substitution DS de 0,15 à 0,5 et un rapport entre les substitutions en C2 et en C6 des unités d'anhydroglucose >/= 8. La substance déshydratante est de préférence de l'amidon hydroxyéthylique avec un poids moléculaire moyen MW de 130.000 plus ou moins 20.000, un degré de substitution MS de 0.38 à 0,45, un degré de substitution DS de 0,32 à 0,40 et un rapport entre les substitutions en C2 et en C6 des unités d'anhydroglucose de 8 à 20. On utilise une concentration d'amidon hydroxyéthylique de 1 à 20 % (en poids/en volume), de préférence de 2 à 15 % (en poids/en volume), tout particulièrement de 7,5 % (en poids/en volume). L'invention concerne également l'utilisation de cet amidon hydroxyéthylique comme substance déshydratante dans la culture de cornées, notamment de la cornée des yeux, et l'utilisation de cet amidon hydroxyéthylique pour préparer un agent déshydratant de cornées, notamment de la cornée des yeux, en culture d'organes.
PCT/EP1998/000324 1997-01-23 1998-01-22 Agent de deshydratation de cornees en culture d'organes Ceased WO1998032839A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP53157298A JP2001510996A (ja) 1997-01-23 1998-01-22 臓器培養内角膜の脱水用試薬
EP98904113A EP1007635A1 (fr) 1997-01-23 1998-01-22 Agent de deshydratation de cornees en culture d'organes
US09/359,374 US6162642A (en) 1997-01-23 1999-07-23 Agent for dehydrating corneas in organ culture

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19702210A DE19702210C2 (de) 1997-01-23 1997-01-23 Verwendung von HES zur Entquellung von Hornhäuten in der Organkultur
DE19702210.3 1997-01-23

Related Child Applications (1)

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US09/359,374 Continuation US6162642A (en) 1997-01-23 1999-07-23 Agent for dehydrating corneas in organ culture

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WO1998032839A1 true WO1998032839A1 (fr) 1998-07-30

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JP (1) JP2001510996A (fr)
DE (1) DE19702210C2 (fr)
WO (1) WO1998032839A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6162642A (en) * 1997-01-23 2000-12-19 Fresenius Ag Agent for dehydrating corneas in organ culture

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0402724A1 (fr) * 1989-06-16 1990-12-19 Fresenius AG Hydroxyéthylamidon comme diluant du plasma et son procédé de préparation
WO1992005693A1 (fr) * 1990-10-01 1992-04-16 Ed Geistlich Söhne Ag Für Chemische Industrie Compositions chimiques

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2908436A1 (de) * 1979-03-05 1980-09-25 Fresenius Chem Pharm Ind Kolloidales gefrierschutzmittel
DE3007913A1 (de) * 1980-03-01 1981-09-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 8000 München Verfahren zum einfrieren von biologischem zellmaterial unter benutzung von frierschutzmitteln, die vor dem einsatz des zellmaterials nicht ausgewaschen werden muessen
US4879283A (en) * 1985-10-03 1989-11-07 Wisconsin Alumni Research Foundation Solution for the preservation of organs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0402724A1 (fr) * 1989-06-16 1990-12-19 Fresenius AG Hydroxyéthylamidon comme diluant du plasma et son procédé de préparation
WO1992005693A1 (fr) * 1990-10-01 1992-04-16 Ed Geistlich Söhne Ag Für Chemische Industrie Compositions chimiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAGENAH, M. ET AL.: "Hydroxyäthylstärke als Entquellungssubstanz in Kurzzeitkulturmedien für Spenderhornhäute", KLINISCHE MONATSBLÄTTER FÜR AUGENHEILKUNDE, vol. 208, no. 2, February 1996 (1996-02-01), pages 107 - 111, XP002068779 *
WALKENBACH, R.J. ET AL.: "The effects of UW solution and its components on corneal thickness during and after storage", CURRENT EYE RESEARCH, vol. 10, no. 12, December 1991 (1991-12-01), pages 1129 - 1136, XP002068780 *

Also Published As

Publication number Publication date
JP2001510996A (ja) 2001-08-07
DE19702210A1 (de) 1998-08-06
EP1007635A1 (fr) 2000-06-14
DE19702210C2 (de) 1999-01-14

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