WO1997014788A1

WO1997014788A1 - Method for inhibiting chymopapain and papain enzyme activity with zinc ions

Info

Publication number: WO1997014788A1
Application number: PCT/US1996/016607
Authority: WO
Inventors: Catherine Lee; Cynthia Zerfass; Tan Thanh Dinh; Minh T. Ma
Original assignee: Baxter International Inc.
Priority date: 1995-10-19
Filing date: 1996-10-18
Publication date: 1997-04-24

Abstract

The activities of chymopapain or papain in enzyme mixtures are inhibited by the addition of zinc ions in concentrations ranging from 0.0004 mM to 2 mM.

Description

METHOD FOR INHIBITING CHYMOPAPAIN AND PAPAIN ENZYME

ACTIVITY WITH ZINC IONS

BACKGROUND OF THE INVENTION

Field of the Invention The present invention relates generally to proteolytic enzyme compositions and procedures for digesting connective tissue. More particularly, the present invention is directed to methods for inhibiting proteolytic enzyme activity. In one of its more particular aspects, this invention relates to methods used to ensure optimum control in reproducibly digesting connective tissue and isolating viable cells from the digested tissue.

Description of Relevant Art

Many cell isolation and connective tissue digestion processes utilize proteolytic enzyme compositions. Similarly, many medical procedures involve the use of proteolytic enzymes to digest and remove unwanted or undesirable tissues such as scars, burn tissue, or herniated discs. To facilitate dissociating cells from connective tissue, a sufficient amount of enzyme must be used to provide the desired degree of digestion. In order to ensure that sufficient digestion occurs, an excess of enzyme may be used. However, the cells which are isolated may be adversely affected by certain enzymatic activity, particularly the activity of proteolytic enzymes characterized by broad specificity. Normal tissue can also be adventitiously degraded due to exposure to enzymes after each procedure. Many enzyme compositions used for cell dissociation and isolation or tissue treatment contain either chymopapain or papain. Both enzymes have activities toward a wide range of proteins.

It is necessary to stop undesired enzymatic activity as quickly as possible once the cells are dissociated from the tissues contaimng them, especially where isolation and purification procedures may continue for an extended period of time. Otherwise, cell integrity and function may be compromised by the continuing enzymatic degradation of cell surfaces which may result in substantial damage to the cells, such as cell membrane degradation or loss of cell surface receptors.

It is equally necessary to reduce enzymatic activity as soon as unwanted 5 tissues are disintegrated to minimize the damage to surrounding normal tissue. Although enzyme catalyzed reactions can, in general, be halted by separ¬ ating the enzyme from the reaction mixture, by changing the pH of the reaction, or by precipitating components of the reaction, these methods may be quite drastic and cause significant changes in reaction components. It would be desir- 10 able to halt the enzymatic action in a manner which had little, if any, effect on dissociated cells or reaction components other than the enzyme or enzymes used.

Accordingly, it is an object of the present invention to provide methods for controlling proteolytic enzyme catalyzed reactions. 15. It is another object of this invention to provide methods for digesting connective tissue in a reproducible and controllable manner.

It is another object of the present invention to provide methods for dissociating and isolating viable cells with predictable and reproducible yields and quality. 20 It is a further object of the present invention to provide viable and efficacious cells for various medical uses.

It is another object of the present invention to minimize damage to other untargeted tissue components.

Further objects, features, and advantages of the present invention will 25 become apparent to those skilled in the art from a consideration of the following detailed description, taken in conjunction with the associated drawings.

SUMMARY OF THE INVENTION The present invention accomplishes the above objectives by providing 30 inhibitors of papain and chymopapain which can be used to halt or reduce enzyme activity when desired. The inhibitors of the present invention are compounds which can release zinc ions in concentrations effective to suppress papain or chymopapain enzymatic activities. The inhibitors of the present invention are highly specific in their inhibitory activity and do not inhibit other sulfhydryl proteases such as clostripain or other broad spectrum proteases such 5 as trypsin.

It is also within the scope of the present invention to provide for isolating viable cells such as hepatocytes or pancreatic islet cells utilizing the inhibition techmques of the present invention. These processes effectively protect cells dissociated from tissue to enable the isolation of highly efficacious and viable

10 cells in high yield.

An exemplary process of the present invention includes enzymatically digesting connective tissue by providing an enzyme composition containing papain or chymopapain, or a mixture of papain and chymopapain essentially free of toxins, in an amount sufficient to hydrolyze connective tissue and dissociate 15. desired viable cells from such tissue. Contacting the connective tissue with the enzyme composition produces a turbid-appearing system indicating substantial tissue hydrolysis.

It is essential to halt or at least substantially slow down the enzymatic activity in the medium containing the isolated viable cells as soon as possible 20 after the cells are dissociated from the tissue in order to preserve the cell integrity. This is accomplished by preventing excessive digestion. The enzyme inhibition process of the present invention can be utilized for this purpose. Followmg addition of an inhibitor in accordance with the present invention, the viability of the isolated cells is greatly reserved and the yield of viable cells is 25 increased.

More specifically, a preferred process of the present invention utilizes the above steps following hydrolysis of connective tissue in order to protect and isolate viable cells such as hepatocytes and pancreatic islet cells. Advantage¬ ously, cells are recovered from the dissociated connective tissue in higher yield 30 and have improved viability when compared with cells which are not protected immediately by utilizing the enzyme inhibition process of the present invention. The process of the present invention is characterized by the increased number of viable healthy cells obtained.

The increased yield, as well as increased viability and integrity of cells isolated according to the processes of the present invention, is readily

5 demonstrated by laboratory testing techniques. Cell function and viability can be demonstrated by their biological function, such as production of insulin by pancreatic islet cells in response to glucose concentration change in culture media. This response is characterized by the ratio of insulin production in the presence of glucose to a base-line value.

10 The greater viability and number of useful cells isolated according to the teachings of the present invention are particularly important for applications which involve various medical procedures such as transplanting hepatocytes or pancreatic islet cells into individuals suffering from liver or pancreatic disease.

In another exemplary process, enzyme inhibition is utilized to protect

15. normal tissue following enzymatic digestion of undesirable tissue in proximity with the normal tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates the activities of human fibroblast cells 24 hours after 20 culturing in the presence of chymopapain and various concentrations of zinc ions; and

Fig. 2 illustrates the activities of human fibroblast cells after varying periods of time culturing in the presence of chymopapain and various concentrations of zinc ions. 25

DESCRIPTION OF EXEMPLARY EMBODIMENTS The present invention provides processes capable of better control in digesting physiological connective tissue in a variety of therapeutic and labor¬ atory applications. These applications range from in vivo therapeutic treatment 30 procedures to techniques which involve dissociating and isolating cells embedded in connective tissue for subsequent laboratory or clinical applications. The processes of the present invention are suitable for reproducibly isolating highly viable cells from tissues made up of proteins, glycoproteins, and extracellular matrix materials. The inhibitors can also be applied to treated areas to prevent over-digestion when these enzymes are used for removal of 5 undesirable tissues. Those skilled in the art will appreciate that the ability to carefully control the hydrolysis of a wide range of proteins and protein mixtures makes the teachings of the present invention widely applicable in a number of tissue removal treatment procedures or in vivo as well as in vitro cell isolation procedures.

10 The processes of the present invention find particular application in cell dissociation procedures including laboratory cell culture methods and related cell isolation techniques. As a feature of the present invention, cells are effectively and reproducibly isolated from a host of different proteinaceous con¬ nective tissues and can be harvested in higher yield with improved preservation

15. of the cell membranes. Moreover, these cells have better viability than cells isolated using prior art processes. For this reason, the compositions and processes of the present invention are particularly suitable for isolating highly viable cells embedded in connective tissues for subsequent utility in various clinical procedures such as transplanting hepatocytes or pancreatic islet cells into

20 individuals suffering from liver or pancreatic diseases.

Preferred exemplary embodiments of the present invention utilize enzyme solutions containing papain or chymopapain or mixtures thereof in a physiologically compatible liquid. Suitable physiologically compatible liquids include phosphate buffered saline solutions and similar buffered electrolyte so-

25 lutions having osmolarities which are compatible with physiological tissue. A particularly suitable commercially available electrolyte solution is Plasmalyte^® electrolyte solution available from Baxter-Hyland, having a buffered pH of 7.4 and an osmolarity of 294 mOsmol/L obtained with controlled concentrations of sodium, potassium, magnesium, chloride, acetate, and gluconate ions. Another

30 suitable electrolyte solution is medium 199. As illustrated below, additives such as human serum albumin and serum are preferred in many applications. Those skilled in the art will appreciate that the concentration or amount of each enzyme present in the solutions will vary with the amount and the type of tissue to be hydrolyzed. The well-known principles of enzyme activity are applicable and basic experimentation involving techniques designed to optimize

5 enzyme concentration and total activity provide necessary information to assure the effective hydrolysis of the amount and type of selected connective tissue.

For applications directed toward digesting connective tissue and isolating hepatocytes and pancreatic islet cells embedded in the tissue, for example, exemplary compositions of the present invention may include a solution of from

10 about 0.15 nkat/ml to about 0.55 nkat/ml purified papain or chymopapain in a suitable pH buffered physiologically compatible liquid. The nkat/ml unit is defined as nanomoles of substrate hydrolyzed per second by 1 ml of enzyme solution under the assay condition used. For purposes of the present invention, enzyme activity assays were conducted at 37°C in Tricine buffer, 50 mM,

15. containing 10 mM Ca l_.

Chymopapain, which is a proteolytic enzyme extracted from papaya latex, is commercially available in a dry lyophilized state from a number of sources including Sigma Chemical of St. Louis, Missouri. Chymopapain is available in crude, partially purified, and more highly purified forms which differ in the

20 amount of papain, lysozyme peptidase A, and sensitizing antigens found in the preparation. Chymopapain suitable for use in the present invention is characterized as having essentially no other proteolytic enzyme contamination as a result of purification processes. Chymopapain from most commercial sources, which has been purified using known chromatographic purification

25 processes, provides chymopapain suitable in the practice of the present invention. Alternatively, purified chymopapain can be prepared using, for example, the process described in U.S. Patent No. 4,719,108. Papain, another papaya-derived enzyme is also commercially available.

An exemplary tissue system for demonstrating the features of the present

30 invention is connective tissue. Generally, connective tissue, which holds cells together, is a complex mixture of collagen, other extracellular proteins, glycoproteins, and mucopolysaccharides.

Thus, the processes of the present invention broadly include providing an enzyme composition and causing the composition to contact selected tissue for

5 a length of time and at a temperature sufficient to substantially hydrolyze the tissue and permit isolation of the desired cells, and inhibiting the enzymatic action after cell release and following cell isolation.

Preferred exemplary processes in accordance with the teachings of the present invention include digesting connective tissue for the purpose of

10 dissociating and isolating cells embedded in the connective tissue. The process of the present invention provides highly viable cells which are particularly useful for gene therapy and transplanting into humans or animals for therapeutic pur¬ poses. For example, pancreatic cells can be isolated from donor pancreases and transplanted into humans or animals for purposes of treating pancreatic related

15. diseases. Additionally, hepatocytes can be isolated from liver in accordance with known procedures utilizing compositions of the present invention.

A most preferred process of the present invention includes providing a mixture of enzymes in which papain or chymopapain is one of the components, contacting connective tissue with the enzyme mixture for a length of time and

20 at a temperature sufficient to substantially hydrolyze the connective tissue and to release viable cells embedded in the tissue, and immediately inhibiting the remaining enzymatic activity by adding a concentration of zinc ions sufficient to halt or substantially slow down such enzymatic activity.

Commercially available chymopapain and papain inhibitors are extremely

25 expensive and, unlike zinc ions, are not natural tissue components. They are toxic and may be immunogenic. Thus, they are not suitable for in vivo use.

The inhibitor can be added before centrifugation and pipeting off the supernatants from the cells, if desired, to reduce damage to cells during this process. Preferred exemplary processes further include rinsing the cells with a

30 physiologically compatible liquid prior to their evaluation and use. The zinc ion inhibitor of the present invention can be furnished in the form of any physiologically acceptable zinc salt, such as zinc acetate or zinc chloride. Concentrations in the range of about 0.0004 mM to 2 mM zinc ions in the medium can be used, preferably concentrations of about 0.01 mM to 1 mM. It has been found that concentrations of 0.05 mM are effective to reduce the enzyme activity of papain or chymopapain by about 75%. Accordingly, concentrations of zinc ions of at least about 0.05 mM are especially preferred.

As generally mentioned above, hepatocytes and pancreatic islet cells isolated from hydrolyzed tissues in accordance with the teachings of the present invention are isolated in higher yields and have greater viability than cells isolated by prior art processes in which enzyme activity is permitted to continue, even to a limited extent, following cell isolation. Moreover, since the enzyme compositions used in the processes of the present invention are purified, in the event that isolated cells are implanted for therapeutic purposes or are subjected to other in vivo uses, any residual cotransplanted enzyme composition will not produce any adverse effect.

The superior physical and functional characteristics of the cells isolated according to the process of the present invention are demonstrated by the higher yield of cells having expected characteristics as determined by known cell- counting methods. Another technique involves use of methods in which a particular substrate is incubated with the cells to convert the substrate to a colored product. The optical density of lysed cells at 570 nm, which is proportional to the number of viable functional cells, is then determined with a spectrophotometer. As described in more detail in the examples which follow, cell activity is found to be higher with increasing zinc ion concentration added in accordance with the process of the present invention.

The resultant superior physical and functional characteristics of cells isolated according to the present invention make them particularly useful for transplanting. The high viability and functional ability of these cells provide a transplant that is less susceptible to functional failure. In another embodiment of the present invention, undesirable tissue can be removed from normal tissue by the steps of providing an aqueous solution of chymopapain, papain, or a mixture thereof, in a physiologically compatible electrolyte solution buffered to a pH of about 7.0 to 7.4; contacting undesirable tissue with the enzyme solution for a length of time and at a temperature sufficient to hydrolyze the undesirable tissue; removing hydrolyzed tissue from normal tissue in contact therewith; and rinsing the normal tissue from which the undesirable tissue has been removed with a solution containing zinc ions in a concentration sufficient to inhibit the chymopapain or papain enzymatic action. The invention will be better understood by reference to the following nonlimiting examples which illustrate the use of various concentrations of zinc ions and other ions in inhibiting various enzymes. In these examples the activities of chymopapain and papain were determined by assay using BAPNA, N-benzoyl-L-arginine-p-nitroanilide synthetic substrate by measuring the increase in absorbance at 415 nm. The activity of clostripain was determined by assay using BAEE, N-benzoyl-Lrarginine ethyl ester synthetic substrate by measuring the increase in absorbance at 253 nm. The activity of trypsin is determined by assay using BAPNA.

The following example demonstrates the reduction in activity of various enzymes in the presence of various metal salts.

Example 1

Protease activity was determined by measuring the reaction rates for the hydrolysis of synthetic substrates for the enzymes trypsin, chymopapain, papain, and clostripain. The increase in absorbance at 415 nm was used for the measurement of the reaction rates for trypsin, chymopapain and papain upon a

BAPNA substrate. The increase in absorbance at 253 nm was used for the measurement of the reaction rates for clostripain. Enzyme activities of trypsin, chymopapain, and papain were determined at 37°C in Tricine buffer, 50 mM, containing 10 mM CaCl_j. Enzyme activities of clostripain were determined at room temperature. Chymopapain, papain, and clostripain were activated with L-cysteine or dithiothreitol reducing agent prior to assay. Concentration of reducing agent in the substrate solution was maintained at 5 mM by addition of reducing agent to the substrate solution. Various salts were tested as inhibitors by dissolving in the assay buffer to a final concentration of 1 mM. The percentage inhibition was obtained from the ratio of enzyme activity in the presence of inhibitor to the activity of a control to which no inhibitor was added. The results are shown in Table I.

TABLE I INHIBITION BY VARIOUS COMPOUNDS

Ti psin Chymopapain Papain

Compound Clostripain % Inhibited % Inhibited % Inhibited % Inhibited

Control 0 0 0 0

CaC^ 0 0 0 0

Mn Acetate 0 5.1 4.8 5.9

FeCNIL^SO^ 7.5 0 0 NA

CoCSCN^ 0 9.7 16.5 0

NiO_ 0 12.5 11.5 NA

MgCl, 0 0 0 1.1

Zn Acetate 0.3 94.4 95 Increased Activity

These results indicate that a simple compound like zinc acetate is effective in inhibiting chymopapain and papain activities. This effect is unique, since other compounds containing metal ions do not produce this effect. These results also indicate that the inhibition effect of zinc acetate on these two enzymes is unique. Other proteases such as trypsin and clostripain are not inhibited. Clostripain in particular is similar to chymopapain and papain in requiring a reducing agent for activation. The following example illustrates the effect of using various concentrations of zinc acetate.

Example 2 The procedure of Example 1 was repeated using various concentrations of zinc acetate.

Zinc acetate was dissolved in the assay buffer to final concentrations of

0.001 mM, 0.002 mM, 0.05 mM, 0.25 mM, and 1 mM. The percentage inhibition was obtained from the ratio of enzyme activity in the presence of inhibitor to the activity of a control to which no inhibitor was added. The results are shown in

Table II.

TABLE π INHIBITION BY ZINC ACETATE

Zinc Acetate Clostripain

Concentration, Trypsin Chymopapain Papain ed % Inhibited mM % Inhibited % Inhibited % Inhibit

0 0 0 0 0

0.001 0 0 0 0

0.002 0 6.3 1.8 0

0.050 0 79.9 77.8 0

0.250 0 91.4 89.8 Increased Activity

1.000 0.3 94.4 95.0 Increased Activity

From the foregoing data it can be seen that whereas a concentration of as low as about 0.05 mM zinc acetate was sufficient to reduce the activities of chymopapain and papain by more than 75% and that, even at concentrations as low as 0.002 mM, inhibiting activity could be detected, zinc acetate had no inhibiting effect on clostripain and was ineffective upon trypsin at all concentrations up to 1 mM. Even at 1 mM, zinc acetate inhibited trypsin only to the extent of 0.3%.

The following example illustrates the use of another zinc salt as the inhibitor.

Example 3 The procedure of Example 1 was repeated except that zinc chloride was used as the inhibitor material. The results are shown in Table lu.

TABLE m INHIBITION BY ZINC CHLORIDE

Zinc Chloride Chymopapain Papain Concentration, mM % Inhibited % Inhibited

- 0 0 0

0.0004 1.6 1.8

0.0020 22.5 19.9

0.0100 49.4 47.1

0.0500 77.2 76.0

0.2500 90.5 89.7

1.0000 94.6 94.1

2.0000 100.0 94.6

The foregoing results show that the inhibition of chymopapain and papain activity by zinc chloride is similar to that displayed by zinc acetate. These results indicate that the observed inhibition is due to the presence of zinc ions, Zn^{+ +} , and not to any particular zinc salt.

In general, any soluble salt of zinc can be used. For use in cell isolation or in therapeutic applications, a physiologically acceptable salt is preferred. The following example illustrates the effect of enzyme inhibition upon cell activity in accordance with the process of the present invention.

Example 4 5 Human fibroblasts were cultured with medium 199 supplemented with bovine serum, trypsinized, collected, and re-suspended in media. A sample of 10,000 fibroblasts was transferred to each well of a microliter plate together with chymopapain to a final concentration of 0.25 nkat/ml to simulate the presence of residual enzyme activity after cell isolation. Zinc acetate in culture media

10 was then added to final concentrations of 0, 10, 25, and 100 μM. The cells were cultured at 37°C in a humidified incubator with 5% CGL.. After two hours, to allow viable cells to attach to the culture surface, the supernatant of each culture was removed and replaced with fresh media without either chymopapain or zinc acetate. The cell activities were measured on day 1 (after 24 hours), day 3 and

15. day 6 using the commercially available Promega method. A substrate solution was added to each culture, and after four hours of incubation at 37°C in the cell culture incubator, a lysing solution was added. Cell samples were mixed one hour later, and the optical density of these solutions at 570 nm were determined with a spectrophotometer. Higher absorbencies indicated the presence of more

20 active cells. The results are shown in Fig. 1 for cell activities after 24 hours and in Fig. 2 for cell activities after 1, 3 and 6 days.

As indicated in Fig. 1, more active fibroblast cells were found in cultures containing higher zinc ion concentrations. These results proved that suppression of chymopapain activity could be used to improve the cell activity or viability.

25 The beneficial effects of zinc ions could be sustained over longer culture periods (as shown in Fig. 2), even when the residual enzyme and inhibitor were removed shortly after the cells were cultured.

Having thus described preferred exemplary embodiments of the present

30 invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments illustrated herein.

Claims

WHAT IS CLAIMED IS:

1. A process for inhibiting the activity of an enzyme selected from the group consisting of chymopapain and papain which comprises adding zinc ions to a medium containing said enzyme.

2. The process of claim 1 wherein the concentration of zinc ions in said medium is in the range of about 0.0004 mM to about 2 mM.

3. The process of claim 1 wherein the concentration of zinc ions in said medium is about 0.01 mM to 1 mM.

4. The process of claim 1 wherein said zinc ions are added to said medium in the form of a zinc salt.

5. The process of claim 4 wherein said zinc salt is zinc acetate.

6. The process of claim 4 wherein said zinc salt is zinc chloride.

7. A process for inhibiting the activity of an enzyme selected from the group consisting of chymopapain and papain which comprises adding zinc ions to a medium containing said enzyme, said zinc ions being added in a concentration of at least about 0.01 mM, whereby the activity of said enzyme is inhibited to the extent of about 50%.

8. A process for isolating viable hepatocytes or pancreatic islet cells from tissue containing viable hepatocytes or pancreatic islet cells, said process comprising the steps of: providing an enzyme composition comprising an aqueous solution of an enzyme selected from the group consisting of chymopapain, papain, and mixtures thereof, said enzyme having an activity of about 0.15 nkat/ml to about 0.55 nkat/ml, in a physiologically compatible electrolyte solution buffered to a pH of about 7.0 to 7.4; contacting tissue containing viable hepatocytes or pancreatic islet cells with said enzyme for a length of time and at a temperature sufficient to hydrolyze said tissue; isolating viable hepatocytes or pancreatic islet cells from the hydrolyzed tissue; and adding zinc ions to a medium containing the isolated viable hepatocytes or pancreatic islet cells, said zinc ions being added in a concentration sufficient to inhibit the enzymatic action of said chymopapain or papain or mixture of chymopapain and papain; thereby improving the yield of viable hepatocytes or pancreatic islet cells and enhancing the cell activity thereof.

9. The process of claim 8 wherein the concentration of said zinc ions is in the range of about 0.0004 mM to about 2 mM.

10. The process of claim 9 wherein said concentration is at least about 0.01 mM and is sufficient to inhibit said enzymatic action to the extent of about 50%.

11. The process of claim 8 wherein said zinc ions are added to said medium as zinc acetate.

12. The process of claim 8 wherein said zinc ions are added to said medium as zinc chloride.

13. A process for removing undesirable tissue from normal tissue, comprising the steps of: providing an enzyme composition comprising an aqueous solution of an enzyme selected from the group consisting of chymopapain, papain, -17-

and mixtures thereof, in a physiologically compatible electrolyte solution buffered to a pH of about 7.0 to 7.4; contacting tissue containing undesirable tissue in contact with normal tissue with said enzyme composition for a period of time and at a temperature sufficient to hydrolyze said undesirable tissue; removing hydrolyzed undesirable tissue from said normal tissue ; and rinsing the normal tissue from which the undesirable tissue has been removed with a solution containing zinc ions, said zinc ions being present in a concentration sufficient to inhibit the enzymatic action of said chymopapain or papain or mixture of chymopapain and papain.

14. The process of claim 13 wherein the concentration of said zinc ions is in the range of about 0.0004 mM to about 2 mM.

15. The process of claim 14 wherein said concentration is at least about 0.01 mM and is sufficient to inhibit said enzymatic action to the extent of about 50%.

16. The process of claim 13 wherein said zinc ions are added to said medium as zinc acetate.

17. The process of claim 13 wherein said zinc ions are added to said medium as zinc chloride.