WO2000040746A1

WO2000040746A1 - Compositions and methods for stabilizing thiol-containing biomolecules

Info

Publication number: WO2000040746A1
Application number: PCT/US2000/000114
Authority: WO
Inventors: Mark H. Smit; Mikhail Bamm
Original assignee: Medical Analysis Systems, Inc.
Priority date: 1999-01-08
Filing date: 2000-01-03
Publication date: 2000-07-13
Also published as: AU2404500A

Abstract

The present invention relates to compositions that are useful for reducing undesirable disulfide bonds in biological molecules. Disclosed herein are compositions that are useful for reactivating oxidized enzymes to restore native activity, as well as compositions that are useful for maintaining thiol compounds such as enzymes in a reduced state. The compositions comprise at least 3 reducing agents with a difference of at least 5 % more or less than the rest nearest agent. Exemplary of the three agents one n-acetyl-1-cysteine, dithiothreitol and methimazole prolonged liquid storage.

Description

WO 00/40746 PCTJUSOO/00114

COMPOSITIONS AND METHODS FOR STABILIZING THIOL-CONTAINING BIOMOLECULES

TECHNICAL FIELD

This invention relates generally to compositions and methods that are useful for stabilizing thiol-containing biomolecules. More particularly, this invention relates to compositions that maintain their reducing potential after extended liquid storage, and their use in reactivating oxidized biomolecules. This invention also relates to methods of preventing the loss of electrons from reduced forms of proteins, as well as methods of making compositions containing liquid stable reducing agents. This invention is particularly useful for reactivating oxidized enzymes for performing diagnostic measurements of such enzymes, the accuracy of which requires full expression of enzymatic activity.

BACKGROUND OF THE INVENTION

Many different types of molecules may contain or be modified to contain disulfide bonds. For example, lipids, polysaccharides, synthetic organic compounds and proteins all may contain disulfide bonds. In complex biological systems, the reduction of disulfide bonds and oxidation of thiols may play an important role in cellular function and metabolism.

Proteins are complex biomolecules that exhibit many different activities in biological systems. Enzymes are highly specialized proteins that act as catalysts of chemical reactions. Because of the complexity of most enzymatic reactions, the catalytic efficiency of an enzyme depends on the integrity of its conformation. If an enzyme is denatured or dissociated into subunits, its enzymatic activity is usually lost. This can be caused by heat, changes in pH, or a variety of other environmental or chemical factors. In particular, many enzymes are subject to oxidation during purification, handling and storage, and must be reduced to restore catalytic activity.

Many different diagnostic tests require the accurate measurement of enzymes in blood and other bodily fluids. For example, the measurement of creatine kinase enzyme in blood is used to assist in the diagnosis of a variety of pathological conditions. Elevated creatine kinase levels in the blood is often associated with myocardial infarction, muscle disease, and various neurological conditions. Thus, the accurate measurement of creatine kinase is an important tool for providing useful diagnostic information.

The most straightforward way of measuring the presence or amount of a particular enzyme in a sample is to measure its enzymatic activity. This is accomplished by providing a substrate for the enzyme and measuring, either directly or indirectly, the resulting catalytic activity. In the case of creatine kinase, one of the most widely recognized assay systems involves the conversion of creatine phosphate and ADP to creatine and ATP (G. Szasz et al, Clin, Chem, 22:650-656 (1976) and M. Harde et al, Eur. J. Clin. Chem. And Clin. Biochem. 29:435-456 (1991)). This reaction is coupled to two additional enzymatic reactions as follows: ( 1 ) Creatine phosphate + ADP -» Creatine + ATP

(2) ATP + Glucose -» ADP + Glucose-6-phosphate

(3) Glucose-6-phosphate + NNDP - 6-phosphogluconate + ΝNDPH Creatine kinase activity is determined by measuring the rate of ΝADPH formation with a spectrophotometer at 340 nm.

Cysteine residues in proteins are labile and can undergo oxidation which may result in undesirable disulfide bond formation. For many enzymes, oxidation of cysteine residues leads to a loss of catalytic activity. For example, creatine kinase has a labile cysteine residue near its catalytic site (R. Furter, et al, Biochemistry, 32:7022-7029; and L.-X. Wang, et al, Biochemistry, 27:5095-5100 (1988)). When this cysteine residue becomes oxidized during storage of clinical samples, creatine kinase activity is diminished. Thus, it is necessary to reactivate the enzyme to restore its native activity. (A. Szasz et al, Clin. Chem., 24:1557-1563 (1978)).

Reactivation is generally accomplished by adding a thiol-containing compound {i.e., "thiol compound") to the sample containing creatine kinase. The thiol compound acts as an electron donor which causes electrons to be transferred to the enzyme to restore the oxidized cysteine residues (i.e., "cystine") to the reduced state. However, because of the propensity for thiol compounds to become oxidized by donating their electrons to electron-poor acceptors, they tend to be unstable in solution. This causes them to lose their effectiveness as reactivators of enzyme activity.

Several different methods have been reported to prevent oxidation of thiol compounds in solution. These methods include the removal of electron acceptors from the solution, as well as the removal of catalysts for electron transfer. For example, it has been recognized that the addition of metal chelators extends the shelf life of creatine kinase reactivating reagents. (G. Szasz et al, Clin. Chem. 25:446-452 (1979)). This stabilizing effect is due to the removal of metal ions, which can function as catalysts for electron transfer.

Other passive methods for stabilizing thiol compounds have also been described. For example, U.S. Patent No. 5,700,653 describes the addition of free radical scavengers to the solution to slow down the oxidation of thiols. In another example, U.S. Patent No. 4,888,289 describes the removal of magnesium, a known catalyst of electron transfer, from a creatine kinase activating reagent to prevent oxidation. However, such mechanisms for stabilization are preventative and passive, and are not fully capable of maintaining a storage stabile reducing environment in which the creatine kinase can eventually be exposed to for reactivation.

The present invention relates to compositions that provide an active method for stabilizing the reduced state of thiol compounds or other reductants in reagents that are used to reduce oxidized disulfide-containing biomolecules such as creatine kinase. The present invention also relates to methods of using such compositions to reduce disulfide-containing biomolecules.

SUMMARY OF THE INVENTION

It has recently been discovered that compositions containing mixtures of reducing agents having different thioreductant potential are capable of retaining their ability to reduce disulfide bond-containing biomolecules during prolonged liquid storage.

Accordingly, one aspect of the present invention pertains to compositions comprising at least three reducing agents having different thioreductant potentials. In a preferred embodiment, all three reducing agents are thiol compounds. Although the range of thioreductant (i.e., "redox") potentials may vary, in a preferred formulation, there is at least a

10% difference in redox potential between reducing agents, such that the redox potential of each agent is at least 5% less or more than the next closest agent.

In another embodiment, the present invention relates to a composition for reducing a disulfide bond in a biological molecule comprising (1) an effective protein reducing agent such as N-acetylcysteine (NAC) as a first reducing agent, (2) a thiol-reductant having a greater thioreductant potential than the protein reducing agent as a second reducing agent, and (3) a supporting thiol agent having a reductant potential at least 5% less than the first reducing agent.

When the composition is to be used to reactivate oxidized creatine kinase, the thiol- reductant compound is preferably dithiothreitol, and the thiol supporting agent is preferably methimazole. In another embodiment, a fourth reducing agent such as an additional thiol- reductant compound (e.g., homocysteine or cysteamine) may also be added for further stabilization. In such compositions, it is useful to enhance stability to maintain the pH between about 5.8 and 6.0. In another aspect of the present invention, the compositions described above remain stable during storage in a liquid medium for up to six months at 4°C, more preferably for up to ten months at 4°C, and most preferably for up to twelve months at 4°C. By "stable," it is intended that the compositions substantially retains its ability to reduce the disulfide bonds of the biological molecule it was designed to reduce. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

OF THE INVENTION

In accordance with the present invention, compositions are prepared that are useful for reducing the disulfide bonds in biological molecules. In particular, these compositions are useful in reactivating thiol-containing enzymes such as creatine kinase that become oxidized during storage. These compositions comprise at least three different reducing agents with varying thioreductant potential. As used herein, the term "thioreductant potential" is used to refer to the extent to which a reducing agent is capable of reducing a disulfide bond. Thioreductant potential, which is also referred to herein as "redox potential", can be measured according to well known chemical principles. For example, glutathione is commonly used as a standard, and the thioreductant potential of individual reducing agents is measured in terms of their ability to reduce glutathione. See, e.g., H. F. Gilbert, Methods in Enzymology, vol. 251, pages 8 to 28, Academic Press, New York, New York (1995).

The compositions of the present invention provide an electron gradient created by combining three or more reducing agents with different redox potentials. The overall reducing potential of such compositions is more stable than compositions containing only one or two reducing agents. A compound is considered to be a "reducing agent" if it is capable of acting as an electron donor to constituents of the composition.

The magnitude of the redox potential of a reducing agent characterizes the agent in terms of its ability to serve as an electron donor. The more negative the redox potential (i.e., a larger negative value), the greater the reducing power. It is generally believed that the best way to preserve the reducing ability of disulfide bond-reducing compositions, and especially enzyme reactivating compositions, is to select a reducing agent with a greater redox potential, or to increase the overall concentration of the reducing agent. However, the need to increase the redox potential of a composition to preserve stability must be balanced with the fact that: (1) the stronger the reducing agent, the more susceptible it is to oxidation during long-term storage; and (2) if the reducing agent is too strong it may be damaging to other components in the final assay mixture. Weaker reducing agents are thus more stable and less inhibitory, but they have a diminished ability to reactivate oxidized enzymes such as creatine kinase.

Therefore, a mixture of reducing agents is optimal to serve the following three important functions: (1) act as an activator of oxidized biological molecules by donating electrons to disulfide bonds to convert them back into thiol groups without inhibiting other liquid reagent components; (2) create a stable reducing environment to minimize oxidation of the principle electron donor; and (3) prevent the terminal loss of electrons from the reducing agent to other components of the solution, such as oxygen. Although the three reducing agents have overlapping roles in the compositions of the present invention, it is generally believed that each of the three (or more) reducing agents will be better adapted to serve one of these three functions. Accordingly, the three-component compositions of the present invention are capable of synergistically maintaining a stable reducing environment by creating a gradient of electron potentials that allows the compositions to remain stable and effective, and serve the function of reducing undesirable disulfide bonds, such as might be necessary to reactivate oxidized enzymes. By employing such an electron gradient mechanism, stability can be improved and the harmful effects of high concentrations of any one reducing agent on other composition components, or on the biological molecule that is to be reduced, can be avoided.

The selection of individual reducing agents for use in the three-component compositions of the present invention depends in part on the characteristics of the disulfide bond-containing biological molecule that the compositions will be used to reduce. First, there is selection of the "primary reducing agent", i.e. the reducing agent that will serve primarily to directly interact with the disulfide bond in the biological molecule to reduce it. In a preferred embodiment, the primary reducing agent is a thiol-containing reducing agent. When the disulfide bond-containing biomolecule is CK, a particularly preferred primary reducing agent is N-acetyl-L-cysteine (NAC).

The primary reducing agent should have a redox potential that is sufficiently negative to allow it to reduce the disulfide bond in the biological molecule. This means that the thiol/disulfϊde exchange reaction between the two species should favor reduction of the disulfide bonds in the biological molecule. See H. F. Gilbert, supra. Second, the liquid environment surrounding the disulfide bond(s) in the biological molecule should be taken into consideration . For example, the primary reducing agent should be capable of direct interaction with the disulfide bond under conditions in which it will be utilized. If the biological molecule is an enzyme such as creatine kinase that is to be measured in a biological sample such as human serum, the selection and concentration of reducing agent should take into account the presence of other substances that may diminish the ability of the reducing agent to reactivate the enzyme in this environment.

Although not necessary for the practice of the present invention, when the disulfide bond-containing biological molecule is a large and complex molecular species such as a protein whose disulfide bonds may be somewhat inaccessible to other large molecules, it is generally preferred that the primary reducing agent be small enough to interact freely with the disulfide bonds. As such, for these types of applications, the primary reducing agent is preferably a small molecule, such as a molecule having a molecular weight less than 2,000 g/mole, and more preferably less than 1000 g/mole, and most preferably less than

200 g/mole.

Once these factors are considered, the selection of primary reducing agent is a matter of routine optimization that can easily be carried out by one of skill in the art by testing reducing agents for their ability to reduce a given disulfide bond. Nfter a primary reducing agent candidate is selected, in order to stabilize the redox potential of the primary agent in solution, two other secondary reducing agents are added to the solution. One secondary reducing agent (also referred to herein as the "second" reducing agent) is added that has a greater redox potential than that of the primary reducing agent. This secondary reducing agent is thought to function by reconverting any oxidized reducing agent back into the reduced state. Another secondary reducing agent (also referred to here as the "third" reducing agent) is added that has a lesser redox potential than that of the primary reducing agent, and is thought to function by preventing electron loss to the surrounding liquid environment. It should be understood that both the second and third reducing agent may also directly interact with the disulfide bonds in the biological molecule to convert them to the reduced state. However, since the compositions of the present invention are specifically designed to perform as an electron gradient in solution, it is less likely that the reducing agent with the lowest redox potential will interact with the biological molecule as readily as the reducing agent(s) with higher redox potentials. Since the third reducing agent serves primarily to prevent loss of electrons to the surrounding liquid medium, its role as a "reducing agent" in the common sense is somewhat minimal.

The redox potential of known reducing agents can be found in standard redox tables in the literature. For example, see P. Wardman, J. Phys. Chem. Ref Data. 18C4 :1637-1755 (1989). Alternatively, redox potentials can be experimentally determined using standard electrochemical techniques. See, for example, H. F. Gilbert, supra. Although the redox potentials of the three reducing agents of the present invention may vary according to the principles outlined above, it is generally believed that the overall spread of redox potentials should preferably be about 0.05V or greater and more preferably 0.1 V or greater.

Examples of the redox potentials of various individual reducing agents are given below in Table I:

TABLE I

*From H. F. Gilbert, supra. The thioreductive potential of some compounds can be estimated based on their comparison to compounds having known thioreductive potentials. For example, the thioreductive potential of methimazole would be expected to be less than that of thionitrobenzoic acid, since it is less efficient at reducing a disulfide-containing substrate, such as dithiobisnitrobenzoic acid (DTNB). In addition, N-acetyl-L-cysteine is expected to have a redox potential greater than that of cysteine, because of the addition of the acetyl group which has known electron donor properties. Accordingly, if the redox potential of a candidate reducing agent cannot be empirically determined, the relative redox potential can be determined by studying the ability of the reducing agent to reduce compounds having known oxidation-reduction characteristics, and three reducing agents can be selected that have the appropriate relative redox potential. For example, reducing agents can be individually tested for their ability to reduce a given disulfide bond in a substrate such as glutathione. Once a primary reducing agent is selected and tested for its ability to reduce the disulfide bond of a biological molecule without causing any undesirable effects on other composition components, secondary reducing agents can be chosen - one with substrate reducing power greater than the primary reducing agent, and one with substrate reducing power less than the primary reducing agent. It is generally believed, although not absolutely necessary, that the relative redox potential of the secondary reducing agents should be at least 5%, and more preferably 10% greater/less than (in terms of absolute values) the primary reducing agent to effect an efficient electron gradient system.

An important aspect of the compositions of the present invention is that they are capable of retaining their redox potential during liquid storage. Monitoring of stability is accomplished by measuring the ability of the composition to reduce a desired disulfide bond- containing biological molecule at different time points after the composition is first formed and under the same (or equivalent) storage conditions as one desires for the selected composition. Preferred compositions are capable of retaining their thioreduction potential for at least 6 months, more preferably at least 10 months, and most preferably at least 12 months at 4°C. The reducing agents of the present invention are preferably small organic molecules. Although thiol-containing compounds are generally preferred, other types of reducing agents such as trivalent phosphorous compounds (see U.S. Patent No. 5,817,467) may also be suitable. In addition, in accordance with the principles described herein, it is also contemplated that larger molecules, such as lipids, polysaccharides, proteins and various synthetic polymers may be designed to serve as reducing agents. It is also possible that a single molecule with three different moieties, each having different redox potentials, may be capable of being utilized in the practice of the present invention.

The compositions of the present invention are useful in a variety of scientific and medical applications, such as components of multi-component diagnostic reagents. For example, when CK is the biological molecule to be reactivated, the composition can be incorporated as part of the "coenzyme" component of the reagent. See Example 2. When the biological molecule is CK, the preferred principle reducing agent is N-acetyl-L-cysteine, or "NAC", because of its known effectiveness at reactivating CK. With NAC as the primary reducing agent, a preferred second reducing agent is dithiothreitol, since it is also a thiol compound that has been shown to be effective at reducing CK, and its greater redox potential is well-suited for enhancing stability of the NAC in solution. A preferred third reducing agent for this combination is methimazole, which is a thiol compound having a relatively low redox potential. In addition to the three reducing compounds, it may also be desirable to include other substances that enhance stability or performance such as metal chelators (e.g., EDTA) antioxidants (e.g., superoxide dismutase or catalase), or buffers. It should also be understood that to enhance stability, the compositions should be manufactured to reduce metal and oxygen contamination. Furthermore, the pH of the composition should be optimized to enhance stability. In general, a greater than neutral pH will cause a loss of thioreduction potential during liquid storage. In addition, a pH that is too low should be avoided to prevent the damaging effects an acidic pH might have on other composition components such as enzymes. Accordingly, a preferred pH is between about 5.5 to 6.5, and more preferably between about 5.8 to 6.0. EXAMPLES

Example 1 Determination of the Relative Effectiveness of Different Reducing Agent Compositions

In Reducing Oxidized Creatine Kinase

The ability of selected thiol-containing reducing agent compositions to activate creatine kinase (CK) was studied as a function of the ability of the compositions to reactivate oxidized CK of different control and clinical samples. A two-part ("substrate" and

"coenzyme") formulation for measuring CK activity was compounded according to the method described by Szasz et al. (Clin. Chem., 22:650-656 (1976)) with minor nonessential modifications. This formulation can be commercially obtained from Medical Analysis Systems, Inc. (MAS), Camarillo, California (Creatine Kinase Reagent, Catalog No. USA 139-162.) In this formulation, creatine phosphate, ADP and glucose are present in the substrate reagent. The coenzyme reagent consists of Mg⁺⁺, NADP+, hexokinase, glucose-6- phosphate, and AMP. All active components are present in sufficient concentrations (in excess) to fully measure CK.

To study the effects of different reducing agents, the coenzyme reagent was divided up into equal aliquots and spiked with the following millimolar concentrations of reducing agent: 5, 10, 20, 50, 75, 100. The reducing agents studied were cysteine, cysteamine, homocysteine, DTT, and N-acetylcysteine.

Each of these coenzyme reagents was then tested for its ability to activate CK using commercially available controls from Medical Analysis Systems, Inc., Camarillo, California. Analyses were performed using an Olympus Reply automated analyzer (Olympus, Inc.,

Irvine, Texas), which mixes equal parts of the substrate and coenzyme reagents, and then adds the sample to be measured. The final concentration of the reducing agent was therefore 2.5, 5,10, 25, 37.5, and 50 millimolar.

The results are given below in Table II, and represent the CK activity in U/L which was measured at 37°C in the presence of different concentrations of reducing agent. These results demonstrate that there are differences between the various activators in terms of their ability to reduce oxidized CK. (The CK activity in the absence of any reducing agent was measured to be 48.6 U/L.)

Table II

Example 2 Stability of the Redox Potential of Different Reducing Agent Compositions

Stability studies were conducted to determine the effectiveness of various reducing agent compositions in reducing oxidized CK after liquid storage at 4°C . Also measured as part of these studies was the stability of the thiol groups as a measure of the ability of the different compositions to retain their redox potential. Thiol stability of the reagent described above containing NAC, DTT, methimazole and cysteamine was measured using the standard thiol reagent 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) according to G.

L. Ellman, Arch. Biochem. Biophys. 82:70 (1959). All studies were carried out under "opened bottle" conditions, i.e. the same storage bottle was repeatedly opened and assayed for CK recovery.

Recovery of CK activity from control samples was measured as described above in Example 1 after storage for 13 months at 4°C. As shown below, the NAC was present in the coenzyme solution at a concentration of 50 mM. All other reducing agents were present at a concentration of 20 mM. Three different studies were performed, each with different reducing agent compositions. The results (in CK, U/L) are shown in Table III.

Table III

These results demonstrate that DTT significantly enhances the CK recovery in NAC- containing formulations. It is also clear from the above results that the presence of three or more reducing agents is unexpectedly superior when compared to compositions containing only two reducing agents. It was also demonstrated that a mixture of NNC, DTT and methimazole provided greater than 50% of the original free thiol of the mixture after 12 months of open-label storage at 4°C. The CK enzymatic activity was undiminished during this period.

The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. Nil publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

CLAIMS What is claimed is:

1. A composition for reducing a disulfide bond in a biological molecule comprising at least three reducing agents having different thioreductant potentials.

2. The composition of claim 1 wherein all three reducing agents are thiol-containing compounds.

3. The composition of claim 1, wherein the composition comprises a first reducing agent having an absolute thioreductant potential of X, wherein X in greater than the reductant potential of the disulfide bond of the biomolecule, a second reducing agent having an absolute thioreductant potential greater than or equal to 1.05X, and a third reducing agent having an absolute thioreductant potential less than or equal to 0.95X.

4. The composition of claim 1 comprising N-acetyl-L-cysteine as a first reducing agent, a thiol-containing compound having a greater thioreductant potential than NAC as a second reducing agent, and a weak reducing agent having a thioreductant potential at least

5% less than that of N-acetyl-L-cysteine as a third reducing agent.

5. The composition of claim 4 wherein the second reducing agent is a thiol- containing compound.

6. The composition of claim 4 wherein the second reducing agent is dithiothreitol and the third reducing agent is methimazole.

7. The composition of claim 6 further comprising either cysteamine or homocysteine.

8. The composition of claim 7, wherein the pH is between about 5.8 and 6.0.

9. The composition of claim 1, wherein the composition remains stable in liquid medium for at least 6 months at 4°C.

10. A method of stabilizing a first reducing agent having a thioreductant potential in a liquid solution comprising adding a second reducing agent to the liquid solution wherein the second reducing agent has a greater thioreductant potential than the first reducing agent, and adding a third reducing agent to the liquid solution wherein the third reducing agent has a lesser thioreductant potential than the first reducing agent.

11. A composition for reducing a disulfide bond in a biological molecule comprising a single reducing agent having at least three moieties, each moiety representing a different redox potential.

12. The composition of claim 11, wherein at least three of the moieties comprises a reducing thiol.

13. The composition of claim 11, wherein a first moiety has a redox potential greater than the redox potential of the disulfide bond of the biological molecule, a second moiety has a redox potential at about 5% greater than the first moiety, and a third moiety has a redox potential at best 5% less than the first moiety.