Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-07-05
2002-03-19
Davenport, Avis M. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S021800, C530S324000, C530S350000, C530S362000, C530S380000, C530S385000, C530S395000, C530S404000
Reexamination Certificate
active
06358918
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to S-nitroso protein preparations.
BRIEF DESCRIPTION OF THE BACKGROUND ART
NO is a gaseous molecule formed in many mammalian cells and involved in a whole series of physiological and pathological processes. Thus, the endothelium-dependent relaxation of the vascular smooth muscles is due to NO.
The exact mechanism of the action of NO still is largely unknown. It is assumed that physiologic carrier substances play an important role.
As such possible carrier substances, i.a. S-nitrosated proteins are assumed (Stamler et al., PNAS 89 (1992), 444-448). Thus, several thiol group-containing proteins of the most varying nature and function have been nitrosated, such as, e.g. serum albumin, t-PA and kathepsin B (Stamler et.al., Hallstrom et al. (in: Shock, Sepsis, and Organ Failure —Nitric oxide, Fourth Wiggers Bernard Conference (1994), pp. 310-321)).
For this, bovine serum albumin (BSA) nitrosation levels of 85% (gtamler et al.,) or of 90 to 95%, respectively, for human albumin (Hallstrom et al.) have been reported. It has, however, been shown that with the methodology applied in these publications for determining the S nitroso-binding not only to S-nitrosations, but also other nitrosations, such as, e.g., N-nitrosations to tryptophane residues, or C-nitrosations (to tyrosine) can be detected.
In particular, the spectroscopic measurement at 335 nm-and a molar extinction coefficient of 3869 mol
−1
cm
−1
proved to be non-specific for the S-nitrosothiol binding. In a publication following upon Stamler et al, the same group could prove by reliable detection |methods i.a. that in the produced nitrosated BSA-preparations the free SH groups on Cysteine 34 were nitrosated to 37% at the most (Zhang et al., Journal of Biological Chemistry 271 (24), 1996, 14271-14279).
This level also matches the known fact that in preparations of proteins having potentially free thiol groups, only 20 to 35% are, in fact, present in free, reduced SH form. Particularly in protein preparations derived from blood or which are contacted with plasma or plasma derivatives in the course of their preparation procedure, the remaining 65 to 80% are blocked, mostly by mixed S—S bonds with small, thiolcarrying compounds, e.g. free L-cysteine or glutathione, respectively (Katachalski et al., J. Am. Chem. Soc. 79 (1957), 4096-4099, DeMaster et al., Biochemistry 34 (1995), 11494-11499).
As regards the sulphur-containing groupings in the respective proteins, basically it must be differentiated between those present in tightly bonded or associated form, e.g. as intramolecular saturated disulfide bonds and which are decisive for the conformation of the proteins, and those representing the potentially free thiol group(s). The latter are a known parameter for the respective protein. Human serum albumin, e.g., in its native state has a single potentially free thiol group per molecule, i.e. cysteine at position 34. These potentially free thiol groups, however, tend to form intermolecular disulfides, and therefore they are also termed mixed disulfides. In plasma, up to 80%. of these thiol groups are present as mixed disulfides and thus are not directly available as free thiol groups.
In tests, in which a preparation of human serum albumin wherein only 20% of cysteine-34 was present in the reduced, and thus free, form, was treated with dithiothreitol (DTT) prior to nitrosation so as to reduce the mixed disulfides of the potentially free SH group, various nitrosation levels could be attained in dependence on the nitrosation agents and reaction conditions (DeMaster et al.).
There is a problem with the reduction of potentially free thiol groups to free reactive thiol groups, if in addition also the conformation-determining disulfide bonds are broken up and thus the nativity of the proteins is lost. A further problem occurs in proteins or protein solutions which have previously been subjected to a treatment for inactivating possibly present pathogens, e.g. a heat treatment. It is then that i.a. a tendency to increased aggregate formation is observed.
SUMMARY OF THE INVENTION
The present invention aims at providing a protein preparation that is nitrosated or that is capable of being nitrosated, as well as a method of preparing the same, whose thiol groups can be nitrosated to a greater extent and which have an increased S-nitrosation level as compared to preparations already disclosed, for the higher the nitrosation level of the protein preparation, the higher the NO-coupled effect, e.g. when pharmaceutically administering such a protein preparation. In particular, a stable, virus-safe pharmaceutical preparation having a high S-nitrosation level is to be provided.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to the invention, the set object is achieved by a preparation comprising thiol-group-containing proteins which are heat-treated, dissolved, and which are characterized in that they are processed. Thus, at least 40% of the thiol groups are capable of being nitrosated.
For, surprisingly it has been found that, in spite of their heat treatment, these virus-inactivated thiol-group-containing protein compositions can be processed such that the potentially free thiol groups are, in fact, available as reactive groups for further reactions. Thus it is possible to enable a high nitrosation level of these proteins even in such virus-inactivated protein compositions, without adversely affecting the nativity of these proteins.
Basically, the pharmaceutical preparation according to the invention may contain any proteins with a “free” thiol group, yet for the purposes of the present invention therapeutically usable proteins are preferred, physiological proteins or human proteins derived from blood, such as albumin, orosomucoid, plasminogen-activator (e.g. tissue-plasminogen activator), fibrinogen, Lys-plasminogen or hemoglobin or also mixtures of such proteins nitrosated or capable of being nitrosated according to the invention, being particularly preferred. According to the present invention, high-molecular proteins are preferred over low-molecular proteins, such as, e.g., glutathione. In particular, the proteins used according to the present invention are those which have a vasodilating and/or anti-aggregating activity on cellular components of the blood. Those proteins which play a decisive role in shock conditions (go-called acute phase proteins) likewise are used according to the invention. In a further preferred embodiment, proteins are used which are suitable as a blood surrogate.
With the method according to the invention, the nitrosation level of the processed thiol group(s) can be varied without any problems, even up to levels corresponding to a complete nitrosation. Yet, since the preparation method according to the invention is the more time-consuming, the higher the nitrosation level is to be, in most cases one has to choose between the nitrosation level and the expenditures of the preparation method, so that preferred preparations are nitrosated to an extent of at least 60%, preferably to an extent of 60 to 95%, in particular 70 to 80%. These levels can be attained by means of the method according to the invention, without higher expenditures.
With the method provided, the preparations according to the invention can be S-nitrosated so specifically that products having a very low N,O,C-nitrosation level of the proteins can be provided. For instance, N-nitroso-compounds are suspected to be cancerogenic, and their delivery kinetics of the NO group is also different from that of S-nitroso-compounds (cf. Zhang et al.), and therefore, in a preferred embodiment of the preparation according to the invention, an N,O,C-nitrosation level of the proteins in the preparation of less than 30, preferably less than 20%, in particular less than 10%, is provided for.
Although it is possible to subject even crude fractions, such as plasma or early plasma fractions or culture liquids, to the processing or nitrosation, respectively, of the invention, the proteins preferably are provid
Gasser Harald
Hallstroem Seth
Schlag Guenther
Baxter Aktiengesellschaft
Davenport Avis M.
Heller Ehrman White & McAuliffe
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