Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-10-23
2003-09-16
Low, Christopher S. F. (Department: 1651)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C530S300000, C530S362000, C530S365000, C530S366000, C530S367000, C530S370000, C530S833000, C435S023000, C435S024000, C435S113000
Reexamination Certificate
active
06620778
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method for the preparation of a mixture of peptides having a cysteine- or cysteine/glycine content between 7-20 w/w %, to preparations comprising said peptides and to the use of such preparations as active compound in a medicament.
Peptides are herein defined as amino acid chains, derived from a protein; the molecular weight of the peptides is preferably between 200D and 8000D, more preferably between 1000D and 5000D.
BACKGROUND OF THE INVENTION
In the art, there is a great demand for cysteine and cysteine/glycine comprising compounds for effective administration of said amino acids to the human or animal body. The availability of especially cysteine and to a lesser extent glycine, is a limiting factor in the syntheses of glutathion. Proper administration of cysteine, but also of glycine is therefore demanded in cases where an elevation of cellular glutathion levels in the human or animal body are needed.
Glutathion (GSH) is a tripeptide-thiol (L-&ggr;-glutamyl-L-cysteinylglycine) having a broad range of vital functions, including protection of cells against oxygen intermediates, free radicals, by products of the oxygen requiring metabolism, and detoxification of xenobiotics. Further, glutathion seems to play a role in the prevention of cataract and oxidative DNA injury. Glutathion is therefore regarded as an important compound against oxidative stress related diseases like myocardial ischemia, cancer and cataract.
In view of the crucial role played by glutathion either in combatting the assaults of free radical injuries or in detoxification of xenobiotics, inclusing drug metabolites (such as cyclophosphamide, paraquat and acetaminophen) and in preventing peroxidation of cell components, a method for maintaining hepatic stores of glutathion, particularly during times of stress to the body, including chemotherapy, is needed.
In the art, various methods are known to increase cellular levels of glutathion. Administration to animals of the glutathion amino acid precursors glutamic acid, cysteine and glycine, may produce an increase in cellular glutathion, but there is a limit to the effectiveness of this procedure.
Cellular concentrations of GSH are dependent on the supply of cysteine, which is often the limiting amino acid, and which is derived from dietary protein and also by trans-sulfuration from methionine in the liver. However, administration of cysteine as free amino acid is not an ideal way to increase GSH concentrations because cysteine is rapidly metabolised and furthermore, appears to be toxic to cells at higher concentrations. Administration to animals of compounds that are transported into cells and converted intracellularly into cysteine is sometimes useful in increasing cellular glutathion levels.
Another way in which tissue GSH concentration may be increased is by administration of gamma glutamylcysteine or of gamma-glutamylcystine. The administered gamma-glutamyl amino acid is transported intact and serves as a substrate of GSH synthetase. It is also known that administration of N-acetyl-L-cysteine can often increase tissue concentrations of GSH. Other reports on using N-mercaptopropionyl glycine for increasing intracellular glutahion are known. A few clinical trials have been done using mercaptopropionyl glycine to elevate intracellular glutathion.
That the administration of glutathion itself might lead to increased glutathion levels has also been considered. However, there is no published evidence that shows that intact glutathion enters cells. In fact, there are several reports on particular biological systems indicating that glutathion itself is not transported into cells. The increase in cellular glutathion sometimes found after administration of glutathion is due to (a) extracellular breakdown of glutathion, (b) transport into cells of free amino acids or dipeptides derived from glutathion extracellularly, and (c) intracellular resynthesis of glutathion.
Apart from these conventional methods for increasing glutathion levels, there have been several attempts to demonstrate how glutathion can be enhanced intracellularly. All these relate to synthetic derivatives or about intact undenatured proteins which are heat labile and none whatsoever to natural derived peptide mixtures. Some of the relevant ones are summarised below:
U.S. Pat. No. 5,869,456 relates to preparation of pure alkyl esters of glutathion (95% pure) and a method for increasing intracellular glutathion levels by administering such alkyl diester of glutathion.
U.S. Pat. No. 5,464,825 describes the method for preparation and use of N-acyl glutathion monoalkyl esters to provide increased intracellular levels of glutathion or glutathion equivalents, e.g. N-acyl glutathion or glutathion monoalkyl esters.
U.S. Pat. No. 5,248,697 describes a method for maintaining and/or enhancing tissue or plasma levels of glutathion. The patent teaches the art of treatment of a mammal with a supranormal amount of glutamine, or a glutamine equivalent, to prevent the reduction in tissue glutathion levels associated with exposure of the mammal to a compound capable of oxidative injury to the tissue.
U.S. Pat. No. 4,665,082 discusses the role of L-2-oxothiazolidine-4-carboxylate, a sulfur analog of 5-oxoproline, cleaved by the enzyme-5-oxo-L-prolinase to form cysteine, thus providing the basis for a cysteine delivery system by the addition of L-2-oxothiazolidine-4-carboxylate to base amino acid solutions or by injecting it directly into in vivo cells.
DE patent No 4,329,857 teaches the use of thiol compounds (cysteine and its derivatives or analogues like N-acetyl cysteine, homocysteine, glutathion, 2-oxothiazolidine-4-carboxylic acid) as an agent for strengthening the immune system and immune reactions.
SUMMARY OF THE INVENTION
According to the present invention, a novel method for the preparation of a mixture of peptides having a cysteine content between 7-20 w/w % from a protein source, comprising cysteine containing proteins is provided. The protein source is preferably a natural protein source. The peptide mixture prepared according to this embodiment of the present invention has the advantage that it is derived from natural protein sources and will not show any adverse side-effects, whereas chemically produced cystein derivatives as mentioned in the prior art, have shown adverse side effects. There has been found that such a preparation of a peptide mixture can be very advantageously used as cysteine source in diet supplements or in medicaments, as will be explained below.
The method is characterized in that it comprises the steps of:
a) cleaving the proteins of the protein source into peptides;
b) digesting the peptides obtained in step a) by at least one exopeptidase, the action of which is at least attenuated at the position of a cysteine in the peptide, therewith forming digested peptides having a terminal cysteine;
c) purifying the digested peptides.
In the first step a) proteins of the protein source are cleaved into smaller peptides. This cleavage can be performed by cleavage reactions, known in the art; preferably, the cleavage is performed by enzymatic hydrolysis of the peptide bonds of the protein by e.g. an endopeptidase, resulting in the peptides of about the desired length, and therewith increasing the amount of substrate for the exopeptidase. In a second step, the peptides as obtained by the cleavage reaction, are digested by at least one exopeptidase. With “at least one exopeptidase” is meant that the digestion reaction can be carried out by one or more different exopeptidases. Exopeptidases release amino acids from the terminal ends of the peptides one by one. The exopeptidase and the digestion reaction conditions are chosen such, that the exopeptidase action is at least attenuated at the position of a cysteine in the peptide. With “at least attenuated” is meant that the exopeptidase does not remove the cysteine from the peptide at the chosen reaction conditions or has very low preference for the cleavage of cysteine, therewith rendering said cleavag
L. Boumans Johannes Wilhelmus
Mallee Leon Franciscus
Nimmagudda Ram
Campina Melkunie B.V.
Kam Chih-Min
Low Christopher S. F.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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