Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing
Reexamination Certificate
1997-07-30
2001-09-04
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
C424S401000
Reexamination Certificate
active
06284246
ABSTRACT:
TECHNICAL FIELD
The present invention relates to modified polypeptides, specifically lipase and protease enzymes, with high activity and reduced allergenicity.
BACKGROUND OF THE INVENTION
An increasing number of commercial products containing active polypeptides are becoming available. The majority of these products utilize an enzyme as the polypeptide. Enzymes are polypeptides which react with a compound, or substrate, to break down that compound. Enzymes are divided into numerous classes based on the class of substrate they react upon. Each class of enzyme generally catalyzes the severing of different chemical bonds resulting in the specific selection of activity. The lipase class of enzymes are known for their ability to hydrolyze ester bonds created between, but not limited to, hydrocarbons and polyalcohol backbone substrates. Examples of these substrates are mono-, di-, and triglyceride—polyglycerol esters. The protease class of enzymes are known for their ability to hydrolyze proteins. Naturally occurring and bio-engineered protease enzymes are incorporated into household cleaning detergents to hydrolyze proteinaceous dirt and stains, into personal care products to remove dirt and dead skin, into oral cleansing products to facilitate plaque removal in the mouth, and medicines to affect undesired proteins in the body.
It is known that current commercial cleansing products are made more effective by the incorporation of protease polypeptides. U.S. Pat. No. 4,261,868 (Hora et al.), U.S. Pat. No. 4,404,115 (Tai), U.S. Pat. No. 4,318,818 (Letton et al.), European Patent Application 130,756 (published Jan. 9, 1985) and U.S. Pat. No. 5,030,378 (Venegas) all disclose the use of protease polypeptides in cleansing or detergent products.
It is also realized, however, that polypeptides are potential antigens, and may cause allergic reactions in humans, under certain conditions. The human immune system can produce specific antibodies upon exposure to polypeptides. This process of producing specific antibodies is referred to as “immunization” when a clinically beneficial response is obtained. When the response leads to hypersensitivity, however, it is referred to as “sensitization”. Allergenic sensitization to polypeptides has been observed in environments where humans are regularly exposed to the polypeptide. Such environments include manufacturing facilities, where workers can be exposed to uncontrolled dust or aerosol containing a polypeptide, or the marketplace, where consumers' repeated use of products containing polypeptides has, on occasion, caused an allergic reaction.
Presently, allergic responses to polypeptides can be minimized by immobilizing, granulating, coating or dissolving the polypeptides to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne polypeptides, still leave the risks associated with extended tissue contact with the finished product and exposure to enzyme-containing dust or aerosol during manufacturing.
Another way of diminishing allergic response has been to select polypeptides of human origin. While this approach minimizes allergenicity problems, it is not a complete solution since it is often not possible to find such a polypeptide which also has the activity properties desired.
A third proposition for decreasing allergenicity has been to reduce the size of the polypeptide molecules (see JP Patent Publication Number 4,112,753). However, size reduction can cause a significant reduction in enzyme activity.
A fourth approach to reduce the allergenicity of polypeptides is through epitope mapping and alteration of the polypeptide amino acid sequence to deliver a polypeptide with reduced allergenicity. This approach usually requires a large investment of development time and money.
In the medical field, suggestions have been made to diminish the immunogenicity of polypeptides through yet another method. This method involves attaching unreactive polymers to the polypeptide. U.S. Pat. No. 4,179,337 (Davis, et al.) relates to polypeptides coupled to substantially straight chain polyethylene glycol (PEG) or polypropylene glycol (PPG) polymer moieties. While PEG/PPG coupling was found to mitigate the allergenicity of the polypeptide, only 15% of the physiological activity was maintained. PCT Application WO 96/17929 (Olsen, et al., published Jun. 13, 1996) relates to the modification of polypeptides by conjugating them with suitable polymers. The Olsen application describes modified polypeptides which demonstrate a reduction in allergenicity of from 25% to 66% compared to the parent polypeptide, while maintaining from 39% to 100% of the activity of the parent.
Monfardini, et al, “A Branched Monomethoxypoly(ethylene glycol) for Protein Modification”, American Chemical Society, 1995) describes efforts to increase the activity of native polypeptides by conjugating branched monomethoxypolyethylene glycol (mPEG) polymers to the reactive enzyme group. Monfardini, et al. teaches conjugation of enzymes with linear mPEG polymers having molecular weight 5000 KD and branched mPEG polymers having a molecular weight of 5000 KD per branch. Conjugation to ribonuclease, catalase, trypsin and asparaginase is shown. Enzymatic activity levels of conjugated enzyme are shown to range from 86% to 133% of the activity of the respective parent enzyme. No allergenicity data is presented.
It would be highly desirable to develop an enzyme-based compound which would virtually eliminate allergenic responses while maintaining the desired high levels of enzymatic activity. If this were accomplished it would provide manufacturers and consumers with safer ways to utilize the benefits of enzyme technology.
It is an object of the present invention to provide a modified enzyme compound which delivers this high activity and yet shows reduced stimulation of and resulting activation of the immune system. It is also an object to provide compositions of use of this modified enzyme compound.
SUMMARY OF THE INVENTION
The present invention relates to a modified polypeptide which has an enzymatic activity level of greater than about 70% of the parent polypeptide and an allergenic response level of less than about 33% of the parent polypeptide. Embodiments of the present invention relate to modified polypeptides with reduced allergenicity and high enzymatic activity comprising the formula:
A-B
n
wherein A is an enzyme selected from the group consisting of lipase enzymes and protease enzymes, and mixtures thereof; B is a twin polymer moiety, having a total molecular weight of from about 0.5 kilodaltons (KD) to about 40 KD, having the formula
conjugated to the enzyme; wherein R
1
and R
2
are essentially straight chain polymers, having a molecular weight ranging from about 0.25 KD to about 20 KD; wherein the ratio of the molecular weights of R
1
and R
2
is from about 1:10 to about 10:1; wherein X is a linking moiety which links the twin moiety to a single site on the enzyme; and n is the number of twin polymer moieties conjugated to the enzyme, and represents an integer from about 1 to about 15.
DETAILED DESCRIPTION OF THE INVENTION
The modified polypeptide of the present invention is represented by the formula:
A-B
n
containing, as essential components, a enzyme, A, and a plurality, n, of twin polymer moieties, B. While not intending to be limited by theory, it is believed that the conjugation of the twin polymer moieties to the enzyme provides a balanced stearic hindrance of the activated surface of the enzyme as to allow for high activity but simultaneously prevent stimulation of the immune system and subsequent antibody formation responsible for allergic reaction.
As used herein, the phrase “amino acid sequence” refers to a specific configuration of the amino acids comprising a polypeptide. The following is a list of abbreviations used herein to describe amino acids:
Three-letter
Amino Acid
Abbreviation
One-letter Symbol
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic Acid
Asp
D
Cysteine
Cys
C
Glutamine
Gln
Q
Glutamic Acid
Glu
Q
Glycine
Gly
G
Histidine
Rubingh Donn N.
Weisgerber David J.
Howard S.
Kendall Dara M.
Murphy Stephen T.
Page Thurman K.
The Procter & Gamble Co.
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