Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1999-08-25
2002-01-01
Stucker, Jeffrey (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C530S350000, C530S370000, C530S412000, C514S002600
Reexamination Certificate
active
06335427
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protein which is a component of bromelain. In particular, the invention relates to a protein which is responsible for the anti-cancer activity of bromelain and which also has activity as an immunostimulant, and an antimicrobial agent.
2. Related Art
Bromelain is the collective name for the proteolytic enzymes found in the tissues of the plant Bromeliaceae. Although fruit bromelain is known, the most common form of bromelain is a mixture of various moieties derived from the stem of the pineapple plant (
Ananas comosus
). Stem bromelain (hereafter called bromelain) is known to contain at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present.
Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea. The use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain detaches pathogens from intestinal receptors.
Taussig et al,
Planta Medica,
1985, 538-539 and Maurer et al,
Planta Medica,
1988, 377-381 both suggest that bromelain may be of use in inhibiting tumour growth. U.S. Pat. No. 5,223,406, DE-A-4302060 and JP-A-59225122 also teach the use of bromelain in the treatment of cancer. U.S. Pat. No. 5,223,406 teaches that bromelain is capable of inducing tumour necrosis factor (TNF) while DE-A4302060 teaches that bromelain can prevent metastasis by the structural modification of the tumour surface protein CD44.
In WO-A-9400147, various experiments were described which demonstrate that proteolytic enzymes and, in particular, bromelain, are capable of inhibiting secretion. The application also discloses that bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST). These observations were explained by the fact that one component of the bromelain mixture, stem bromelain protease, appears to be capable of modulating cyclic nucleotide pathways and this is discussed further in WO-A-9500169. In addition, bromelain has also been demonstrated to inhibit secretion caused by the calcium dependent pathway.
WO-A-9600082 also relates to bromelain and discloses that crude bromelain is capable of interfering with signalling pathways which are important for growth, in particular, signalling pathways which lead to the production of growth factors such as interleukin-2 (IL-2), platelet derived growth factor (PDGF) and insulin like growth factor (IGF). This document teaches that, as a consequence of its ability to block signalling pathways, bromelain is capable of acting as an anti-cancer agent. In addition, bromelain can be used either as an immunosuppressive agent or an immunostimulant depending on the type of cell being treated and whether the cell has previously been activated.
From the prior art, it is clear that bromelain is a mixture which has a variety of different physiological effects. Not all of the components of the bromelain mixture have been characterised and so, except for stem bromelain protease, whose activity we have described, it is not clear which of the components is responsible for which of the various different effects of bromelain. This is, of course, a major disadvantage if the bromelain mixture is to be administered as a pharmaceutical because while one component of bromelain might give the desired effect, there may well be unwanted side effects arising from the action of some other component of the bromelain mixture.
SUMMARY OF THE INVENTION
It would therefore be beneficial if individual components of bromelain giving rise to particular medicinal activities could be isolated and administered separately so as to lessen the possibility of side effects.
The present inventors have now isolated, purified and characterised one such component of bromelain which is distinct from other known components of the mixture and which has been found to have anti-cancer activity.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect of the present invention there is provided a protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE, has an isoelectric point of 3.8 to 4.79 and has the amino terminal sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr Gly Ala Val Asn Glu Val Lys Asn (SEQ ID NO:1)
and, additionally, contains the following sequences:
Gly Gly Trp Glu Phe Lys (SEQ ID NO: 2)
Lys Ala Val Asn Gly (SEQ ID NO: 3)
Tyr Trp Ile Val Arg (SEQ ID NO: 4)
Asn Ser Trp Gly Ser Ser Trp Gly Glu Gly Gly Tyr Val Arg (SEQ ID NO:5)
Thr Ser Leu Asn His Ala Ile Thr Ile Ile Val Tyr (SEQ ID NO: 6)
Leu Pro Glu Phe (Gln) Pro (Gln) Val Leu Asp-Ala- (SEQ ID NO: 7)
Gly Val Ser Ser Ser Ser Gly Ala Cys Gly Ile Ala Met Ser Pro Leu-Thr- (SEQ ID NO: 8)
Gly Gly Val Phe Ser Gly Pro Ala Gly (SEQ ID NO: 9)
Asn Asn Ala Tyr (SEQ ID NO: 10)
Ser Ser Gly Thr Lys Tyr Trp-Val- (SEQ ID NO: 11);
where the bracketed amino acids represent alternatives to the preceding amino acid and a “-” represents an unidentified amino acid.
The protein of the present invention (designated CCX2 by the inventors) is largely responsible for the anti-cancer activity of bromelain since other known components such as stem bromelain protease, ananain, comosain, and F9 have all been found to have reduced anti-cancer activity. Also, because it is a single molecule, the protein of the invention does not have the disadvantages of multi-component mixtures when used as a pharmaceutical agent.
The protein may be isolated from the bromelain mixture by conventional methods, for example by chromatography. High performance liquid chromatograpy (HPLC) is suitable for the purpose and particularly good separation of bromelain proteins may be achieved by fast protein liquid chromatography (FPLC™) using a column packing material such as SP-sepharose. As will be described in more detail in the examples, in chromatography on SP-sepharose using a linear gradient of 0 to 0.8M sodium chloride in acetate buffer over 300 ml, the protein of the present invention was contained in the fraction represented by the second sharp peak off the column. This fraction was designated the CCX fraction. The protein of the invention was the major component of the CCX fraction and was isolated from the minor components by conventional methods which will be described in greater detail below.
Therefore, in a second aspect of the invention, there is provided a protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE and is obtainable by the following method:
i. dissolving bromelain in acetate buffer at pH 5.0;
ii. separating the components of bromelain by fast flow high performance liquid chromatography on S-sepharose eluting with a linear gradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;
iii. collecting the fraction corresponding to the second sharp peak off the column; and
iv. isolating the major protein from the fraction collected in (iii) by anion chromatography and hydrophobic interaction chromatography.
It is known from, for example, U.S. Pat. No. 5,223,406 and WO-A-9600082 that the bromelain mixture has anti cancer activity but it has previously been assumed that one of the known components of bromelain, probably stem bromelain protease, was responsible for this activity. It has now been found that this is not the case and that the protein of the present invention is an anti-cancer agent whereas stem bromelain protease has no anti-cancer activity.
The protein of the present invention has a high degree of homology with fruit bromelain protease, an enzyme isolated from fruit
Engwerda Christian
Mynott Tracey Lehanne
Peek Keith
Provalis UK Limited
Sterne Kessler Goldstein & Fox P.L.L.C.
Stucker Jeffrey
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