Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-12-06
2004-08-24
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S013800, C514S014800, C514S017400, C530S352000, C530S360000, C424S049000, C424S052000, C424S057000
Reexamination Certificate
active
06780844
ABSTRACT:
The present invention relates to novel complexes in which amorphous calcium phosphates are stabilised by phosphopeptides. These complexes have anti-cariogenic effects, and may also be used as dietary supplements to increase calcium bioavailability and to heal or prevent diseases associated with calcium deficiencies. Methods of making the complexes of the invention and of treatment or prevention of dental caries, calcium malabsorption, and bone diseases are also provided
BACKGROUND
Dental Caries
Dental caries is initiated by the demineralisation of hard tissue on the teeth by organic acids produced from fermentation of dietary sugar by dental plaque odontopathogenic bacteria
Even though the prevalence of dental caries has deceased through the use of fluoride in most developed countries, the disease =as a major public health problem. The estimated economic burden of treating dental caries in Australia in 1991 was $471 million, being higher an that for other diet-related diseases including coronary heart disease, hypertension or stroke.
In developing countries where the availability of industrialised food products is increasing, prevalence of dental caries is also increasing. Recent studies have highlighted a number of socio-demographic variables associated with the risk of developing caries; high risk is associated with ethnicity and low socio-economic status. The level of high-risk individuals has remained constant even though the overall severity and prevalence of disease in the community has decreased. Dental caries is therefore, still a major public health problem, particularly in ethnic and lower socioeconomic groups. This highlights the need for a non-toxic, anticariogenic agent that could supplement the effects of fluoride to fit lower the incidence of dental caries. An agent which would reduce the dose of fluoride required to reduce the incidence of caries would be particularly desirable in view of community anxiety about fluoride, and in view of the fact that fluorosis can develop even at currently used doses.
The food group most recognised as exhibiting anticaries activity is dairy products (milk, milk concentrates, powders and cheeses). U.S. Pat. No. 5,130,123 discloses the component responsible for this anticariogenic activity as casein. However, the use of casein as an anticariogenic agent is precluded by adverse organoleptic properties and the very high levels required for activity.
Preliminary investigations determined that tryptic casein phosphopeptides contributed to the anticariogenic activity and this was made subject of U.S. Pat. No. 5,015,628. In particular, peptides Bos &agr;
s1
-casein X-5P (f59-79) (SEQ ID NO: 1, Bos &bgr;-casein X-4P (f1-25) (SEQ ID: NO: 2), Bos &agr;
s2
-casein X-4P (f46-70) (SEQ ID NO: 3) and Bos &agr;
s2
-casein X-4P (f1-21) (SEQ ID NO: 4) were disclosed in U.S. Pat. 5,015,628 as follows:
(SEQ ID NO: 1) Gln
59
-Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Gln-Lys
79
. &agr;
s1
(59-79)
(SEQ ID NO: 2) Arg
1
-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg
25
. &bgr;(1-25)
(SEQ ID NO: 3) Asn
46
-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys
70
. &agr;
s2
(46-70)
(SEQ ID NO: 4) Lys
1
-Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-Tyr-Lys
21
. &agr;
s2
(1-21)
The preliminary determination of the above phosphopeptides for use in combination with CaHPO
4
and hydroxyaptite provided novel peptides having anticariogenic properties. However, subsequent investigations have determined that the Ser(P) cluster sequence motif within the previous disclosed phosphopeptides have the unexpected ability to stabilize their own weight in amorphous calcium phosphate. The ability of the above phosphopeptides and in particular the Ser(P) motif to stabilise amorphous calcium phosphate was quite unexpected and neither disclosed or taught in any publications known to the Applicants. We have now found that the amorphous form of calcium phosphate Ca
3
(PO
4
)
1.87
(HPO
4
)
0.2
xH
2
O where x≧1 stabilised by the casein phosphopeptides is the most soluble, basic form of non-crystalline calcium phosphate and a superior form of calcium phosphate which prevents caries and increases calcium bioavailability. Amorphous calcium phosphate (ACP) must be formed by careful titration of Ca ions (eg CaCl
2
) and phosphate ions (eg Na HPO
4
) while maintaining the pH above 7 (preferably 9.0) in the presence of the phosphopeptide. As the ACP is formed, the phosphopeptide binds to the nascent nuclei and stabilises the ACP as a phosphopeptide-ACP complex. Without the phosphopeptide, the ACP will precipitate out of solution and transform within minutes into, the most stable calcium phosphate phase, crystalline hydroxyapatite (HA). HA, by being insoluble has limited anticariogenic activity and presents calcium in a poorly bioavailable form. The acidic phase of calcium phosphate CaHPO
4
, while certainly being more soluble than hydroxyapatite, is poorly bound by the phosphopeptide and poorly localised at the tooth surface and therefore also has limited anticariogenic activity. The unexpected ability of the aforementioned phosphopeptides and in particular Ser(P) cluster motif to stabilize amorphous calcium phosphate was not disclosed or taught in U.S. Pat. No. 5,015,628 and provides for the first time a reliable and effective method of producing a stabilized amorphous calcium phosphate complex having distinct and novel advantages in calcium treatments and delivery. U.S. Pat. No. 5,015,628 does not disclose the unique amorphous calcium fluoride phosphate phase Ca
8
(PO
4
)
5
F x H
2
O where x≧1 which we have now found to be stabilised by the above phosphopeptides and can be localised at the tooth surface to provide superior anticaries efficacy. This unexpected ability to stabilize amorphous calciun phosphate forms the basis of the instant invention.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a stable calcium phosphate complex, comprising amorphous calcium phosphate or a derivative thereof stabilized by a phosphopeptide, wherein said phosphopeptide comprises the sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu-(SEQ ID NO: 5).
In one embodiment, the complex may include phosphopeptide stabilized amorphous calcium fluoride phosphate.
The phosphopeptide (PP) may be from any source; it may be obtained by tryptic digestion of casein or other phospho-acid rich proteins such as phosphitin, or by chemical or recombinant synthesis, provided that it comprises the core sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-(SEQ ID NO: 5). The sequence flanking this core sequence may be any sequence. However, those flanking sequences in &agr;
s1
(59-79) (SEQ ID NO: 1), &bgr;(1-25) (SEQ ID NO: 2), &agr;
s2
(46-70 (SEQ ID NO: 3) and &agr;
s2
(1-21) (SEQ ID NO: 4) are preferred. The flanking sequences may optionally be modified by deletion, addition or conservative substitution of one or more residues. The amino acid composition and sequence of the flanking region are not critical as long as the conformation of the peptide is maintained and that all phosphoryl and carboxyl groups interacting with calcium ions are maintained as the preferred flanking regions appear to contribute to the structural action of the motif.
When the complex takes the form of phosphopeptide stabilized amorphous calcium fluoride phosphate, the calcium fluoride phosphate may be of the approximate formula [Ca(PO
4
)
5
Fx H
2
O] wherein x≧1.
The complex may firer include HPO
4
as a minor optional component to the complex. The HP04 is believed to act as a coating for the ACP cluster. When the complex takes the alternative form of a stable soluble alkaline calcium phosphate complex including stabilized amorphous calcium phosphate, the amorphous calcium phosphate may be of the approximate formula [Ca
3
(PO
4
)
2
x H
2
O] wherein x≧1.
The complex may firther include HPO
4
as a minor optional component. The
Foley & Lardner
Low Christopher S. F.
Lukton David
The University of Melbourne
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