USE OF LACTOFERRIN FRAGMENTS AND HYDROLYSATES
The present invention relates to use of at least one lactoferrin fragment or a lactoferrin hydrolysate or a mixture thereof for stimulating skeletal growth, inhibiting bone resorption, stimulating chondrocyte proliferation, stimulating osteoblast proliferation, inhibiting osteoclast development or treating or preventing a skeletal, joint or cartilage disorder.
1 . A method of treating or preventing a skeletal, joint or cartilage disorder comprising administering to a subject in need thereof an effective amount of at least one lactoferrin fragment or a lactoferrin hydrolysate or a mixture thereof, wherein the lactoferrin fragment is a fragment selected from, or the lactoferrin hydrolysate comprises at least one fragment selected from
(a) a truncated lactoferrin polypeptide, or
(b) an N-lobe fragment of lactoferrin, or
(c) a C-lobe fragment of lactoferrin, or
(d) a lactoferricin, or
(e) a lactoferrampin, or
(f) a fragment of SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(g) a functional variant of any of (a) to (f), or
(h) a functional fragment of any of (a) to (g), or
(i) a mixture of any two or more fragments selected from (a) to (h).
2 . A method according to claim 1 , wherein the lactoferrin is, or the lactoferrin hydrolysate comprises a truncated lactoferrin polypeptide or a functional variant or a functional fragment thereof.
3 . A method according to claim 1 , wherein the lactoferrin fragment is a fragment selected from, or the lactoferrin hydrolysate comprises at least one fragment selected from
(a) an N-lobe fragment of lactoferrin, or
(b) a lactoferricin, or
(c) a functional variant of (a) or (b), or
(d) a functional fragment of (a) or (b) or (c), or
(e) a mixture of any two or more fragments selected from (a) to (d).
4 . A method according to claim 1 , wherein the lactoferrin fragment is a fragment selected from, or the lactoferrin hydrolysate comprises at least one fragment selected from
(a) a C-lobe fragment of lactoferrin, or
(b) a lactoferrampin, or
(c) a functional variant of (a) or (b), or
(d) a functional fragment of (a) or (b) or (c), or
(e) a mixture of any two or more fragments selected from (a) to (d).
5 . A method according to claim 1 , wherein the lactoferrin fragment is a fragment selected from, or the lactoferrin hydrolysate comprises at least one fragment selected from
(a) a polypeptide of SEQ ID NO. 7, 8, 11, 18, 19, 21, 23, 30, 31, 32 or 33, or
(b) a functional variant of (a), or
(c) a functional fragment of (a) or (b), or
(d) a mixture of any two or more fragments selected from (a) to (c).
6 . A method according to claim 1 , wherein the lactoferrin fragment is a fragment selected from, or the lactoferrin hydrolysate comprises at least one fragment selected from
(a) SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(b) a functional variant of SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(c) a functional fragment SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(d) a mixture of any two or more fragments selected from (a) to (c).
7 . A method according to claim 1 , wherein the hydrolysate is a full or partial enzyme hydrolysate, a full or partial microorganism hydrolysate, a full or partial acid hydrolysate, a full or partial cyanogen bromide hydrolysate, or a mixture thereof.
8 . A method according to claim 2 , wherein the truncated lactoferrin polypeptide is a polypeptide selected from SEQ ID NO. 1, 2, 3 and 4, truncated by at least about 10 amino acids at the N-terminus, the C-terminus or at both the N-terminus and C-terminus of the polypeptide.
9 . A method according to claim 2 , wherein the truncated lactoferrin polypeptide is a polypeptide of SEQ ID NO. 20, 24 or 26, or a mixture thereof.
10 . A method according to claim 3 , wherein N-lobe fragment or functional fragment thereof is a polypeptide selected from SEQ ID NO. 5, 6, 9, 10, 12, 25, 27 and 29, or a mixture of any two or more thereof.
11 . A method according to claim 4 , wherein the C-lobe fragment or functional fragment thereof is a polypeptide selected from SEQ ID NO. 7, 8, 11, 18, 19, 21 and 23, or a mixture of any two of more thereof.
12 . A method according to claim 3 , wherein the lactoferricin fragment or functional fragment thereof is a polypeptide selected from SEQ ID NO. 13, 14, 15, 16, 17 and 28, or a mixture of any two or more thereof.
13 . A method according to claim 4 , wherein the lactoferrampin fragment is a polypeptide selected from SEQ ID NO. 30, 31, 32 and 33, or a mixture of any two or more thereof.
14 . A method according to claim 7 , wherein the lactoferrin hydrolysate comprises at least one fragment selected from
(a) SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(b) a functional variant of SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(c) a functional fragment SEQ ID NO.s 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, or
(d) a mixture of any two or more fragments selected from (a) to (c).
15 . A method according to claim 7 , wherein the enzyme is selected from a protease, trypsin, chymotrypsin, chymosin, plasmin, pepsin, papain, a peptidase, an aminopeptidase or a mixture thereof.
16 . A method according to claim 7 , wherein the enzyme is trypsin and the lactoferrin is a polypeptide having the amino acid sequence of SEQ ID NO. 2; or wherein the enzyme is pepsin and the lactoferrin is a polypeptide having the amino acid sequence of SEQ ID NO. 2.
17 . A method according to claim 7 , wherein the hydrolysate comprises the peptides
(1) ADAVTLDGGMVF, ADAVTLDGGMVFEAGR, ADRDQYELL, ANEGLTWN, ANEGLTWNSLK, APVDAFK, CLQDGAGDVAFVK, DGKEDLIWK, DLLFKDSALGFLR, DSALGF, DSALGFLR, EKYYGYTGAFR, EPLQGAVAK, EPYFGYSGAFK, ESPQTHYY, ESPQTHYYAVAVVK, ETTVFENLPEK, FENLPEK, FGYSGAFK, FKDSALGFLR, GEADALNLDGGY, GEADALNLDGGYIY, GILRPYLSWTESLEPLQGAVAK, GSNFQLDQLQGR, GTEYVTAIANLKK, GYSGAFK, IIPMGILRPYLSWTESLEPLQGAVAK, IPSKVDSALYLGSR, KADAVTLDGGMVF, KADAVTLDGGMVF, KANEGLTWNSLK, KDSALGFLR, KGSNFQLDQLQGR, KPVTEAQSCHLAVAPNHAVVSR, LAQVPSHAVVAR, LAVAPNHAVVSR, LAVAVVK, LFGSPPGQR, LFKDSALGFLR, LGAPSITCVR, LGGRPTYEEY, LGGRPTYEEYLGTEY, LGGRPTYEEYLGTEYVTAIANLK, LGGRPTYEEYLGTEYVTAIANLKK, LGTEYVTAIANLK, LHQQALFGK, LLHQQALFGK, LRPVAAEIY, LRPVAAEIYGTK, LSWTESLEPLQGAVAK, LQGAVAK, NFQLDQLQGR, NLLFNDNTECLAK, PLQGAVAK, PQTHYYAVAVVK, PSKVDSALYLGSR, PTEGYLAVAVVK, PTYEEYLGTEYVTAIANLK, PVAAEIYGTK, PYLSWTESLEPLQGAVAK, QLDQLQGR, QVLLHQQALF, QVLLHQQALFGK, QVLLHQQALFGKNGK, SAGWIIPMGILRPY, SAGWIIPMGILRPYLSWTESLEPLQGAVAK, SFQLFGSPPGQR, SVDGKEDLIWK, SWTESLEPLQGAVAK, TESLEPLQGAVAK, TVFENLPEK, VFENLPEK, VLLHQQALFGK, VTAIANLK, WTESLEPLQGAVAK, YAVAVVK, YFGYSGAFK, YYGYTGAF and YYGYTGAFR, or a selection thereof that is able to stimulate osteoblast proliferation or inhibit osteoclast development; or
(2) AEIYGTKESPQTHY, AENRKSSKYSSL, AKLGGRPTYE, AKLGGRPTYEE, AKNLNRED, AKNLNREDF, AQEKFGKNKSRS, ARSVDGKEDL, AVVKKANEGLTWNSL, DGGMVFEAGRDPYKLRPVA, DRDQYEL, DRTAGWNIPMGL, EAGRDPYKLRPVA, EAGRDPYKLRPVAA, EAGRDPYKLRPVAAE, EIYGTKESPQTHY, EKKADAVTL, ENLPEKADRDQ, ENLPEKADRDQY, ENLPEKADRDQYE, ENLPEKADRDQYEL, ESLEPLQG, ESLEPLQGA, ESLEPLQGAV, FEAGRDPYKLRPVA, FEAGRDPYKLRPVAA, FGKNKSRS, FGSPPGQRDL, FGSPPGQRDLL, FGSPPGQRDLLF, FKCLQDGAGDVAF, FKDSALGF, FKSETKNLL, FNDNTECL, FQLFGSPPGQRDLL, FRCLAEDVGD, GSPPGQRDLL, IAEKKADAVT, IAEKKADAVTL, IPMGI, IWKLLSKAQEKFGKNKSRS, IWKLLSKAQEKFGKNKSRSFQL, IYGTKESPQTHY, KAQEKFGKNKSRS, KDSALGF, KGEADALNL, KKADAVTL, KSETKNLL, KYYGYTGA, LECIRA, LFGSPPGQRDLL, LFKDSALGF, LKNLRE, LKNLRETAE, LNLDGGY, LPEKADRDQYE, LRIPSKVD, LRIPSKVDSA, LRIPSKVDSAL, LSKAQEKFGKNKSRS, LSKAQEKFGKNKSRSFQL, LTTLKNLRE, LTTLKNLRETAE, NLDGGY, NLDGGYI, NLNREDFRL, NLPEKADRDQ, NREDFRL, PEKADRDQ, PEKADRDQYE, PEKADRDQYEL, PPGQRDLL, PYKLRPVA, QLFGSPPGQRDLL, RSDRAAHVKQVL, RSVDGKEDL, RTAGWNIPMGL, SWTESLEPLQG, TESLEPLQG, TTLKNLRETAE, VARSVDGKEDL, VFEAGRDPYKLRPVA, VFEAGRDPYKLRPVAA, VFEAGRDPYKLRPVAAE, VKETTVF, VLKGEADAL, VSRSDRAAHVKQ, VTLDGGM, VTLDGGMV, VTLDGGMVF, VVARSVDGKEDL, VVKKANEGLTW, VVKKANEGLTWNSL, VVSRSDRAAHVKQ, VVSRSDRAAHVKQVL, WAKNLNRE, WAKNLNRED, WAKNLNREDF, WIIPMGI, WNIPMGL, YGTKESPQTHY and YLGSRY, or a selection thereof that is able to stimulate osteoblast proliferation or inhibit osteoclast development.
18 . A method according to claim 7 , wherein the microorganism is selected from the genera Bacillus, Bifidus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionbacter, Pseudomonas, Streptococcus or a mixture thereof, or wherein the acid is selected from trifluoro acetate and hydrochloric acid.
19 . A method according to claim 1 , wherein the disorder is osteoporosis, rheumatoid arthritis, osteoarthritis, hepatic osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, chronic renal disease, sarcoidosis, glucocorticoid-induced osteoporosis, idiopathic hypercalcemia, Paget's disease, or osteogenesis imperfecta.
20 . A method according to claim 19 , wherein the disorder is osteoporosis, rheumatoid arthritis or osteoarthritis.
21 . A method according to claim 1 , wherein the lactoferrin fragment comprises a metal ion binding site that is bound to a metal ion or the lactoferrin fragment comprises two metal ion binding sites that are independently empty or bound to a metal ion.
22 . A method according to claim 21 , wherein the metal ion is selected from a bismuth ion, an iron ion, copper ion, chromium ion, cobalt ion, manganese ion, zinc ion, or a mixture thereof.