Source: http://www.google.de/patents/US4761471?hl=de
Timestamp: 2013-05-18 10:43:20
Document Index: 756221436

Matched Legal Cases: ['arts 22', 'art 14', 'arts 18', 'arts 22', 'arts 18', 'art 14']

Patent US4761471 - Bone morphogenetic protein composition - Google PatenteSuche Bilder Maps Play YouTube News Gmail Drive Mehr » Erweiterte Patentsuche | Webprotokoll | Anmelden Erweiterte Patentsuche PatenteBMP compositions including the human factor and bovine factor thereof, the process of isolating BMP compositions and factors, and the use of such factors and compositions to induce bone formation in animals....http://www.google.de/patents/US4761471?utm_source=gb-gplus-sharePatent US4761471 - Bone morphogenetic protein composition Ver�ffentlichungsnummerUS4761471 APublikationstypErteilung Anmeldenummer06/899,020 Ver�ffentlichungsdatum2. Aug. 1988Eingetragen22. Aug. 1986 Priorit�tsdatum4. Aug. 1980 ErfinderMarshall R. UristUrspr�nglich Bevollm�chtigterThe Regents Of The University Of California US-Klassifikation530/350530/840530/417514/8.8Internationale KlassifikationC07K14/435C07K14/51A61L27/00A61L27/22A61K38/00 UnternehmensklassifikationC07K14/51A61K38/00A61L27/227 Europ�ische KlassifikationC07K 14/51A61L 27/22RReferenzenNichtpatentzitate (2) Referenziert von (134)Externe LinksUSPTO USPTO-Zuordnung EspacenetBone morphogenetic protein compositionUS 4761471 A Zusammenfassung BMP compositions including the human factor and bovine factor thereof, the process of isolating BMP compositions and factors, and the use of such factors and compositions to induce bone formation in animals.
2. The bone morphogenetic protein composition of claim 1, wherein the BMP factor is human BMP factor having a molecular weight of 17.5.+-5 dKa, and the BMP associated proteins are human BMP associated proteins having molecular weights of about 14-, 22-, 24- and 34-kDa.
3. The bone morphogenetic protein composition of claim 1, wherein the BMP factor is bovine BMP factor having a molecular weight of 18.5.+-5 kDa, and the BMP associated proteins are bovine BMP associated proteins having molecular weights of about 14-, 16.5-, 17-, 17.5, 22-, 24-, and 34-dKa.
4. The aggregate of: (a) hBMP factor having a molecular weight in the range of about 17.5.+-5 kDa, and comprised, as determined by amino acid analysis, of lysine, histidine, arginine, aspartate, threonine, serine, glutamate proline, glycine, alanine, cystine, leucine, tyrosine and phenylalanine; and (b) at least one BMP-associated protein selected from a group consisting of BMP-associated proteins having molecular weights of about 14-, 22-, 24-, and 34-kDa.
5. The aggregate of: (a) bBMP factor having a molecular weight in the range of about 18.5.+-5 kDa and and comprised, as determined by amino acid analysis, of lysine, histidine, arginine, aspartate, threonine, serine, glutamate, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine; and (b) at least one BMP associated protein selected from a group consisting of BMP associated proteins having molecular weights of about 14-, 16.5-, 17.0-, 22-, 24-, and 34-kDa.
6. The aggregate of claim 5 consisting essentially of about 7 parts 22-kDa and about 1 part 14-kDa BMP associated proteins, and about 2 parts 18.5.+-5-kDa bBMP factor.
9. The aggregate of claim 8, said hBMP factor having a molecular weight in the range of about 17.5.+-5 kDa and being trypsin and chymotrypsin labile, alkali sensitive, partially degradable by pepsin and papain, and resistant to chondroitinases A, B and C; amylase; neuroamidase; hyaluronidase; alkaline phosphates; acid phosphatase; chymopapain; collagenase; tyrosinase; thermolysine; RNase and DNase.
10. The aggregate of claim 9, said hBMP factor having a molecular weight of about 17.5.+-5 kDa and being an acidic polypeptide, bindable to hydroxyapetite, having an isoelectric point of 5+-2, being insoluble in chloroform, methanol, absolute alcohol, and acetone and being degraded by acid alcohol solutions.
Basically, the process of this invention comprises purifying and isolating the new BMP composition by the steps of further purifying the BMP obtained by the process of the aforementioned patent and application. The further process steps begin with the precipitate formed when the urea (or guanidine) solution has been dialyzed against water. With reference to the Example 1 of said patent, the precipitate formed during dialysis against water is that point in the process described up to column 5, line 57 of the patent. This is also shown in the drawing as the 8th process box wherein water is added to the BMP/urea solution and dialyzed against water to remove urea, CaCl.sub.2 and other impurities, thereby providing a BMP precipitate.
BMP factor is an acidic protein embedded in a complex assortment of intra- and extracellular protein aggregates derived from dentin, bone and osteosarcoma tissues. BMP composition is solubilized under dissociative conditions by either a CaCl.sub.2 /urea inorganic-organic solvent mixture, or 4M GuHCl, or the two solvents in sequence.
Table 1 discloses the amino acid composition of a 17.5.+-5-kDa protein isolated and purified in accordance with this invention from human femoral and tibial cortical bone obtained soon after death. This protein has been determined to be the active protein factor of BMP derived from human bone sources and is referred to herein a human BMP (hBMP) factor. It is a component of the associated proteins that form human BMP composition.
TABLE 1__________________________________________________________________________AMINO ACID ANALYSIS OF hBMP FACTORISOLATED BY SDS EXTRACTION OF PAGE SLICES               Residue WeightAmino Acids  Nanomoles*          Mole %               17,000    Mole Protein__________________________________________________________________________Lys    2.05    4.09 7.16(7)   897.19His    0.83    1.66 2.91(3)   411.42Arg    3.51    7.00 12.25(12) 1874.16Asp    4.00    7.98 13.97(14) 1611.12Thr    1.54    3.07 5.37(5)   505.50Ser*   4.03    8.04 14.07(14) 1218.98Glu    5.10    10.17               17.80(18) 2323.98Pro    1.86    3.71 6.49(6-7) 582.66Gly    12.45   24.83               43.45(43) 2453.15Ala    3.40    6.78 11.87(12) 852.841/2 Cys*  0.98    1.95 3.41(3-4) 309.42Val*   2.20    4.39 7.68(8)   793.04Met    0.48    0.96 1.68(2)   262.38Ileu*  1.55    3.09 5.41(6)   678.90Leu    3.40    6.78 11.87(12) 1357.80Tyr    1.45    2.89 5.06(5)   815.85Phe    1.31    2.61 4.57(5)   735.85  50.14   100.00               175-177 + Trp +                         17,684 + Trp +               CH.sub.2 O)X                         (CH.sub.2 O)X__________________________________________________________________________ *Nanomoles for each amino acid equals the average of 24 and 72hour hydrolysates or the extrapolated value (Ser) or the 72hour value only (Va and Ileu) or the value from the hydrolysate of the performic acidoxidized sample (1/2 Cys). The margin of accuracy was about .+-.3%.
Variable quantities of other relatively low molecular weight proteins found in the human BMP composition with assigned molecular weights of 14-, 22-, 24-, and 34-kDa are dissassociated from hBMP by differential precipitation, or in some instances by ultrafiltration. Each of the associated proteins can be removed from hBMP without loss of BMP activity. However, the isolated 17.5-kDa hBMP factor is more rapidly adsorbed by tissue than the aggregates of 17.5-kDa hBMP factor and the other associated proteins, and must be implanted in greater quantities to produce the same yield of new bone. In one example, the bovine BMP (bBMP) factor which was isolated and purified as described herein, and which had an assigned molecular weight of 18.5.+-5-kDa, yielded approximately 1 gram of new bone for each milligram of bBMP implanted in the absence of the other proteins. The isolated BMP factor induces a lower yield of bone when implanted in the absence of the associated proteins.
bBMP factor is an 18.5.+-5-kDa molecular weight molecule derived from a non-collagenous protein aggregate that induces differentiation of mesenchymal type cells into cartilage and bone. bBMP factor is difficult to separate from insoluble proteins associated therewith that have assigned molecular weights of 34-, 24-, 22-, 17.5-, 17.0-, 16.5-, and 14-kDa and which may include traces of ferritin. The bBMP aggregate becomes soluble in aqueous media when it is separated from the 14-kDA protein by ultrafiltration. (For example, 2 liter batches of CaCl.sub.2 /urea soluble bovine BMP composition in 0.01M phosphate buffer, pH 7, were processed through an H1 P-10-8 hollow fiber cartridge (Amicon), 10-kDa approximate cutoff.) The 34-kDa protein is separated by extraction with Triton X-100. In the absence of 34-kDa, the 22-kDa is collected in the unbound fraction by hydroxyapatite chromatography. The 24- and 22-kDa proteins are separated by precipitation in 1.0-1.5M GuHCl. BMP fractions produced an electrophoretic pattern with a broad band for proteins with an assigned molecular weight of 17- to 18-kDa. Further resolution of these proteins by means of hydroxyapatite chromatography produces three components with apparent molecular weights of 18.5-, 17.5-, and 17.0-kDa. Although relatively small in quantity (&lt;0.001 percent of the wet weight of cortical bone), the isolated 18.5.+-5-kDa protein induces bone formation independent of other matrix components. The 34-, 24-, 22-, 17.5-, 17.0-, 16.5-, and 14-kDa components do not induce bone formation. The 17.5-kDa has the N-terminal amino acid sequence of histone H2B. Insofar as the N-terminal amino acid was unblocked, the 17.5-kDa associated BMP (bovine) derived from bBMP composition was selected for amino acid sequencing. The first 26 amino acids are in the following sequence: Pro GlutA Pro Ala Lys Phe Ala Pro Ala Pro.sup.10 Lys Lys Gly Phe Lys Lys Ala Val Tyr (Tyr).sup.20 Ala GluN (lys) Asp (Phe).sup.26.
TABLE 3______________________________________PHYSIOCHEMICAL PROPERTIES OF hBMP Factor______________________________________Apparent MW 17.5 .+-. 0.5-kDa.Acidic Polypeptide.Binds to hydroxyapatite.Isoelectric point (pI) 5.0 .+-. 0.2.Carbohydrate, none detected.Soluble in neutral salt solution at pH 7.2.Degraded by acid alcohol solutions.Alkali sensitive.Insolubilized by forming aggregates with associated 14-kDaproteins.Contains carboxyglutamic acid, about 3 residues/170 residues.May contain hydroxyproline.Insoluble in chloroform, methanol, absolute alcohol, acetone.BMP 14-kDa complex with hBMP factor: insoluble in TritonX-100 (non-ionic detergent); insoluble in 0.6 N HCl; soluble in6 M urea or 4 M guanidine HCl; soluble in 0.1% SDS; solublein 0.02 N HCl; and partially soluble in ethylene glycol.Trypsin and chymotrysin labile.Resistant to: chondroitinases A, B, and C; amylase;neuroamidase; hyaluronidase; alkaline phosphates; acidphosphatase; chymopapain; collagenase; tyrosinase;thermolysin; and nuclease (RNAase and DNAase).Partially degraded by pepsin and papain.______________________________________
The dose-response curve to implants to BMP (human) composition, prepared by the procedure described herein, is shown in the accompanying FIG. 2 of the drawings. The yield of calcified new bone is shown to be in direct proportion to the quantity of BMP (human) composition implanted in muscle in the mouse thigh. The incorporation of .sup.45 Ca into histologically valid deposits of new bone was directly proportional to the weight of implanted BMP (human) composition, ranging from 22 to 102 cpm .sup.45 Ca contralateral muscle emitted only 3 cpm. The correlation coefficient of the BMP (human) composition to induced bone formation was 0.9924. Control implants of human tendon collagen and serum albumin invariably failed to induce bone formation.
The water insoluble precipitate of relatively crude BMP composition was solubilized in a 6M urea solution containing 0.5M CaCl.sub.2 and dialyzed against water three times. The resultant precipitate was centrifuged to remove water and redissolved in 4.0M guanidine hydrochloride. The BMP solution was then dialyzed against 1.0M guanidine hydrochloride and the supernatant was dialyzed against cold water, leaving a precipitate. The precipitate was then redissolved in 4.0M guanidine hydrochloride that was formed was washed thrice with cold water and again dissolved in 4.0M guanidine hydrochloride.
EXAMPLE 2 Ten kilogram batches of 1 year old steer long bones were obtained from abbatoire. After the epiphyseal ends were cut away with a band saw, the diaphyses were mechanically scraped clean of soft tissues, and extensively washed in a cold water solution of sodium azide (NaN.sub.3), 3 mmoles/l. The washed bone was frozen in liquid nitrogen, ground in a Wiley mill to a particular size of 1 mm, defatted in 1:1 chloroform methanol, and again washed in 10 liters of cold water (Step I, see Table 4).
The bone particles were demineralized in 0.6N HCl at 4 hrs. and again extensively rewashed in NaN.sub.3 solution (Step II). The demineralized washed bone particles were chemically extracted to remove soluble non-collagenous proteins (i.e., sialoproteins, plasma proteins, gla proteins, and phosphoproteins) simultaneously converting the collagen to insoluble bone matrix gelatin (Step III) by procedures described in U.S. Pat. No. 4,294,753. Ten kilograms of whole wet bone produced approximately 1.4 kgs. of freeze dried insoluble bone matrix gelatin.
The BMP was extracted from the insoluble bone matrix gelatin in an inorganic-organic solvent mixture of 0.5M CaCl.sub.2 in 6M urea at 28 endogenous degradative enzymes.
The undissolved matrix and other substances were removed by centrifugation at 10,000 g for 30 minutes. The supernatant solution was decanted, diluted with 23 volumes of deionized water at 4 overnight while a precipitate formed. The precipitate was collected by centrifugation (Sorvall RC-5B Refrigerated Superspeed) at 40,000 g for 20 minutes, and washed in cold deionized water, and weighed (Step IV).
Sixty grams of the fraction obtained by Step IV were redissolved in the original 0.5M CaCl.sub.2, 6M urea solution and dialyzed against 0.25M citrate buffer, pH 3.1, at 4 precipitate was collected by centrifugation at 40,000 g for 1 hour. The precipitate was extensively washed, defatted in 1:1 chloroform-methanol, and evaporated to dryness (Step V).
Twenty-two grams of the fraction obtained by Step V were redissolved in 4M GuHCl and diluted about 4 times to about 1.5M GuHCl at 28 12 hours or until formation of a precipitate. The 1.5M GuHCl soluble fraction was washed, dialyzed against water for 24 hours, until precipitation was complete. The water insoluble precipitate was centrifuged, extensively washed in cold water, lyophilized and weighed (Step VI). The 1.5M GuHCl insoluble fraction was centrifuged at 50,000 g for 1 hour, washed in cold water, lyophilized, and weighed (Step VII).
The protein fraction obtained by Step VII was redissolved in 0.5M CaCl.sub.2 in 6M urea, and dialyzed against 0.2% Triton X-100 in 0.10M Tris HCl buffer solution, pH 7.2, for 24 hours, at 28 dialysis was continued for an additional 12 hours. (Step VIII).
In Step IV, 60 grams of 0.5M CaCl.sub.2, 6M urea soluble, 0.25M urea insoluble proteins were obtained from 1.4 kgs. of bone matrix gelatin; this protein fraction produced bone formation in 16/20 implants and the yield was three times greater in volume than the yield from bone matrix gelatin. In Step V, the gelatin peptides were separated from the 0.5M CaCl.sub.2 -6M urea soluble proteins by dialysis against 0.5M citrate buffer at pH 3.4. This reduced the quantity of protein with high BMP activity from 60 to 22 grams but further increased the incidence in yields of bone formation to 18/20 implants.
For preparative tube gel electrophoresis and amino acid analysis of individual proteins, 6-mm slabs and 1-cm tubes of 15% PA were loaded with a 0.2% SDS and 0.12% urea solution of hBMP; 1.7 μg/tube was run at 15 mA.tube for 18 hours. Gels were sliced with a knife across molecular weight zones corresponding to the 34-, 24-, 22-, 17.5, and 14-kDa protein using duplicates stained for orientation. Gel slices containing the 17.5-kDa bBMP were extracted with 0.2% SDS, dialyzed against water, and lyophilized; 200-ug samples were hydrolyzed under vacuum for 24 hours at 110 (equipped with a Spectrum Physics data reduction system). Similarly prepared 35-, 24, 22-, and 145-kDa protein fractions were analyzed for comparison.
EXAMPLE 3 Hydroxyapatite (HA) columns of Bio-Gel HTP (Bio-Rad Laboratories, California), were prepared from 15 g./100 ml of 0.05M NaP.sub.i /6M urea. After 10 minutes the solvent was decanted and the HA was resuspended to give a settled bed height of 9 cm (volume, approximately 55 ml). The column was equilibrated with 100 ml starting buffer (at a flow rate of 0.6 ml/min.) the flow rate of the column by gravity; 100 mg of the preparation obtained by Step VIII of Example 2 was dissolved in 6M urea, applied to the column, and separated into fractions. Each fraction was dialyzed against deionized water (cold), frozen and lyophilized. Weighed samples were incubated for 24 hours in 0.1M sodium phosphate buffer containing 2M urea and 0.1% SDS, pH 7.2, for (PAGE); 5 μl (2.5 mg/1 ml) of each sample were applied to 8.5% gel and electrophoresed at 25 mA, and stained with 0.044% Coomassie Brilliant Blue R-250. The electrophoretic mobilities were measured with the aid of two sets of standard proteins.
The protein consistently associated with BMP activity was white in color and fluffy in texture with an apparent molecular weight of 18.5.+-5-kDa. Two other proteins with molecular weights of 17.5 and 17.0-kDa did not induce bone formation.
EXAMPLE 4 The individual proteins isolated by preparative gel electrophoresis were hydrolyzed at 110 sealed tubes. Analysis for tryptophan and cysteine were performed following hydrolysis at 110 acid analysis was performed on an amino acid analyzer (Beckman 119 C) equipped with a Spectra Physics 4000 data reduction system.
Analyses of the samples of 22-, 18.5.+-5-kDa, and 14-kDa bovine BMP fractions, isolated by elution of SDS PAGE disc gel slices had the composition of an acidic polypeptide, and are shown in Table 2. Not shown in Table 2, is 1 residue of hydroxyproline and three residues of gla in the 18.5.+-5-kDa protein.
EXAMPLE 7 When the 18.5.+-5-kDa bBMP factor was present, implants of protein fractions isolated by hydroxyapatite chromatography induced bone formation in the mouse hind quarter muscles, and produced regeneration in trephine defects in rat and dog skulls. Implants of 10 mg or more of 14-, 17.5-, 17.0-, 22-, or 34-kDa proteins alone, or in various combinations failed to induce bone formation. The association with the 14-kDa decreased solubility in vitro, increased resorption time in vivo and improved both incidence and the yield of new bone from implants of as little as 1 mg. Implants of 34-kDa combinations with 17.5-kDa did not improve and may even have suppressed the bone formation. Implants of 7 parts 22-kDa, 2 parts 18.5.+-5-kDa and 1 part 14-kDa weighing 3 mg consistently induced formation of large deposits of bone.
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