text:
This application is a divisional of U.S. patent application Ser. No. 09/370,084, filed Aug. 6, 1999 (now U.S. Pat. No. 6,576,235), which claims the benefit of U.S. Provisional Application No. 60/219,318, filed Aug. 6, 1998, the disclosures of which are incorporated herein by reference in their entireties.
A portion of the research described in this application was made with support from Grant DK48529 from the National Institutes of Health. Accordingly, the U.S. government may have certain rights in this invention.
The present invention relates to chemical modification of proteins to prolong their circulating lifetimes and reduce their immunogenicity. More specifically, the invention relates to conjugation of poly(ethylene glycols) or poly(ethylene oxides) to urate oxidases, which substantially eliminates urate oxidase immunogenicity without compromising its uricolytic activity.
Statements contained in this background section do not constitute an admission of prior art, but instead reflect the inventors' own subjective comments on and interpretations of the state of the art at the time the invention was made. These interpretations may include personal, heretofore undisclosed, insights of the inventors, which insights were not themselves part of the prior art.
Urate oxidases (uricases; E.C. 188.8.131.52) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantoin, a purine metabolite that is more readily excreted. Humans do not produce enzymatically active uricase, as a result of several mutations in the gene for uricase acquired during the evolution of higher primates. Wu, X, et al., (1992) J Mol Evol 34:78-84. As a consequence, in susceptible individuals, excessive concentrations of uric acid in the blood (hyperuricemia) and in the urine (hyperuricosuria) can lead to painful arthritis (gout), disfiguring urate deposits (tophi) and renal failure. In some affected individuals, available drugs such as allopurinol (an inhibitor of uric acid synthesis) produce treatment-limiting adverse effects or do not relieve these conditions adequately. Hande, K R, et al., (1984) Am J Med 76:47-56; Fam, A G, (1990) Bailli�re's Clin Rheumatol 4:177-192. Injections of uricase can decrease hyperuricemia and hyperuricosuria, at least transiently. Since uricase is a foreign protein in humans, however, even the first injection of the unmodified protein from Aspergillus flavus has induced anaphylactic reactions in several percent of treated patients (Pui, C-H, et al., (1997) Leukemia 11: 1813-1816), and immunologic responses limit its utility for chronic or intermittent treatment. Donadio, D, et al., (1981) Nouv Presse M�d 10:711-712; Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic 50:553-554.
The sub-optimal performance of available treatments for hyperuricemia has been recognized for several decades. Kissel, P, et al., (1968) Nature 217:72-74. Similarly, the possibility that certain groups of patients with severe gout might benefit from a safe and effective form of injectable uricase has been recognized for many years. Davis, F F, et al., (1978) in G B Broun, et al., (Eds.) Enzyme Engineering, Vol. 4 (pp. 169-173) New York, Plenum Press; Nishimura, H, et al., (1979) Enzyme 24:261-264; Nishimura, H, et al., (1981) Enzyme 26:49-53; Davis, S, et al., (1981) Lancet 2(8241):281-283; Abuchowski, A, et al., (1981) J Pharmacol Exp Ther 219:352-354; Chen, R H-L, et al., (1981) Biochim Biophys Acta 660:293-298; Chua, C C, et al., (1988) Ann Int Med 109:114-117; Greenberg, M L, et al., (1989) Anal Biochem 176:290-293. Uricases derived from animal organs are nearly insoluble in solvents that are compatible with safe administration by injection. U.S. Pat. No. 3,616,231. Certain uricases derived from plants or from microorganisms are more soluble in medically acceptable solvents. However, injection of the microbial enzymes quickly induces immunological responses that can lead to life-threatening allergic reactions or to inactivation and/or accelerated clearance of the uricase from the circulation. Donadio, et al., (1981); Leaustic, et al., (1983). Enzymes based on the deduced amino acid sequences of uricases from mammals, including pig and baboon, or from insects, such as, for example, Drosophila melanogaster or Drosophila pseudoobscura (Wallrath, L L, et al., (1990) Mol Cell Biol 10:5114-5127), have not been suitable candidates for clinical use, due to problems of immunogenicity and insolubility at physiological pH.
Covalent modification of proteins with poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), has been used to increase protein half-life and reduce immunogenicity. U.S. Pat. Nos. 4,179,337, 4,766,106, and 4,847,325; Saifer, M G P, et al., (1994) Adv Exp Med Biol 366:377-387. The coupling of PEG of high molecular weight to produce conjugates with prolonged circulating lifetimes and/or decreased immunogenicity, while conserving functional activity, was previously demonstrated for another enzyme, superoxide dismutase (Somack, R, et al., (1991) Free Rad Res Commun 12-13:553-562; U.S. Pat. Nos. 5,283,317 and 5,468,478) and for other types of proteins, e.g., cytokines (Saifer, M G P, et al., (1997) Polym Preprints 38:576-577; Sherman, M R, et al., (1997) in J M Harris, et al., (Eds.), Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680 (pp. 155-169) Washington, D.C.: American Chemical Society). Conjugates of uricase with polymers other than PEG have also been described. U.S. Pat. No. 4,460,683.
In nearly all of the reported attempts to PEGylate uricase (i.e. to covalently couple PEG to uricase), the PEG was attached primarily to amino groups, including the amino-terminal residue and the available lysine residues. In the uricases commonly used, the total number of lysines in each of the four identical subunits is between 25 (Aspergillus flavus (U.S. Pat. No. 5,382,518)) and 29 (pig (Wu, X, et al., (1989) Proc Natl Acad Sci USA 86:9412-9416)). Some of the lysines are unavailable for PEGylation in the native conformation of the enzyme. The most common approach to reducing the immunogenicity of uricase has been to couple large numbers of strands of low molecular weight PEG. This has invariably resulted in large decreases in the enzymatic activity of the resultant conjugates.
Previous investigators have used injected uricase to catalyze the conversion of uric acid to allantoin in vivo. See Pui, et al., (1997). This is the basis for the use in France and Italy of uricase from the fungus Aspergillus flavus (Uricozyme�) to prevent or temporarily correct the hyperuricemia associated with cytotoxic therapy for hematologic malignancies and to transiently reduce severe hyperuricemia in patients with gout. Potaux, L, et al., (1975) Nouv Presse M�d 4:1109-1112; Legoux, R, et al., (1992) J Biol Chem 267:8565-8570; U.S. Pat. Nos. 5,382,518 and 5,541,098. Because of its short circulating lifetime, Uricozyme� requires daily injections. Furthermore, it is not well suited for long-term therapy because of its immunogenicity.
A single intravenous injection of a preparation of Candida utilis uricase coupled to 5 kDa PEG reduced serum urate to undetectable levels in five human subjects whose average pre-injection serum urate concentration was 6.2 mg/dL, which is within the normal range. Davis, et al., (1981). The subjects were given an additional injection four weeks later, but their responses were not reported. No antibodies to uricase were detected following the second (and last) injection, using a relatively insensitive gel diffusion assay. This reference reported no results from chronic or subchronic treatments of human patients or experimental animals.
A preparation of uricase from Arthrobacter protoformiae coupled to 5 kDa PEG was used to temporarily control hyperuricemia in a single patient with lymphoma whose pre-injection serum urate concentration was 15 mg/dL. Chua, et al., (1988). Because of the critical condition of the patient and the short duration of treatment (four injections during 14 days), it was not possible to evaluate the long-term efficacy or safety of the conjugate.
In this application, the term "immunogenicity" refers to the induction of an immune response by an injected preparation of PEG-modified or unmodified uricase (the antigen), while "antigenicity" refers to the reaction of an antigen with preexisting antibodies. Collectively, antigenicity and immunogenicity are referred to as "immunoreactivity." In previous studies of PEG-uricase, immunoreactivity was assessed by a variety of methods, including: 1) the reaction in vitro of PEG-uricase with preformed antibodies; 2) measurements of induced antibody synthesis; and 3) accelerated clearance rates after repeated injections.
Previous attempts to eliminate the immunogenicity of uricases from several sources by coupling various numbers of strands of PEG through various linkers have met with limited success. PEG-uricases were first disclosed by F F Davis and by Y Inada and their colleagues. Davis, et al., (1978); U.S. Pat. No. 4,179,337; Nishimura, et al., (1979); Japanese Patents 55-99189 and 62-55079. The conjugate disclosed in the '337 patent was synthesized by reacting uricase of unspecified origin with a 2,000-fold molar excess of 750 dalton PEG, indicating that a large number of polymer molecules was likely to have been attached to each uricase subunit. The '337 patent discloses the coupling of either PEG or poly(propylene glycol) with molecular weights of 500 to 20,000 daltons, preferably about 500 to 5,000 daltons, to provide active, water-soluble, non-immunogenic conjugates of various polypeptide hormones and enzymes including oxidoreductases, of which uricase is one of three examples. In addition, the '337 patent emphasizes the coupling of 10 to 100 polymer strands per molecule of enzyme, and the retention of at least 40% of enzymatic activity. No test results were reported for the extent of coupling of PEG to the available amino groups of uricase, the residual specific uricolytic activity, or the immunoreactivity of the conjugate.
Data from 13 citations relating to PEGylation of uricase are summarized in Table 1. Some of these results are also presented graphically in
Among the studies summarized in Table 1, the immunoreactivity of uricase was reported to be decreased by PEGylation in seven of them and eliminated in five of them. In three of the latter five studies, the elimination of immunoreactivity was associated with profound decreases in uricolytic activity—to at most 15%, 28%, or 45% of the initial activity. Nishimura, et al., (1979) (15% activity); Chen, et al., (1981) (28% activity); Nishimura, et al., (1981) (45% activity). In the fourth report, PEG was reported to be coupled to 61% of the available lysine residues, but the residual specific activity was not stated. Abuchowski, et al., (1981). However, a research team that included two of the same scientists and used the same methods reported elsewhere that this extent of coupling left residual activity of only 23-28%. Chen, et al., (1981). The 1981 publications of Abuchowski et al., and Chen et al., indicate that to reduce the immunogenicity of uricase substantially, PEG must be coupled to approximately 60% of the available lysine residues (Table 1). The fifth publication in which the immunoreactivity of uricase was reported to have been eliminated does not disclose the extent of PEG coupling, the residual uricolytic activity, or the nature of the PEG-protein linkage. Veronese, F M, et al., (1997) in J M Harris, et al., (Eds.), Poly(ethylene glycol) Chemistry) and Biological Applications. ACS Symposium Series 680 (pp. 182-192) Washington, D.C.: American Chemical Society.
Conjugation of PEG to a smaller fraction of the lysine residues in uricase reduced but did not eliminate its immunoreactivity in experimental animals. Tsuji, J, et al., (1985) Int J Immunopharmacol 7:725-730 (28-45% of the amino groups coupled); Yasuda, Y, et al., (1990) Chem Pharm Bull 38:2053-2056 (38% of the amino groups coupled). The residual uricolytic activities of the corresponding adducts ranged from <33% (Tsuji, et al.) to 60% (Yasuda, et al.) of their initial values. Tsuji, et al., synthesized PEG-uricase conjugates with 7.5 kDa and 10 kDa PEGs, in addition to kDa PEG. All of the resultant conjugates were somewhat immunogenic and antigenic, while displaying markedly reduced enzymatic activities (Table 1;
A PEGylated preparation of uricase from Candida utilis that was safely administered twice to each of five humans was reported to have retained only 11% of its initial activity. Davis, et al., (1981). Several years later, PEG-modified uricase from Arthrobacter protoformiae was administered four times to one patient with advanced lymphoma and severe hyperuricemia. Chua, et al., (1988). While the residual activity of that enzyme preparation was not measured, Chua, et al., demonstrated the absence of anti-uricase antibodies in the patient's serum 26 days after the first PEG-uricase injection, using an enzyme-linked immunosorbent assay (ELISA).
As summarized in Table 1, previous studies of PEGylated uricase show that catalytic activity is markedly depressed by coupling a sufficient number of strands of PEG to decrease its immunoreactivity substantially. Furthermore, most previous preparations of PEG-uricase were synthesized using PEG activated with cyanuric chloride, a triazine derivative (2,4,6-trichloro-1,3,5-triazine) that has been shown to introduce new antigenic determinants and to induce the formation of antibodies in rabbits. Tsuji, et al., (1985).
Characteristics of PEG-uricases from Previous Studies
Percent of
Lysines with
Antigenicity or
Source of
Weight of
PEG (kDa)
Not reported
Not reported
Not reported
Not reported
U.S. Pat. No. 4,179,337
% of "98":
Antigenicity with rabbit serum
Nishimura, et al., 1979
(% of that of the unmodified
2 � 5
Nishimura, et al., 1981
Five men tolerated two injections
Davis, et al., 1981
in 30 days.
Not reported
Similar immunogenicity in birds
Abuchowski, et al.,
to native uricase
to Chen
Not reported
Immunogenicity negative
et al., 1981
Porcine liver
Accelerated clearance in mice
Chen, et al., 1981
Accelerated clearance in mice
Constant Clearance
(half-life ca. 8 hours)
Constant Clearance
(half-life ca. 8 hours)
Not reported
PEG decreased the immuno-
Savoca, K V, et al.,
genicity in rabbits.
(1984) Int Arch
to Chen,
Not reported
PEG decreased the immuno-
Allergy Appl Immunol
et al., 1981
genicity in rabbits.
Not reported
Not reported
PEG-uricase was given orally to
Nishida, Y, et al.,
chickens in liposomes (once).
(1984) J Pharm
Pharmacol 36:354-355
Immunogenicity was reduced, but
Tsuji, et al., 1985
positive in rabbits.
(Antibodies are not to uricase;
they cross react with PEG-
superoxide dismutase.)
Antigenicity tested with guinea
pig antibodies was reduced.
Not reported
Not reported
No antibodies were detected by
Chua, et al., 1988
ELISA 26 days after the first of
four PEG-uricase injections.
2 � 5
Not reported
Yasuda, et al., 1990
Not reported
Not reported
Not reported
Antigenicity tested with rabbit
serum was reduced by 75%.
2 � 5
Single injection. PEG increased
Fujita, et al., 1991
the half-life from ca. 1 h to ca. 8 h
in mice. PEG blocked clearance
by liver, spleen and kidney (24-h
study duration).
Not reported
Not reported
Not reported
Immunogenicity in mice was
Veronese, et al., 1997
Reported to
Not reported
decreased by 98% (PEG) or 100%
Linkage not
be tbe same
as for PEG
Japanese Patent 3-148298 to A Sano, et al., discloses modified proteins, including uricase, derivatized with PEG having a molecular weight of 1-12 kDa that show reduced antigenicity and "improved prolonged" action, and methods of making such derivatized peptides. However, there are no disclosures regarding strand counts, enzyme assays, biological tests or the meaning of "improved prolonged." Japanese Patents 55-99189 and 62-55079, both to Y Inada, disclose uricase conjugates prepared with PEG-triazine or bis-PEG-triazine (denoted as PEG2 in Table 1), respectively. See Nishimura, et al., (1979 and 1981). In the first type of conjugate, the molecular weights of the PEGs were 2 kDa and 5 kDa, while in the second, only 5 kDa PEG was used. Nishimura, et al., (1979) reported the recovery of 15% of the uricolytic activity after modification of 43% of the available lysines with linear 5 kDa PEG, while Nishimura, et al., (1981) reported the recovery of 31% or 45% of the uricolytic activity after modification of 46% or 36% of the lysines, respectively, with PEG2.
Previous studies teach that when a significant reduction in the immunogenicity and/or antigenicity of uricase is achieved by PEGylation, it is invariably associated with a substantial loss of uricolytic activity. The safety, convenience and cost-effectiveness of biopharmaceuticals are all adversely impacted by decreases in their potencies and the resultant need to increase the administered dose. Thus, there is a need for a safe and effective alternative means for lowering elevated levels of uric acid in body fluids, including blood and urine. The present invention provides a substantially non-immunogenic PEG-uricase that retains all or nearly all of the uricolytic activity of the unmodified enzyme.
One embodiment of the present invention is a conjugate of urate oxidase (uricase) that retains at least about 75% of the uricolytic activity of unconjugated uricase and has substantially reduced immunogenicity. This embodiment includes a purified uncase in which each subunit may be covalently linked to an average of 2 to 10 strands of PEG, which may be linear or branched, wherein each molecule of PEG may have a molecular weight between about 5 kDa and 100 kDa. The uricase of this aspect of the invention may be recombinant. Whether recombinant or not, the uricase may be of mammalian origin. In one aspect of this embodiment, the uricase may be porcine, bovine or ovine liver uricase. In another aspect of this embodiment, the uricase may be chimeric. The chimeric uricase may contain portions of porcine liver and/or baboon liver uricase. For example, the chimeric uricase may be pig-baboon chimeric uricase (PBC uricase) or porcine uricase containing the mutations R291K and T301S (PKS uricase) (see sequences in
Another embodiment of the present invention is a pharmaceutical composition for lowering uric acid levels in body fluids, containing any of the PEG-uricase conjugates described above and a pharmaceutically acceptable carrier. The composition may be stabilized by lyophilization and also may dissolve promptly upon reconstitution to provide solutions suitable for parenteral administration.
The present invention also provides a method for lowering uric acid levels in body fluids and tissues of a mammal. The method includes administering to a mammal an effective uric acid-lowering amount of PEG-uricase. The PEG-uricase may be a purified uricase of two or more subunits in which each subunit may be covalently linked to an average of 2 to 10 strands of linear or branched PEG, wherein each molecule of PEG may have a molecular weight between about 5 kDa and 100 kDa, in a pharmaceutically acceptable carrier. The mammal may be a human. The administering step may be, for example, injection by intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal routes or inhalation of an aerosolized preparation. The elevated uric acid levels may be in blood, urine and/or other body fluids and tissues, and may be associated with gout, tophi, renal insufficiency, organ transplantation or malignant disease.
Other embodiments of the present invention are a method for isolating a tetrameric form of uricase from a solution containing multiple forms of uricase and the product of that method. Initially, the solution may contain tetrameric uricase and uricase aggregates. The method may include the steps of: applying the solution to at least one separation column at a pH between about 9 and 10.5, such as, for example, 10.2; recovering fractions of the eluate and identifying those that may contain isolated tetrameric uricase, wherein the fractions are substantially free of uricase aggregates; and pooling the fractions of the isolated tetrameric uricase. The separation column may be based on ion exchange, size exclusion, or any other effective separation property. The method may also include analysis of the fractions to determine the presence of tetrameric uricase and/or the absence of uricase aggregates. For example, such analysis may include high performance liquid chromatography (HPLC), other chromatographic methods, light scattering, centrifugation and/or electrophoresis. In one aspect of this embodiment, the purified tetrameric uricase may contain less than about 10% uricase aggregates.
The present invention provides improved conjugates of water-soluble polymers, preferably poly(ethylene glycols) or poly(ethylene oxides), with uricases. The invention also provides pharmaceutical compositions of the improved conjugates. These conjugates are substantially non-immunogenic and retain at least 75%, preferably 85%, and more preferably 95% or more of the uricolytic activity of the unmodified enzyme. Uricases suitable for conjugation to water-soluble polymers include naturally occurring urate oxidases isolated from bacteria, fungi and the tissues of plants and animals, both vertebrates and invertebrates, as well as recombinant forms of uricase, including mutated, hybrid, and/or truncated enzymatically active variants of uricase. Water-soluble polymers suitable for use in the present invention include linear and branched poly(ethylene glycols) or poly(ethylene oxides), all commonly known as PEGs. Examples of branched PEG are the subject of U.S. Pat. No. 5,643,575. One preferred example of linear PEG is monomethoxyPEG, of the general structure CH3O—(CH2CH2O)nH, where n varies from about 100 to about 2,300.
One preferred mammalian uricase is recombinant pig-baboon chimeric uricase, composed of portions of the sequences of pig liver and baboon liver uricase, both of which were first determined by Wu, et al., (1989). One example of such a chimeric uricase contains the first 225 amino acids from the porcine uricase sequence (SEQ ID NO: 1) and the last 79 amino acids from the baboon uricase sequence (SEQ ID NO: 2) (pig-baboon uricase, or PBC uricase; see
The cDNAs for various mammalian uricases, including PBC uricase, PKS uricase and a recombinant baboon-like uricase, were subcloned and the optimal conditions were determined for expression in E. coli, using standard methods. See Erlich, H A, (Ed.) (1989) PCR Technology. Principles and Applications for DNA Amplification. New York: Stockton Press; Sambrook, J, et al., (1989) Molecular Cloning. A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. The recombinant uricases were extracted, purified and their stability and activity were assessed using a modification of standard assays. See Fridovich, I, (1965) J Biol Chem 240:2491-2494; Nishimura, et al., (1979), and Example 1.
In one embodiment of the invention, uricase may be conjugated via a biologically stable, nontoxic, covalent linkage to a relatively small number of strands of PEG. Such linkages may include urethane (carbamate) linkages, secondary amine linkages, and amide linkages. Various activated PEGs suitable for such conjugation are available commercially from Shearwater Polymers, Huntsville, Ala.
For example, urethane linkages to uricase may be formed by incubating uricase in the presence of the succinimidyl carbonate (SC) or 4-nitrophenyl carbonate (NPC) derivative of PEG. SC-PEG may be synthesized using the procedure described in U.S. Pat. No. 5,612,460, which is hereby incorporated by reference. NPC-PEG may be synthesized by reacting PEG with 4-nitrophenyl chloroformate according to methods described in Veronese, F M, et al., (1985) Appl Biochem Biotechnol 11:141-152, and in U.S. Pat. No. 5,286,637, which is hereby incorporated by reference. The methods described in the '637 patent are adapted to PEGs of higher molecular weight by adjusting the concentrations of the reactants to maintain similar stoichiometry. An alternative method of synthesis of NPC-PEG is described by B�ttner, W, et al., East German Patent Specification DD 279 486 A1.
Amide linkages to uricase may be obtained using an N-hydroxysuccinimide ester of a carboxylic acid derivative of PEG (Shearwater Polymers). Secondary amine linkages may be formed using 2,2,2-trifluoroethanesulfonyl PEG (tresyl PEG; Shearwater Polymers) or by reductive alkylation using PEG aldehyde (Shearwater Polymers) and sodium cyanoborohydride.
In conjugates containing PEGs with molecular weights between 5 kDa and 30 kDa, the maximum number of strands of PEG that were coupled per subunit, while retaining at least 75% of the uricolytic activity of the unmodified enzyme, ranged from an average of 2 strands for soybean uricase to more than 10 strands for PBC uricase (see assay conditions in Example 1 and results in
There are several factors that may affect the choice of the optimal molecular weight and number of strands of PEG for coupling to a given form of uricase. In general, the reduction or elimination of immunogenicity without substantial loss of uricolytic activity may require the coupling of relatively more strands of PEG of lower molecular weight, compared to relatively fewer strands of PEG of higher molecular weight. For example, either 6 strands of 20 kDa PEG per subunit or 4 strands of 30 kDa PEG per subunit might be optimally effective. Likewise, each different form of uricase may have a different optimum with respect to both the size and number of strands. See
PEG conjugation rendered all of the tested uricases soluble and stable in buffers at physiological pH, without the addition of a substrate analog or inhibitor, such as 8-azaxanthine that is used as a stabilizer in the fungal uricase (Uricozyme�) sold by Sanofi Winthrop in France and Italy. Two different conjugates of PBC uricase, one containing approximately 6 strands of 10 kDa PEG per subunit and the other containing approximately 2 strands of 19 kDa PEG per subunit, retained significant activity after incubation in mouse serum for more than one month at 37� C. In addition, several of the conjugates of this invention had circulating half-lives in mice that were greater than two days, in contrast to the approximately 8-hour or 24-hour half-lives previously reported for PEG-modified mammalian and microbial uricases. Chen, et al., (1981); Fuertges, F, et al., (1990) J Contr Release 11: 139-148; Fujita, T, et al., (1991) J Pharmacobiodyn 14:623-629. Longer half-lives of injected protein drugs make them more cost-effective and can lead to improved patient compliance. Prolonged half-life is also indicative of products that are better tolerated by the body.
When PEG conjugates of PBC uricase were prepared from the purified tetrameric form of the enzyme (four 35 kDa subunits), they displayed profoundly reduced immunogenicity in mice (
Injections of fully active conjugates of PBC uricase with 10 kDa PEG (
Purified preparations of naturally occurring and recombinant uricases usually contain a mixture of aggregates of the enzyme, in addition to the tetrameric (140 kDa) form. The percentage of each uricase preparation that is in the tetrameric form generally varies from approximately 20% to 90%. Despite evidence that unPEGylated aggregates of several other proteins are highly immunogenic (see, e.g., Moore, W V, et al., (1980) J Clin Endocrinol Metab 51:691-697), previous studies of PEG-uricase do not describe any efforts to limit the content of aggregates, suggesting that the potential immunogenicity of the PEG-modified aggregates was not considered. On the basis of the observations of the present inventors, it appears likely that such aggregates were present in the enzyme preparations used for previous syntheses of PEG-uricase. Their presence may have rendered the task of preparing non-immunogenic conjugates more difficult. It also appears that the large losses of uricolytic activity observed in previous efforts to PEGylate uricase were related to the large number of strands of low molecular weight PEG that were coupled. On the other hand, the methods of uricase purification and PEGylation described herein permit the covalent attachment of as many as 10 strands of PEG per subunit while retaining more than 75% of the uricolytic activity, at least for certain uricases, e.g., pig-baboon chimeric uricase and the enzyme from A. flavus (see
In another preferred embodiment, substantially all aggregates of the tetrameric form of the enzyme may be removed by ion-exchange or size-exclusion chromatography at a pH between about 9 and 10.5, preferably 10.2, prior to PEG conjugation of the resulting substantially tetrameric preparation of uricase. The molecular weight of the unease in each fraction from the preparative column may be monitored by any size-dependent analytical technique, including, for example, HPLC, conventional size-exclusion chromatography, centrifugation, light scattering, capillary electrophoresis or gel electrophoresis in a non-denaturing buffer. For tetrameric unease isolated using size-exclusion chromatography, fractions containing only the 140 kDa form of the enzyme may be pooled and used for conjugation to PEG. For tetrameric uricase isolated using ion-exchange chromatography, fractions from the ion-exchange column may be analyzed with respect to size to determine which fractions contain substantial amounts of the tetrameric form without detectable aggregates. Of the uricase thus pooled, at least 90% may be in the tetrameric form; the undesirable aggregates may thus constitute as little as about 10%, 5%, 2%, or less, of the total isolated uncase.
The results presented herein indicate that, even when extensively PEGylated, forms of PBC uricase larger than the tetramer are highly immunogenic in mice (
In another embodiment of the present invention, a mutated baboon liver uricase of unexpectedly increased potency, relative to that of the unmutated enzyme, is provided. This improved primate uricase was prepared by conventional recombinant DNA techniques. It was particularly unexpected that the substitution of a single amino acid residue (histidine for tyrosine at position 97) in baboon uricase would result in a substantial increase in specific enzymatic activity. When expressed in E. coli, this mutant protein was found to have at least 60% higher specific activity than the recombinant baboon enzyme from which it was derived.
In another embodiment, the specific activity is increased and/or the solubility of the unPEGylated enzyme is improved by expressing truncated variants of porcine or porcine-baboon chimeric uricases from which at least the first six amino acids at the amino terminal and/or at least the last three amino acids at the carboxyl terminal are deleted from the expressed proteins (see
The PEG-uricase conjugates of the present invention are useful for lowering the levels of uric acid in the body fluids and tissues of mammals, preferably humans, and can thus be used for treatment of elevated uric acid levels associated with conditions including gout, tophi, renal insufficiency, organ transplantation and malignant disease. PEG-uricase conjugates may be injected into a mammal having excessive uric acid levels by any of a number of routes, including intravenous, subcutaneous, intradermal, intramuscular and intraperitoneal routes. Alternatively, they may be aerosolized and inhaled. See Patton, J S, (1996) Adv Drug Delivery Rev 19:3-36 and U.S. Pat. No. 5,458,135. The effective dose of PEG-uricase of the present invention will depend on the level of uric acid and the size of the individual. In one embodiment of this aspect of the invention, PEG-uricase is administered in a pharmaceutically acceptable excipient or diluent in an amount ranging from about 10 μg to about 1 g. In a preferred embodiment, the amount administered is between about 100 μg and 500 mg. More preferably, the conjugated uricase is administered in an amount between 1 mg and 100 mg, such as, for example, 5 mg, 20 mg or 50 mg. Masses given for dosage amounts of the embodiments refer to the amount of protein in the conjugate.
Pharmaceutical formulations containing PEG-uricase can be prepared by conventional techniques, e.g., as described in Gennaro, A R (Ed.) (1990) Remington's Pharmaceutical Sciences, 18th Edition Easton, Pa.: Mack Publishing Co. Suitable excipients for the preparation of injectable solutions include, for example, phosphate buffered saline, lactated Ringer's solution, water, polyols and glycerol. Pharmaceutical compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions, or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. These formulations may contain additional components, such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, buffers, antioxidants and diluents.
PEG-uricase may also be provided as controlled-release compositions for implantation into an individual to continually control elevated uric acid levels in body fluids. For example, polylactic acid, polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate, gellan gum, chitosan, agarose, multilamellar liposomes and many other conventional depot formulations comprise bioerodible or biodegradable materials that can be formulated with biologically active compositions. These materials, when implanted or injected, gradually break down and release the active material to the surrounding tissue. For example, one method of encapsulating PEG-uricase comprises the method disclosed in U.S. Pat. No. 5,653,974, which is hereby incorporated by reference. The use of bioerodible, biodegradable and other depot formulations is expressly contemplated in the present invention. The use of infusion pumps and matrix entrapment systems for delivery of PEG-uricase is also within the scope of the present invention. PEG-uricase may also advantageously be enclosed in micelles or liposomes. Liposome encapsulation technology is well known in the art. See, e.g., Lasic, D, et al., (Eds.) (1995) Stealth Liposomes. Boca Raton, Fla.: CRC Press.
The PEG-uricase pharmaceutical compositions of the invention will decrease the need for hemodialysis in patients at high risk of urate-induced renal failure, e.g., organ transplant recipients (see Venkataseshan, V S, et al., (1990) Nephron 56:317-321) and patients with some malignant diseases. In patients with large accumulations of crystalline urate (tophi), such pharmaceutical compositions will improve the quality of life more rapidly than currently available treatments.
The following examples, which are not to be construed as limiting the invention in any way, illustrate the various aspects disclosed above. These examples describe PEG-uricases prepared by coupling activated (i.e., electrophilic) PEG derivatives of several sizes and compositions with naturally occurring porcine, fungal or bacterial uricases, or with recombinant soybean, porcine or pig-baboon chimeric uricases. Results of activity, solubility, stability, pharmacokinetic, pharmacodynamic and immunological studies are included. The data in
The tetrameric form of uricase (molecular weight ca. 140 kDa) was purified from a solution of porcine liver uricase by preparative size-exclusion or ion-exchange chromatography, followed by analytical size-exclusion chromatography. Porcine liver uricase was obtained from Sigma-Aldrich, St. Louis, Mo., catalog No. U3250 or U3377; or Boehringer Mannheim, Indianapolis, Ind.
Preparative and analytical size—exclusion chromatography were performed at pH 10-10.5, preferably 10.2, in 10 mM sodium carbonate buffer containing 0.1 M NaCl on Superdex 200 columns that had been previously calibrated with proteins of known molecular weight. Superdex was obtained from Amersham Pharmacia, Piscataway, N.J. Any buffer may be used that is capable of maintaining the desired pH and that is compatible with the chemistry to be used for subsequent PEG coupling. Such buffers are well known in the art. The ultraviolet absorbance of the eluate from the preparative column was monitored at 280 nm, and uricase-containing portions of the eluate corresponding to the molecular weight of the desired tetrameric form, but free of higher molecular weight species, were collected for use in synthesizing substantially non-immunogenic PEG-uricase as described in Example 2. Alternatively, tetrameric forms of uricase can be isolated using other size-exclusion media such as, for example, Superose 12 (Amersham Pharmacia) or any other medium that is compatible with mildly alkaline solutions and that has an appropriate size fractionation range. Such media are readily available and are well known in the art.
Ion-exchange chromatography was performed at pH 10-10.5, preferably 10.2, on Mono Q columns (Amersham Pharmacia, Piscataway, N.J.) that had been equilibrated with 0.1 M sodium carbonate buffer. Any buffer that is compatible with the chemistry of PEG coupling and that is capable of maintaining the desired pH may be used at sufficiently low ionic strength to permit the adsorption of uricase to the column. Such buffers are well known in the art. The ultraviolet absorbance of the eluate was monitored at 280 nm during elution of the uricase from the ion-exchange resin by increasing the ionic strength of the applied buffer solution, e.g. by a linear gradient of 0 to 0.5 M NaCl in the sodium carbonate buffer. Size-exclusion HPLC was then used to identify the fractions of the eluate containing the desired tetrameric form of uricase, without detectable aggregates, for the synthesis of substantially non-immunogenic PEG-uricase. Alternatively, the tetrameric form of uricase can be isolated using other ion-exchange media, such as Q-Sepharose (Amersham Pharmacia) or any other medium that is compatible with mildly alkaline solutions. Such media are readily available and are well known in the art.
Uricase activity was assayed using a modification of standard methods. See, e.g., Fridovich (1965); Nishimura, et al., (1979). Solutions of uric acid were prepared fresh daily in 50 mM sodium borate buffer, pH 9.2, to provide final concentrations in the assay of 6-150 μM. Uricase preparations were diluted in this borate buffer containing bovine serum albumin (Sigma-Aldrich, St. Louis, Mo., catalog No. A-7030), so that the final concentration of albumin in the assay was 0.1 mg/mL. After mixing various dilutions of the enzyme with the substrate in the wells of a microtiter plate in a microplate reader, the rate of disappearance of uric acid at 25� C. was monitored at 292 nm every 4 seconds for 3 minutes. From samples in which between 10% and 40% of the substrate was consumed within 3 minutes, at least 20 data points were used to calculate the maximal rate of decrease in the absorbance per minute. One international unit (IU) of uricase activity is defined as the amount of enzyme that consumes one micromole of uric acid per minute; specific activities are expressed as IU/mg protein. Some of the data for relative uricase activities in
V 100=100�V max/(K M+100)
where KM is expressed in micromolar units.
To a solution of tetrameric uricase in 0.1 M sodium carbonate buffer, pH 10.2, 10-200 moles of an activated derivative of monomethoxyPEG, e.g., the 4-nitrophenyl carbonate (NPC-PEG), of various sizes (5 kDa to 30 kDa) were added for each mole of uricase subunit (molecular weight 35 kDa). These and other suitable activated PEGs are available from Shearwater Polymers. Instructions for coupling these PEGs to proteins are given in the catalog of Shearwater Polymers, on the Internet at www.swpolymers.com, and in J M Harris, et al., (Eds.) (1997) Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680, Washington, D.C.: American Chemical Society. The coupling reaction was allowed to proceed at 0-8� C. until the extent of PEG coupling no longer changed significantly with time. Unreacted PEG was then removed from the reaction product by chromatography and/or ultrafiltration.
The number of strands of PEG coupled per subunit of uricase was determined by an adaptation of the methods described by Kunitani, M, et al., (1991) J Chromatogr 588:125-137; Saifer, et al., (1997) and Sherman, et al., (1997). Briefly, aliquots of the PEGylation reaction mixtures or fractions from the preparative ion-exchange or size-exclusion columns were characterized by analytical size-exclusion HPLC on a TSK 5,000 PWXL column at room temperature in 10 mM sodium carbonate buffer, pH 10.2, containing 0.1 M NaCl. The HPLC column was obtained from TosoHaas, Montgomeryville, Pa. Proteins and PEGs were monitored by ultraviolet absorbance and refractive index detectors. The amount of protein in the conjugate was calculated from the ultraviolet absorbance relative to that of the appropriate unmodified uricase standard. The amount of PEG in the conjugate was then calculated from the area of the refractive index peak, corrected for the contribution of the protein to refractive index, relative to the area of the refractive index peak of the appropriate PEG standard.
Recombinant pig-baboon chimeric (PBC) uricase cDNA was subcloned into the pET3d expression vector (Novagen, Madison, Wis.) and the resultant plasmid construct was transformed into and expressed in a strain of Escherichia coli BL21(DE3)pLysS (Novagen). These procedures were carried out using methods well known in the art of molecular biology. See Erlich (1989); Sambrook, et al., (1989); Ausubel, F, et al., (Eds.), (1997) Short Protocols in Molecular Biology. New York: John Wiley & Sons.
The tetrameric form of PBC uricase was isolated and coupled to PEGs of various molecular weights as described in Examples 1 and 2. Conjugates prepared with 5 kDa, 10 kDa, 19 kDa or 30 kDa PEG contained up to 10 strands of PEG per subunit. Those prepared with PEGs of at least 10 kDa retained more than 95% of the initial specific activity of the recombinant uricase (
The following properties of a conjugate of tetrameric PBC uricase with approximately 6 strands of 10 kDa PEG per subunit are illustrated in the indicated figures: the lack of immunogenicity (
In addition to the results summarized in
Uricase from Candida utilis was obtained from either Sigma-Aldrich (St. Louis, Mo.; catalog No. U1878) or Worthington Biochemical Corporation (Freehold, N.J.; catalog No. URYW). Proceeding as described in Examples 1 and 2, the tetrameric form was isolated and PEG conjugates were synthesized with 5 kDa, 10 kDa or 30 kDa PEG (
As shown in
Uricase from Aspergillus flavus was obtained from Sanofi Winthrop (Gentilly Cedex, France). Proceeding as described in Example 2, conjugates with PEGs of various molecular weights were synthesized (
Recombinant uricase from soybean root nodule (also called nodulin 35) was prepared and purified as described by Kahn and Tipton (Kahn, K, et al., (1997) Biochemistry 36:4731-4738), and was provided by Dr. Tipton (University of Missouri, Columbia, Mo.). Proceeding as described in Examples 1 and 2, the tetrameric form was isolated and conjugates were prepared with PEGs of various molecular weights (
Uricase from Arthrobacter globiformis was obtained from Sigma-Aldrich (catalog No. U7128). See Japanese Patent 9-154581. Proceeding as described in Examples 1 and 2, the tetrameric form was isolated and conjugates with 5 kDa and 30 kDa PEG were prepared. While conjugates with an average of more than 3 strands of 5 kDa PEG per subunit retained less than 60% of the initial specific activity, conjugates with an average of approximately 2 strands of 30 kDa PEG per subunit retained at least 85% of the initial specific activity.
Recombinant porcine and PBC uricases from which the first six amino acids at the amino terminal are deleted are expressed in and purified from E. coli by standard techniques, as described in Example 3. Proceeding as described in Examples 1 and 2, PEG conjugates of the amino-truncated uricases are synthesized to produce substantially non-immunogenic conjugates that retain at least 75% of the initial specific activity.
Recombinant porcine and PBC uricases from which the last three amino acids at the carboxyl terminal are deleted are expressed in and purified from E. coli by standard techniques, as described in Example 3. This carboxyl-terminal deletion may enhance the solubility of the unmodified enzymes, since it removes the peroxisomal-targeting signal. See Miura, et al., (1994). Proceeding as described in Examples 1 and 2, PEG conjugates of the carboxyl-truncated uricases are synthesized to produce substantially non-immunogenic conjugates that retain at least 75% of the initial specific activity. The sequence of recombinant PBC uricase truncated by six residues at the amino terminal and by three residues at the carboxyl terminal (PBC-NT-CT) is shown in
Recombinant porcine uricases are prepared as described in Example 3, in which the potential number of sites of PEG attachment is increased by replacing one or more arginine residues with lysine. See Hershfield, M S, et al., (1991) Proc Natl Acad Sci USA 88:7185-7189. The amino acid sequence of one example of such a mutant (PKS uricase), in which the arginine at residue 291 is replaced by lysine and the threonine at residue 301 is replaced by serine, is shown in
Using standard methods of molecular biology, as in Example 3, recombinant baboon uricase is constructed having an amino acid substitution (histidine for tyrosine) at position 97 (see baboon sequence in
Uricase from Candida utilis, Aspergillus flavus, and Arthrobacter globiformis are obtained as described in Examples 4, 5, and 7, respectively. Proceeding as described in Examples 1 and 2, PEG conjugates are synthesized with 5 kDa, 10 kDa, 20 kDa or 30 kDa PEG. The immunogenicity of these conjugates is substantially reduced or eliminated.
Cited Patent | Filing date | Publication date | Applicant | Title |
US3616231 | Nov 14, 1968 | Oct 26, 1971 | Boehringer Mannheim Gmbh | Process for the production of uricase |
US4179337 | Jul 28, 1977 | Dec 18, 1979 | Davis Frank F | Non-immunogenic polypeptides |
US4460683 | Jul 2, 1982 | Jul 17, 1984 | Boehringer Mannheim Gmbh | Soluble liver uricase with a process for the preparation thereof and with the use thereof |
US4766106 | Jan 25, 1988 | Aug 23, 1988 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
US4847325 | Jan 20, 1988 | Jul 11, 1989 | Cetus Corporation | Conjugation of polymer to colony stimulating factor-1 |
US4917888 | Dec 11, 1987 | Apr 17, 1990 | Cetus Corporation | Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation |
US5286637 | Jul 26, 1990 | Feb 15, 1994 | Debiopharm, S.A. | Biologically active drug polymer derivatives and method for preparing same |
US5382518 * | Jul 13, 1990 | Jan 17, 1995 | Sanofi | Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells |
US5428128 | Sep 27, 1994 | Jun 27, 1995 | Mensi-Fattohi; Nahla | Site specific synthesis of conjugated peptides |
US5541098 | Sep 28, 1994 | Jul 30, 1996 | Sanofi | Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells |
US5612460 | Feb 17, 1994 | Mar 18, 1997 | Enzon, Inc. | Active carbonates of polyalkylene oxides for modification of polypeptides |
US5643575 | Oct 27, 1993 | Jul 1, 1997 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
US5653974 | Nov 30, 1994 | Aug 5, 1997 | Board Of Regents,The University Of Texas System | Preparation and characterization of liposomal formulations of tumor necrosis factor |
US5880255 | Apr 14, 1995 | Mar 9, 1999 | Polymasc Pharmaceuticals Plc | Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct of PEG and granulocyte-macrophage colony stimulating factor |
US5919455 | Mar 20, 1997 | Jul 6, 1999 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
US5932462 | May 17, 1995 | Aug 3, 1999 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US5955336 | Jun 26, 1992 | Sep 21, 1999 | Toyo Boseki Kabushiki Kaisha | DNA sequence for uricase and manufacturing process of uricase |
US6201110 | Jul 6, 2000 | Mar 13, 2001 | Novo Nordisk A/S | Polypeptide with reduced respiratory allergenicity |
DD279486A1 | Title not available | |||
JPH09154581A | Title not available | |||
JPS6255079A | Title not available | |||
JPS55135590A | Title not available | |||
WO1994019007A1 | Feb 14, 1994 | Sep 1, 1994 | Enzon, Inc. | Ribosome inactivating protein compositions having reduced antigenicity |
WO2000007629A2 | Aug 2, 1999 | Feb 17, 2000 | Mountain View Pharmaceuticals, Inc. | Peg-urate oxidase conjugates and use thereof |
WO2000008196A2 | Aug 5, 1999 | Feb 17, 2000 | Duke University | Urate oxidase |
WO2001059078A2 | Feb 7, 2001 | Aug 16, 2001 | Mountain View Pharmaceuticals, Inc. | Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates |
Reference | ||
1 | Abuchowski et al., (1976), Effect of Covalent Attachment of Polyethylene Glycol on Immunogenicity and Circulating Life of Bovine Liver Catalase, The Journal of Biochemical Chemistry 252:3582-3586. | |
2 | Alvares, K., et al., "Rat urate oxidase produced by recombinant baculovirus expression: Formation of peroxisome crystalloid core-like structures," Proc. Natl. Acad. Sci. USA 89:4908-4912, National Academy of Sciences (1992). | |
3 | Alvares, K., et al., "The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase," Biochem. Biophys. Res. Commun. 158:991-995 (1989) (abstract only). | |
4 | Alvares, K., et al., "The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase," Biochem. Biophys. Res. Commun. 158:991-995 (1989). | |
5 | Braun, A., and Alsenz, J., "Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-alpha) Formulations," Pharm. Res. 14:1394-1400, Plenum Press (Oct. 1997). | |
6 | Braun, A., and Alsenz, J., "Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations," Pharm. Res. 14:1394-1400, Plenum Press (Oct. 1997). | |
7 | Braun, A., et al., "Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-alpha) in Normal and Transgenic Mice," Pharm. Res. 14:1472-1478, Plenum Press (Oct. 1997). | |
8 | Braun, A., et al., "Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice," Pharm. Res. 14:1472-1478, Plenum Press (Oct. 1997). | |
9 | Burnham, Nora, (1994), Polymers for Delivering Peptides and Proteins, Am. J Hosp Pharm. 51:210-218. | |
10 | Caliceti, P., et al., "Biopharmaceutical Properties of Uricase Conjugated to Neutral and Amphiphilic Polymers," Bioconj. Chem. 10:638-646, American Chemical Society (1999). | |
11 | Chen, R.H.-L., et al., "Properties of Two Urate Oxidases Modified by the Covalent Attachment of Poly(Ethylene Glycol)," Biochim. Biophys. Acta 660:293-298, Elsevier/North-Holland Biomedical Press (1981). | |
12 | Chinese Second Office Action for Chinese Application No. 01807750.1, issued Mar. 21, 2008, Chinese Patent Office, Beijing, China. | |
13 | Chua et al., (1988), Use of Polyethylene Glycol-Modified Uricase (PEG-Uricase) to Treat Hyperuricemia in a Patient with Non-Hodgkin Lymphoma, Annals of Internal Medicine 109:114-117. | |
14 | Colloc'h, N., et al., "Crystal Structure of the protein drug urate oxidase-inhibitor complex at 2.05 � resolution," Nature Struct. Biol. 4:947-952, Nature America, Inc. (Nov. 1997). | |
15 | Colloc'h, N., et al., "Crystal Structure of the protein drug urate oxidase-inhibitor complex at 2.05 Å resolution," Nature Struct. Biol. 4:947-952, Nature America, Inc. (Nov. 1997). | |
16 | Conley, T.G., and Priest, D.G., "Thermodynamics and Stoicheiometry of the Binding of Substrate Analogues to Uricase," Biochem. J. 187:727-732, The Biochemical Society (1980). | |
17 | Davis et al., (1978), Enzyme-Polyethylene Glycol Adducts: Modified Enzymes with Unique Properties, Enzyme Engineering 4:169-173. | |
18 | Davis et al., (1981), Hypouricaemic Effect of Polyethyleneglycol Modified Urate Oxide, The Lancet pp. 281-283. | |
19 | Donadio et al., (1981), Manifestation De Type Anaphylactique Apres Injection Intra-Veineuse D'urate-Oxydase Chez Un Enfant Asthmatique Atteint De Leucemie Aigue, La Nouvelle Presse Medicale, 28:711-712. | |
20 | European Examination Report for related European Application No. 01 923 265.1, mailed Dec. 13, 2007, European Patent Office, Munich, DE. | |
21 | Fam (1990), Strategies and Controversies in the Treatment of Gout and Hyperuricaemia. Clinical Rheumatology International Practice and Research 4:177-192. | |
22 | Francis, G.E., et al., "PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques," Int. J. Hematology 68:1-18, Elsevier Science Ireland Ltd. (1998). | |
23 | Fuertges, F., and Abuchowski, A., "The Clinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins," J. Contr. Rel. 11:139-148, Elsevier Science Publishers (1990). | |
24 | Hande et al., (1984), Severe Allopurinol Toxicity, The American Journal of Medicine 76:47-58. | |
25 | Hedlund et al., (1991), Magnetic Resonance Microscopy of Toxic Renal Injury Induced by Bromoethylamine in Rats, Fundamental and Applied Toxicology 16:787-797. | |
26 | Henney, C.S., and Ellis, E.F., "Antibody Production to Aggregated Human UpsilonG-Globulin in Acquired Hypogammaglobulinemia," N. Engl. J. Med. 278:1144-1146, Massachusetts Medical Society (1968). | |
27 | Henney, C.S., and Ellis, E.F., "Antibody Production to Aggregated Human ΥG-Globulin in Acquired Hypogammaglobulinemia," N. Engl. J. Med. 278:1144-1146, Massachusetts Medical Society (1968). | |
28 | * | Herbst et al. Biochemistry, (May 5, 1998) 37 (18) 6586-97. |
29 | Hershfield, M.S., et al., "Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol," Proc. Natl. Acad. Sci. USA 88:7185-7189, National Academy of Sciences (1991). | |
30 | * | Ishino et al. Agricultural and Biological Chemistry (1980), 44(6), 1259-66. |
31 | Ito, M., et al., "Identification of an Amino Acid Residue Involved in the Substrate-Binding Site of Rat Liver Uricase by Site-Directed Mutagenesis," Biochem. Biophys. Res. Commun. 187:101-107, Academic Press, Inc. (1992). | |
32 | Kahn, et al., (1997), Kinetic Mechanism and Cofactor Content of Soybean Root Nodule Urate Oxidase, American Chemical Society 36:4731-4738. | |
33 | Kelly, S.J., et al., "Diabetes Insipidus in Uricase-Deficient Mice: A Model for Evaluating Therapy with Poly(Ethylene Glycol)-Modified Uricase," J. Am. Soc. Nephrol. 12:1001-1009, Lippincott Williams & Wilkins (2001). | |
34 | Kito, M., et al., "A Simple and Efficient Method for Preparation of Monomethoxypolyethylene Glycol Activated with p-Nitrophenylchloroformate and Its Application to Modification of L-Asparaginase," J. Clin. Biochem. Nutr. 21:101-111, Institute of Applied Biochemistry (1996). | |
35 | Kral, L.G., et al., "Cloning a cDNA for Drosophila melanogaster urate oxidase," Gene 45:131-137, Elsevier Science Publishers B.V. (1986). | |
36 | Kunitani et al., (1991), On-Line Characterization of Polyethylene Glycol-Modified Proteins, Journal of Chromatography 588:125-137. | |
37 | Kunitani, M., et al., "Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection," J. Pharm. Biomed. Anal. 16:573-586, Elsevier Science (Dec. 1997). | |
38 | Leach et al., (1998), Efficacy of Urate Oxidase (Uricozyme) in Tumor Lysis Induced Urate Nephropathy, Blackwell Science Limited 20:169-172. | |
39 | Lee, C.C., et al., "Generation of cDNA Probes Directed by Amino Acid Sequence: Cloning of Urate Oxidase," Science 239:1288-1291, American Association for the Advancement of Science (1988). | |
40 | Legoux et al., (1991), Cloning and Expression in Escherichia coli of the Gene Encoding Aspergillus flavus Urate Oxidase The Journal of Biological Chemistry 267:8565-8570. | |
41 | Mahler, H.R., et al., "Studies on Uricase: I. Preparation, Purification, and Properties of a Cuproprotein," J. Biol. Chem. 216:625-641, American Society of Biological Chemists, Inc. (1955). | |
42 | Mahmound of al., (1998). Advances in the Management of Malignancy-Associated Hyperuricaemia, British Journal of Cancer 77:18-20. | |
43 | * | Malakhova et al. (1991), Biologicheskie Membrany 8(5), 453-9 (abstract only). |
44 | Miura et al., (1994), Urate Oxidase is Imported into Peroxisomes Recognizing the C-terminal SKL Motif of Proteins, Eur. J. Biochem 223:141-146. | |
45 | Montalbini, P., et al., "Isolation and characterization of uricase from bean leaves and its comparison with uredospore enzymes," Plant Sci. 147:139-147, Elsevier Science Ireland Ltd. (Sep. 1999). | |
46 | Montalbini, P., et al., "Uricase from leaves: its purification and characterization from three different higher plants," Planta 202:277-283, Springer-Verlag (Jul. 1997). | |
47 | Moore, W.V., and Leppert, P., "Role of Aggregated Human Growth Hormone (hGH) in Development of Antibodies to hGH," J. Clin. Endocrinol. Metab. 51:691-697, The Endocrine Society (1980). | |
48 | * | Motojima et al. JBC 283 (32) : 16677-16681 (1988). |
49 | Mountain View Pharmaceuticals, Inc., "PURICASE�," U.S. Trademark Registration No. 2,246,623 (report obtained from U.S. Trademark Electronic Search System (TESS), Dec. 5, 2001). | |
50 | NCBI Entrez Protein (PRF) Database, deposited sequence for rat urate oxidase (NP 446220), National Library of Medicine, National Institutes of Health, Accession No. 20127395, available online at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Search&DB=protein (accessed Dec. 10, 2003). | |
51 | Nishimura et al., (1981), Improved Modification of Yeast Uricase with Polyethylene Glycol, Accompanied with Nonimmunoreactivity towards Anti-Uricase Serum and High Enzymic Activity, Enzyme 26:49-53. | |
52 | Nucci et al., (1991), The Therapeutic Value of Poly(Ethylene Glycol)-Modified Proteins, Advanced Drug Delivery Reviews 6:133-151. | |
53 | Osman, A.M., et al., "Liver Uricase in Camelus dromedarius: Purification and Properties," Comp. Biochem. Physiol. 94B:469-474, Pergamon Press (1989). | |
54 | Palleroni, A.V., et al., "Interferon Immunogenicity: Preclinical Evaluation of Interferon-alpha2a," J. Interferon Cytokine Res. 17 (Suppl 1):S23-S27, Mary Ann Liebert, Inc. (Jul. 1997). | |
55 | Palleroni, A.V., et al., "Interferon Immunogenicity: Preclinical Evaluation of Interferon-α2a," J. Interferon Cytokine Res. 17 (Suppl 1):S23-S27, Mary Ann Liebert, Inc. (Jul. 1997). | |
56 | Patent Abstract of Japan, English language abstract of JP 55-135590 A (Document FP1), (1980). | |
57 | Patent Abstract of Japan, English language abstract of JP 62-55079 A (Document FP2), (1987). | |
58 | Porstmann, B., et al., "Comparison of Chromogens for the Determination of Horseradish Peroxidase as a Marker in Enzyme Immunoassay," J. Clin. Chem. Clin. Biochem. 19:435-439, Walter De Gruyter & Co. (1981). | |
59 | Pui et al., (1997), Urate Oxidase in Prevention and Treatment of Hyperuricemia Associated with Lymphoid Malignancies, Leukemia 11:1813-1816. | |
60 | R&D Focus Drug News (DataStar File IPNR/IPNA), Accession No. 1998:2984 DRUGNL, "PEG-uricase BioTechnology General, Duke University, Mountain View licensing agreement," Aug. 24, 1998. | |
61 | Saifer, et al., (1994), Plasma Clearance and Immunologic Properties of Long-Acting Superoxide Dismutase Prepared Using 35,000 to 120,000 Dalton Poly-Ethylene Glycol, Advances in Experimental Medicine and Biology 366:377-387. | |
62 | Sartore et al., (1991), Enzyme Modification by MPEG with an Amino Acid or Peptide as Spacer Arms, Applied Biochemistry and Biotechnology 27: 45-54. | |
63 | Savoca, K.V., et al., "Induction of Tolerance in Mice by Uricase and Monomethoxypolyethylene Glycol-Modified Uricase," Int. Archs. Allergy Appl. Immunol. 75:58-67, S. Karger Medical and Scientific Publishers (1984). | |
64 | Shearwater Polymers, Inc. (1997-1998), Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Shearwater Polymers, Inc. Catalog. 27, 47, 48. | |
65 | * | Sigma Catalog (1993), page No. 1002, Product Nos. U 3250, 292-8, U3500, U 9375 or U 3377. |
66 | Suzuki, H., and Verma, D.P.S., "Soybean Nodule-Specific Uricase (Nodulin-35) Is Expressed and Assembled into a Functional Tetrameric Holoenzyme in Escherichia coli," Plant Physiol. 95:384-389, American Society of Plant Physiologists (1991). | |
67 | * | Treuheit et al. Pharmaceutical research, (Apr. 2002) 19 (4) 511-6. |
68 | Tsuji, J.-I., et al., "Studies on Antigenicity of the Polyethylene Glycol (PEG)-Modified Uricase," Int. J. Immunopharmac. 7:725-730, Elsevier Science (1985). | |
69 | Venkataseshan et al., (1990), Acute Hyperuricemic Nephropathy and Renal Failure after Transplantation, Nephron 56:317-321. | |
70 | Veronese at al., (1985), Surface Modification of Proteins, Applied Biochemistry and Biotechnology 11:141-152. | |
71 | Veronese et al., (1997), New Synthetic Polymers for Enzyme and Liposome Modification in Poly(ethylene Glycol) Chemistry and Bioloaical Applications, Chapter 13:182-192. | |
72 | Wang, L.-W.C., and Marzluf, G.A., "Purification and Characterization of Uricase, a Nitrogen-Regulated Enzyme, from Neurospora crassa," Archs. Biochem. Biophys. 201:185-193, Academic Press, Inc. (1980). | |
73 | Wang, X., et al., "Rat urate oxidase: cloning and structural analysis of the gene and 5'-flanking region," Gene 97:223-229 (1991). | |
74 | Wang, X., et al., "Rat urate oxidase: cloning and structural analysis of the gene and 5′-flanking region," Gene 97:223-229 (1991). | |
75 | Wu et al, (1984), Hyperuricemia and Urate Nephropathy in Urate Oxidase-Deficient Mice Proc. Natl. Acad. Sci. USA 91:742-746. | |
76 | * | Wu et al. [PNAS, USA, vol. 86, pp. 9412-9416, Dec. 1989]. |
77 | Wu et al., (1989), Urate Oxidase: Primary Structure and Evolutionary Implications Proc. Natl. Acac. Sci. USA 86:9412-9416. | |
78 | Wu, X., et al., "Two Independent Mutational Events in the Loss of Urate Oxidase During Hominoid Evolution," J. Mol. Evol. 34:78-84, Springer-Verlag (1992). | |
79 | Yasuda et al., (1990), Biochemical and Biopharmaceutical Properties of Macromolecualr Conjugates of Uricase with Dextran and Polyethylene Glycol. Chem. Pharm. Bull. 38:2053-2056. | |
80 | Yeldandi, A.V., et al., "Human Urate Oxidase Gene: Cloning and Partial Sequence Analysis Reveal a Stop Codon within the Fifth Exon," Biochem. Biophys. Res. Commun. 171:641-646, Academic Press, Inc. (1990). |
Citing Patent | Filing date | Publication date | Applicant | Title |
US7927852 | Aug 3, 2007 | Apr 19, 2011 | Mountain View Pharmaceuticals, Inc. | Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates |
US8067553 | Apr 28, 2010 | Nov 29, 2011 | Mountain View Pharmaceuticals, Inc. | PEG-urate oxidase conjugates and use thereof |
US8618267 | Apr 8, 2011 | Dec 31, 2013 | Mountain View Pharmaceuticals, Inc. | PEG-urate oxidase conjugates and use thereof |
US8921064 | Apr 13, 2011 | Dec 30, 2014 | Mountain View Pharmaceuticals, Inc. | Method for purifying urate oxidase tetramers and octamers |
US8940861 | Apr 8, 2011 | Jan 27, 2015 | Georgia Tech Research Corporation | Variants of ancestral uricases and uses thereof |
US9017980 | Aug 21, 2013 | Apr 28, 2015 | Crealta Pharmaceuticals Llc | Variant forms of urate oxidase and use thereof |
US9377454 | Jun 25, 2010 | Jun 28, 2016 | Crealta Pharmaceuticals Llc | Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy |
US20050014240 * | Aug 30, 2004 | Jan 20, 2005 | Mountain View Pharmaceuticals, Inc. | Aggregate-free protein compositions and methods of preparing same |
US20060188971 * | Feb 21, 2006 | Aug 24, 2006 | Duke University | Urate oxidase |
US20080057048 * | Aug 3, 2007 | Mar 6, 2008 | Mountain View Pharmaceuticals, Inc. | Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates |
US20100323422 * | Apr 28, 2010 | Dec 23, 2010 | Williams L David | PEG-Urate Oxidase Conjugates and Use Thereof |
US20100323423 * | Apr 28, 2010 | Dec 23, 2010 | Williams L David | PEG-Urate Oxidase Conjugates and Use Thereof |
US20120149083 * | Nov 29, 2011 | Jun 14, 2012 | Duke University | PEG-Urate Oxidase Conjugates and Use Thereof |
U.S. Classification | 435/190, 435/191, 435/440, 536/23.2 |
International Classification | A61K47/48, C12N9/02, A61K38/44, C12N9/96, C12N9/06, A61K38/00, C12N9/04, C12N1/21 |
Cooperative Classification | C12Y107/03003, C12N9/96, A61K38/00, A61K47/48215, C12N9/0048, C12N9/0093, C12N9/0046 |
European Classification | A61K47/48K6P, C12Y107/03003, C12N9/00P36, C12N9/00P8H3, A61K38/44, C12N9/96 |
Date | Code | Event | Description |
Nov 28, 2001 | AS | Assignment | Owner name: DUKE UNIVERSITY, NORTH CAROLINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HERSHFIELD, MICHAEL S.;KELLY, SUSAN J.;REEL/FRAME:012320/0572 Effective date: 19991006 Owner name: DUKE UNIVERSITY,NORTH CAROLINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HERSHFIELD, MICHAEL S.;KELLY, SUSAN J.;REEL/FRAME:012320/0572 Effective date: 19991006 |
Nov 30, 2001 | AS | Assignment | Owner name: MOUNTAIN VIEW PHARMACEUTICALS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WILLIAMS, L. DAVID;SAIFER, MARK G. P.;SHERMAN, MERRY R.;REEL/FRAME:012320/0564 Effective date: 19990929 Owner name: MOUNTAIN VIEW PHARMACEUTICALS, INC.,CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WILLIAMS, L. DAVID;SAIFER, MARK G. P.;SHERMAN, MERRY R.;REEL/FRAME:012320/0564 Effective date: 19990929 |
Nov 22, 2013 | FPAY | Fee payment | Year of fee payment: 4 |
Jul 21, 2015 | AS | Assignment | Owner name: JEFFERIES FINANCE LLC, AS COLLATERAL AGENT, NEW YO Free format text: SECURITY INTEREST;ASSIGNOR:CREALTA PHARMACEUTICALS LLC;REEL/FRAME:036142/0931 Effective date: 20150720 |
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Nov 18, 2016 | AS | Assignment | Owner name: HORIZON PHARMA USA RHEUMATOLOGY LLC, ILLINOIS Free format text: CHANGE OF NAME;ASSIGNOR:CREALTA PHARMACEUTICALS LLC;REEL/FRAME:040648/0361 Effective date: 20160113 Owner name: HORIZON PHARMA RHEUMATOLOGY LLC, ILLINOIS Free format text: CHANGE OF NAME;ASSIGNOR:HORIZON PHARMA USA RHEUMATOLOGY LLC;REEL/FRAME:040648/0367 Effective date: 20160115 |


text:
Cedar Master Sock
Cedar Architecture: Cuff down.
Arch expansion is around ankle.
Any yarn, any gauge, plug-in your measurements.
New Pathways for Sock Knitters, which I published in 2007, is by far my most ambitious and comprehensive sock book. It contains 8 unique architectures, each one introduced with a quick little baby or toddler sock, followed by a collection of adult designs, and a Master pattern so that you can literally knit an infinite number of variations on each architecture.
Hundreds of designers have used these architectures as a leaping-off point for their own innovations, with the Riverbed, Upstream, and Cedar architectures appearing most often. This was my hope—that the new fields I plowed would become gardens in the minds and hands of others, including you!
This digital version of the book insures that it can remain a resource for knitters and designers forever.
Cedar architecture is a top-down method with increases distributed around the foot between the ankle and the base of the foot, like a skirt. This Master Pattern lets you design your own, using any yarn for any foot in the universe!
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- Last updated: September 29, 2015 …
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subset: commoncrawl


text:
I don't want to buy them all to find out which is most suited to the majority of my fishing, so maybe you can help me decide on the one best suited overall line for both rods. I have a Z-Axis 590-4 and Winston BIIX 690-4. Used 99% nymphing and 99% river fishing for large trout, but the dries come out when the noses do. Pretty set on RIO (have had good luck, like dealing with them when I have issues). Here's the question: Have the 5wt Grand, like it, but have nothing to compare it to. Never owned a Gold, told I should be using it. Rio Grand or Rio Gold? (don't like the nymph line) 5wt or 6wt?
I am a big fan of the Rio Gold lines. Great lines that cast nicely. I don't own many new faster graphite, but I'd imagine they'd be good on those rods. I'm not a big fan of overlining so I'd probably go with a 5wt Gold and fish it on both. That's the route I'd go.
I have the 590 Z. I tried the Rio gold and grand, a SA line, and the 444. Liked the gold the best. For the past couple of years It seems whenever I try several lines on a rod the gold comes out as my favorite.
A used Gold came with a reel I bought last year. It was a 5wt and was perfect for my med action Sage Vantage 5wt. I thought it was great for both dries and nymphing. I have trouble keeping a lot of line in the air, and it helped. A 6wt should mate well with the z-axis.
I own both rods. I prefer a 5 wt rio gold on the Z and 6 wt rio gold on the biix. I have tried multiple lines on each and prefer the gold lines on each of these rods.
My favorite line for a Winston BIIX 490-4 is an airflow ridge double taper; My favorite line for a Winston BIIX 710-4 is a RIO Steelhead and atlantic Salmon for all around fishing; with an airflow 40 plus extreme distance a close second. These are lighter and heavier rods than what you are asking about but the line at the appropriate weight should be very similar on the same brand of rods---by far the favorite combo is the airflow ridge DT on the four wt. This combo will shoot a lot of line, and handle a large variety of terminal gear. I would certainly try to find an ariflow ridge to try. The only Rio Gold that I have is for a 2 wt. and I haven't used it on a fish yet, (waiting for June unless I can find a bluegill pond close by before then).
I doubt a Gold 5wt will load the 6wt B2X enough. You probably already have the right 5.5 weight line to kinda-sorta make both rods work. If that line doesn't work, you need two different lines, and as suggested above, Gold 5wt and 6wt are good places to start. Honestly, those two rods are already very similar, so why not sell one? The Z-Axis is a tippy 5.5wt, while the B2X is a tippy true 6wt. Sell one, then get two spare spools for the reel and add a sink tip and intermediate line for the remaining rod.
I don't understand this statement..... That goes against a true gear whore's mantra. While your recommendation makes perfect sense it is hard to justify that rational to a giddy gear whore.
6wt Gold it is. Most of my fishing in close in short line nymphing, so the heavier line really should be the better fit floppin' out the 'cator. Thanks for the help.


text:
Amazon Cloud Player
Amazon's impressed us with the strength of its MP3 catalog, and the frequent deals it offers on albums. A lot of times, we find ourselves going there for music instead of iTunes, especially because the whole process has been made so simple. Now it's taking a crack at a music playback app for OS X, essentially replicating the "tunes" part of iTunes. Amazon Cloud Player (Free) was previously accessible over the web, but now it can be downloaded directly to your Mac.
Cloud Player hooks up to your Amazon account, and automatically identifies any music you've downloaded through its digital storefront. As a bonus, this includes the song files of many physical CDs bought through the company, meaning you don't have to worry about ripping past purchases. From there, you can play music, make playlists, or do anything else you'd expect from a music app. Additionally, you can upload your own music, including songs purchased from iTunes, to the cloud. The first 250 songs are free, while you can do up to 250,000 for $25 a year.
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About us
Statistics Norway is the national statistical institute of Norway and and the main producer of official statistics. We are responsible for collecting, producing and communicating statistics related to the economy, population and society at national, regional and local levels.
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In addition to having primary responsibility for meeting the need for statistics on Norwegian society, Statistics Norway is also a driving force in the international statistical cooperation.
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Statistics Norway (The Central Bureau of Statistics) was formally established as an independent entity in 1876.


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Shawn Parr: How To Use The F Word In Business To Deliver Better Results
Focus is a key word to keep in mind when determining the purpose of a new initiative, why it matters, and how it connects to the mission of a business.
I've seen the curse of the "whack-a-mole" raise its ugly head more frequently than normal in recent months. While it's the perfect game for a little stress relief, it's a distracting behavior that senior executives often adopt without realizing it—and more importantly—understanding the disruptive spin cycle it can create company-wide. The curse of one-off tactical initiatives and the search for the new sliver bullet can put an unnecessary drain on the performance of an entire organization. The challenge we often see is that there's little thought put into how initiatives strategically ladder to the purpose of the business, how they build the brand over time or how they impact the operational teams and their ability to execute their current imperatives.
One of the biggest challenges we see working closely with senior teams on brand turnarounds, product innovation and the mobilization of people is how easily senior teams can be distracted by a competitor's move, the latest business book espousing a newfound philosophy or a newly surfaced trend. While it's critical to understand and respect the competition and to be agile to relevant emerging trends, it's easy to overlook how these must-do-now initiatives disrupt the flow of strategic business.
That's where the F word comes in. Focus for goodness sakes. The simple act of asking, "What is the focus of this new initiative? Why does it matter? And how does it connect to the mission of the business?" helps to eradicate the whack-a-mole syndrome and validate the time and resources invested in it. How does the initiative tie into the brand's purpose and business strategy? Every executive and leader in the company should understand the focus and purpose of the initiatives they're leading.
Sony is a beautiful brand with a history of being a category-creating innovator—The Walkman, the Trinitron and the PlayStation, to name a few. Their founders are famous for saying; "You never ask the consumer what they want because they don't know until you show it to them." Steve Jobs admired Sony greatly for their design and disruptive thinking–so much so that it was rumored he wanted to buy Sony at one point. Fast forward to today… Sony's electronics business has been in steady decline over the past 10 years because leadership has had their focus on the wrong things. They've spent too much time worrying about the competition instead of focusing on what made them Sony. Bold innovation, beauty, function and form were all part of their magical formula, yet their culture got in their way and allowed upstarts like GoPro, Beats and Vizio and majors like Samsung and Apple to give them a painful run for their money. Not using the F word got Sony into trouble.
While I realize this is an oversimplification, the Sony culture has put their admired brand very close to the edge, and their leadership's inability to focus on the right things could fuel the brand's demise in the electronics business. If they are going to make a comeback, it's time for them to focus on being Sony.
Here are a few Uncommon Sense suggestions for putting the F word to work in your everyday operations:
How much time is wasted before the problem you're trying to solve is clearly and succinctly stated? Ask yourself and your team to put this question front and center: What's the problem we're trying to solve or what's the opportunity we're trying to discover?
This may seem like an elementary question, but in the context of the problem or opportunity identified, it's imperative to revisit and restate what business you're in. It puts clarity and context around the initiative. Ask: What businesses are we in, and are we clear how this initiative supports our mission?
Defining the potential impact to the business at the start of the initiative is an important step that's often overlooked. And, while it's not always easy to quantify financial impact, understanding what success looks like and designing the path to deliver on that is a grounding step. Ask: What does success look like? What does the outcome look like?
People solve problems, and the right people find opportunity in any situation. Carefully selecting who's on your project team and knowing why you have selected them is a key step. Ask: Have I picked the best team for the job? What's the role of each person on the team? Does each person understand how this initiative ties to our business purpose?
Spontaneity is the spice of life; however, mapping a deliberate project path is a basic step that must not be overlooked with must-do-now initiatives. Ask: What's the project path the team is going to follow to the finish line? Does each person understand the path we're going to follow?
Shawn Parr is the Guvner & CEO of Bulldog Drummond, an innovation and design consultancy headquartered in San Diego whose clients and partners have included Starbucks, Diageo, Jack in the Box, Taco Bell, Adidas, MTV, Nestle, Pinkberry, American Eagle Outfitters, Ideo, Sony, Virgin, Disney, Nike, Mattel, Heineken, Annie's Homegrown, Kashi, The Michael J. Fox Foundation for Parkinson's Research, CleanWell, The Honest Kitchen, and World Vision. Follow the conversation at @BULLDOGDRUMMOND.
[Image: Flickr user Mark Krajnak]


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This Gif entitled Water Bottle was created from the youtube video 3 years ago by MrSync. It uses 12 frames per second and is displayed at 360px resolution. This animated image plays for 4 seconds, was built with normal quality and an estimated weight of 1.17 MB for download. This animation was originally created from the video from 02:08 to 02:12.
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text: There are some immediate benefits for contractors in the United States who begin right now to use the metric system for measurements of length without troubling to adopt other metric measurements such as weights, strengths, and temperatures. Indeed the other units would probably be bothersome at the present time. The practical consequences of using the metre now are the main subject of this article. The United States is almost alone in the way it measures and controls its production and construction. Virtually all the rest of the world has converted its weights and measures to the metric system. The United States faces enormous disadvantages in international commerce, business, and finance because of its inefficient system. Our system imposes disadvantages in domestic construction as well. Eventually we must convert. The longer we wait the more problems there will be and the greater will be the cost. The contractor would perhaps continue to use English units in making the estimates. For convenience in making job measurements, however, all dimensions shown on the drawings would be converted to metric in the contractor's office, entered in color next to their English equivalents and further distinguished by encircling the numbers. Conversion to a partial system is not particularly difficult. As a matter of fact, if a law were passed tomorrow calling for complete conversion to the metric system, the problems would be much less severe for the construction industry than for others. Complete conversion will be a benefit to all of us, once these conversion problems have been solved and we have begun to think and calculate in metric.


text:
Er zijn geen Nederlandse reviews over Hotel Bellevue Palace Bekijk reviews in alle andere talen. Of schrijf zelf een review Voeg een review toe
vertalen Location, service, quality!!! 10 Richard, 25 juni 2009 Ligging - Service - Prijs / kwaliteit - Eten - Kamers - Kindvriendelijk - Zwembad - I'm in the hotel and conventions business and my expectations were high. I was not disappointed. Bellevue Palace has that old world elegance and feel, but has all the up to date amenities and connectivity I need as a business and leisure traveler. Service levels are those you would find in only the finest hotels in the world. The location is just steps from the main clock tower which is just about dead center of the "walking" district of arcaded shops and restaurants. It could not be a more convenient base with views over the River Aare. Direktor (GM) Urs Buhrer was the connsumate hotelier in greeting guests and making the rounds at dinner in La Terrasse making sure his guests were enjoying their stay. Switzerland is definietly not cheap, but Bellevue Palace is worth the price.


text: Big thanks go out to everyone who has donated in response to our funding appeal this year! We've raised around 15 million THB ($ US 500,000) since June, which has enabled us to pay for essential medicines, supplies, food and stipends for our health, education and child protection programmes. We're still 3 million THB (100,000 USD) short of our appeal total, so if you are looking to make a gift before the end of the year, please visit our donate page! Thanks!


text:
Popham Beach Artists' Cottage, Great View, Private Beach Access,
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- Minimum stay:7 nights
- Sleeps:4
- Bedrooms:2
- Bathrooms:1
- Property type:Cottage
- Internet:Yes
- Pets considered:Yes
- Wheel chair accessible:No
About the Property
Ocean-view, Beach-access Cottage, 2 Bedroom,1 Bath (Sleeps 3-4)
This small white cape at Popham Beach, in Phippsburg, ME, was built about 60 years ago by Robert D. Stevens. It has fantastic ocean views and easy beach access. Seguin Island and light house, Fox Island, and Wood Island can all be seen from the windows and from the large deck. Dine inside, or outside: in the sun or under the oak tree that the large deck was built around. Listen to the surf from your bed as you drift to sleep.... wake up and see the ocean from your bed... (or you might hear the fog horns!) You can walk to Fox Island at low tide! (and some summers, walk to Wood Island as well.) We have beach chairs, and beach toys. Our planters will with any luck be over-flowing with petunias, and usually herbs too. A private boardwalk accesses the beach at Popham Beach State Park. The walk might take less than a minute even if you're carrying a picnic basket and beach umbrella. The cottage has two small bedrooms with hand painted beds: both with full mattresses. The mural-painted bathroom has a new mosaic floor, a shower, and there's also an outdoor shower. The kitchen is small and has a stove, refrigerator, microwave oven, toaster, coffee and cappuccino makers, and a good variety of pots, pans, and colorful vintage dishes for cooking and dining. It's usually equipped with a few condiments, teas, sugar, and spices. The living room has a dining table, several chairs, and a sofa. We have WIFI, and a phone for unlimited US calls, a DVD player, a few DVDs, lots of books and games, some art materials for you to use, (and art work by family members.) We have pillows and plenty of blankets; guests bring their own sheets and towels. There is also some 'fowl weather gear' in case of rain. If you're looking for spacious and slick, with many amenities this is NOT the place for you. If you like colorful, cozy, slightly rustic charm, petunias, SAND and THE OCEAN, you might love spending time in our charming cottage as do we!
FOR 1 TO 4 PEOPLE. There is parking available for 2 automobiles. To access the cottage from the driveway one needs to walk up hill about 20 steps including stairs.
Approximately 1 mile down the road your cell phones generally work well. There you can also find 2 restaurants with groceries and sundries available, a small library, a church, a hiking trail, and two forts to explore. Popham Beach is 3 miles long with islands you can walk to at low tide. Many people enjoy fishing from the beach, swimming, sun bathing, shell collecting, and and surfing at times. There are boat trips available. Popham is part of the town of Phippsburg, which has other beautiful peninsulas to see, a golf course, many hiking trails, places to kayak, more restaurants, BnBs, and a lush organic farm & garden store to visit. There are museums, a theater, many antique stores and other types of shopping approximately 1/2 hour away in the Bath /Brunswick area. Freeport, which offers outlet shopping and L.L. Bean is a 45 minute drive. Traveling to Portland or Boothbay Harbor can take 1 hour or more, depending on traffic. (3 hours to Boston.)
We sometimes allow dogs, however, the State Park does NOT allow them on the beach. 1 mile away, in the village, there is beach access where you can take dogs and there is a wooded trail between our neighborhood and Fort Baldwin, which is also a great place to walk dogs.
We don't begin begin to schedule our bookings for the next season, until Dec. or more likely: after Jan. 1. Please wait until after Thanksgiving, to inquire for the following season. Thank you!
Keywords: Popham Beach, at Popham Beach State Park, Popham, Phippsburg, ME, cottage rental, beach, on the ocean, off-season rates, ocean beach, beach access, sandy beach, ocean view, 2 bedroom, ocean view, island view, view, scenic, quaint, quiet, artist, cozy, small, shells, white clapboard, sand beach, scenic, unspoiled, uncrowded, hiking, dunes, coastal, mid-coast, available cottage, cabin.
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Member since: 2011
Property Type
- Cottage
House Rules
- Max. occupancy 4
- Children Welcome
- Non Smoking Only
- Pets Considered
Accommodation Type
- Vacation Rental
- Guests Provide Their Own Meals
- Alternative Lifestyle
- Wheelchair Inaccessible
Bedrooms: 2 Bedrooms, Sleeps 4, Beds for 4
- Away From It All
- Family
- Historic
- Romantic
- Churches
- Library
- Museums
- Restaurants
- Ruins
Local Services & Businesses
- ATM/Bank
- Groceries
Leisure Activities
- Antiquing
- Beachcombing
- Bird Watching
- Boating
- Scenic Drives
- Shopping
- Sight Seeing
- Walking
Location Type
Beach View:
sandy beach with surf visible, boats, occasionally surfers ...moreless
Popham Beach State Park, approximately three miles to walk....moreless
Ocean View:
open ocean and islands, Sequin Lighthouse, and Pond Island lighthouses visible....moreless
- Oceanfront
- Water View
Sports & Adventure Activities
- Deepsea Fishing
- Fishing
- Golf
- Hiking
- Kayaking
- Sailing
- Surf Fishing
- Surfing
- Swimming
- Wind-Surfing
- Dining Area
- Seating for 6 people
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Currency ConversionDates | Nightly | Weekend Night | Weekly | Monthly * | Event |
My Standard Rate 7 night min stay |
$1,600 | ||||
Additional information about rental rates
- Tax Rate 9.5%
one half upon reservation, second half 30 days before check in.
Owner's cancellation policy:
50% refund for cancellations more than 7 days before check-in date.
100% refund for cancellations more than 14 days before check-in date.
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The property's calendar has not been updated in over 8 months. You can contact the owner for availability or look for properties with confirmed availability
Owner info
About the owner: The cottage is owned by two sisters who are both Maine artists. They and their families have been spending time at Popham all of their lives and they are descended from folks who settled in this area around 1620.
Why the Owner Chose Phippsburg, ME, USA:
Popham is arguably the nicest beach in the world! This cottage is cozy, colorful, and comfortable. Our part of the beach is utilizable at high and low tide!
The Unique Benefits at this Cottage:
Jane: ' At Popham I receive inspiration, rest, and a renewed sense of intimate connection to the natural world and to God. ' ' ...love to walk to the Popham village via the beach, especially at dusk, eat an ice cream, and then walk back to the cottage ... and I often go down to the beach in the late afternoon carrying several books and magazines, plop down onto the warm sand, watch the ocean for a few minutes, and read only a few pages before falling asleep!'
Lately we've really been enjoying barbecuing and dining out on the deck, especially at night. I'm going to install tiny lights on the deck's tree.
Contact us
Send email
- Primary: 207 6260738
- Click Here to see All My Rental Listings
Artists' Cottage, Popham, ME. available for portions of May-Oct.
(Popham Beach)living room, 3 windows face ocean, (we've redecorated since.)
part of the view from the deck. (Popham Beach, ME)
Popham Beach,ME., Artists' Cottage
Its fun to cook here! (Popham Beach, Artists' Cottage)
This listing was first published here in 2011.
Date last modified - Monday, March 27, 2017
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Atlanta, GA
All Time
> beef ribs
Top Dishes >
Restaurant Cuisine
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Este site dedica-se às aves e a relação com o Homem. O canto do Timbrado, do Harz,do Malinois. O objectivo de site é contar as aventuras e os arcos de descobertas. This site is dedicated to birds.singing canaries song,canaries,canaries de chant. carmelita=carmelo=jardim
6 de abril de 2015
NASA - A Week in the Life of Rain
Rain, snow, hail, ice, and every mix in between make up the precipitation that touches everyone on our planet. But precipitation doesn't fall equally in all places around the world, as seen in NASA's new animation that captures every shower, snowstorm and tropical cyclone over a six-day period in August 2014. The time lapse was created from data captured by the Global Precipitation Measurement (GPM) satellite mission, now just over a year old, which scientists are using to better understand freshwater resources, natural disasters, crop health and more.