Modified human four helical bundle (4HB) polypeptides and uses thereof are provided.

The growth hormone (GH) supergene family (Bazan, F. Immunology Today 11: 350-354 (1991); Mott, H. R. and Campbell, I. D. Current Opinion in Structural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represents a set of proteins with similar structural characteristics. Each member of this family of proteins comprises a four helical bundle, the general structure of which is shown in FIG. 1. Family members are referred to herein as "four helical bundle polypeptides" or "4HB" polypeptides. While there are still more members of the family yet to be identified, some members of the family include the following: growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-1, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, epsilon interferon, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) ("the GH supergene family"). Members of the GH supergene family have similar secondary and tertiary structures, despite the fact that they generally have limited amino acid or DNA sequence identity. The shared structural features allow new members of the gene family to be readily identified. The general structures of family members hGH, EPO, IFNα-2, and G-CSF are shown in FIGS. 2, 3, 4, and 5, respectively.
One member of the GH supergene family is human growth hormone (hGH). Human growth hormone participates in much of the regulation of normal human growth and development. This naturally-occurring single-chain pituitary hormone consists of 191 amino acid residues and has a molecular weight of approximately 22 kDa. hGH exhibits a multitude of biological effects, including linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic effects, among others (Chawla, R., et al., Ann. Rev. Med. 34:519-547 (1983); Isaksson, O., et al., Ann. Rev. Physiol., 47:483-499 (1985); Hughes, J. and Friesen, H., Ann. Rev. Physiol., 47:469-482 (1985)).
The structure of hGH is well known (Goeddel, D., et al., Nature 281:544-548 (1979)), and the three-dimensional structure of hGH has been solved by x-ray crystallography (de Vos, A., et al., Science 255:306-312 (1992)). The protein has a compact globular structure, comprising four amphipathic alpha helical bundles, termed A-D beginning from the N-terminus, which are joined by loops. hGH also contains four cysteine residues, which participate in two intramolecular disulfide bonds: C53 is paired with C165 and C182 is paired with C189. The hormone is not glycosylated and has been expressed in a secreted form in E. coli (Chang, C., et al., Gene 55:189-196 (1987)).
A number of naturally occurring mutants of hGH have been identified. These include hGH-V (Seeberg, DNA 1: 239 (1982); U.S. Pat. Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated by reference herein) and a 20-kDa hGH containing a deletion of residues 32-46 of hGH (Kostyo et al., Biochem. Biophys. Acta 925: 314 (1987); Lewis, U., et al., J. Biol. Chem., 253:2679-2687 (1978)). In addition, numerous hGH variants, arising from post-transcriptional, post-translational, secretory, metabolic processing, and other physiological processes, have been reported (Baumann, G., Endocrine Reviews 12: 424 (1991)).
The biological effects of hGH derive from its interaction with specific cellular receptors. The hormone is a member of a family of homologous proteins that include placental lactogens and prolactins. hGH is unusual among the family members, however, in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D., et al., Nature 330:537-543 (1987)) or prolactin (Boutin, J., et al., Cell 53:69-77 (1988)) receptor. Based on structural and biochemical studies, functional maps for the lactogenic and somatogenic binding domains have been proposed (Cunningham, B. and Wells, J., Proc. Natl. Acad. Sci. 88: 3407 (1991)). The hGH receptor is a member of the hematopoietic/cytokine/growth factor receptor family, which includes several other growth factor receptors, such as the interleukin (IL)-3, -4 and -6 receptors, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin (EPO) receptor, as well as the G-CSF receptor. See, Bazan, Proc. Natl. Acad. Sci. USA 87: 6934-6938 (1990). Members of the cytokine receptor family contain four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine motif positioned just outside the transmembrane region. The conserved sequences are thought to be involved in protein-protein interactions. See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490 (1992). The interaction between hGH and extracellular domain of its receptor (hGHbp) is among the most well understood hormone-receptor interactions. High-resolution X-ray crystallographic data (Cunningham, B., et al., Science, 254:821-825 (1991)) has shown that hGH has two receptor binding sites and binds two receptor molecules sequentially using distinct sites on the molecule. The two receptor binding sites are referred to as Site I and Site II. Site I includes the carboxy terminal end of helix D and parts of helix A and the A-B loop, whereas Site II encompasses the amino terminal region of helix A and a portion of helix C. Binding of GH to its receptor occurs sequentially, with Site I binding first. Site II then engages a second GH receptor, resulting in receptor dimerization and activation of the intracellular signaling pathways that lead to cellular responses to the hormone. An hGH mutein in which a G120R substitution has been introduced into site II is able to bind a single hGH receptor, but is unable to dimerize two receptors. The mutein acts as an hGH antagonist in vitro, presumably by occupying receptor sites without activating intracellular signaling pathways (Fuh, G., et al., Science 256:1677-1680 (1992)).
Recombinant hGH is used as a therapeutic and has been approved for the treatment of a number of indications. hGH deficiency leads to dwarfism, for example, which has been successfully treated for more than a decade by exogenous administration of the hormone. In addition to hGH deficiency, hGH has also been approved for the treatment of renal failure (in children), Turner's Syndrome, and cachexia in AIDS patients. Recently, the Food and Drug Administration (FDA) has approved hGH for the treatment of non-GH-dependent short stature. hGH is also currently under investigation for the treatment of aging, frailty in the elderly, short bowel syndrome, and congestive heart failure.
Recombinant hGH is currently sold as a daily injectable product, with five major products currently on the market: Humatrope™ (Eli Lilly & Co.), Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer) and Saizen/Serostim™ (Serono). A significant challenge to using growth hormone as a therapeutic, however, is that the protein has a short in vivo half-life and, therefore, it must be administered by daily subcutaneous injection for maximum effectiveness (MacGillivray, et al., J. Clin. Endocrinol. Metab. 81: 1806-1809 (1996)). Considerable effort is focused on means to improve the administration of hGH agonists and antagonists, by lowering the cost of production, making administration easier for the patient, improving efficacy and safety profile, and creating other properties that would provide a competitive advantage. For example, Genentech and Alkermes formerly marketed Nutropin Depot™, a depot formulation of hGH, for pediatric growth hormone deficiency. While the depot permits less frequent administration (once every 2-3 weeks rather than once daily), it is also associated with undesirable side effects, such as decreased bioavailability and pain at the injection site and was withdrawn from the market in 2004. Another product, Pegvisomant™ (Pfizer), has also recently been approved by the FDA. Pegvisomant™ is a genetically-engineered analogue of hGH that functions as a highly selective growth hormone receptor antagonist indicated for the treatment of acromegaly (van der Lely, et al., The Lancet 358: 1754-1759 (2001). Although several of the amino acid side chain residues in Pegvisomant™ are derivatized with polyethylene glycol (PEG) polymers, the product is still administered once-daily, indicating that the pharmaceutical properties are not optimal. In addition to PEGylation and depot formulations, other administration routes, including inhaled and oral dosage forms of hGH, are under early-stage pre-clinical and clinical development and none has yet received approval from the FDA. Accordingly, there is a need for a polypeptide that exhibits growth hormone activity but that also provides a longer serum half-life and, therefore, more optimal therapeutic levels of hGH and an increased therapeutic half-life.
Interferons are relatively small, single-chain glycoproteins released by cells invaded by viruses or exposed to certain other substances. Interferons are presently grouped into three major classes, designated: 1) leukocyte interferon (interferon-alpha, α-interferon, IFN-α), 2) fibroblast interferon (interferon-beta, β-interferon, IFN-β), and 3) immune interferon (interferon-gamma, γ-interferon, IFN-γ). In response to viral infection, lymphocytes synthesize primarily α-interferon (with omega interferon, IFN-ω), while infection of fibroblasts usually induces production of β-interferon. IFNα and IFNβ share about 20-30 percent amino acid sequence homology. The gene for human IFN-β lacks introns, and encodes a protein possessing 29% amino acid sequence identity with human IFN-α, suggesting that IFN-α and IFN-β genes have evolved from a common ancestor (Taniguchi et al., Nature 285 547-549 (1980)). By contrast, IFN-γ is synthesized by lymphocytes in response to mitogens. IFNα, IFNβ and IFNω are known to induce MHC Class I antigen expression and are referred to as type I interferons, while IFNγ induces MHC Class II antigen expression, and is referred to as type II interferon.
A large number of distinct genes encoding different species of IFNα have been identified. Alpha interferons fall into two major classes, I and II, each containing a plurality of discrete proteins (Baron et al., Critical Reviews in Biotechnology 10, 179-190 (1990); Nagata et al., Nature 287, 401-408 (1980); Nagata et al., Nature 284, 316-320 (1980); Streuli et al., Science 209, 1343-1347 (1980); Goeddel et al., Nature 290, 20-26 (1981); Lawn et al., Science 212, 1159-1162 (1981); Ullrich et al., J. Mol. Biol. 156, 467-486 (1982); Weissmann et al., Phil. Trans. R. Soc. Lond. B299, 7-28 (1982); Lund et al., Proc. Natl. Acad. Sci. 81, 2435-2439 (1984); Capon et al., Mol. Cell. Biol. 5, 768 (1985)). The various IFN-α species include IFN-αA (IFN-α2), IFN-αB, IFN-αC, IFN-αC1, IFN-αD (IFN-α1), IFN-αE, IFN-αF, IFN-αG, IFN-αH, IFN-αI, IFN-αJ1, IFN-αJ2, IFN-αK, IFN-αL, IFN-α4B, IFN-α5, IFN-α6, IFN-α74, IFN-α76 IFN-α4a), IFN-α88, and alleles thereof.
Interferons were originally derived from naturally occurring sources, such as buffy coat leukocytes and fibroblast cells, optionally using inducing agents to increase interferon production. Interferons have also been produced by recombinant DNA technology.
The cloning and expression of recombinant IFNαA (IFNαA, also known as IFNα2) was described by Goeddel et al., Nature 287, 411 (1980). The amino acid sequences of IFNαA, B, C, D, F, G, H, K and L, along with the encoding nucleotide sequences, are described by Pestka in Archiv. Biochem. Biophys. 221, 1 (1983). The cloning and expression of mature IFNβ is described by Goeddel et al., Nucleic Acids Res. 8, 4057 (1980). The cloning and expression of mature IFNγ are described by Gray et al., Nature 295, 503 (1982). IFNω has been described by Capon et al., Mol. Cell. Biol. 5, 768 (1985). IFNτ has been identified and disclosed by Whaley et al., J. Biol. Chem. 269, 10864-8 (1994).
Interferons have a variety of biological activities, including anti-viral, immunoregulatory and anti-proliferative properties, and have been utilized as therapeutic agents for treatment of diseases such as cancer, and various viral diseases. As a class, the interferon-α's have been shown to inhibit various types of cellular proliferation, and are especially useful for the treatment of a variety of cellular proliferation disorders frequently associated with cancer, particularly hematologic malignancies such as leukemias. These proteins have shown anti-proliferative activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response Modifiers 3:580; Oldham, R. K. (1985) Hospital Practice 20:71).
Specific examples of commercially available IFN products include IFNγ-1b (Actimmune®), IFNβ-1a (Avonex®, and Rebif®), IFNβ-1b (Betaseron®), IFN alfacon-1 (Infergen®), IFNα-2 (Intron A®), IFNα-2a (Roferon-A®), Peginterferon alfa-2a (Pegasys®), and Peginterferon alfa-2b (PEG-Intron®). Some of the problems associated with the production of PEGylated versions of IFN proteins are described in Wang et al. (2002) Adv. Drug Deliv. Rev. 54:547-570; and Pedder, S. C. Semin Liver Dis. 2003; 23 Suppl 1:19-22. Wang et al. characterized positional isomers of PEG-Intron®, and Pedder at al. compared Pegasys® with PEG-Intron® describing the lability of the PEGylation chemistries used and effects upon formulation. Despite the number of IFN products currently available on the market, there is still an unmet need for interferon therapeutics.
Another member of the GH supergene family is human Granulocyte Colony Stimulating Factor (G-CSF). Naturally-occurring G-CSF is a glycoprotein hormone of about 177 amino acids, having a molecular weight of about 20 kiloDaltons (kDa). The crystal structure of G-CSF is known (Hill et al., (1993) Proc. Natl. Acad. Sci. USA 90:5167-71), and a crystal structure of G-CSF bound to its receptor is also known (Aritomi et al., (1999) Nature, 401:713-717). The three dimensional structure of G-CSF is known at the atomic level. From the three-dimensional structure of G-CSF, predictions of how changes in the amino acid composition of a G-CSF molecule may result in structural changes can be made. These structural characteristics or changes may be correlated with biological activity to design and produce G-CSF analogs.
G-CSF is a pharmaceutically active protein which regulates proliferation, differentiation, and functional activation of neutrophilic granulocytes (Metcalf, Blood 67:257 (1986); Yan, et al. Blood 84(3): 795-799 (1994); Bensinger, et al. Blood 81(11): 3158-3163 (1993); Roberts, et al., Expt'l Hematology 22: 1156-1163 (1994); Neben, et al. Blood 81(7): 1960-1967 (1993); Welte et al. PNAS-USA 82: 1526-1530 (1985); Souza et al. Science 232: 61-65 (1986) and Gabrilove, J. Seminars in Hematology 26:2 1-14 (1989)). G-CSF was purified to homogeneity from cell culture supernatants of the human bladder carcinoma cell line 5637 (Welte et al., Proc. Natl. Acad. Sci. (1985) 82:1526-30). The sequence of the cDNA coding for native G-CSF is known from Souza et al., Science (1986) 232:61-65. As a consequence of alternative splicing in the second intron two naturally occurring forms of G-CSF exist with 204 or 207 amino acids of which the first 30 represent a signal peptide (Lymphokines, IRL Press, Oxford, Washington D.C., Editors D. Male and C. Rickwood). The mature protein was shown to have a molecular weight of about 19 kDa and has 5 cysteine residues which can form intermolecular or intramolecular disulfide bridges. Binding studies have shown that G-CSF binds to neutrophilic granulocytes. Little to no binding is observed with erythroid, lymphoid eosinophilic cell lines as well as with macrophages.
In humans, endogenous G-CSF is detectable in blood plasma (Jones et al. Bailliere's Clinical Hematology 2:1 83-111 (1989)). G-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in two forms of G-CSF mRNA, one version coding for a protein of 177 amino acids, the other coding for a protein of 174 amino acids (Nagata et al. EMBO J 5: 575-581 (1986)), and the form comprised of 174 amino acids has been found to have the greatest specific in vivo biological activity. G-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine or monkey, sustained neutrophil leukocytosis is elicited (Moore et al. PNAS-USA 84: 7134-7138 (1987)).
Human G-CSF can be obtained and purified from a number of sources. Natural human G-CSF (nhG-CSF) can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology, see, for instance, U.S. Pat. No. 4,810,643 (Souza) incorporated herein by reference, has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression.
G-CSF has been found to be useful in the treatment of indications where an increase in neutrophils will provide benefits. G-CSF can mobilize stem and precursor cells from bone marrow and is used to treat patients whose granulocytes have been depleted by chemotherapy, or as a prelude to bone marrow transplants. For example, for cancer patients, G-CSF is beneficial as a means of selectively stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. Other indications include treatment of various infectious diseases and related conditions, such as sepsis, which is typically caused by a metabolite of bacteria. G-CSF is also useful alone, or in combination with other compounds, such as other cytokines, for growth or expansion of cells in culture, for example, for bone marrow transplants.
The G-CSF receptor (G-CSFR) is a member of the hematopoietic/cytokine/growth factor receptor family, which includes several other growth factor receptors, such as the interleukin (IL)-3, -4 and -6 receptors, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin (EPO) receptor, as well as the prolactin and growth hormone receptors. See, Bazan, Proc. Natl. Acad. Sci. USA 87: 6934-6938 (1990). Members of the cytokine receptor family contain four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine motif positioned just outside the transmembrane region. The conserved sequences are thought to be involved in protein-protein interactions. See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490 (1992). The G-CSF receptor consists of a single peptide chain with a molecular weight of about 150 kD (Nicola, Immunol. Today 8 (1987), 134).
Glycosylated hG-CSF has been compared with de-glycosylated hG-CSF, prepared by in vitro enzymatic digestion with neuraminidase and endo-α-N-acetylgalactosaminidase, with respect to its stability as a function of pH and temperature (Oh-eda et al., 1990, J. Biol. Chem. 265 (20): 11432-35). The de-glycosylated hG-CSF, dissolved at a concentration of 1 μg/mL in 20 mM phosphate buffer containing 0.2 M NaCl and 0.01% Tween 20 was rapidly inactivated within the pH range of from about pH 7 to about pH 8 after a two-day incubation at 37° C. In contrast, glycosylated hG-CSF retained over 80% of its activity under the same conditions. Furthermore, evaluation of the thermal stability of both forms of hG-CSF, measured by biological assay and calorimetric analysis, indicated that de-glycosylated hG-CSF was less thermally stable than the native form of hG-CSF.
A number of approaches have been taken in order to provide stable, pharmaceutically acceptable G-CSF compositions. One approach to improving the composition stability of G-CSF involves the synthesis of derivatives of the protein. U.S. Pat. No. 5,665,863 discloses the formation of recombinant chimeric proteins comprising G-CSF coupled with albumin, which have new pharmacokinetic properties. U.S. Pat. Nos. 5,824,784 and 5,320,840, disclose the chemical attachment of water-soluble polymers to proteins to improve stability and provide protection against proteolytic degradation, and more specifically, N-terminally modified G-CSF molecules carrying chemically attached polymers, including polyethylene glycol.
An alternative approach to increasing stability of G-CSF in composition involves alteration of the amino acid sequence of the protein. U.S. Pat. No. 5,416,195 discloses genetically engineered analogues of G-CSF having improved composition stability, wherein the cysteine residue normally found at position 17 of the mature polypeptide chain, the aspartic acid residue found at position 27, and at least one of the tandem proline residues found at positions 65 and 66, are all replaced with a serine residue. U.S. Pat. No. 5,773,581 discloses the genetically engineered G-CSF analogues of G-CSF that have been covalently conjugated to a water soluble polymer.
Another member of the GH supergene family is human erythropoietin (hEPO). Naturally-occurring erythropoietin (EPO) is a glycoprotein hormone of molecular weight 34 kilo Daltons (kDa) that is produced in the mammalian kidney and liver. EPO is a key component in erythropoiesis, inducing the proliferation and differentiation of red cell progenitors. EPO activity also is associated with the activation of a number of erythroid-specific genes, including globin and carbonic anhydrase. See, e.g., Bondurant et al., Mol. Cell Biol. 5:675-683 (1985); Koury et al., J. Cell. Physiol. 126: 259-265 (1986).
The erythropoietin receptor (EpoR) is a member of the hematopoietic/cytokine/growth factor receptor family, which includes several other growth factor receptors, such as the interleukin (IL)-3, -4 and -6 receptors, the G-CSF receptor (G-CSFR), the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor as well as the prolactin and growth hormone receptors. See, Bazan, Proc. Natl. Acad. Sci USA 87: 6934-6938 (1990). Members of the cytokine receptor family contain four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine motif positioned just outside the transmembrane region. The conserved sequences are thought to be involved in protein-protein interactions. See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490 (1992).
U.S. Pat. Nos. 5,441,868; 5,547,933; 5,618,698; and 5,621,080 describe DNA sequences encoding human EPO and the purified and isolated polypeptide having part or all of the primary structural conformation and the biological properties of naturally occurring EPO.
The biological effects of hEPO derive from its interaction with specific cellular receptors. The interaction between hEPO and extracellular domain of its receptor (hEPObp) is well understood. High-resolution X-ray crystallographic data has shown that hEPO has two receptor binding sites and binds two receptor molecules sequentially using distinct sites on the molecule. The two receptor binding sites are referred to as Site I and Site II. Site I includes the carboxy terminal end of helix D and parts of helix A and the A-B loop, whereas Site II encompasses the amino terminal region of helix A and a portion of helix C. Binding of EPO to its receptor occurs sequentially, with site I binding first. Site II then engages a second EPO receptor, resulting in receptor dimerization and activation of the intracellular signaling pathways that lead to cellular responses to the hormone.
Recombinant hEPO is used as a therapeutic and has been approved for the treatment of human subjects. hEPO deficiency leads to anemia, for example, which has been successfully treated by exogenous administration of the hormone.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a method of increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are of importance. In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N-terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule. As a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem., 271:21969-21977. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule.
Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure. Proteins, including enzymes, are composed of various sequences of alpha-amino acids, which have the general structure H2N—CHR—COOH. The alpha amino moiety (H2N—) of one amino acid joins to the carboxyl moiety (—COOH) of an adjacent amino acid to form amide linkages, which can be represented as —(—CHR—CO)n—, where the subscript "n" can equal hundreds or thousands. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
For example, in the case of the amino acid lysine, there exists an —NH2 moiety in the epsilon position as well as in the alpha position. The epsilon —NH2 is free for reaction under conditions of basic pH. Much of the art in the field of protein derivatization with PEG has been directed to developing PEG derivatives for attachment to the epsilon —NH2 moiety of lysine residues present in proteins. "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1-17. These PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins. This can be a significant limitation in instances where a lysine residue is important to protein activity, existing in an enzyme active site for example, or in cases where a lysine residue plays a role in mediating the interaction of the protein with other biological molecules, as in the case of receptor binding sites.
A second and equally important complication of existing methods for protein PEGylation is that the PEG derivatives can undergo undesired side reactions with residues other than those desired. Histidine contains a reactive imino moiety, represented structurally as —N(H)—, but many chemically reactive species that react with epsilon —NH2 can also react with —N(H)—. Similarly, the side chain of the amino acid cysteine bears a free sulfhydryl group, represented structurally as —SH. In some instances, the PEG derivatives directed at the epsilon —NH2 group of lysine also react with cysteine, histidine or other residues. This can create complex, heterogeneous mixtures of PEG-derivatized bioactive molecules and risks destroying the activity of the bioactive molecule being targeted. It would be desirable to develop PEG derivatives that permit a chemical functional group to be introduced at a single site within the protein that would then enable the selective coupling of one or more PEG polymers to the bioactive molecule at specific sites on the protein surface that are both well-defined and predictable.
In addition to lysine residues, considerable effort in the art has been directed toward the development of activated PEG reagents that target other amino acid side chains, including cysteine, histidine and the N-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated by reference herein, and "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1-17. A cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactive functional groups. This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein. Thus, it would be desirable to have a means to introduce a chemical functional group into bioactive molecules that enables the selective coupling of one or more PEG polymers to the protein while simultaneously being compatible with (i.e., not engaging in undesired side reactions with) sulfhydryls and other chemical functional groups typically found in proteins.
As can be seen from a sampling of the art, many of these derivatives that have been developed for attachment to the side chains of proteins, in particular, the —NH2 moiety on the lysine amino acid side chain and the —SH moiety on the cysteine side chain, have proven problematic in their synthesis and use. Some form unstable linkages with the protein that are subject to hydrolysis and therefore decompose, degrade, or are otherwise unstable in aqueous environments, such as in the bloodstream. See Pedder, S. C. Semin Liver Dis. 2003; 23 Suppl 1:19-22 for a discussion of the stability of linkages in PEG-Intron®. Some form more stable linkages, but are subject to hydrolysis before the linkage is formed, which means that the reactive group on the PEG derivative may be inactivated before the protein can be attached. Some are somewhat toxic and are therefore less suitable for use in vivo. Some are too slow to react to be practically useful. Some result in a loss of protein activity by attaching to sites responsible for the protein's activity. Some are not specific in the sites to which they will attach, which can also result in a loss of desirable activity and in a lack of reproducibility of results. In order to overcome the challenges associated with modifying proteins with poly(ethylene glycol) moieties, PEG derivatives have been developed that are more stable (e.g., U.S. Pat. No. 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Pat. No. 6,610,281, which is incorporated by reference herein). There is clearly a need in the art for PEG derivatives that are chemically inert in physiological environments until called upon to react selectively to form stable chemical bonds.
Recently, an entirely new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which has enabled the incorporation of non-genetically encoded amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. Comm. 1-10. These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups, such as ketone groups, alkyne groups and azide moieties, that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.
The ability to incorporate non-genetically encoded amino acids into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon —NH2 of lysine, the sulfhydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages. Azide and acetylene groups, for example, are known in the art to undergo a Huisgen [3+2]cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe, et al., (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. By introducing an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxyl groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.
The present invention addresses, among other things, problems associated with the activity and production of four helical bundle (4HB) polypeptides, and also addresses the production of a 4HB polypeptide with improved biological or pharmacological properties, such as improved therapeutic half-life.

Modified human four helical bundle polypeptides and their uses


A carrier sheet includes two blank librarian's card forms, a plurality of spine labels, and two standard library book pockets all positioned to be printed in a single computer printing operation. The spine labels and the book pockets have pressure sensitive adhesive backings and are adapted to be peeled off of the carrier sheet for attachment to an associated book.

The normal record keeping procedures of libraries includes a librarian's card for each book in the library, a book pocket for each book, and an identification label which is affixed to the spine of each book and which indicates the classification of the particular book according to the Dewey Decimal system. Usually, the name of the author, the title of the book, and the Dewey decimal classification number are printed on the upper portion of the librarian's card which is then kept in a file and used to indicate the name of the borrower of the book and the date when the book is due back at the library. In some cases a bar code number which identifies the book is also printed on the librarian's card. The same information identifying the book which is printed on the librarian's card is also printed on the front face of the book pocket, and where a universal bar code is used, the bar code may also be printed directly on the bottom portion of the book pocket.
The librarian's cards, the book labels, and the book pockets are generally purchased by the libraries as blank forms, and the information identifying the individual books is then typed and printed on the cards and pockets as new books are added to the inventory of the library. In the past the typing of the information on the book pockets, on the labels and on the librarian's cards has entailed two separate typing and printing operations. To solve the problem specially constructed book pockets have been attached to a librarian's card, but this has not proven to be successful because it entails the use of special printers. Also, these pockets are less satisfactory than the standard book pocket which has been in use for the last several decades and has proven to be durable and easy to use.
It would be desirable to provide a new and improved kit of forms which incorporates one or more standard book pockets in conjunction with one or more standard librarian's cards in such a manner as to permit both the book pocket and the associated librarian's card to be typed and printed in a single operation, thereby facilitating the use of the book pocket and the librarian's card. It would be further desirable to incorporate in the kit the necessary book labels which could thus be printed at the same time that the librarian's card and the book pocket are printed and later removed for attachment to the spine of the associated book.


The present invention relates to a glove and storage box assembly (1) which is located in vehicles, into which articles are placed, opened slowly and prevented from vibrating after being opened, has low production cost, does not resist while being dosed and does not make a sound during opening and closing. The inventive glove and storage box assembly (1) essentially comprises at least one shell (2) which has at least one clearance (3) providing access to the chamber formed in the vehicle cockpit in order that articles are placed on thereof and at least one cover (4) which is disposed on the shell (2) and ensures to access the clearance (3) from out-side by opening and closing.

Glove and storage box assemblies known in the art are opened by free fall method. The moving part and the fixed part collide when fully open position is reached as a result of transformation of the potential energy into the kinetic energy because of the weight during opening. Various methods are used in the technique in order to damp the reactive power which will occur due to the said collision. These methods are briefly as follows: an additional deceleration mechanism which is composed of a rotational damper and gearwheel system, a deceleration and locking mechanism which is composed of a leaf spring produced from spring steel and plastic drive arm on the lower part of the storage box, a mechanism which prevents friction that will occur as a result of the motion by providing friction to the fixed part of the storage box via an angled gusset addition made on the moving part of the storage box. Cost of the gearwheel system which is used to connect the linear and rotational dampers and the said dampers to the storage box is very high. In addition, the resistance force performed while the storage box is opened in the said system is also performed while it is closed and this increases the burden of closing the storage box. The mechanism which is formed by using the leaf spring and the drive arm on the lower part of the storage box increases the cost and the burden of closing as well. In the mechanism wherein the application of angled gusset is used, unwanted noises and deformations arising from plastic friction occur.
The Japanese patent document no. JP2006069240 discloses a glove box cover which can be used in the shape of a tray and which is not vibrated during traveling. At the same time, it is mentioned that the system is quiet since it is not vibrated.
The U.S. Pat. No. 4,828,236 discloses a glove and storage box cover which can reach to a totally opened position from a totally closed or partially opened position with its own weight and prevents sudden openings by means of a damper making an effect contrary to the direction of movement. In the system, the damper size and the place that it occupies are minimized by using a spring instead of using rack and pinion and the service life thereof is prolonged since the opening speed of the cover can be controlled.
The Korean patent document no. KR20050017459 discloses elastic shock absorbers which are installed to the ends of dampers in order to prevent the cover of the vehicle glove box to make a sound during impacts that may occur while it is being opened and closed.
The European patent document no. EP1350675A2 discloses a glove and storage box assembly, which is equipped with an operating mechanism to secure, respectively, release and lock the lid of the glove compartment.

Tofas Turk Otomobil Fabrikasi Anonim Sirketi


A medical appliance is provided that includes a self-expanding stent; a sheath adapted to enclose the self-expanding stent in an interior space during deployment; and an external body comprising a bioactive agent attached to the self-expanding stent and adapted to lie outside the interior space of the sheath when the self-expanding stent is enclosed in the sheath. The medical appliance may be deployed in a lumen of a human body and the external body may release the bioactive agent. A method of producing a medical device is provided that includes attaching an external body to a stent and coupling the stent to a delivery arrangement. The external body includes a bioactive agent. A medical device is provided that includes a tissue scaffold; a sheath adapted to enclose the tissue scaffold in an interior space during deployment; and an external body comprising endothelial cells attached to the tissue scaffold and adapted to lie outside the interior space of the sheath when the tissue scaffold is enclosed in the sheath.

Medical devices may be coated so that the surfaces of such devices have desired properties or effects. For example, it may be useful to coat medical devices to provide for the localized delivery of therapeutic agents to target locations within the body, such as to treat localized disease (e.g., heart disease) or occluded body lumens. Localized drug delivery may avoid some of the problems of systemic drug administration, which may be accompanied by unwanted effects on parts of the body which are not to be treated. Additionally, treatment of the afflicted part of the body may require a high concentration of therapeutic agent that may not be achievable by systemic administration. Localized drug delivery may be achieved, for example, by coating balloon catheters, stents and the like with the therapeutic agent to be locally delivered. The coating on medical devices may provide for controlled release, which may include long-term or sustained release, of a bioactive material.
Aside from facilitating localized drug delivery, medical devices may be coated with materials to provide beneficial surface properties. For example, medical devices are often coated with radiopaque materials to allow for fluoroscopic visualization while placed in the body. It is also useful to coat certain devices to achieve enhanced biocompatibility and to improve surface properties such as lubriciousness.
Coatings have been applied to medical devices by processes such as dipping, spraying, vapor deposition, plasma polymerization, spin-coating and electrodeposition. Although these processes have been used to produce satisfactory coatings, they have numerous, associated potential drawbacks. For example, it may be difficult to achieve coatings of uniform thicknesses, both on individual parts and on batches of parts. Further, many conventional processes require multiple coating steps or stages for the application of a second coating material, or may require drying between coating steps or after the final coating step.
The spray-coating method has been used because of its excellent features, e.g., good efficiency and control over the amount or thickness of coating. However, conventional spray-coating methods, which may be implemented with a device such as an airbrush, have drawbacks. For example, when a medical device has a structure such that a portion of the device obstructs sprayed droplets from reaching another portion of the device, then the coating becomes uneven. Specifically, when a spray-coating is employed to coat a stent having a tube-like structure with openings, such as stents described in U.S. Pat. Nos. 4,655,771 and 4,954,126 to Wallsten, the coating on the inner wall of the tube-like structure may tend to be thinner than that applied to the outer wall of the tube-like structure. Hence, conventional spraying methods may tend to produce coated stents with coatings that are not uniform. Furthermore, conventional spraying methods are inefficient. In particular, generally only 5% of the coating solution that is sprayed to coat the medical device is actually deposited on the surface of the medical device. The majority of the sprayed coating solution may therefore be wasted.
In the spin-dipping process, a medical device is coupled to a spinning device, and then, while rotating about a central axis, the medical device is dipped into a coating solution to achieve the desired coating. This process also suffers from many inefficiencies including the unevenness of the coated layer and a lack of control over the coated layer's thickness.
In addition to the spray coating and spin-dipping methods, the electrostatic deposition method has been suggested for coating medical devices. For example, U.S. Pat. Nos. 5,824,049 and 6,096,070 to Ragheb et al. mention the use of electrostatic deposition to coat a medical device with a bioactive material. In the conventional electrodeposition or electrostatic spraying method, a surface of the medical device is electrically grounded and a gas may be used to atomize the coating solution into droplets. The droplets are then electrically charged using, for example, corona discharge, i.e., the atomized droplets are electrically charged by passing through a corona field. Since the droplets are charged, when they are applied to the surface of the medical device, they will be attracted to the surface since it is grounded.
One disadvantage of conventional electrostatic spraying is that it requires a complicated spraying apparatus. In addition, because conventional electrostatic systems use a gas to move the droplets from a source to a target, controlling the gas pressure is crucial for accurate coating. However, it is not easy to control the gas pressure so that the target surface is evenly and sufficiently coated without losing much of the coating solution.
Another method of coating a device can be achieved with electrohydrodynamic spraying. Using this method, a gas is not needed to disperse the coating. Electrohydrodynamic coating is accomplished by forcing a compatible solution through a nozzle assembly that has been electrically charged. The coating solution passes through the charged nozzle where it is electrically charged. As the solution exits the nozzle, the solution atomizes as the charged particles repel each other. This action forms the spray mist. The charged particles are attracted to the device to be coated when the device is connected to an electrical ground.
Devices may be coated by a gas assisted spraying process. A polymer/drug combination may be dissolved in a solvent mixture. The solution may be sprayed onto the devices and a polymer/drug film may be formed when the solvents evaporate. The ability to apply thin coatings on products may be limited by the capabilities of a gas assisted spraying process. The coating may flow on the medical device prior to drying, thereby creating an uneven concentration of bioactive agent on the surface of the device. A gas assisted spraying process may have a high variability for thin coatings.
Conventional methods of coating stents or devices with a drug-polymer layer, such as spraying or dipping, may require a solution of the drug-polymer to physically wet the surface of the stent. Spraying or dipping may cause uneven and unpredictable wetting, and distribution and evaporation of the solvent molecules may result in a non-uniform coating. The drying of the coating may lead to cracking and/or points of stress in the coating. A non-uniform coating may lead to the unit failing agent release requirements, drug uniformity and coating thickness specifications.
During deployment and loading of self-expanding (SE) stents, there may be significant friction between the stent surface and the sheath. Longer stents may have higher friction forces. These shear forces may be especially damaging in relation to coated SE stents. As the application of drug eluting coatings allows the use of longer stents, the problems resulting from this frictional interaction may increase. Similarly, during deployment of balloon-expandable stents, there may be a significant friction between the unfolding balloon and the stent surface.
Additionally, due to the large weight of a stent in relation to a coating weight (with SIBS\paclitaxel coating weight to stent weight having a ratio of approximately 1 to 1000), it may be difficult to measure the exact amount of drug coating on a stent.
Furthermore, due to the complex shape of a stent, it may be difficult to produce a homogeneous uniform coating on the stent surface. Similarly, the deposition of a drug coating that has a predefined pattern over the stent surface (for example, a 20% increased drug concentration at both ends of the stent in order to anticipate edge effects) may be difficult.
There is, therefore, a need for a cost-effective method of linking drug release coatings to the medical device which is not sensitive to the loading and deployment forces. The method should assure defect-free coatings and uniform drug dose per unit device. The method would provide better control of the agent release profile of the device, including increasing or decreasing the release of the bioactive agent. The method would also improve the quality of the coating of the device by allowing drug concentration variations as well as different drugs on different parts of the device. The method would thus allow for better control of the sensitivity of the bioactive material and would reduce variations in the coating properties. Each of the references cited herein is incorporated by reference herein for background information.

System and method for deploying a drug-eluting external body and tissue scaffold


There is provided a novel carbamoyloxy arylalkanoyl arylpiperazine derivative compound having abundant racemic or enantiomeric characteristics, represented by the Formula 1, and pharmaceutically available salts or hydrates thereof. Also, there are provided a pharmaceutical composition for treating pain, anxiety or depression including an effective amount of the compound, and a method for treating pain, anxiety or depression in mammals by administering an effective amount of the compound to the mammals in need of treatment thereof.

Up to now, arylpiperazine compounds were proven to be effective to a variety of indications in the field of central nervous system. In particular, U.S. Pat. No. 3,002,976 reported that the following thiophene-engrafted arylpiperazine compound has a pharmacological effect to treat depression. In this formula, R represents hydrogen, methyl group or halogen.

Also, it has been known that effects of buspirone and its structurally related compounds on the treatment of anxiety is due to their selective activities in serotonin (5-hydroxytryptamine: 5HT) sub-type receptor represented by a receptor 5-HT1A. In particular, U.S. Pat. No. 4,988,814 discloses piperazine derivatives showing affinity to the 5-HT1A receptor characterized as therapeutic agents to treat depression and anxiety.

wherein, R1 is alkyl having carbon atoms of 1 to 6; R2 and R3 are each independently alkyl having carbon atoms of 1 to 6, or R2 and R3 are taken together to form polymethylene having carbon atoms of 2 to 12 or to form a 5-norbornen-2-yl residue with carbon atoms bound to the radicals R2 and R3; X is selected from the group consisting of —CO2—, —OCO—, —OCO2—, —N(R7)CO—, —NHNHCO—, —ON(R7)CO—, —CON(R7)—, —N(R7)CO2—, —OCON(R7)— and —N(R7)CON(R8) (wherein, R7 and R8 are each independently is selected from the group consisting of hydrogen; alkyl having carbon atoms of 1 to 6; phenyl; benzyl; and phenyl or benzyl substituted by halo, alkyl having carbon atoms of 1 to 6, alkoxy having carbon atoms of 1 to 6, cyano, nitro or perhalomethyl); R4 is hydrogen or alkyl having carbon atoms of 1 to 6; R5 is selected from the group consisting of hydrogen; alkyl having carbon atoms of 1 to 8; hydroxyalkyl having carbon atoms of 1 to 3; phenyl; benzyl; and phenyl or benzyl substituted by hydroxy, halo, alkyl having carbon atoms of 1 to 6, alkoxy having carbon atoms of 1 to 6, trifluoromethyl, nitro, cyano, carbalkoxy having carbon atoms of 2 to 7, carboxamido, amino, alkylamino having carbon atoms of 1 to 6 or dialkylamino having carbon atoms of 2 to 12; R6 is phenyl, benzyl, 2-, 3- or 4-pyridinyl, 2-pyrimidinyl or 2-pyrazinyl that may be substituted by at least one substituents selected from the group consisting of hydroxy, halo, alkyl having carbon atoms of 1 to 6, alkoxy having carbon atoms of 1 to 6, trifluoromethyl, nitro, cyano, carbalkoxy having carbon atoms of 2 to 7, carboxamido, amino, alkylamino having carbon atoms of 1 to 6, and dialkylamino having carbon atoms of 2 to 12; n is one integer selected from the group consisting of 0, 1, 2, 3, 4 and 5, provided that R6 is not 2-pyrimidinyl when X is —CON(R7)— (wherein, R7 is alkyl), and R6 is not 3,5-di(trifluoromethyl)phenyl when X is CO2, R1, R2 and R3 are methyl and n is 1.
The present inventors have confirmed that an arylpiperazine structure is correlated with an effect to treat pains as well as anxiety and depression, conducted comprehensive researches on the arylpiperazine structure, and found that novel carbamoyloxy arylalkanoyl arylpiperazine compounds have a medical effect in various pain-induced animal models. In particular, the present inventors have found that the novel carbamoyloxy arylalkanoyl arylpiperazine compounds show their therpeutic effects to treat a wide scope of pains including acute pain, chronic pain, neuropathic pain, post-surgery neuropathic pain, diabetic pain, postherpetic neuralgia, inflammatory pain, joint pain, migraine headache and the like, anxiety and depression. Therefore, the present invention was completed on the basis of the above-mentioned facts.

This patent is subject to a terminal disclaimer.

Method for treating pain, anxiety or depression using carbamoyloxy alkanoyl piperazine compound


A rotary printing press is used for printing standard pages and for double truck printing of the centerfold page. This is accomplished by mounting side by side printing plates to locking devices in the rotary printing press. The printing plates are positioned by two registrations which are selectively movable toward and away from each other to one of at least two predetermined fixed positions. In one of the fixed positions, the standard printing takes place, and in the other fixed position, the printing press is used for double truck printing.

Rotary printing presses are used for various purposes including newspaper printing. Each section of a newspaper is comprised of standard pages wherein a margin exists along the sides of each page. The centerfold page however does not require a margin and in fact it is sometimes desirable that there be no margin so that various printed material could span the central area of the centerfold page and print a continuous image. Heretofore, the prior art has printed newspapers by utilizing conventional techniques for printing the standard pages. When the double truck page is printed, however, it is necessary to have each half of the centerfold butted together. This is conventionally done manually with special equipment and involves such manual activities as sheering, trimming and punching. Such equipment is quite costly, being about $40,000, in addition to requiring the great manual effort.

Double truck printing registration system for a rotary printing press


Stable velocity control operations are made possible independent of a circuit constant or the like in digital circuit arrangement. A velocity controlling apparatus comprises a track cross detector, a counter for counting track cross pulses, a sampling device for periodically sampling an output of the counter, and a relative velocity detector for detecting a relative velocity between a light spot and tracks, based upon a difference between a currently sampled value and a prior value sampled by the sampling device prior to the sampling of the currently sampled value. The sampling device periodically samples the output of the counter to count the number of tracks crossed, and a velocity is detected based on the difference, whereby information on detecting velocity may be readily obtained in digital value and the control may be conducted with less errors.

1. Field of the invention
The present invention relates to a velocity detecting apparatus and a velocity controlling apparatus in a seek circuit of an optical disk apparatus or the like. More particularly, the invention relates to a velocity detecting apparatus and a velocity controlling apparatus which enable a stable velocity detection operation and a velocity control operation free from influence of circuit constant or the like, in arrangement of digital circuit.
2. Description of the Related Art
A conventional optical disk apparatus is provided with seek means comprising a track pulse generator, which generates a certain number of track pulses every time a light spot focused on an optical disk crosses a track on the optical disk, and a velocity detector for detecting a moving velocity of the light spot from a frequency of the track pulses (see for example Japanese Patent Laying Open (Kokai) Application No. 2-33731).
The conventional optical disk apparatus of this type normally employs a so-called F/V converter, which is a circuit for converting the frequency of track pulses periodically obtained in accordance with the relative movement between the light spot and the tracks, into a voltage in order to detect a relative velocity between the light spot and the tracks on the optical disk.
FIG. 1 is a functional block diagram to show an example of main structure of the F/V converter. In FIG. 1, reference numeral 11 designates a mono multivibrator (MM), 12 an amplifier, 13 a resistor, 14 a condenser, IN an input signal, MMO an output of the mono multivibrator 11, and OUT an output signal of the amplifier 12.
FIG. 2 is a time chart to illustrate an operation of the F/V converter as shown in FIG. 1. Reference characters given for respective signal waves correspond to locations of the characters in FIG. 1.
As shown by MMO in FIG. 2, the mono multivibrator 11 is triggered at each leading edge of the input signal IN to provide an output of pulse with a constant width.
The output MMO of the mono multivibrator is amplified by a subsequent amplifier 12, and the amplifier 12 generates an output signal as shown by OUT in FIG. 2.
The F/V converter is, however, an analog circuit, an output of which may include considerable error elements risen from a dispersion of analog circuit constant and from an offset or a gain error of the amplifier.
Such errors may cause a problem of unstable seek velocity control in the F/V converter.
In addition, the seek velocity control loop itself is an analog circuit basically composed of a an F/V converter, a subtracter, and an amplifier, which is likely to cause errors in hardware and which is also unsuitable for stable velocity control.


A hydraulic booster for a vehicle and more particularly an improved hydraulic booster with improved brake-relieving action, among other improvements.

1. Field of the Invention
This invention relates to a hydraulic booster for vehicle which assists the vehicle driver with the operation of the master cylinder.
2. Description of the Prior Art
Japanese Patent Application No. 217892/1988 (Japanese Patent Opening Gazzette No. 68258/1990) discloses a hydraulic booster in which a second valve apparatus is arranged at a reaction chamber formed adjacent to a top end of an input member in order to reduce the initial drive force required to develop master cylinder pressure. The second valve apparatus prevents pressure-fluid from flowing into the reaction chamber until the pressure of fluid in the pressure-servo chamber becomes higher than a predetermined pressure. Thus, a force applied to the input member before the master cylinder pressure starts to rise, can be smaller. When the second valve apparatus is opened, there occurs the so-called "Jump-up phenomenon" in which the master cylinder pressure steeply rises up to a certain pressure.
When the pressure of the servo-chamber is lowered to relieve the braking action, the pressurized fluid from the servo-chamber is returned into the fluid pressure-releasing source arranged at the downstream side of the second valve apparatus. Accordingly, the flow amount of the pressurized fluid is increased, since the pressurized fluid is discharged also from the reaction chamber. Abnormal sound is made from the second valve apparatus. Further, since the fluid amount is much, there is the disadvantage that the brake-relieving action lags.


A foot brake valve apparatus is provided for a heavy vehicle braking system. The foot brake valve apparatus comprises a valve body having a first side face and a second side face opposite the first side face. The apparatus further comprises a primary supply pressure port associated with a primary braking circuit and adjoining the first side face, and a primary delivery pressure port associated with the primary braking circuit and adjoining the first side face. The apparatus also comprises a secondary supply pressure port associated with a secondary braking circuit and adjoining the first side face, and a secondary delivery pressure port associated with the secondary braking circuit and adjoining the first side face.

The present application relates to heavy vehicle braking systems, and is particularly directed to a foot brake valve apparatus for a heavy vehicle braking system, such as a truck braking system.
One type of foot brake valve apparatus includes a foot brake valve having a valve body and parallel supply pressure ports on one side of the valve body and delivery pressure ports on an opposite side of the valve body. A primary supply pressure port and a primary delivery pressure port are provided for a primary braking circuit. A secondary supply pressure port and a secondary delivery pressure port are provided for a secondary braking circuit. The primary and secondary supply pressure ports are on the one side of the valve body, and the primary and secondary delivery pressure ports are on the opposite side of the valve body. A drawback in this known type of foot brake valve apparatus is that a single pressure sensing package is unable to be installed on the valve body to access the four different pressure ports to measure pressure at each of the four pressure ports. It would be desirable to provide a foot brake valve apparatus in which a single pressure sensing package can be installed on the valve body to access all four pressure ports so that pressure measurements can be obtained at each of the pressure ports.

Foot brake valve apparatus for a heavy vehicle braking system


The present invention provides taxonomy system for enterprise data management and analysis. Building a taxonomy is based on defining taxonomy themes to solve different aspects of enterprise data management. For each theme, a taxonomy is created by classifying data related to the theme. Taxonomy nodes include enterprise data management policies and data analysis methods to be applied to data stored at the nodes. The amount of data that companies need to access and manage today is complex and voluminous. Because of computational requirements and time constraints, it is very difficult to classify and process all data. In many cases, current solutions ignore a sizable amount of data and process a small subset of it. This leads to incomplete and inaccurate results as ignored data may contain valuable information. The present invention provides taxonomies that encompass all data. Voluminous data is processed first to eliminate irrelevant data that is not related to the theme of a taxonomy. This in effect reduces the amount of data used to create the taxonomy without impacting its accuracy.

The invention relates to taxonomy system for enterprise data management and analysis.
The concept of taxonomy evolved from the life sciences. In the scientific community, taxonomies were conceived as a way to organize and categorize life forms into a structured and controlled hierarchy. In this approach, a plant or animal is placed in a single spot describing its hierarchical relationship to other plants and animals.
Classification or categorization of animals and plants is the arrangement of entities in a hierarchical series of classes. A class is defined as a collection of similar entities, where the similarity consists of the entities having attributes or traits in common. Corporate taxonomy, sometimes referred to as business taxonomy, is a hierarchy of categories used to classify documents, digital assets and other information. Taxonomies is can cover virtually any type of physical or conceptual entities (products, processes, knowledge fields, human groups, etc.).
The application of taxonomy as a means to organize business content is a complex issue. Unlike the categorizing of life forms, categorizing business documents can be ambiguous. A document could be placed in multiple categories depending on the business context or expertise of the user. This added level of classification complexity in the business setting makes the design and construction of taxonomy structures more challenging. Corporate taxonomies are increasingly used in information systems, particularly content management and knowledge management systems, as a way to allow instant access to the right information within exponentially growing volumes of enterprise data.
Enterprise data management is the development and execution of policies, practices and procedures that properly manage enterprise data. The typical enterprise has data stored in a variety of data sources. Some aspects of data management are: security and risk management, legal discovery, storage resource management (SRM), information lifecycle management (ILM) and content-based archiving. In addition, some companies have their own internal management policies. A policy is an action or a set of actions to be performed on data. For instance, a policy action could be deletion of a category of data. Another aspect of data management is data auditing. Data auditing allows enterprises to validate compliance with federal regulations and insures that data management objectives are being met.
Data analysis is the process of studying, culling, summarizing data to extract useful information. It is used by business, financial organizations, academic institutions and government agencies to acquire meaningful insights from data and predict futuristic trends. It helps to cut costs, improve revenue, reduce risks and enhance performance.
In many aspects of enterprise data management it is not enough to find a document that contains a term. What is needed is the relationship between documents and the ability to track changes in the relationship when new documents are added or existing documents are modified. For instance, when investigating payments plans, it is important to find documents that include modifications to payment plans and related contracts. Management policies should be applied to all related documents. When a policy is to retain, restore, or protect documents about payment plans, the policy should be applied to all related documents. Another example is when categorizing documents under "international commerce", there is need to find all documents that contain terms such as foreign trade, import, export and free trade. The relationship between documents also helps an enterprise to perform data analysis that leads to good business decisions. For instance, by finding documents related to payment plans, an enterprise can analyze the data and modify contracts to maximize revenue generation or reduce risks. A solution to the problem of determining the relationship between documents and their inter-dependencies is to provide a taxonomy that organizes and classifies enterprise data in a systematic and structured manner.
Categorization of data is based on metadata or full text search. Metadata consist of information that characterizes data. Sometimes it is referred to as "data about data". Categorization rules specify how data is classified into different groups. Data categorization methods, based on metadata, group data according to information extracted from its metadata. A few examples of such information are: the time a document was last accessed, its owner, its type and its size. There are many methods for accessing and extracting information from metadata. Some methods utilize file system utilities. File system utilities can only extract file system metadata, sometimes referred to as file system attributes. Document parsers, sometimes called filters, are used for extracting metadata from documents, such as Microsoft Word, Microsoft PowerPoint and PDF files. Examples of such metadata are: document type, publisher, author, subject, etc. The three top commercial parsers being used now are: Stellent, KeyView and iFitler. Some software developers write their own parsers or use open source parsers such as Apache POI. Results of extracting metadata are stored in a repository accessible during classification. Classification based on full text utilizes a search index built by a search engine. Full text search is used to identify documents that contain specific terms, phrases or combination of both. The result of the search is used to categorize data. One of the widely used open source search engines is Lucene.
There are many classification technologies for building corporate taxonomies. The most common ones are: Rule-based, Bayesian, Linguistic and Semantic, Support Vector Machine and neural networks. Bayesian, Linguistic and Semantic, Support Vector Machine, neural networks, and similar methods are collectively referred to as non-rule-based classification methods.
Rule-based systems define the criteria by which a document is classified. The rule measures how well a given document meets the criteria for membership in a particular class. The rule can be applied to the content or the metadata. Boolean logic is used to combine words and phrases using the words AND, OR, and NOT (otherwise known as Boolean operators) to define classification more accurately. For example, a rule could be defined as all documents in a content that include the terms "San Francisco," OR "Los Angeles," OR "San Diego" AND "California" be included in a category called "Cities, California". Besides the content of documents, rules can be applied to metadata. For instance, a rule may specify that only Microsoft Word and Excel documents owned by marketing OR sales, and created during the current fiscal year be included in a category called "Marketing and Sales, Current Fiscal Year". Rules must be carefully articulated and made as unambiguous as possible. A user may elect to provide a complex query all at once, or through a series of interactions with the system, thus potentially broadening or narrowing the scope of the query.
Bayesian probability theory is a means of quantifying uncertainty. The theorem defines a rule for refining a hypothesis by factoring in additional evidence and background information, and leads to a number representing the degree of probability that the hypothesis is true. It uses statistical models from words in training sets, and uses pattern analysis to assign the probability of correlation.
Linguistic and semantic approach is language dependent. Documents are clustered or grouped depending on meaning of words using thesauri, custom dictionaries (e.g. a dictionary of abbreviations), parts-of-speech analyzers, rule-based and probabilistic grammar, recognition of idioms, verb chain recognition, noun phrase identifiers, and linguistic or semantic clustering.
Support Vector Machine (SVM) calculates the maximum separation, in multiple dimensions of one document from another. Each document is represented as a vector. SVM continuously analyses documents and separates them into either the relevant space or the irrelevant space.
A neural network utilizes artificial intelligence to build an interconnected system of processing elements, each with a limited number of inputs and outputs. Rather than being programmed, these systems learn to recognize patterns. Neural networks are an information processing technique based on the way biological nervous systems, such as the brain, process information.
Bayesian, Linguistic and Semantic, Support Vector Machine and neural networks classification methods often require complex computation and could be referred to as computationally intensive classification methods. The complexity is exacerbated by the intricacy and size of enterprise data. In this document, the terms "computationally intensive classification" and "non-rule-based classification" are used interchangeably.
Because of data explosion, some enterprises and organizations have hundreds of millions of documents of different formats stored in hundreds of terabytes of storage. Classification of voluminous data is very costly because of the computational resources needed and the time it takes to process all data. The time required to perform data classification can run into weeks, in particular when computationally intensive classification methods are used. For this reason many existing solutions process only a subset of the data that staff members in the information technology (IT) department think it contains all relevant information. This subset is usually a small percentage, sometimes in the order of 5% of the total data. The assumption here is that excluded data does not include relevant information. This assumption is difficult to validate and is risky. Many times enterprises were fined by regulators because of their failure to comply with government regulations related to protection of all private data. The failure is due to the fact that the subset of data processed does not include all data that needs protection. In conclusion, processing a subset of the data results in inaccurate classification and that in effect results in imprecise and incomplete taxonomies.
During the last few years, many enterprises have adopted cloud computing. Cloud computing has many definitions. According to National Institute of Standards and Technology (NIST), cloud computing is a model for enabling ubiquitous, convenient, on-demand network access to a shared pool of configurable computing resources (e.g., networks, servers, storage, applications, and services) that can be rapidly provisioned and released with minimal management effort or service provider interaction. Cloud computing adoption is accelerating. For this reason it is imperative that data in a cloud computing environment should be included in taxonomies.
The procedure for building taxonomies can be divided in two approaches: rule-based classification and non-rule-based classification. The non-rule-based classification is sometimes referred to as machine learning. In the first approach, a domain expert decides on the structure of the taxonomy tree and assigns a rule-based classification to each node of the tree. Once a rule is set up, large numbers of incoming documents are automatically gauged against the rule for membership of a taxonomy node. In machine learning based classification, an administrator presents the system with positive and negative sample documents for membership in a node. Positive sample documents are expected to be included in a node. Negative sample documents are not included. From these samples the system learns and builds a model to be used in data classification, which is then used to decide whether a subsequently presented document belongs to a node. The effort for training can be significant, as the administrator may have to provide several hundred to thousands of documents for training the system. A variety of mathematical models and algorithms are used in automatic classifiers based on machine learning, including several flavors of Bayesian inference, Bayesian networks, neural networks, and Support Vector Machines.
The complexity of enterprise data and the information explosion has reached the point where no information architect within an organization can build a comprehensive taxonomy that fully describes the data in a manner similar to life science taxonomy. The problem is exacerbated by the ambiguity of languages. Even when using automated classification methods, information architects lack adequate grasp of all the various themes and topics represented in an enterprise data and for this reason cannot be relied on to extract a hierarchy of concepts and label them into a comprehensive taxonomy. Even within a single organization, it is extremely difficult, to build a single taxonomy tree that covers all enterprise data and captures important relationships between all documents. Such trees are non-existent.
Because of the prohibitive difficulty of building a single taxonomy tree that covers all enterprise data, in particular when data is voluminous, it is time for a new pragmatic approach that includes all data. The approach is based on defining taxonomy themes to solve different aspects of enterprise data management and data analysis. For each theme, a taxonomy is built by classifying data related to the theme. When data is voluminous, irrelevant data that is not related to the theme and does not impact the accuracy of the taxonomy is filtered out first. This results in less data to be processed by the taxonomy.

Taxonomy system for enterprise data management and analysis


A strap-tensioning device includes a base that receives a rotatable drum rigidly connected to toothed wheels, and configured to engage a holding pawl; tensioning lever rotatably mounted by hinging onto the drum, and provided with a pawl for driving the toothed wheels; an unlocking pawl configured to release the toothed wheels from the respective engagement thereof with the holding pawl and with the driving pawl, rotatably mounted, independently of the rotation of the tensioning lever, by hinging onto the drum. The unlocking lever is rigidly connected to two parallel cams having the same profile, and configured to simultaneously engage, respectively, the holding pawl and the driving pawl, so as to release the rotation of the drum.

"Ratchet" systems are now in widespread use for tensioning straps. In the main, they traditionally comprise a base rigidly connected to the end of a strap, the base comprising an axis on which is mounted a rotatable drum around which the other end of the strap, or even another strap, is intended to be wound. This drum is rotated by means of a lever, known as a tensioning lever, that actuates the rotation of the drum in order to allow the other end of the strap to be wound thereupon, thereby tensioning the strap. The axis of the drum is provided with toothed wheels intended to engage with holding pawls mounted on the base, and driving pawls mounted on the tensioning lever, in order respectively to keep the strap tensioned and to drive the drum when the tensioning lever is actuated.
One of the major difficulties with this kind of device lies in releasing the tension, when it is required to release the secured product. Indeed, to effect the release of the rotation of the drum, prior art devices are basically provided with an unlocking lever rigidly connected to the tensioning lever, which needs to be pulled first, in order to release the drum from the driving pawl. Consequently, it is also necessary to fully open the tensioning lever, in other words, move it away from the base to its extreme position, in order to release the drum from the holding pawl. This last stage is not that simple, and short of finding the knack, few users are able to correctly release the drum. This dual operation thus causes a problem precisely because of the tension being exerted on the drum when the strap is stretched.
The targeted objective of this invention is to simplify this unlocking operation.


The invention is a bis-BCB compound of the structure: ##STR1## a method for making same and polymers made therefrom. The novel compounds of this invention are useful in preparing polymers that can form thin film coatings for multichip midules (MCMs) and integrated circuits (ICs). These compounds can be isolated as liquids at room temperature, and can form polymers that are hydrophobic and have low dielectrical and dissipative properties.

This invention relates to bisbenzocyclobutene compounds (bisBCBs) containing hydrophobic substituents. An extensive review of benzocyclobutenes is Kirchhoff and Bruza's monograph "Benzocyclobutenes in Polymer Synthesis" (Prog. Polym. Sci., Vol 18, 85-185, 1993), herein incorporated by reference.
Polymers derived from benzocyclobutenes (BCBs) (of which bisBCBs are a subset) are described in U.S. Pat. No. 4,540,763. These polymers are prepared by subjecting BCBs to temperatures sufficient for polymerization. The polymers exhibit properties that make them useful for preparing composites, coatings, and films.
One useful property is a low dielectric constant. It is possible, as Kirchoff reports (pg 92 of the above-cited monograph), to prepare low dielectric constant hydrophobic benzocyclobutene polymers by starting with precursor monomers that contain hydrocarbon or siloxane groups interspersed between the reactive moieties of the monomer molecule. For example, the polymer of the divinylbenzene bisbenzocyclobutene compound shown below has a dielectric constant of 2.7.
Unfortunately, this compound has a melting point of 150.degree.-152.degree. C. which makes processing prior to polymerization time-consuming and costly. ##STR2##
Schrock addressed this deficiency in the art (U.S. Pat. No. 4,812,588) by preparing the liquid, tetramethyldivinyldisiloxane bisbenzocyclobutene (DVS-bisBCB), which is represented by the following structure: ##STR3##
The polymer formed from DVS-bisBCB, poly-DVS-bisBCB has a low dielectric constant (2.55 at 1 MHz) a low dissipation factor (0.0008 at 1 MHz and <0.002 at 10 GHz), and low water absorption (0.25% at 100.degree. C. for 24 hours). (See Kirchhoff et al., J. Macromol. Sci.-Chem., A28(1&12), pp. 1094 and 1104-1106.) Thus, poly-DVS-bisBCB is especially electrically resistive and hydrophobic.
One disadvantage of DVS-bisBCB is that it is not isolated as a single compound, but rather as a complex mixture of compounds. Since the composition of this mixture may vary from batch to batch, the nature of the polymer derived from the DVS-bisBCB monomer may also vary. Thus, there is a potential problem of variability in the properties of the polymer.
In view of the deficiency in the art, it would be desirable to have bisbenzocyclobutene compounds that were liquids at room temperature. At the same time, it is essential that the same desirable properties of the prior art polymers--low dielectric constants, low dissipation factors, and low water uptake--not be compromised.

Bisbenzocyclobutene compounds with hydrophobic substituents, a method for making same