COMPLEXES AND COMBINATIONS OF FETUIN WITH THERAPEUTIC AGENTS

Abstract: There is disclosed a complex and a combination of the glycosylated polypeptide fetuin and a therapeutically active small molecule compound having a net positive charge at physiological pH. The presence of fetuin as a drug complex or in combination with the therapeutically active small molecule compound enhances therapeutic activity of the small molecule compound. The present invention further provides a means for screening for therapeutically active small molecule compounds by means of binding to fetuin.

Description: COMPLEXES AND COMBINATIONS OF FETUIN WITH THERAPEUTIC AGENTS Technical Field of the Invention The present invention provides a complex and a combination of the glycosylated polypeptide fetuin and a therapeutically active small molecule compound having a net positive charge at physiologic pH. The presence of fetuin as a drug complex or in combination with the therapeutically active small molecule compound enhances therapeutic activity of the small molecule compound. The present invention further provides a means for screening for therapeutically active small molecule compounds by means of binding to fetuin. Background of the Invention A class of aromatic guanylhydrazones have been described in United States Patent Application 08/463,568 filed on 05 June 1995, the disclosure of which is incorporated by reference herein. These compounds have been shown to have therapeutic activity, primarily as anti-inflammatory agents. The activity is based upon an ability to suppress proinflammatory cytokine synthesis in activated macrophages and other cells. The preferred compound, called "CNI-1493" (N,N'-bis[3,5-bis [l(aminoiminomethyl)hydrazono]ethyl]phenyl]decanediamide tetrahydrochloride), inhibits TNF (tumor necrosis factor) translation and suppresses the production of IL-1, IL-6, MlP-lα and MlP-lβ (proinflammatory cytokines) in human peripheral blood mononuclear cells. In vivo, CNI-1493 protects mice against lethal effects of endotoxin (lipopolysaccharide, LPS) and is anti-inflammatory as evidenced by suppression of rat paw edema and inflammation following carageenan administration. Therefore, there is always a need to improve the therapeutic properties of drag candidates such as CNI-1493 and related aromatic guanylhydrazone compounds. Macrophages play important roles in fundamental immune protection, including, for example, phagocytosis, antigen presentation, microbiocidal and tumoricidal activities (Adams, 1984), and release of a large number of factors during host defense and inflammation (Currie 1978). Upon stimulation by interferon γ (INF-γ) and potent macrophage activators such as bacterial endotoxin, macrophages secrete large amounts of reactive nitrogen intermediates (RNI), and several cytokines including TNF-α, IL-1, IL-6, MlP-lα, MlP-lβ that augment the inflammatory response during bacterial infection. Generation of RNI and tumor cell killing by macrophages are both dependent on L-arginine (Keller et al., Cancer Res. 50:1421-1424, 1990; Hibbs et al, J. Immunol. 140:550-565, 1987; Drapier et al., J Immunol. 140:2829-2838, 1988), a common substrate shared by arginase and nitric oxide synthase (iNOS). Arginase (EC 3.5.3.1.) is an enzyme active in converting L-arginine to L-ornithine and urea, which plays an essential role in the urea cycle, as well as the biosynthesis of proline and polyamine (Janne et al., Ann. Med. 23:241, 1991). Nitric oxide synthase is an enzyme active in catalyzing the formation of nitric oxide (NO). Nitric oxide synthase is an important molecule implicated in antimicrobial, cytotoxic, and inflammatory processes mediated by macrophages, as well as in blood pressure regulation and neurotransmission in the nervous system (Hibbs et al., Biochem. Biophys. Res. Comm. 157:87-94, 1988; Moncada et al., Proc. Natl. Acad. Set USA 88:2166- 2170, 1991, Nathan, FASEB J. 6:3015-3064, 1992; Moncada et al, N Engl. J. Med. 329:2001- 20012, 1993). Both arginase (All) and NO synthase (iNOS) are LPS-inducible in macrophages (Granger et al., J. Clin. Invest. 85:264-273, 1990; Currie, Nature 273:758-759, 1978; Corraliza, J. Immunol. Methods 174:231-235, 1994; Stuehr et al, Proc. Natl. Acad. Sci. USA 82:7738- 7742, 1985; Steuhr et al, J. Immunol. 139:518-525, 1987; Modlell, Immunology Lett. 40:139- 146, 1994). Although a biological role for urea and NO 2 7NO 3 " synthesis by cells of the immune system is not yet known, it appears to represent a quantitative feature in the activation of macrophages. Arginase activity potentially controls the NO synthesis of macrophages by substrate depletion as in the absence of arginine the production of NO is dramatically reduced (Vodovotz, J. Immunol. 152:4110-4118, 1994). Thus, all factors controlling the relative rates of flux of L-arginine between arginase and NOS may be important in the regulation of macrophage' s cytotoxic activity. L-arginine transport in macrophages and other cells also increases in response to agents that induce iNOS (Bogel, Biochem J. 284:15-18, 1992; Sato et al, Biochem. Biophys. Acta 1069:46-52, 1992; and Cendan, Surgery 117:312-219, 1995). CNI-1493 and related compounds inhibit cytokine-induced arginine transport (IC 50 = 59 μM), and NO production (IC 50 = 5 μM. The cytokine-suppressive effects of CNI-1493 are possibly not mediated by inhibition of NO or L-arginine transport, nor by generalized suppression of protein synthesis or RNA synthesis. Fetuin is a globular 341 -amino acid protein containing 20-25% carbohydrate (by weight) and 6 internal disulfide bonds. The human fetuin sequence (also known as α2-HS glycoprotein) is provided herein as SEQ ID NO. 1 and SEQ ID NO. 2. Fetuin was first identified over 50 years ago as a major protein component of bovine fetal serum but its biological function remains unclear. Bovine fetuin is a globular 341 amino acid polypeptide with six internal disulfide bonds and three N-linked and two O-linked oligosaccharide chains. Primary amino acid sequence and the position of cysteine residues are well conserved in human, bovine, sheep, rat and mouse fetuin homologs (Dziegielewska et al., J. Biol. Chem. 265:4354, 1990; Rauth et al., Eur. J. Biochem. 205:321,1992; Lee et al., Proc. Natl. Acad. Sci. USA 84:4403, 1987; and Brown et al, Eur. J. Biochem. 205:321, 1992). Fetuin levels in human plasma are regulated in a manner of a negative acute phase reactant (Lebreton et al., J. Clin. Invest. 64:1118, 1979). IL-1 was shown to suppress fetuin transcript levels in cultured hepatocytes (Akhoundi et al., J. Biol. Chem. 268:15925, 1994). Fetuin appears to be expressed in bone because transcripts have been detected in both chondrocytes and osteoblasts (Yang et al., Blood 12:7, 1991). The polypeptide α2-HS glycoprotein is a human homolog of fetuin and is secreted in high levels by adult liver into the peripheral circulation (Triffitt et al., Nature 262:226, 1976). . Human fetuin has 3 N-linked oligosaccharide chains (attached to the amine nitrogen atom of asparagine), and 2 O-linked oligosaccharide chains (attached to the oxygen atom of serine or theronine). The sugar moiety directly attached to the fetuin polypeptide is usually a Ν-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a Ν-acetylneuraminic acid (ΝAΝA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from fetuin by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. It is also a carrier protein for growth factors. Fetuin is sometimes referred to as α2-HS-glycoprotein. Thus, it is considered that fetuin' s biological effects on cultured cells are related to its carrier function for molecules with growth-promoting properties. Fetuin is secreted by adult liver into the peripheral circulation and accumulates to high levels in bone. The synthesis of human α2-HS-glycoprotein is down-regulated by cytokines (hIL-lβ, hIL-6) (Lebreton et al, J. Clin. Invest. 64:1118-1129, 1979). Human fetuin levels are decreased (25-50%) in trauma patients (van Oss et al., J. Trauma 15:451, 1975). Therefore, there is a need in the art to find a utility for fetuin and to understand fetuin' s physiological role and the importance of its many negatively charged (at physiologic pH) sialic acid residues. Summary of the Invention The present invention provides a complex of a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and a positively charged (at physiologic pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R X 2 ^ X ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine, C,. 6 alkyl-substituted purine, xanthine, pyrimidine, C,_ 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of ΝH 2 (CHΝ)-ΝH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein "Z" is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 . ]0 alkyl, straight or branched C 2 . 10 alkenyl, -A- straight or branched C 2 _ 10 alkyl -A-, -A- straight or branched C 2. , 0 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X,, X'„ X 2 and X' 2 are each NH 2 (CHN)- NH-N=C(CH 3 )-. The present invention further provides a pharmaceutical composition comprising: (A) an active component consisting essentially of a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and (B) a positively charged (at physiologic pH) therapeutic compound selected from the formula: wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C,. 6 alkyl-substituted purine, xanthine, pyrimidine, C,_ 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 _ 10 alkyl, straight or branched C 2 _ 10 alkenyl, -A- straight or branched C 2 . 10 alkyl -A-, -A- straight or branched C 2.10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-; and (C) a pharmaceutically acceptable carrier. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X,, X',, X 2 and X' 2 are each NH 2 (CHN)-NH-N=C(CH 3 )-. The present invention further provides a therapeutic combination of a glycosylated polypeptide and a positively charged (at physiologic pH) therapeutic compound: wherein the glycosylated polypeptide is selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and wherein the positively charged (at physiologic pH) therapeutic compound selected from the formula: wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C,. 6 alkyl-substituted purine, xanthine, pyrimidine, C,_ 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 _ ]0 alkyl, straight or branched C 2.10 alkenyl, -A- straight or branched C 2 . ]0 alkyl -A-, -A- straight or branched C 2 ]0 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X„ X'„ X 2 and X' 2 are each NH 2 (CHN)- NH-N=C(CH 3 )-. The present invention further provides a method for finding therapeutically active small molecule compounds having a net positive charge at physiologic pH, comprising: (a) providing human fetuin glycoprotein in a buffer solution at physiologic pH, wherein the fetuin glycoprotein oligosaccharide moiety contains a plurality of sialic acid residues; (b) contacting a small molecule compound in solution or a library of multiple small molecule compounds in solution to the fetuin glycoprotein at physiologic pH; and (c) measuring whether the small molecule compound or a small molecule compound from the library of small molecule compounds had bound to the fetuin glycoprotein. Preferably, the fetuin glycoprotein is bound to a solid substrate support. Preferably, the fetuin glucoprotein has a human sequence. Brief Description of the Drawings Figure 1 shows an initial finding that launched the investigation detailed in the examples section. Specifically, RAW264.7 cells (murine macrophage-like cells) precultured under low serum conditions were not responsive to treatment with compound CNI-1493 (N,N'- bis[3 ,5-bis [ 1 (aminoiminomethyl)hydrazono]ethyl]phenyl]decanediamide tetrahydrochloride). An indication of nonresponsiveness is a failure by compound CNI-1493 to inhibit TNF (tumor necrosis factor) levels in culture supernatants in response to LPS (endotoxin or lipopolysaccharide) challenge. Figure 2 shows that when the supernatant from responsive cell cultures was added to non-responsive cell cultures, therapeutic activity of CNI-1493 was restored. Figure 2 specifically shows therapeutic activity for CM- 1493 in non-responsive cell cultures only when the supernatant fraction ("+F") from responsive cell cultures was added. The therapeutic activity was a measure of inhibition of TNF response to LPS (endotoxin) challenge. Figure 3 shows that the accumulation of CNI-1493 activity enhancing activity in cell culture medium is inducible over time. The inducible "factor" appears within a few hours after challenge with LPS and also appears to be made constitutively. Figure 4 shows a purification scheme used to isolate the "factor" (i.e., fetuin) responsible for making non-responsive cell cultures responsive. Figure 5 shows three measures of LPS-stimulated macrophage activation (i.e., production of TNF-α, urea and nitrite) that each independently show an unresponsive state with respect to expected inhibition by compound CNI-1493 and a state of restored responsiveness to compound CNI-1493 when a 30-100 kDa fraction of LPS-stimulated RAW 264.7 cell conditioned medium was added. Figure 6 shows a protein elution profile (A 280 ) of LPS-stimulated RAW 264.7 cell culture supernatant (serum-free) over a MONO-Q™ column eluted with NaCl. S Figure 7 shows the corresponding activity profile of the Figure 6 MONO Q™ fractions. Activity was measured as the relative potency for enhancing TNF suppressive activity in LPS- stimulated cell cultures by compound CNI-1493. Figure 8A shows several intracellular and extracellular fractions may or may not enhance CM- 1493 therapeutic activity. Figure 8B shows the corresponding SDS-PAGE analysis of the Figure 8 A fractions which shows enrichment of a single band on the gel. Figure 9 shows a comparison of urea concentration from LPS-stimulated non- responsive cells (or no LPS in the far left bar) showing that the addition of 2.5 μM CNI-1493 showed a small therapeutic activity by lowering the urea concentration but that two doses of the 30- 100 kDa fraction (2.5 μl and 7.5 μl) from LPS-stimulated responsive RAW 264.7 cells increased CM- 1493 therapeutic activity in a concentration-dependent fashion. Figure 10 shows that asialofetuin (fetuin glycoprotein treated to remove terminal sialic acid residues) at concentrations of 1.0 and 2.5 μg/ml was not nearly as active as native fetuin (complete glycoprotein) at the same 1.0 and 2.5 μg/ml concentrations for enhancing CNI-1493 therapeutic activity (measured by suppression of urea production in LPS stimulated nonresponding RAW 264.7 cells). Figure 11 shows that an extremely low concentration of C - 1493 (50 nM) was therapeutically active by suppressing TNF production in LPS-stimulated human PBMCs (peripheral blood mononuclear cells) and that this therapeutic activity was enhanced, in a dose- dependent fashion, by adding human fetuin glycoprotein (commercially-prepared, Sigma) at concentrations of either 1.0 μg/ml or 10 μg/ml along with CM- 1493. Figure 12 shows a comparison of CNI-1493 therapeutic activity in LPS -challenged mice as measured by TNF serum levels. The greater therapeutic activities of CNI-1493 exhibited lower TNF concentrations following LPS challenge. The uniform CNI-1493 dose was 2.5 mg/kg (ip) and the LPS challenge concentration was 13 mg/kg which is the approximate LD 50 for LPS in mice. The fetuin pretreatment dose was 20 mg/kg. These data show that fetuin pretreatment greatly enhanced CNI-1493 therapeutic activity, as measured by suppression of TNF levels. Figure 13 shows a comparison of CM- 1493 activity in RAW 264.7 cells measuring nitrite production, comparing CNI-1493 alone (2.0 μM concentration for all CNI-1493 doses) and when adding 2.5 μg/ml of fetuin or other glycoproteins (glycophorin, αl-acid glycoprotein, albumin, or human glycoprotein). These data show that fetuin was the only glycoprotein tested that was effective for augmenting CNI-1493 therapeutic activity. Detailed Description of the Invention The present invention provides a complex of a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and a positively charged (at physiologic pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R N ^ X X 2 wherein R is independently selected from the group consisting of phenyl, pyrimidine, pyridine, amino-substituted pyridine, purine, oxo-substituted purine, xanthine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X„ X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 _ 10 alkyl, straight or branched C 2 _ 10 alkenyl, -A-straight or branched C 2 _ 10 alkyl- A-, - A-straight or branched C 2 _ 10 alkenyl-A-, and -A-, wherein A is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X,, X',, X 2 and X' 2 are each NH 2 (CHN)- NH-N=C(CH 3 )-. In RAW264.7 macrophages, lipopolysaccharide (LPS) substantially stimulated production of NO, urea, and pro-inflammatory cytokine TNF-α over baseline control levels. CNI- 1493 is a tetravalent guanylhydrazone-substituted aromatic compound that promoted a dose-dependent suppression of urea, NO, and TNF-α production from the LPS-activated macrophages. However, when RAW264.7 macrophages cells were pre-cultured under low- serum conditions, their response to CNI- 1493 was markedly impaired, although their response to LPS was not affected. Such cell cultures are called non-responsive cell cultures because they did not respond to the therapeutic activity of CNI- 1493 but did respond to endotoxin. When given concurrently with CNI- 1493, a 30-100 kDa macrophage protein fraction obtained from CNI- 1493 -responsive cells by ultrafiltration, dose-dependently increased CNI- 1493- mediated inhibition of urea and TNF production from LPS-activated macrophages. This CNI- 1493 -enhancing activity was found to accumulate in RAW 264.7 cell-conditioned media, with a substantial increase by 4 hours in the presence of LPS at 1 μg/ml. Further fractionation by ion-exchange chromatography, and SDS-PAGE gel revealed that the CNI- 1493 -enhancing activity associated with a ~ 60-70 kDa protein that was eluted in high-salt fractions. These results indicated involvement of a macrophage-secreted protein in an inactivation of LPS- stimulated RAW 264.7 macrophages by CNI- 1493. A 30-100 kDa fraction from RAW 264.7 macrophage culture supernatants, cultured under normal conditions ("responsive cells") potentiated the cells' ability to respond to CNI- 1493. A similar 30-100 kDa fraction from macrophage RAW 264.7 cell culture supernatants, cultured under low serum conditions, ("non-responsive cells") failed to potentiate the cells' ability to respond to CNI- 1493. These data indicated that CNI- 1493 -mediated signaling pathway might be impaired in RAW 264.7 macrophages cultured under low-serum conditions. The CNI- 1493 enhancing activity was isolated from macrophage whole-cell lysates by ultrafiltration with membranes with defined Mr cut-off ranges. Ultrafiltration of whole-cell lysate on membranes with Mr cut-off of 100,000 dalton distributed CNI-1493-enhancing activity predominantly to the filtrate. Subsequent ultrafiltration of this filtrate on a membrane with a Mr cut-off of 30,000 daltons (Centricon®-30 ) distributed CNI- 1493 -enhancing activity entirely to the retentate fraction (that is, the 30-100 kDa fraction). These data suggested that CNI- 1493 enhancing activity existed as a polypeptide with molecular weight less than 100 kDa but greater that 30 kDa. The CNI- 1493 -enhancing activity was released from macrophage cell cultures into the medium. In minimal medium (OPTI-MEM) conditioned by RAW 264.7 macrophages, CNI- 1493 -enhancing activity was detected by as early as 2 hours in a 30-100 kDa fraction. A further increase of the CNI-1493-enhancing activity in RAW 264.7-conditioned medium was seen by 4 hours in the presence of LPS. Four hours after LPS stimulation, a corresponding decrease of CNI-1493-enhancing activity was detected in the 30-100 kDa intracellular fraction. These data indicate that protein(s) contributing to CNI- 1493 -enhancing activity are secreted into the medium, and this accumulation is further stimulated by treatment with bacterial endotoxin (LPS). The 30-100 kDa fraction (obtained from the above-described ultrafiltration process) was further purified by ion-exchange chromatography (see Figure 4). The protein(s) contributing to CNI- 1493 -enhancing activity in the macrophage intracelluar and extracellular fraction were the same protein(s) because: i) the proteins coeluted at an identical concentration range (1.8-2.0 M) of a lineal salt gradient from a cationic (MONO-Q™) ion exchange column; and ii) the proteins co-migrated on a SDS-PAGE gel as two polypeptides, which also correlated with the fraction exhibiting therapeutic activity of the MONO-Q™ fractions. Further N-terminal sequence analysis of the protein and comparison to existing databases revealed that the protein of interest had a sequence similar to bovine fetuin. CNI-1493 is a tetravalent guanylhydrazone compound (N,N'-bis[3,5-bis [l(aminoiminomethyl)hydrazono]ethyl]phenyl]decanediamide tetrahydrochloride) that carries four positive charges at physiological pH. It suppressed LPS-induced production of cytokines, NO, and urea from LPS-activated macrophages. Without being bound by theory, the following is a hypothesis of the mechanism of action of CNI- 1493 and the activity potentiation by a complex or combination with fetuin and similar sialic acid-containing glycoproteins: i) Macrophages can release negatively-charged glycoprotein factors that can act to potentiate CNI-1493 therapeutic activity; ii) Cell membrane can bind CNI-1493 and fetuin complex or combination, which complex or combination then transduces signal to the cell. Signal is transduced either through having the complex or CNI- 1493 small molecule compound bind a specific receptor on the cytoplasmic cell membrane, or through facilitory uptake of CNI- 1493 into the target cell. Macrophage effector functions are highly regulated, presumably to limit damage to normal tissues. In the context of cell-mediated reactivity to ongoing antigenic stimulation, as occurs in chronic infection, both positive and negative macrophage stimuli are usually generated. CNI- 1493 likely negatively regulates an LPS-mediated activation pathway. Macrophages can release negative charged protein factors that potentiate CNI- 1493 enhancing activity, potentially by binding to the polycationic compound and facilitating its presentation to a cell or to an intracellular signaling pathway of a cell. The CNI- 1493 fetuin complex or compbination may transduce signal to the cell. Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising: (A) an active component consisting essentially of a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and (B) a positively charged (at physiologic pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R Y- 2 X ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C,_ 6 alkyl-substituted purine, xanthine, pyrimidine, C,_ 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X„ X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2.]0 alkyl, straight or branched C 2.]0 alkenyl, -A- straight or branched C 2 _ 10 alkyl -A-, -A- straight or branched C 2.10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-; and (C) a pharmaceutically acceptable carrier. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X,, X',, X 2 and X' 2 are each NH 2 (CHN)-NH-N=C(CH 3 )-. The present invention further provides a therapeutic combination of a glycosylated polypeptide and a positively charged (at physiologic pH) therapeutic compound: wherein the glycosylated polypeptide is selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and wherein the positively charged (at physiologic pH) therapeutic compound selected from the formula: Xi X ' l R - Z - R X 2 X ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C, 6 alkyl-substituted purine, xanthine, pyrimidine, C,. 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2.10 alkyl, straight or branched C 2 _ 10 alkenyl, -A- straight or branched C 2 _ 10 alkyl -A-, -A- straight or branched C 2.10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. Preferably, the glycosolated polypeptide is human fetuin. Preferably, X,, X',, X 2 and X' 2 are each NH 2 (CHN)- NH-N=C(CH 3 )-. Pharmaceutical Formulations The inventive pharmaceutical complex or inventive pharmaceutical combination can be administered to a patient either by itself (complex or combination) or in pharmaceutical compositions where it is mixed with suitable carriers and excipients. The inventive complex or combination can be administered parenterally, such as by intravenous injection or infusion, intraperitoneal injection, subcutaneous injection, or intramuscular injection. The inventive complex or combination can be administered orally or rectally through appropriate formulation with carriers and excipients to form tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like. The inventive complex or combination can be administered topically for various dermatologic disorders where an anti-inflammatory agent is appropriate. The inventive complex or combination is formulated into topical creams, liquids or gels suitable to topical application to skin or mucosal membrane surfaces. The inventive complex or combination can be administered by inhaler to the respiratory tract for local treatment of various respiratory inflammatory diseases. The dosage of the inventive complex or combination suitable for use with the present invention can be determined by those skilled in the art from this disclosure. The pharmaceutical composition will contain an effective dosage (depending upon the route of administration and pharmacokinetics of the active agents) of the inventive complex or combination and suitable pharmaceutical carriers and excipients, which are suitable for the particular route of administration of the formulation (i. e. , oral, parenteral, topical or by inhalation). The active complex or combination is mixed into the pharmaceutical formulation by means of mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical formulations for parenteral administration include aqueous solutions of the active complex or combination in water- soluble form. Additionally, suspensions of the active complex or combination may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may optionally contain stabilizers or agents to increase the solubility of the complex or combination to allow for more concentrated solutions. Pharmaceutical formulations for oral administration can be obtained by combining the active complex or combination with solid excipients, such as sugars (e.g., lactose, sucrose, mannitol or sorbitol), cellulose preparations (e.g., starch, methyl cellulose, hydroxypropylmethyl cellulose, and sodium carboxymethyl cellulose), gelaten, gums, or polyvinylpyrrolidone. In addition, a desintegrating agent may be added, and a stabilizer may be added. Research Applications The present invention further provides a method for finding therapeutically active small molecule compounds having a net positive charge at physiological pH, comprising: (a) providing human fetuin glycoprotein in a buffer solution at physiologic pH, wherein the human fetuin glycoprotein oligosaccharide moiety contains a plurality of sialic acid residues; (b) contacting a small molecule compound in solution or a library of multiple small molecule compounds in solution to the fetuin glycoprotein at physiologic pH; and (c) measuring whether the small molecule compound or a small molecule compound from the library of small molecule compounds had bound to the human fetuin glycoprotein. Preferably, the human fetuin glycoprotein is bound to a solid support matrix to provide for better isolation and a determination of fetuin binding to a candidate compound. The amino acid sequence for human fetuin glycoprotein is found in SEQ ID NO: 1 and SEQ ID NO: 2. The binding assay is useful to screen potential drag compounds whose therapeutic activity can by augmented by either co-administration with fetuin or by forming a drag-fetuin complex prior to administration. Example 1 This example illustrates the initial observation that there are macrophage-like cell cultures which are responsive and non-responsive to the therapeutic effects of compound CNI- 1493. Prior studies have demonstrated that pre-treatment of RAW 264.7 cell cultures with CNI-1493 suppressed LPS-stimulated production of NO, TNF, and the cytokines IL-1 β, IL-6, MlP-lα, and MIP-1 β. The effect of CNI- 1493 on inducible secreted arginase and iNOS activity was investigated, because arginase and inducible nitric oxide synthase (iNOS) activity in macrophages have been posited to play important roles in the pathogenesis of human disease, and can be stimulated by potent inflammatory agents, such as bacterial lipopolysaccharide (LPS). RAW 264.7 murine macrophages were obtained from the American Tissue Culture Collection (ATCC), and cultured in RPMI 1640 containing 10% fetal bovine seram (FBS) and 2 mM glutamine. Cultured were maintained in 150-mm plates with 30 mis culture medium until they reached 80-90% confluence, and subjected to different experimental protocols. Cultures were split at 1 :4 and initially cultured in RPMI 1640 containing 10% FBS/1% ' / glutamine for 24 hours. Subsequently, old medium was replaced by fresh medium (RPMI 1640) containing 5% FBS/1% glutamine to create "serum depleted" cultures. The incubation was continued for another 24 hours before subjecting the cultures to further experiments. RAW 264.7 cells were harvested by gentle scraping, washed, resuspended in RPMI 1640/10% FBS/1 % glutamine at 10 6 cell per ml in 24-well tissue culture plates, and precultured for 2 hours at 37 °C in 5% CO 2 /95% air to allow the cells to readhere. LPS (endotoxin) was sonicated for 10 minutes and added to media at a final concentration of 100 ng/ml. Urea and NO concentrations in culture supernatants were assayed 20 hours after LPS treatment. Drug was added to the medium one hour before LPS. Protein fractions were added concurrently with drug in RPMI 1640 medium containing 10% fetal bovine seram and 1% glutamine. Accumulation of urea in the cell culture medium was measured as a functional assay of LPS-inducible arginase II activity. Urea concentrations were measured spectrophotometrically 20 hours after LPS treatment using Sigma Diagnostics Urea Nitrogen reagents as instructed by the manufacturer (Ca. No. 535-A, Sigma, St. Louis, MO USA). The optical density at 535 nm was measured, and urea concentrations were calculated by comparison with OD 535 of standard dilutions of urea prepared in culture medium. Percent of inhibition (I) for urea production was calculated by the formula, I = 100 x (A-B)/ A, where A and B are the urea concentrations corresponding to treatment with either LPS alone, or LPS plus CNI- 1493, respectively. Inducible nitric oxide synthase activity (iNOS) was assayed indirectly by measuring NO 2" production. NO 2" was measured by a colorimetric assay based on the Griess reaction. Briefly, 100 μl aliquots of macrophage-conditioned medium were incubated with 900 ul aliquots of Griess reagent (1% sulfanilamide/0.1% naphtylethylene diamine dihydrochloride 2.5% H 3 PO 4 ). The optical density was read at 560 nm after 10 min, and NO 2" concentration was determined with reference to a standard curve by using concentrations from 1.5 to 50 μM sodium nitrite in culture medium. TNF concentrations were determined on the supernatants collected from stimulated RAW 264.7 cell cultures four hours after LPS stimulation by ELISA (Genzyme) performed in 96-well micro titer plates (the minimum detectable concentration [MDC] = 10 pg/ml). Addition of CNI- 1493 promoted a dose-dependent suppression of urea production by LPS-stimulated RAW 264.7 cell cultures. Approximate 5 μM, corresponding to a >95% decrease compared with control (p < 0.01), was the CNI- 1493 dose. The IC 50 (concentration of 50% inhibition) of CNI- 1493 to urea production was estimated to be <2.0 μM, which was approximate to the C 50 of CNI- 1493 for NO and TNF production. An assay for arginine uptake found the IC 50 for CNI- 1493 to be about 10 μM. The level of urea and nitrite produced by LPS-activated macrophage cultures correlated well to level of TNF. Thus, the level of urea and nitrite produced from LPS-activated macrophages was employed as a useful index of LPS- inducible secreted arginase and iNOS activity, and the state of LPS-induced macrophage activation when compared with appropriate controls. 13- When RAW 264.7 macrophage cultures were cultured in RPMI- 1640 medium containing 10% FBS/1% glutamine for extended periods of time (>56 hours after 1 :4 split as opposed to <48 hours, for example), significant levels of urea, NO, and TNF could still produced from LPS-activated macrophages. However, the levels of urea and/or TNF were not reduced when CNI- 1493 was added even at a concentration as high as 5 μM. Macrophage cultures that failed to respond to CNI- 1493 ("the CNI- 1493 non-responsive cells" ) still retained their ability to respond to LPS, as they accumulated the same concentrations of urea, NO, and TNF-α after LPS stimulation as did shorter term cultures. A comparison of responsive and non-responsive RAW 264.7 cell cultures is provided in Figure 1 with different concentrations of the drag compound CNI- 1493. We tested if we could reproduce the above phenomenon by manipulating the seram concentration of the medium in which RAW 264.7 macrophages were precultured. Preculturing the RAW 264.7 cells for one day in medium containing 5% FBS substantially affected the response of the cultures to CNI-1493, but not to LPS. At 2.5 μM, CNI-1493 revealed a less than 10% suppression to urea, NO, and TNF production from the "serum starved" macrophages, as opposed to a larger than 50% suppression to urea and NO, and > 90% suppression of TNF production from the responsive cells that were cultured. These data suggest that serum-starvation affected the response of RAW 264.7 macrophage cell cultures ability to CNI- 1493, which was independent of the LPS activation pathway. Example 2 This example illustrates a procedure used to discover that fetuin was the protein responsible for restoring non-responsive macrophage cell cultures to responsive cells for the therapeutic activity of CNI- 1493. The fractionation scheme used is shown in Figure 4. Cells from responsive RAW 264.7 cell cultures were harvested by gentle scraping and washed three times in phosphate buffer saline (PBS), collected by centrifugation (1000 rpm, 10 min. at 4 °C), and subjected to three rounds of freeze-thaw cycles to lyse the cells. Whole-cell lysate preparations were fractionated by sequential ultrafiltration over Amicon Centricon®-100, and Centricon®-30 filters, respectively. Different fractions, including a filtrate of Centricon®-100 and the corresponding retentate of Centricon®-30, were then assayed for their biological activity with respect to the production of urea, NO, and TNF from LPS-stimulated macrophage cultures also treated with CNI- 1493, described in example 1. CNI-1493-enhancing activity was detected in terms of arginase activity, 1 unit of arginase activity representing the amount of activity necessary to produce 2 picomole of urea/16 hour/10 6 cells. Ultrafiltration of RAW 264.7 macrophage cultures whole-cell lysate eliminated the vast majority (>90%) of proteins in the macrophage whole-cell lysate. When added concurrently with CNI- 1493, the retentate of Centricon®-100 did not affect the response of macrophage "seram depleted" cultures (i.e., CNI- 1493 non-responsive) to CNI- 1493. Nor were the effects of CNI- 1493 on urea, nitrite, and TNF production by LPS-stimulated macrophage cultures affected by the co-addition of whole-cell lysate, which lysate comprises a wide variety of /3 components with the potential to specifically or non-specifically influence urea, nitrate and TNF production in vitro. However, when the filtrate fraction from Centricon®-100 was concentrated by Centricon®-30, and added concurrently with CNI- 1493, the level of urea and TNF produced from the LPS-stimulated "seram depleted" macrophage cultures (i.e., CNI- 1493 non-responsive) was substantially reduced as compared to identical cultures treated in parallel with CNI-1493 alone. The 30-100 kDa fraction did not affect the level of urea, nitrate, and TNF production from LPS-stimulated RAW 264.7 macrophage cultures poteins not otherwise treated. These data suggest that a 30-100 kDa macrophage cellular protein fraction potentiated the suppressing activity of CNI- 1493 on macrophage activation. We also examined the dose effect of the 30-100 kDa fraction on the suppression of CNI- 1493 with respect to urea and TNF production from LPS-stimulated RAW 264.7 macrophage cultures. As shown in Figure 2, in combination with CM-1493, the 30-100 kDa fraction promoted a dose-dependent further inhibition of urea and TNF production, with a maximal > 65% total inhibition of LPS-stimulated urea, nitrite, and TNF production from RWA 264.7 macrophage cultures. We further investigated whether CNI- 1493 -enhancing activity could be isolated from "seram deprived" non-responsive cells in the corresponding 30-100 kDa macrophage intracellular fraction. The 30-100 kDa fraction from "seram deprived" non-responsive cell cultures did not reveal any activity to potentiate the CNI- 1493 -mediated inhibition of LPS- induced urea, nitrite, and TNF production by RAW 264.7 macrophage cell cultures. In other cells or tissues examined, including kidney cells and liver extract, no CNI- 1493 -enhancing activity was detected in their respective 30-100 kDa fraction. RAW 264.7 cells were split (1 :4) into RPMI 1640 containing 10% FBS/1% glutamine, and incubation continued until confluence reached to about 80% to isolate proteins in RAW 264.7-conditioned media. Cells were washed twice with pre-warmed OPTI-MEM medium, and then cultured in OPTI-MEM medium (20 ml per 150-mm tissue culture dish) in the presence or absence of LPS at 1 μg/ml. At specified time points, medium and cells were separately harvested, proteins were fractionated by sequential ultrafiltration through Centricon®-100 and Centricon®-30 filters, as described above, and subjected to biological assay. Figure 3 shows a time course of accumulation of CNI- 1493 -enhancing activity in RAW 264.7-conditioned media, where medium and cells were separately harvested and analyzed at each time point. Proteins in medium conditioned by RAW 264.7 cells were concentrated and fractionated by ultrafiltration following the procedure described above and assayed for their CNI- 1493 -effect enhancing activity. As shown in Figure 3, no CNI- 1493 -effect enhancing activity was detected in medium that was conditioned for less than 5 minutes ("0 hour" time point). However, a readily detectable level of CNI-1493-enhancing activity was measured in RAW 264.7-conditioned medium by 2 hours. Moreover, at a concentration of 1 μg/ml, LPS caused a marked increase in CNI- 1493 -enhancing activity by 4 hours, after which time the rate i of CNI- 1493 -effect enhancing activity accumulation markedly declined. Determination of "recoverable" CNI- 1493 -effect enhancing activity in the 30-100 kDa intracellular fraction of the corresponding cells revealed a marked decrease in CNI- 1493 -effect enhancing activity by 4 hours of LPS activation, which corresponded with the increase level of the CNI- 1493 -effect enhancing activity observed in the culture medium. These observations suggest that the markedly increased CNI- 1493 -enhancing activity in the macrophage culture medium is attributeable to a LPS-stimulated release of protein factor(s) and that this release can be stimulated with LPS. Thus, cultured macrophages stimulated with LPS possess a 30-100 kDa protein fraction that potentiates the activity of CNI- 1493 to inhibit the production of urea and TNF by these cells in a dose-dependent manner (Figure 5). The 30-100 kDa fraction itself showed no inhibitory effect on the LPS-induced urea and TNF production except in the presence of CNI- 1493. Figure 9 shows a comparison of urea concentration from LPS-stimulated non-responsive cells (or no LPS in the far left bar) showing that the addition of 2.5 μM CNI- 1493 showed a small degree of inhibitory activity by lowering the urea concentration, but that doses of the 30- 100 kDa fraction (2.5 μl and 7.5 μl) from LPS-stimulated responsive RAW 264.7 cell cultures increased CNI- 1493 activity in a dose-dependent fashion. Proteins were concentrated from large volumes of RAW 264.7-conditioned media by sequential ultrafiltration on either YM-100, or YM-30 membranes (Amicon Corp., Lexington, MA) under N 2 at 4 °C with slow stirring. Proteins concentrated either from macrophage intracellular lysates, or from RAW 264.7-cell conditioned medium were loaded onto a 1-ml MONO-Q® ion exchange column re- equilibrated in buffer "B" (50 raM Tris-HCl pH 7.5, 150 mM NaCl) at 0.5 ml/min. The ion exchange column was washed with buffer B until the A 280 dropped below 1 % of its maximum (Figure 6). Bound protein was eluted in 1.0 ml fractions over 25 min with a linear gradient of NaCl increasing to 2 M in buffer B. Fractions were concentrated by Amicon Centricon®-30 ultrafilter, and aliquots were assayed for CNI-1493-effect enhancing activity, protein content and polypeptide migration on SDS-PAGE (Figure 7). Samples were assayed biologically at each stage of the preparative sequence. Protein fractions were mixed with one volume of solubilization buffer (2% SDS, 10% B-mercaptethanol, 0.03% bromophenol blue, 1.25 M Tris-HCl, pH 7.0), boiled for 5 min, and subjected to electrophoresis on a 4 - 20% premade gradient SDS-PAGE gel according to the manufacturer's instructions (Bio-Rad). After electrophoresis, the gel was stained either by Coomasse blue or silver staining using the Silver Plus kit as instructed by the manufacture (Bio-Rad). Molecular weight of the protein contributing to the CNI- 1493 -effect enhancing activity was determined. MONO-Q® fractions containing CNI- 1493 -enhancing activity were fractionated on a 10% SDS-PAGE gel. After electrophoresis, gel was evenly sliced ( 2.0 mm in width) and proteins was eluted from each gel slice (Figure 8). Briefly, gel slices were rinsed /S three times with sterile water, once with PBS, crushed into small fragments in 1.0 ml of PBS, and incubated at room temperature overnight with gentle shaking. Gel fragments were subsequently pelleted by a brief centrifugation (2 min, 14,000 rpm), and the isolated proteins were concentrated over a Centricon®-10 filter, and subject to biological assay. For both macrophage intracellular and extracellular protein fractions, only one peak of CNI- 1493 -effect enhancing activity, as revealed by the urea and/or nitrite bioassay, was eluted from the MONO-Q column between 1.8 to 1.9 M NaCl (Figure 7). The activity was confirmed both by visual examination of the cultures for cell growth (i.e., cell counting and culture growth rates), and by TNF ELISA for TNF production. A substantial decrease in number of cells detached from the bottom of plates was consistently observed in wells treated with CNI- 1493 and high-salt fractions. Furthermore, all detectable CNI- 1493 -enhancing activity was abolished by treatment of high-salt fractions from both macrophage intracellular and extracellular fraction with protease. The most highly purified sub-fraction contained a mixture of material with a molecular weight ranging from 90,000 to 7,000 as defined by silver-staining of SDS-polyacrylmide gels. Two major polypeptides with the molecular weight of 67 and 57 kDa, respectively, co-eluted with the peak of CNI- 1493 -effect enhancing activity. Moreover, two polypeptides isolated from the macrophage intracellular and extracellular protein co-migrated at about a 4-20% gradient SDS-polyacrylmide gel. It confirmed that the CNI- 1493 -effect enhancing activity in the macrophage intracellular fraction and extracellular fraction were the same protein(s), as they share the same charge property at pH 7.4, same molecular weight, and same biological activities. Treatment of the ultrafiltrate from macrophages with protease abolished CNI-1493- effect enhancing activity. Proteins corresponding to the CNI-1493-effect enhancing activity were eluted from the preparative SDS-PAGE gel and subjected to N-terminal sequencing analysis. The N terminal sequence found (SEQ. ID NO. 3) and shown in the top row below as matched to the N-terminal sequence of fetuin (SEQ. ID NO. 4) shown in the bottom row below. Query ( SEQ . I D NO . 3 ) ? PLDPVAGYKEPA? DE ?ETEQAALA I Mil! bovine Fetuin ( SEQ . I D NO . 4 ) I PLDPVAGYKEPACDDPDTEQAALA Example 3 This example illustrates further characterization of the importance of a complex and combination of fetuin or its corresponding human homolog α2-HS glycoprotein, and a small molecule having a positive charge at physiological pH. CNI- 1493 and the genus of structurally related compounds are positively charged at physiological pH by virtue of the presence of multiple guanylhydrazone moieties as provided in the chemical formula herein. Fetuin and α2- HS glycoprotein are negatively charged by virtue of being highly glycosylated and having multiple terminal sialic acid residues in many of the N-linked and O-linked oligosaccharide chains. Therefore, a complex forms between the positively charged small molecule having the / fa guanylhydrazone moiety and the negatively charged sialic acid moieties of fetuin and α2-HS glycoprotein. Figure 10 shows that asialofetuin (fetuin lacking terminal sialic acid residues) at concentrations of 1.0 and 2.5 μg/ml was not nearly as active as fetuin at the same 1.0 and 2.5 μg/ml concentrations in enhancing CNI-1493 (2.5 μM)therapeutic activity (measured by suppression of urea production in LPS stimulated non-responsive RAW 264.7 cell cultures. Figure 11 shows that low concentrations of CNI- 1493 (e.g., 50 nM) were active in suppressing TNF production in LPS-stimulated human PBMCs (peripheral blood mononuclear cells) and that this activity was enhanced, in a dose-dependent fashion, by co-administration of human fetuin (Sigma) at concentrations of either 1.0 μg/ml or 10 μg/ml along with CNI- 1493. These data show the synergistic importance of the inventive combination of the positively charged small molecule guanylhydrazone-containing compound (in this case a multi-valent guanylhydrazone) in combination with the negatively charged fetuin or α2-HS glycoprotein at physiological pH. Figure 12 shows a comparison of CNI- 1493 therapeutic activity in LPS-challenged mice as measured by TNF seram levels. LPS-challenged mice pretreated with human fetuin at concentrations and followed by CNI- 1493 exhibited lower TNF concentrations following LPS challenge. The CNI-1493 dose was 2.5 mg/kg (ip) and the LPS challenge concentration was 13 mg/kg which is the approximate LD 50 of LPS in mice. The human fetuin pretreatment dose was 20 mg/kg. These data show that fetuin pretreatment greatly enhanced CNI- 1493 therapeutic activity as measured by suppression of TNF levels in an in vivo model and support the therapeutic importance of the inventive complex and combination in an in vivo predictive therapeutic model. Figure 13 shows a comparison of CNI- 1493 activity in RAW cells measuring nitrite production comparing CNI- 1493 alone (2.0 μM for all CNI- 1493 concentrations) and when adding 2.5 μg/ml of fetuin or other glycoproteins (glycophorin, αl-acid glycoprotein, albumin, or human glycoprotein). These other glycoproteins are also net negatively charged at physiological pH. These data show that only fetuin was effective in augmenting CNI- 1493 therapeutic activity, proving the specific importance of fetuin and α2-HS glycoprotein, as opposed to other net negatively charged glycoprotein. SEQUENCE LISTING (1) GENERAL INFORMATION (I) APPLICANT: Tracey, Kevin et al . (ii) TITLE OF INVENTION: Complexes and Combinations of Fetuin with Therapeutic Agents (iϋ) NUMBER OF SEQUENCES: 4 (IV) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Oster, Jeffrey B. (B) STREET: THE PICO ER INSITTUTE FOR MEDICAL RESEARCH (C) 350 Community Drive (C) CITY: Manhasset (D) STATE: New York n (E) ZIP: 11030 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Pentium PC (C) OPERATING SYSTEM: Windows 95 (D) SOFTWARE: Word (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) ATTORNEY INFORMATION: (A) NAME: Oster, Jeffrey B. (B) REGISTRATION NUMBER: 32,585 (C) REFERENCE/DOCKET NUMBER: 0106 (viii) TELECOMMUNICATION INFORMATION (A) TELEPHONE: (516) 562 9404 (B) TELEFAX: (516) 365 7919 ) INFORMATION FOR SEQ ID NO: 1: (1) SEQUENCE CHARACTERISTICS : (A) LENGTH: 359 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: protein (iϋ ) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: N-terminal fragment ( i) ORIGINAL SOURCE: (A) ORGANISM: human (ix) SEQUENCE DESCRIPTION: SEQ ID NO: : MKSFVLLFCL AQLWGCHSIP LDPVAGYKEP ACDDPDTEQA ALAAVDYINK HLPRGYKHTL NQIDSVKVWP RRPTGEVYDI EIDTLETTCH VLDPTPLANC 1 SVRQQTQHAV EGDCDIHVLK QDGQFSVLFT KCDSSPDSAE DVRKLCPDCP 1 LLAPLNDSRV VHAVEVALAT FNAESNGSYL QLVEISRAQF VPLPVSVSVE 1 FAVAATDCIA KEVVDPTKCN LLAEKQYGFC KGSVIQKALG GEDVRVTCTL 1 FQTQPVIPQP QPDGAEAEAP SAVPDAAGPT PSAAGPPVAS VVVGPSVVAV 1 PLPLHRAHYD LRHTFSGVAS VESSSGEAFH VGKTPIVGQP SIPGGPVRLC 1 PGRIRYFKI 359 INFORMATION FOR SEQ ID NO: 2: (1) SEQUENCE CHARACTERISITCS : (A) LENGTH: 367 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: N-terminal fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: human (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: MKSLVLLLCL AQLWGCHSAP HGPGLIYRQP NCDDPETEEA ALVAIDYINQ 1 # 1 NLPWGYKHTL NQIDEVKVWP QQPSGELFEI EIDTLETTCH VLDPTPVARC 01 SVRQLKEHAV EGDCDFQLLK LDGKFSVVYA KCDSSPDSAE DVRKVCQDCP 51 LLAPLNDTRV VHAAKAALAA FNAQNNGSNF QLEEISRAQL VPLPPSTYVE 01 FTVSGTDCVA KEATEAAKCN LLAEKQYGFC KATLSEKLGG AEVAVTCTVF 51 QTQPVTSQPQ PEGANEAVPT PVVDPDAPPS PPLGAPGLPP AGSPPDSHVL 01 LAAPPGHQLH RAHYDLRHTF MGVVSLGSPS GEVSHPRKTR TVVQPSVGAA 51 AGPVVPPCPG RIRHFKV 367 (2) INFORMATION FOR SEQ ID NO: 3: (1) SEQUENCE CHARACTERISITCS : (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: uncertain (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: PLDPVAGYKE PAXDEXETEQ AALA 24 (2) INFORMATION FOR SEQ ID NO: 4: (1) SEQUENCE CHARACTERISITCS: (A) LENGTH: 25 amino acids (B) TYPE: amino t acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: bovine (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 1 IPLDPVAGYK EPACDDPDTE QAALA 25 l°l

Claims: We claim: 1. A complex comprising: a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and a positively charged (at physiological pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R^^ X 2 ^ X ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,^ alkyl-substituted purine, xanthine, pyrimidine, C,. 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH,(CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 _ 10 alkyl, straight or branched C 2-10 alkenyl, -A- straight or branched C 2 . 10 alkyl -A-, -A- straight or branched C 2 . 10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. 2. The complex of claim 1 wherein the glycosolated polypeptide is human fetuin. 3. The complex of claim 1 wherein X,, X'„ X 2 and X\ are each NH 2 (CHN)-NH- N=C(CH 3 )-. 4. A pharmaceutical composition comprising: (A) an active component consisting essentially of a glycosylated polypeptide selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and (B) a positively charged (at physiological pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R X 2 X ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C,_ 6 alkyl-substituted purine, xanthine, pyrimidine, C,_ 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH= H-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X„ X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 . 10 alkyl, straight or branched C 2 . 10 alkenyl, -A- straight or branched C 2-10 alkyl -A-, -A- straight or A O branched C 2 . 10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-; and (C) a pharmaceutically acceptable carrier. 5. The pharmaceutical composition of claim 4 wherein the glycosolated polypeptide is human fetuin. 6. The pharmaceutical composition of claim 4 wherein X l5 X' l5 X 2 and X' 2 are each NH 2 (CHN)-NH-N=C(CH 3 )-. 7. A therapeutic combination of a glycosylated polypeptide and a positively charged (at physiological pH) therapeutic compound: wherein the glycosylated polypeptide is selected from the group consisting of a mammalian fetuin, α2-HS glycoprotein, and combinations thereof; and wherein the positively charged (at physiological pH) therapeutic compound selected from the formula: Xi X ' ι R - Z - R X 2 ' 2 wherein R is independently selected from the group consisting of phenyl, pyridine, amino- substituted pyridine, purine, oxo-substituted purine,C,. 6 alkyl-substituted purine, xanthine, pyrimidine, C,. 6 alkyl-substituted pyrimidine, and oxo-substituted pyrimidine; wherein X 2 is selected from the group consisting of NH 2 (CHN)-NH=CH-, NH 2 (CHN)-NH-N=C(CH 3 )-, and H-; wherein X,, X', and X' 2 are independently selected from the group consisting of NH 2 (CHN)-NH=CH -, and NH 2 (CHN)-NH-N=C(CH 3 )-; wherein Z is selected from the group consisting of phenyl, pyrimidine (C 5 NH 3 ), -NH(CO)NH-, straight or branched C 2 . 10 alkyl, straight or branched C 2 . 10 alkenyl, -A- straight or branched C 2 _ ]0 alkyl -A-, -A- straight or branched C 2 . 10 alkenyl -A-, and -A-, wherein "A" is independently selected from the group consisting of -NH(CO)-, -(CO)NH-, -NH(CO)NH-, -NH-, and -O-. 8. The therapeutic combination of claim 7 wherein the glycosolated polypeptide is human fetuin 9. The therapeutic combination of claim 7 wherein X,, X',, X 2 and X' 2 are each NH 2 (CHN)-NH-N=C(CH 3 )-. 10. A method for finding therapeutically active small molecule compounds having a net positive charge at physiologic pH, comprising: (a) providing a fetuin glycoprotein in a buffer solution at physiologic pH, wherein the fetuin glycoprotein oligosaccharide moiety contains a plurality of sialic acid residues; (b) contacting a small molecule compound in solution or a library of multiple small molecule compounds in solution to the fetuin glycoprotein at physiologic pH; and (c) measuring whether the small molecule compound or a small molecule compound from the library of small molecule compounds had bound to the fetuin glycoprotein. 11. The method of claim 10 wherein the fetuin glycoprotein is bound to a solid substrate support. 12. The method of claim 10 wherein the small molecule compound is bound to a solid substrate support. 13. The method of claim 10 wherein the method allows a compound to discover proteins that bind to the small molecule compound. 4

Inventors: TRACEY KEVIN J, WANG HAICHAO, TRACEY, KEVIN, J., WANG, HAICHAO
Applicants: PICOWER INST MED RES, THE PICOWER INSTITUTE FOR MEDICAL RESEARCH
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, A61K45, C07K14