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Publication number | US5637311 A |
Publication type | Grant |
Application number | US 08/496,810 |
Publication date | Jun 10, 1997 |
Filing date | Jun 29, 1995 |
Priority date | Jun 29, 1995 |
Fee status | Lapsed |
Also published as | WO1997001559A1 |
Publication number | 08496810, 496810, US 5637311 A, US 5637311A, US-A-5637311, US5637311 A, US5637311A |
Inventors | Alexander J. Pallenberg |
Original Assignee | Procyte Corporation |
Export Citation | BiBTeX, EndNote, RefMan |
Patent Citations (2), Non-Patent Citations (20), Referenced by (19), Classifications (16), Legal Events (6) | |
External Links: USPTO, USPTO Assignment, Espacenet | |
This invention is generally directed to zinc(II) complexes and methods relating to the use thereof and, more specifically, to zinc(II) complexed by a multi-dentate ligand.
Zinc is found in both plants and animals and over 100 zinc-containing proteins and enzymes have been identified (Chaney, Textbook of Biochemistry with Clinical Correlations, pp. 1115-1147, Devlin (ed.), New York, New York, Wiley-Liss, 1992). Examples of zinc-containing enzymes include carbonic anhydrase (hydration of carbon dioxide in red blood cells), carboxypeptidase A (pancreatic peptidase), NAD-dependent dehydrogenases (alcohol dehydrogenase in liver), leucine aminopeptidase (peptidase in kidney and gastric mucosa), pyruvate carboxylase (citric acid cycle component), and leukotriene A4 hydrolase (synthesis of lipid mediators in neutrophils) (White et al., Principles of Biochemistry, New York, N.Y. McGraw-Hill Book Company, 1973). Zinc also serves as an important structural component of many proteins such as DNA binding proteins in a structure commonly termed "zinc fingers" (Schultz et al., Textbook of Biochemistry with Clinical Correlations, pp. 91-134, Devlin (ed.), New York, N.Y., Wiley-Liss, 1992).
Zinc compounds, primarily zinc salts, have shown utility in a number of areas. Examples are wound healing (Agren, Acta. Derm. Venereol. Supp. (Stockh) 154: 1-36, 1990; Pastorfide et al., Clin. Ther. 11: 258-63, 1989), healing of gastric ulcers (Frommer, Med. J. Aust. 2: 793-96, 1975), inhibition of leukotriene A4 hydrolase (prevention of the formation of lipid mediators of inflammation) (Wetterholm et al., Arch. Biochem. Biophys. 311: 263-71, 1994), and the inhibition of certain viruses such as human immunodeficiency virus (HIV) (Bridget et al., J. Med. Chem. 38: 366-78, 1995), inhibition of the HIV protease (Zhang et al., Biochemistry 30: 8717-21, 1991), herpes virus (Kumel et al., J. Gen. Virol. 71: 2989-97, 1990; Fridlender et al., Virology 84: 551-54, 1978; Gordon et al., Antimicrob. Agents Chemother. 8: 377-80, 1975), vaccinia virus (Katz et al., Antimicrob. Agents Chemother. 19: 213-17, 1981; Zaslavsky et al., J. Virol. 29: 405-48, 1979), foot and mouth disease virus (Firpo et al., Arch. Virol. 61: 175-81, 1979), and rhino virus(Korant et al., J. Virol. 71: 2989-97, 1976).
In addition to zinc(II) salts, a number of zinc(II) complexes have been made and characterized. However, in most instances such zinc(II) complexes have merely been studied to determine the coordination geometry of the metal, or luminescence thereof. For example, Jordan et al. (Inorg. Chem. 30: 4588-93, 1991) report the structural dependence of the luminescence from bis(substituted benzenethiolato) (2,9-dimethyl-1,10-phenanthroline) zinc(II) coplexes, while Monge (Acta Cryst. B33: 2329-31, 1977) reports the crystal structure of a (dicyanide)(2,9-dimethyl-1,10-phenan-throline) zinc(II) complex. Other researchers have reported zinc(II) complexes with 1,10-phenanthroline, but not for use as biologically active compounds (Fitzgerald et al., J. Chem. Soc. Dalton Trans. 141-49, 1985; Romero et al., Polyhedron 10: 197-202, 1991; Bell et al. (Inorganica Chimica Acta. 156: 205-11, 1989; Reimann et al., Inorg. Chem. 5: 1185-89, 1966; Bencini et al., Inorg. Chem. 28: 1963-69, 1989; Hu and Liu, Acta Cryst. C47: 2326-33, 1991; Cremers et al., Acta Cryst. B36: 3097-99, 1980).
While the use of zinc salts appear promising for use in certain therapeutic areas, there is still a need in the art for additional zinc-containing compounds, complexes or compositions which possess biological activity. The present invention fulfills this need, and provides further related advantages.
This invention is generally directed to zinc(II) complexes and methods relating thereto. More specifically, the zinc(II) complexes of the present invention comprise zinc(II) complexed by a multi-dentate ligand.
The zinc(II) complexes have utility for the inhibition of lipid mediators of inflammation, and for inhibiting viral activity and infection, including (but not limited to) HIV replication in an HIV-infected animal. Methods of the present invention comprise administering to an animal in need thereof an effective amount of a zinc(II) complex.
Other aspects of this invention will become evident upon reference to the attached figures and following detailed description. All references identified herein are hereby incorporated by reference in their entirety
FIG. 1 is the x-ray crystal structure of a representative zinc(II) complex of this invention (i.e., zinc(II):neocuproine (2:1)).
FIG. 2 is the absorption spectrum of a further representative zinc(II) complex of this invention (i.e., zinc(II):bathocuproine disulfonic acid ("BCDS") (2:1)), and presents the absorption spectrum of BCDS for comparison.
This invention is generally directed to zinc(II) complexes. Zinc is almost always found in the +2 oxidation state, in which it is invariably quite stable. (See, generally, Cotton and Wilkinson, Advanced Inorganic Chemistry, 5th ed., John Wiley & Sons, New York, pp. 503-527, 1988). Zinc(II) has previously been reported to have four coordination sites, and to generally possess a tetrahedral configuration.
In general, chelating agents are coordination compounds in which a single ligand occupies more than one coordination position of a metal ion. If the ligand occupies two coordination positions, it is considered a bi-dentate ligand; if more than two coordination positions are occupied by the ligand, it is considered a poly-dentate ligand (such as a tri-dentate ligand or a tetra-dentate ligand). As used herein, a "multi-dentate ligand" is a bi-, tri- or tetra-dentate ligand which occupies two, three or four coordination sites, respectively, of zinc(II).
Any multi-dentate ligand which chelates zinc(II) to yield a zinc(II) complex is suitable in the practice of this invention. However, in a preferred embodiment, the multi-dentate ligands of this invention are selected from the following general structures I through VII: ##STR1## wherein A and B represent heteroatoms which may occupy coordination sites of zinc(II), and are preferably selected from nitrogen, oxygen, sulfur and phosphorous.
The rings of structures I through VII may be aromatic, non-aromatic or a mixture of both aromatic and non-aromatic rings. For example, the following structures are representative of such combinations: ##STR2##
Representative examples of multi-dentate ligands of this invention having structures I through VII are set forth in Table 1. Specifically, Table 1 identifies the structure of the representative multi-dentate ligand, lists the corresponding chemical name, identifies the Chemical Abstracts Registration Number ("CA Reg. No."), and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the identified multi-dentate ligand.
TABLE 1__________________________________________________________________________Structure Name CA Reg. No. Reference__________________________________________________________________________ ##STR3## benzo (2,1-b:3,4-b) dithiophene 211-53-0 Sturaro et al., Heterocycl. Chem. 27:1867, 1990 ##STR4## benzo (2,1-b:3,4-b) difuran 211-47-2 Rene et al., Eur. J. Med. Chem.-Chim. Ther. 13:435, 1978 ##STR5## thieno (3,2-g) benzofuran 438-31-9 Cagniant and Kirsch, Hebd. Seances Acad. Sci. C. 282: 465, 1976 ##STR6## 2H-furo (3,2-g) indole 103671-62-1 Lawrence Jr., Eur. Pat. Appl. EP 173,520, 1986 ##STR7## 2H-benzo (2,1-b:3,4-b') dipyrrole 112149-08-3 Berlin et al., J. Chem. Soc. Chem. Commun. (15):1176, 1987 ##STR8## 1H-cyclopenta (2,1-b:3,4-b') bipyridine 42262-29-3 ##STR9## 1,10-phenanthroline 66-71-7 ##STR10## furo (3,2-h) quinoline 234-28-6 ##STR11## 2,2'-bipyridyl 366-18-7__________________________________________________________________________
In structures I through VII above, further ring substitutions with heteroatoms are permitted. Preferably, such heteroatoms are selected from nitrogen, oxygen, sulfur, and phosphorus. For example, the compounds listed in Table 2 illustrate further representative multi-dentate ligands of the present invention having additional ring substitutions. As with Table 1, Table 2 identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the identified multi-dentate ligand.
TABLE 2__________________________________________________________________________Structure Name CA Reg. No. Reference__________________________________________________________________________ ##STR12## furano (3,2-g) benzoxazole 25885-39-6 ##STR13## furano (2,3-e) benzoxazole 66037-80-1 Turin et al., Fr. Demande 2,338,041, 1977 ##STR14## thieno (3,2-g) benzoxazole 58188-85-5 Iddon et al., J. Chem. Soc., Perkin Trans. I 17:1686, 1975 ##STR15## thieno (3,2-g) benzothiazole 72121-58-5 ##STR16## thieno (2,3-e) benzothiazole 211-36-9 ##STR17## benzo (1,2-d:3,4-d') bis (1,3) dioxide 211-50-7 Dallacker and Weiner, Justus Liebigs Ann. Chem. 725:99, 1969 ##STR18## benzo (1,2-d:3,4-d') diimidazole 211-10-9 ##STR19## pyrrolo (2,3-e) benzimidazole 53068-46-5 Chetverikov et al., U.S.S.R. 425,906, 1974 ##STR20## benzo (2,1-d:3,4-d') bis (1,3) oxathiole 211-54-1 ##STR21## 2H-imidazo (4,5-e) benzothiazole 42341-40-2 ##STR22## 2H-imidazo (4,5-g) benzothiazole 211-23-4 ##STR23## 1,3-dioxolo (4,5-e) benzothiazole 77482-58-7 Foerster et al., Ger. Offen. 2,903,966, 1980 ##STR24## benzo (1,2-d:3,4-d') bisthiazole 211-37-0 ##STR25## benzo (2,1-d:3,4-d') bisthiazole 23147-19-5 ##STR26## benzo (1,2-d:4,3-d') bisthiazole 10558-80-2 Grandolini et al., Ann. Chim. 58:91, 1968 ##STR27## thiazolo (5,4-e) benzoxazole 211-35-8 ##STR28## thiazolo (5,4-g) benzoxazole 51273-21-3 ##STR29## thiazolo (4,5-e) benzoxazole 315-47-9 ##STR30## thiazolo (4,5-f) benzoxazole 67239-73-0 Fridman et al., Ikr. Khim. Zh. 44:399, 1978 ##STR31## benzo (2,1-d:3,4-d') bisoxazole 211-19-8 ##STR32## benzo (1,2-d:3,4-d') bisoxazole 211-20-1 ##STR33## benzo (1,2-d:4,3-d') bisoxazole 54935-19-2 Barker et al., J. Chem. Res. Synop. (9):328, 1986 ##STR34## furo (2,3-d) thieno (3,2-b) pyridine 110665-19-5 ##STR35## 1H-imidazo (4,5-d) thieno (3,2- b)-pyridine 111163-54-3 Takada et al., Eur. Pat. Appl. EP 223,420, 1987 ##STR36## dithieno (3,2-b:2',3'-d) pyridine 40826-38-8 Yang et al., Synthesis 2:130, 1989; Heeres et al., Syn. Commun. 2:365, 1972 ##STR37## 5H-oxazolo (4,5-e) thiazolo (3,2-c) pyrimidine 211-46-1 ##STR38## dithieno (3,2-c:2',3'-e) pyridazine 51974-92-6 Nonciaux et al., Bull. Soc. Chim. Fr. 12 Pt 2, 3318, 1973 ##STR39## 1H-(1,2,4) triazolo (5,1-b) purine 387-96-2 ##STR40## bis (1,2,4) triazolo (1,5-d:5', 1'-c) pyrazine 55366-22-8 Vercek et al., Tetrahedron Lett. (51/52):4539, 1974 ##STR41## benzo (2,1-b:3,4-b') dipyran 231-29-8 Monatsch 80:743, 1949 ##STR42## benzo (1,2-b:4,3-b') bis (1,4)- oxathiin 231-34-5 ##STR43## benzo (1,2-e:3,4-e') dipyrazine ##STR44## benzo (1,2-d:3,4-d') diimidazole 211-10-9 ##STR45## pyrazino (2,3-f) quinoxaline 231-23-2 Shim et al., Synthesis 2:116, 1980; Nasielski-Hinkins et al., J. Chem. Soc. Perkin Trans. 1:1229, 1975 ##STR46## bis (1,2,4) oxadiazolo (2,3-d:3', 2'-c) pyrazine 74382-83-5 ##STR47## (1,2,4)-oxadiazolo (3,2-i) purine 56248-95-4 Miura et al., Chem. Pharm. Bull. 23:464, 1975 ##STR48## bis (1,2,4) triazolo (1,5-b:5',1'-f) pyridazine 51519-32-5 Polanc et al., J. Org. Chem. 39:2143, 1974 ##STR49## bis (1,2,4) triazolo (1,5-d:1', 5'-c) pyrimidine 76044-62-7 Brown and Shinozuka, Aust. J. Chem. 33:1147, 1980__________________________________________________________________________
General structures I through VII identified above may possess further chemical moieties covalently attached to the structural backbone, as illustrated below: ##STR50## wherein R1 through R8 are the same or different, and are selected from the following chemical moieties: --H, --OH, --X, --OX, --XOH, --COOH, --COOX, --CHO, --CXO, --F, --Cl, --Br, --I, --CN, --NH2, --NHX, --NX2, --PX2, --SO3 H, --SO3 Na, --SO3 K, --SO3 X, --PO3 H, --OPO3 H, --PO3 X, --OPO3 X and --NO2. As used herein, "X" represents and an alkyl moiety or an aryl moiety. An "alkyl moiety" is a straight chain or branched, cyclic or noncyclic, saturated or unsaturated, substituted or unsubstituted carbon chain containing from 1-20 carbon atoms; and an "aryl moiety" is a straight chain or branched, cyclic or noncyclic, saturated or unsaturated, substituted or unsubstituted carbon chain containing at least one substituted or unsubstituted aromatic moiety and containing from 6-20 carbon atoms. Such chemical moieties may also be covalently attached to the ring fusion atoms. Representative examples of the chemical moieties of this invention include, but are not limited to, the moieties identified in Table 3 below.
TABLE 3__________________________________________________________________________H CH3 CH2 BrCH2 OH CH2 Cl CBr3CH2 C6 H5 C6 H5 (CH2)1-12 CH3Cl CHO COOHCOOMe CHNOH CH2 NH2CH2 CCH CHCH2 P(C6 H5)2CH2 CH(CO2 H)2 CON(CH2 COOH)2 CH2 N(CH2 COOH)2 ##STR51## ##STR52## ##STR53##PhSO3 Na__________________________________________________________________________
Representative examples of the multi-dentate ligands possessing further chemical moieties covalently attached to the structural backbone of structures I through VII are presented in Table 4. In particular, Table 4 identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the multi-dentate ligand.
TABLE 4__________________________________________________________________________Structure Name CA Reg. No. Reference__________________________________________________________________________ ##STR54## 2,2'-bipyridine-4,4'-dicarboxylic acid 6813-38-3 ##STR55## 2,2'-bis (4,5-dimethylimidazole) 69286-06-2 J. Organomet. Chem. 307:39, 1986 ##STR56## 2,3-bis (2-pyridyl) pyrazine 25005-96-3 (Aldrich: 28,164-16) ##STR57## 5,5'-dimethyl-2,2'-bithiophene 16303-58-5 ##STR58## 6,6'-dimethyl-2,2'-dipyridine 4411-80-7 Kauffmann et al., Chem. Ber. 109:3864, 1976__________________________________________________________________________
The chemical moieties covalently attached to the structural backbone may be joined to yield an aromatic or nonaromatic cyclic chemical moiety. Representative examples of such cyclic chemical moieties are set forth in Table 5, which identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the multi-dentate ligand.
TABLE 5__________________________________________________________________________Structure Name CA Reg. No. Reference__________________________________________________________________________ ##STR59## 6,7-dihydro-5,8-dimethyl dibenzo (b) (1, 10) phenanthroline 5298-71-5 ##STR60## bibenzimidazole 123067-51-6 ##STR61## 2,2'-bisquinoline 119-91-5 (Aldrich: B3,540-7)__________________________________________________________________________
The synthesis of representative examples of the multi-dentate ligands of this invention are disclosed in Table 6 and Table 7 below. Specifically, in these tables the structure of the multi-dentate ligands are identified along with their CA Reg. No. and one or more references disclosing their synthesis and/or chemistry.
TABLE 6__________________________________________________________________________Synthesis of Ligands for Representative Zinc (II) Complexes Having theStructure: ##STR62##R1 R8 CA Reg. No. Reference__________________________________________________________________________CH3 CH3 484-11-7 O'Reilly et al., Aust. J. Chem. 13:145, 1960CH2 Br CH2 Br 78831-37-5 Weijen et al., J. Org. Chem. 57:72358, 1992; Jukkala et al., Helv. Chim. Acta. 75:1621, 1992; Chandler et al., J. Heterocycl. Chem. 18:599, 1981CH2 Br CH2 OH 142470-16-4 Weijen et al., J. Org. Chem. 57:7258, 1992CBr3 CBr3 Chandler et al., J. Heterocycl. Chem. 18:599, 1981CH2 Cl CH2 Cl Newkome et al., J. Org. Chem. 50:3807, 1985; Newcome et al., J. Org. Chem. 48:5112, 1983CCl3 CCl3 Chandler et al., J. Heterocycl. Chem. 18:599, 1981; Newcome et al., J. Org. Chem. 48:5112, 1983CN CN 57709-63-4 Chandler et al., J. Heterocycl. Chem. 18:599, 1981; Sjoegren et al., Organometallics 11:3954, 1992CH2 C6 H5 CH2 C6 H5 223-20-1 Sjoegren et al., Organo- metallics 11:3954, 1992(CH2)11 CH3 (CH2)11 CH3 Menger et al., J. Am. Chem. Soc. 113:4017, 1991(CH2)3 CH3 (CH2)3 CH3 85575-93-5P Sugihara et al., JP 02096578 A2, Jpn. Kokai Tokkyo Koho 113(15):132159v(R3 = R6 = H, Ph(CH2)3 CH3 (CH2)3 CH3 Delton et al., EP 339973 A1, Eur. Pat. Appl. 112(21):19835p, 1989(R4 = R5 = CH3)Cl Cl 29176-55-4 Sjoegren et al., Organo- metallics 11:3954, 1992; Delton et al., EP 33993 A1, Eur. Pat. Appl. 112(21):19835p, 1989CH2 OH CH2 OH 78831-36-4 Chandler et al., J. Heterocycl. Chem. 18:599, 1981; Delton et al., EP 339973 A1, Eur. Pat. Appl. 112(21):19835p, 1989; Newcome et al., J. Org. Chem. 48:5112, 1983CHO CHO 57709-62-3 Ziessel, Tetrahedron Lett. 30: 463, 1989; Toner, EP 288256 A2, Eur. Pat. Appl. 111(15): 130322c; Bell et al., J. Inclu- sion Phenom. 5:149, 1987COOH COOH Chandler et al., J. Heterocycl. Chem. 18:599, 1981COOMe COOMe Chandler et al., J. Heterocycl. Chem. 18:599, 1981; Newcome et al., J. Org. Chem. 48:5112, 1983CHNOH CHNOH Chandler et al., J. Heterocycl. Chem. 18:599, 1981CH2 NH2 CH2 NH2 Chandler et al., J. Heterocycl. Chem. 18:599, 1981CHO H 33795-37-8 Toner, EP 288256 A2, Eur. Pat. Appl. 111(15):130322cCOOH H 1891-17-4 Toner, EP 288256 A2, Eur. Pat. Appl. 111(15):130322cCH2 CCH CH2 CCH Sjoegren et al., Organo- metallics 11:3954, 1992C6 H5 C6 H5 Dietrich-Buchecker et al., Tetrahedron Lett. 23:5291, 1982Cl CH3 Newcome et al., J. Org. Chem. 54:1766, 1989CHCH2 CHCH2 Newkome et al., J. Org. Chem. 50:3807, 1985P(C6 H5)3 P(C6 H5)3 Ziessel, Tetrahedron Lett. 30:463, 1989CH2 CH(CO2 H)2 CH2 CH(CO2 H)2 Newcome et al., Inorg. Chem. 24:811, 1985 ##STR63## ##STR64## Weijen et al., J. Org. Chem. 57:7258, 1992 ##STR65## ##STR66## Weijen et al., J. Org. Chem. 57:7258, 1992CH2 OH ##STR67## Weijen et al., J. Org. Chem. 57:7258, 1992 ##STR68## ##STR69## Weijen et al., J. Org. Chem. 57:7238, 1992CH2 N(CH2 COOH)2 CH2 N(CH2 COOH)2 Mukkala et al., Helv. Chim. Acta 75:1621, 1992; Toner, EP 288256 A2, Eur. Pat. Appl. 111(15):130322cCON(CH2 COOH)2 CON(CH2 COOH)2 Toner, EP 288256 A2, Eur. Pat. Appl. 111(15):130322cCH3 CH3 52698-84-7 Blair et al, Talanta 7:163, 1961(R3 = R6 = PhSO3 Na__________________________________________________________________________
TABLE 7__________________________________________________________________________Synthesis of Representative Zinc (II) Complexes Having the Structure: ##STR70##R1 R8 CA Reg. No. Reference__________________________________________________________________________CN CN 4411-83-0 Sjoegren et al., Organometallics 11:3954, 1992CH2 Cl CH2 Cl 74065-64-8 Bell et al., J. Inclusion Phenom. 5:149, 1987CHO CHO Newkome et al., J. Org. Chem. 50:3807, 1985CHCH2 CHCH2 Newkome et al., J. Org. Chem. 50:3807, 1985(R1 and R2 = (R7 and R8 = 119-91-5 (Aldrich: B3,540-7)benzo moiety) benzo moiety)__________________________________________________________________________
In one embodiment of this invention, the multi-dentate ligands are selected from the following structures: ##STR71## wherein R1 through R8 are the same or different, and are selected from hydrogen, an alkyl moiety and an aryl moiety.
In a preferred embodiment, the multi-dentate ligand is 6,6'-dimethyl-2,2'-dipyridine having structure Id: ##STR72##
In a further preferred embodiment, the multi-dentate ligand is neocuproine (2,9-dimethyl-1,10-phenanthroline) having structure IId, or is bathocuproine disulfonic acid ("BCDS") having one of the isomeric structures IIe, IIe', IIe" or IIe"': ##STR73## Unless otherwise indicated, BCDS refers to a physical mixture of the above isomers (i.e., IIe, IIe', IIe" and IIe"'). Typically, the ratio of the various isomers (i.e., IIe:IIe':IIe") vary depending upon the commercial source of BCDS as follows: Aldrich Chemical Co., Inc. (Milwaukee, Wis.) 9.1:38.6:41.2; Spectrum Chemical Manufacturing Corp. (Gardena, Calif.) 8.5:39.7:45.2; GFS Chemicals (Columbus, Ohio) 8.4:38.5:45.3; Janssen Pharmaceutica (subsidiary of Johnson & Johnson) (Beerse, Belgium) 4.6-8.7:36.4-39.4:44.4-55.9; with the IIe"' isomer present in commercial sources in only trace amounts (i.e., typically about 1%).
As discussed above, zinc(II) complexes of this invention may be made by contacting a multi-dentate ligand with a zinc(II) source. The multi-dentate ligands may be obtained from commercial sources, or may be synthesized by known organic synthesis techniques from commercially available reagents. Preferably, water soluble multi-dentate ligands are complexed with the zinc(II) in aqueous solution, employing Zn(NO3)2 or Zn(SO4) as the zinc(II) source. The resulting zinc(II) complex may then be recovered by evaporation of solvent to yield the zinc(II) complex. Alternatively, if the multi-dentate ligand is not readily soluble in water, zinc(II) complexes may be formed by the above procedure employing a suitable non-aqueous (e.g., organic) solvent.
In the practice of this invention, the ratio of the multi-dentate ligand to zinc(II) may be any ratio which results in a zinc(II) complex. Preferably, the ligand to zinc ratio is at least 1:1, and more preferably at least 2:1. In a further embodiment, the ligand to zinc(II) ratio ranges from 1:1 to 3:1 (including 2:1). Such zinc(II) complexes may be made by the procedures identified in the preceding paragraph by reacting the appropriate molar ratios of the multi-dentate ligand and the zinc(II) ion source.
In the case of zinc(II):neocuproine, the molar ratio of zinc to neocuproine is preferably in excess of 1:1, and more preferably at least 2:1.
When administered to an animal for therapeutic, prophylactic or cosmetic purposes, the zinc(II) complexes of this invention may be readily formulated by techniques known to those skilled in the art. (Preferred zinc(II) complexes of this invention are colorless, which is highly desirable for consumer appeal and acceptance.) For example, the zinc(II) complexes may be combined with one or more suitable carriers or diluents to yield a pharmaceutical preparation suitable for topical, oral or parenteral application- Such diluents or carriers, however, should not interact with the zinc(II) complex to significantly reduce the effectiveness thereof. Effective administration will preferably deliver a dosage of approximately 0.01 to 100 mg of the zinc(II) complex per kg of body weight.
Methods for encapsulating compositions (such as in a coating of hard gelatin) for oral administration are well known in the art (see, e.g., Baker, Richard, Controlled Release of Biological Active Agents, John Wiley and Sons, 1986)(incorporated herein by reference). Suitable carriers for parenteral application (such as intravenous, subcutaneous or intramuscular injection) include sterile water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol) and phosphate-buffered saline. The zinc(II) complexes may be topically applied in the form of liquids, containing acceptable diluents (such as saline and sterile water) or may be applied as lotions, creams or gels, containing additional ingredients to impart the desired texture, consistency, viscosity and appearance. Such additional ingredients are familiar to those skilled in the art and include emulsifying agents such as non-ionic ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, organic or inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, hydrophilic beeswax derivatives, hydrocarbon oils such as palm oil, coconut oil, mineral oil, cocoa butter waxes, silicon oils, pH balancers and cellulose derivatives.
Topical administration may by accomplished by applying an amount of the preparation directly to the desired area. The required dosage will vary according to the particular condition to be treated, the severity of the condition, and the duration of the treatment. Preferably, when the zinc(II) complex is topically applied in the form of a lotion, cream or gel, the preparation may contain about 1% to about 20% of a penetration enhancing agent. Examples of penetration enhancing agents include dimethylsulfoxide (DMSO), urea and eucalyptol. In the case of a liquid preparations for topical application, the concentration of penetration enhancing agent (such as DMSO) may comprise about 30% to about 80% of the preparation.
The zinc(II) complexes of the present invention posses utility as anti-viral agents, and are particularly effective in the inhibition of the AIDS virus. Human acquired immunodeficiency syndrome or "AIDS" is a fatal disease for which there is presently no cure. The disease is believed to be caused by a virus known as the human immunodeficiency virus, commonly referred to as "HIV." The virus is transmitted by HIV-infected individuals through the exchange of bodily fluids. HIV infection results most commonly from sexual contact with an infected partner and the sharing among intravenous drug users of hypodermic syringes previously used by an infected individual. A pregnant HIV-infected mother may infect her unborn child by trans-placental transmission, and HIV-contaminated blood is a possible source of infection for individuals subject to blood transfusion.
HIV infection causes a suppression of the immune system. The immune suppression renders the infected individual vulnerable to a variety of opportunistic infections and conditions that are otherwise kept in balance by a healthy immune system. Fatalities result from HIV infection due to the inability of AIDS patients to respond to treatment of the opportunistic infections and conditions as a consequence of their compromised immune systems. Because the virus may often remain dormant, the manifestation of AIDS from HIV infection may take as long as ten years.
One approach to the treatment of AIDS has targeted the opportunistic infections or conditions which result from HIV infection. The treatment of such infections or conditions, however, is ultimately ineffective and, while prolonging the life of the infected individual, does not treat the underlying HIV infection. A second approach to the treatment of AIDS targets the cause of the disease itself. Because AIDS results from viral infection, it is believed that viral inactivation may ultimately provide a cure. (Materials which are capable of viral inactivation or inhibition are referred to herein as "antiviral agents.")
To understand the mode of action of antiviral agents in the treatment of AIDS, an understanding of the process of HIV infection is necessary. HIV chronically infects specific immune cells known as T-helper cells, which are required for normal immune response. The HIV infected T-helper cells serve as hosts to the virus and facilitate the reproduction of the virus (the process of viral reproduction is commonly referred to as "replication"). After HIV infection, the infected host cell eventually dies, the replicated HIV virus is released, and the infection spreads to additional cells. This cycle continues unabated, depleting the population of T-helper cells and, in time, weakens the immune system to the onset of AIDS symptoms. Because T-helper cells are continuously produced by the body, the population of these cells may be reestablished in the absence of further HIV infection. Therefore, the progression of HIV infection (and the subsequent onset of AIDS) may be arrested by the prevention or inhibition of viral replication, and antiviral agents capable of inhibiting or preventing the replication of HIV should be effective in the treatment of AIDS.
At the genetic level, HIV replication requires the insertion of vital deoxyribonucleic acid ("DNA") into the genome of the host cell. The genome of the host cell consists of the cell's own DNA, and is responsible for the synthesis of materials essential to the cell's own function and proliferation. Once the viral DNA is inserted into the host genome, the host facilitates replication of HIV. The inserted viral DNA is an enzymatic product derived from vital ribonucleic acid ("RNA") and the action of an enzyme known as HIV reverse transcriptase. Inhibition of HIV reverse transcriptase precludes the formation of viral DNA required for insertion into the genome of the host. Vital replication is prevented by the absence of vital DNA in the host cell genome. Antiviral agents which inhibit HIV reverse transcriptase are thus potential therapeutic drugs for treatment of AIDS.
Accordingly, in yet another embodiment of the present invention, antiviral agents are disclosed for inhibiting HIV replication, as well as methods relating to the administration thereof to an HIV-infected patient. The antiviral agents of this invention are the zinc(II) complexes discloses above, and the methods include administration of a therapeutically effective amount of a composition which includes a zinc(II) complex in combination with a pharmaceutically acceptable carrier or diluent. Although not limited by the following theory, the zinc(II) complexes of this invention may enhance transport of zinc(II) into HIV infected cells which, in turn, may inhibit or inactivate the HIV protease and thus inhibit the replication of HIV. As used herein, the term "HIV" includes the various strains of the virus (such as HIV-1 and HIV-2).
Administration of the zinc(II) complexes of the present invention may be accomplished in any manner which will result in a systemic dose of a therapeutically effective amount of the zinc(II) complex to an HIV-infected animal or patient (including human patients). For example, such administration may be by injection (intramuscular, intravenous, subcutaneous or intradermal), oral, nasal, or suppository applications. Typically, preparations of the present invention include zinc(II) complexes in solution for various forms of injection, or in preparations which are formulated for the sustained release of the zinc(II) complexes for oral, nasal, or suppository dosage application and generally include one or more inert, physiological acceptable carriers. As used herein, the term "effective amount" means an amount of the zinc(II) complex which inhibits HIV replication in the patient. Suitable dosages may range from approximately 0.01 to 100 mg of zinc(II) complex per kg body weight.
The zinc(II) complexes of this invention may be screened for their ability to inhibit HIV replication using known techniques. For example, HIV virus replication may be monitored using the Cytopathic Effect (CPE) assay disclosed by Bergeron et al. (J. Virol. 66: 5777-5787, 1992). In this assay, the degree of infection is monitored by the appearance of fused cellular membranes ("syncitium"). Alternatively, assays directed to activity of HIV protease may be employed. For example, the assays and techniques disclosed in the following references may be employed: Ashorn et al., Proc. Natl. Acad. Sci. U.S.A. 87: 7472-7476, 1990; Schramm et al., Biochem. Biophys. Res. Commun. 179: 847-851, 1991; Sham et al., Biochem. Biophys. Res. Commun. 175: 914-919, 1991; and Roberts et al., Science 248: 358-361, 1990.
Inhibition of viral replication by the zinc(II) complexes of this invention may also be due to inhibition and/or prevention of viral entry into a cell. With respect to HIV, for example, the zinc(II) complexes may prevent viral entry by interfering with CD4 receptor binding and membrane fusion. This may be illustrated by observing the inhibitory effect of a zinc(II) complex of this invention on syncytium formation using a virus-free, genetically engineered syncytium formation assay (Fu et al., J. Virol. 7: 3818, 1993). This assay relies upon the molecular recognition of gp120, gp41 and the CD4 receptor to create syncytium. Inhibition of syncytium formation in this assay indicates that the zinc(II) complexes inhibit HIV replication by preventing viral entry, presumably by interacting with the viral proteins gp120 and gp41, and thus prevent and/or inhibit gp120 and gp41 function related to viral binding and membrane fusion. Thus, the zinc(II) complexes of the present invention have utility in preventing and/or inhibiting the spread of HIV to uninfected cells.
Accordingly, in this aspect of the present invention, zinc(II) complexes may be formulated in a manner suitable for application to, for example, the vaginal or rectal mucosa, as well as the penis. Suitable formulations include, but are not limited to, solutions, creams, gels, ointments, foams, suppositories and powders, and may include a variety of additional components such as lubricants, preservatives, carriers and diluents, as well as other active ingredients such as spermacides. Such formulations contain a sufficient quantity of the zinc(II) complex, and are applied to the epithelium of the vaginal mucosa, cervix, anus and/or penis in an amount sufficient to prevent and/or inhibit vital transmission.
In this embodiment, the zinc(II) complexes of the present invention may also serve to prevent and/or inhibit the transmission of sexually transmitted diseases in addition to HIV, including human herpes virus and Hepatitis virus (as well as Chlamydia). The zinc(II) complexes of this invention may also have contraceptive activity.
The zinc(II) complexes of this invention, in addition to inhibiting HIV replication, may also inhibit replication of other viruses. Such viruses include, but are not limited to, human T-cell leukemia (HTLV) I and/or II, human herpes virus (HSV1 and 2), cytomegalo virus (human, hCMV, and murine, mCMV), encephalomyocarditis viruses (HAV, HBV, HCV (EMCV), Epstein Barr virus (EBV), human hepatitis virus (e.g., hepatitis B virus, HBV), Varicella Zoster virus, Rhinovirus, rubella virus, respiratory syncytium virus (RSV), influenza viruses A and B, parainfluenza viruses and adenovirus. One skilled in the art could readily assay the zinc(II) complexes of this invention for their inhibitory activity with regard to these viruses, as well as other viruses.
The following examples are offered by way of illustration, and not by way of limitation.
The examples which follow illustrate the preparation, characterization and utility of certain exemplary embodiments of the zinc(II) complexes of the present invention. To summarize the examples that follow: Example 1 illustrates the synthesis and characterization of zinc(II):neocuproine (2:1); Example 2 illustrates the synthesis and characterization of zinc(II):BCDS (2:1); Example 3 discloses methods for monitoring zinc (II) transport into cells by the zinc(II) complexes of this invention; Example 4 discloses methods for assaying the zinc(II) complexes of this invention for their ability to inhibit synthesis of lipid mediators of inflammation; and Example 5 discloses methods for assaying the zinc(II) complexes of this invention for their ability to function as antiviral agents.
Neocuproine hydrate was used as received from Aldrich Chemical Company ("Aldrich"), having the following properties: mp161� C.-163� C.; 1 H NMR (500MHz, DMSO-d6) δ8.32 (2H, d, J=8.2), 7.85 (2H, s), 7.60 (2H, d, J=8.1), 2.79 (6H, s); 13 C NMR (125MHz, DMSO-d6) δ158.0, 144.6, 136.1, 126.4, 125.3, 123.1, 24.9.
A solution of neocuproine hydrate (4.53 g, 20.0 mmol) in methanol (20 mL) was added to a stirred solution of zinc nitrate hydrate (2.97 g, 10.0 mmol) in methanol (20 mL). A white precipitate formed immediately. This material was collected on a filter, washed with methanol, and dried under vacuum to give 5.66 g (91%) of the monohydrate as a white solid. Recrystallization from aqueous ethanol yielded zinc(II):neocuproine (2:1) as clear plates: mp225.5� C.-226� C.; UV-vis λmax (H2 O) 226 nm (ε=5,185M-1 cm-1), 276 nm (s=2,992), 298 nm (s=1,196); MS m/z (relative intensity) 546 (M(68 Zn)-(NO3)-)+ (7), 545 (M(67 Zn)-(NO3)-)+ (5), 544 (M(66 Zn)-(NO3)-)+ (10), 542 (M(64 Zn)-(NO3)-)+ (15), 334 (30), 209 (100); HRMS calcd for C28 H24 N5 O3 68 Zn (M+ less NO3) 546.1128, found 546.1108; calcd for C28 H24 N5 O3 67 Zn (M+ less NO3) 545. 1150, found 545. 1136; calcd for C28 H24 N5 O3 66 Zn (M+ less NO3) 544.1140, found 544.1119; calcd for C28 H24 N5 O3 64 Zn (M+ less NO3) 542.1171, found 542.1138. Anal. calcd. for C28 H26 N6 O7 Zn: C, 53.90; H, 4.20; N, 13.47. Found: C, 54.15; H, 3.99; N, 13.46.
A clear plate of zinc(II):neocuproine (2:1) was mounted on a glass pin with epoxy and transfered to the diffractometer in a nitrogen stream where collection was done at -90� C. The crystals remained clear with little deterioration. The crystal had approximate dimensions of 0.08 mm�0.36 mm�0.36 mm. Twenty five reflections in the range of 24-32 degrees in two-theta were found, and an orientation matrix was determined providing a unit cell with a volume of 2947 Å3. Reduction of data was carried out by the program XCAD4 and all further work was performed using the PC version of Siemens SHELX. The Laue merging R factor was 1.5% for 504 equivalent reflections with a density of 1.37 with four molecules in the unit cell.
The structure was solved by direct methods, and agreed with the heavy metal location as given by a Patterson function, and the structure was determined from Fourier difference maps. One counter-ion nitrate was found, as well as one nitrate bonded to the zinc atom, giving a five-coordinate complex as illustrated in FIG. 1. Structure determination parameters are summarized in Table 8.
TABLE 8______________________________________Structure Determination Summary______________________________________A. Crystal Data Empirical Formula C30 H32 N6 O4 Zn Color; Habit Clear Plates Crystal Size (mm) 0.08 � 0.36 � 0.36 Crystal System Monoclinic Space Group P2(1)/n Unit Cell Dimensions a = 11.385(2) Å b = 15.440(3) Å c = 17.475(3) Å β = 106.37(3)� Volume 2947.3(15) Å3 Z 4 Formula Weight 606.0 Density (calc.) 1.366 Mg/m3 Absorption Coefficient 0.878 mm-1 F(000) 1264B. Data Collection Diffractometer Enraf-Nonius CAD4 Radiation MoKα (λ = 0.71073 Å) Temperature (K.) 183 Monochromater Highly oriented graphite crystal 2θ Range 2 to 50� Scan Type ω Scan Speed Variable; 1.5 to 5.5�/min in ω Scan Range (ω) 0.06� Background Measurement Stationary crystal and stationary counter at beginning and end of scan, each for 0.5% of total scan time Standard Reflections 2 measured every 2 hr. Index Range 0 ≦ h ≦ 13, -1 ≦ k ≦ 18, -20 ≦ 1 ≦ 19 Reflections Collected 5634 Indep. Reflections 4949 (Rint = 1.85%) Observed Reflections 3419 (F > 4.0σ (F)) Absorption Corrections Semi-EmpiricalC. Solution and Refinement System Used Siemens SHELXTL PLUS Solution Direct Methods Refinement Method Full-Matrix Least-Sqrs. Quantity Minimized Σw (Fo - Fc)2 Extinction Correction X = -0.00018(12), where F* = F[1 + 0.002XF2 /sin(2θ)]-1/4 Hydrogen Atoms Riding model, fixed isotropic U Weighting Scheme w-1 = σ2 (F) + 0.0035F2 No. Parameters Refined 434 Final R Indices (obs. data) R = 4.59%, wR = 6.81% R Indices (all data) R = 7.47%, wR = 8.01 Goodness-of-Fit 1.05 Largest and Mean Δ/σ 1.515, 0.041 Data-to-Parameter Ratio 7.9:1 Largest Difference Peak 0.52 eÅ-3 Largest Difference Hole -0.50 eÅ-3______________________________________
A stock solution of bathocuproine disulfonic acid disodium salt (BCDS) was prepared by dissolving 27.96 mg of bathocuproine disulfonic acid disodium salt (Aldrich) in 100 ml of HPLC-grade methanol. This solution was further diluted 1:10 in methanol for a final concentration of 0.0495 mM.
Stock Zn(NO3)2 was prepared by dissolving 1.0764 g of Zn(NO3)2 �6H2 O (Aldrich) in 25 ml HPLC-grade methanol followed by two consecutive 1:10 dilutions in methanol for a final concentration of 2.274 mM.
Bathocuproine disulfonic acid disodium salt (2.0 mL, 0.0495 mM) was placed in a quartz cuvette equipped with a magnetic stir bar and the absorption spectrum was acquired. To the cuvette was added 55 ml of 2.274 mM Zn(NO3)2 in methanol (1.25 equivalents) using a microliter syringe. The absorption spectrum was acquired with a Hewlett Packard 8452A diode array spectrophotometer and then normalized to correct for the increase in volume.
A spectrum of methanolic Zn(NO3)2 was also acquired as a control. This spectrum did not show any absorbance at wavelengths greater than 260 nm (data not shown).
The spectra of bathocuproine disulfonic acid disodium salt and Zn-bathocuproine disulfonic acid disodium salt (normalized to the same concentration) are presented in FIG. 2. These data indicates that the observed shift in λmax and the corresponding increase in the extinction coefficient at the λmax (at wavelengths greater than 260 nm) is due to complexation of the metal atom by bathocuproine disulfonic acid disodium salt.
Detection of metal ion transport into intact cells may be determined utilizing one or more suitable cell lines (such as RBL-1 cells available from American Type Culture Collection, Rockville, Md.). Cells are cultured under established conditions with and without the addition of a zinc(II) complex of this invention. After 24 or 48 hours incubation with the zinc(II) complex of this invention, the culture media is withdrawn and the cells are harvested and washed several times with fresh media or phosphate buffered saline by centrifugation. The resulting cell pellet is then analyzed for total zinc(II) content by, for example, atomic absorption spectroscopy. Based on this procedure, administration of zinc(II) complexes of this invention are shown to enhance and/or facilitate zinc transport into cells.
Inhibition of leukotriene synthesis can be conveniently shown by incubating enzymes or cell fractions containing enzymes with compounds which can be converted to leukotrienes by the action of these enzymes. For example, Leukotriene A4 (LTA4) hydrolase (also known as LTA4 synthase) is involved in the formation of LTB4 from LTA4. Leukotriene A4 (LTA4) hydrolase is prepared as a crude fraction from rat lung tissue. The sodium salt of LTA4 is used as a substrate and incubated with the enzyme fraction and a zinc(II) complex of this invention for 1 minute at 37� C. The reaction is terminated by the addition of ice cold methanol. The formation of LTB4 is assessed by a specific RIA (Raclmark et al., J. Biol. Chem. 259: 12339-12345, 1984; Kuhl et al., Prostaglandins 31: 1029-1048, 1986; Izumi et al., Biochem. Biophys. Res. Comm. 135: 139-145, 1986).
The inhibition of the formation of lipid mediators of inflammation can also be determined in intact cells. This assay involves incubation of the rat basophilic leukemia cell line RBL-1 with the zinc(II) complexes of this invention, followed by radioimmunoassay (RIA) analysis for a prostaglandin product of the cyclooxygenase (PGF2α) and a leukotriene product of the 5-lipoxygenase (LTB4). Positive and negative controls are also run in each assay (Boschelli et al., J. Med. Chem. 36: 1802-1810, 1993). Using the above methodology, zinc(II) complexes of this invention are shown to inhibit the formation of lipid mediators of inflammation.
A. Inhibition of HIV
The zinc(II) complexes of this invention may be screened for their ability to inhibit human immunodeficiency virus (HIV) replication using known techniques. For example, HIV virus replication may be monitored using the Cytopathic Effect (CPE) assay disclosed by Bergeron et al. (J. Virol. 66: 5777-5787, 1992). In this assay, the degree of infection is monitored by the appearance of fused cellular membranes ("syncitium"). Alternatively, assays directed to activity of HIV protease may be employed. For example, the assays and techniques disclosed in the following references may be employed: Ashorn et al., Proc. Natl. Acad. Sci. U.S.A. 87: 7472-7476, 1990; Schramm et al., Biochem. Biophys. Res. Commun. 179: 847-851, 1991; Sham et al., Biochem. Biophys. Res. Commun. 175: 914-919, 1991; and Roberts et al., Science 248: 358-361, 1990.
In another method, PHA-stimulated peripheral blood mononuclear cells (PBMC) are infected by HIVIIIB in the presence of the zinc(II) complexes of this invention and cultured in the presence of the zinc(II) complexes of this invention for two weeks. The extent of HIV replication is assayed at 1 and 2 weeks by a p24 antigen capture ELISA assay. More specifically, PBMC are stimulated with PHA for 24 to 72 hours in basal medium, containing RPMI-1640, 10% fetal bovine serum, and 50 μg/mL gentamicin, and then cultured overnight in the presence of 250 units/ml IL-2. Treated PBMC are pelleted by centrifugation and resuspended to 0.75�106 /mL in basal medium with appropriate dilutions of the zinc(II) complexes of this invention or with no zinc(II) complexes of this invention added (i.e., control). To each 0.5 mL aliquot of cells, 0.5 mL of appropriate HIV dilution is added. The virus-cell mixture is incubated for 2 hours at 37� C. in a 5% CO2 humidified atmosphere. Following the incubation period, the PBMC are washed twice in phosphate-buffered saline. Cells are resuspended in 5 mL to 7�104 cells/mL in basal medium with (or without) the zinc(II) complexes of this invention. Each cell aliquot is dispensed into four replicate wells of a 48 well tissue culture plate. Cells are fed twice a week with appropriate medium.
At one week and two week culture timepoints the extent of HIV replication is assayed by a p24 antigen capture assay kit (Coulter Corp., Hialeah, Fla.). PBMC are treated with buffered detergent to release viral proteins. The cell extract is absorbed to immunoassay titer plates and p24 is detected by binding of a monoclonal anti-p24 antibody coupled to an enzyme. Following the addition of a chromogenic substrate, the amount of p24 is quantified spectrophotometrically.
B. Inhibition of Other Viruses
Inhibition of other viruses by the zince(II) complexes of this invention may be assayed by similar techniques as described below:
1. VZV (Varicella Zoster Virus)
HFF cells are maintained in monolayer culture. VZV infections are measured by the plaque reduction technique, in which the virus is suspended in a agarose and distributed over the monolayer of HFF. Areas of destruction (plaques) are determined by removing the agarose and staining the remaining monolayer. Virus yield (plaque forming units) is calculated, and the effect of the test drug is reported as EC50, the concentration which reduces viral yield by 50%. Cytotoxicity (CC50 for this assay is determined by neutral red (vital dye) uptake on confluent HFF monolayers or by counting cells in rapidly proliferating HFF cells.
2. MV (Measles Virus)
CV-1 cells are cultured in monolayer, and the cytopathic effect (CPE) of the virus id quantitated by vital dye (Neutral Red Uptake). This quantitative method is used for the determination of the CC50 also. The point of 50% dye uptake is determined for both the infected (EC50 and the uninfected CC50 cells), and the ratio determines the SI.
3. PIF-3 (Parainfluenza Type 3 Virus)
MA-104 cells are used with this virus and the same Neutral Red technique for determining (EC50, CC50 cells, and SI is used as is for MV.
4. RSV (Respiratory Syncytium Virus)
MA-104 cells, and the same Neutral Red Assays are used for RSV as is for MV and PIF-3.
5. IF-A (Influenza A Virus)
The MDCK cell line is used with the same Neutral Red technique as for MV, PIF-3 and RSV.
6. IF-B (Influenza B Virus)
The same technique is used to determine the antiviral effects as with IF-A virus.
7. HCMV (Human Cytomegalovirus)
The same Neutral Red technique is used for HCMV as is for RSV, MV, PIF-3 and the influenza viruses, except that the cells used are HFF.
8. HSV-2 (Herpes Simplex Type 2)
Same technique as HCMV; HFF Cells, Neutral Red for Both EC50 and CC50, with ratio of CC50 /EC50 =SI
9. HBV (Hepatitis B Virus)
The HBV virions released by the cells are quantitated (Korba and Milman, Antiviral Res. 19: 55, 1992) via DNA hybridization to specific radiolabeled HBV DNA fragments. In this technique, the EC90 is determined as the effective concentration of the drug which reduces the yield of HBV DNA by 90%. The CC50 is determined for the drug on uninfected cells, and the SI is the ratio CC50 /EC50.
In the above examples, the abbreviations used are as follows:
HFF--Human Foreskin Fibroblasts, primary fibroblast cultures derived from newborn circumcision skin samples, cultured in vitro;
CV-1--African Green Monkey Kidney Cells cultured in vitro;
MA-104--Embryonic Rhesus Monkey Kidney Cells cultured in vitro;
MDCK--Madin-Darby Canine Kidney Cells cultured in vitro;
2.2.15--Specialized cells for expression of HBV (Korba and Milman, Antiviral Res. 15: 217, 1991), 2.2.15 cells are a derivative of Hep G2 Cells (Human hepatocellular carcinoma) which is engineered with a plasmid containing tandem copies of HBV genomes (Sells et al., PNAS 84: 1005, 1988; and Sells et al., J. Virology 62: 2836, 1987);
EC50 --Effective Concentration50, concentration of a drug which causes a half-maximal biological response (i.e., antiviral effect--the concentration of test substance which restores the culture to 50% viability);
EC90 --Effective Concentration90, concentration of a drug which produces a 90% maximal biological response (for antiviral activity, the concentration which restores the culture to 90% viability);
CC50 --Cytotoxic Concentration50, the concentration of a drug which produces 50% loss of viability, used to describe the cytotoxic or deleterious potential of a drug (also referred to as IC50 or Inhibitory Concentration50); and
SI--Selectivity Index, used to compare compounds with respect to a therapeutic potential by dividing the EC50 by either the CC50 or the CC90.
The zinc(II) complexes of this invention inhibit viral replication in one or more of the above assays, and thus function generally as antiviral agents, and more specifically as antiviral agents to a specific virus. While specific pathogenic viruses are disclosed above for purposes of illustration, other viruses may be assayed by one skilled in the art by known techniques.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.
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US20110183375 * | Jan 25, 2011 | Jul 28, 2011 | Life Technologies Corporation | Metal binding compounds and their use in cell culture medium compositions |
US20110212902 * | May 4, 2011 | Sep 1, 2011 | David Bar-Or | Metal-binding compounds and uses therefor |
US20110212903 * | May 4, 2011 | Sep 1, 2011 | David Bar-Or | Metal-binding compounds and uses therefor |
EP2327713A1 | Sep 29, 2000 | Jun 1, 2011 | DMI Biosciences, Inc. | Metal-binding compounds and uses therefor |
WO2001025265A1 | Sep 29, 2000 | Apr 12, 2001 | Dmi Biosciences, Inc. | Metal-binding compounds and uses therefor |
U.S. Classification | 424/434, 424/641, 556/135, 424/435, 514/332, 424/489, 424/436, 424/443, 424/433, 556/134 |
International Classification | C07D471/04, A61K31/315 |
Cooperative Classification | A61K31/315, C07D471/04 |
European Classification | A61K31/315, C07D471/04 |
Date | Code | Event | Description |
Jan 16, 1996 | AS | Assignment | Owner name: PROCYTE CORPORATION, WASHINGTON Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PALLENBERG, ALEXANDER J.;REEL/FRAME:007771/0252 Effective date: 19950829 |
Dec 8, 2000 | FPAY | Fee payment | Year of fee payment: 4 |
Dec 10, 2004 | FPAY | Fee payment | Year of fee payment: 8 |
Dec 15, 2008 | REMI | Maintenance fee reminder mailed | |
Jun 10, 2009 | LAPS | Lapse for failure to pay maintenance fees | |
Jul 28, 2009 | FP | Expired due to failure to pay maintenance fee | Effective date: 20090610 |


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The Style on How to Get Away With Murder Is So Good, It's Criminal
October 7, 2016
Celebrity Style, How to Get Away With Murder, Screen Style, Uncategorized, Viola Davis
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Full disclosure: while we'll have a bottle of red on hand to toast Olivia Pope's many perfect outfits when Scandal returns on air, right now we'll focus on the cast of How to Get Away With Murder. It's not just the lineup of characters, the thrilling plot twists, or sexy hookups that have us glued to the couch. Like Olivia, Shonda Rimes's latest HBIC, Annalise …

subset: commoncrawl


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Deb, I'm so glad your young man enjoyed this; that's a real vote of confidence! And sorry for the delayed response …
Hearty Vegetable Pot Pie
Everyone loves the nostalgic classic, vegetable pot pie. Many VegKitchen readers have let us know that this mild dish of hearty veggies enclosed in pie crust is on their regular cool-weather meal rotation; it's also a crowd-pleasing vegan option for a Thanksgiving or Christmas menu. Using prepared whole-grain pie crusts makes pot pies a snap to prepare. Adapted from The Vegetarian Family Cookbook by Nava Atlas. Photo credit: Sang An. This recipe makes two pies, for at least 12 servings. For a smaller crowd, to make one pie, halve the recipe.
- 8 medium potatoes
- 2 tablespoons extra-virgin olive oil
- 1 large onion, quartered and finely chopped
- 3 cups diced vegetables of your choice (choose 3 or 4 from among cauliflower, broccoli, carrots, leeks, peas, corn kernels, zucchini, yellow summer squash, mushrooms, kale, etc.)
- 2 tablespoons unbleached white flour
- 1 cup vegetable stock (homemade or store bought)
- ¼ cup nutritional yeast (optional but highly recommended)
- 1½ tablespoons all-purpose seasoning blend (such as Frontier or Mrs. Dash)
- 1 teaspoon dried thyme
- ¼ cup minced fresh parsley
- Salt and freshly ground pepper to taste
- Two 9-inch prepared good-quality pie crusts, preferably whole grain
- 1 cup fine whole grain bread crumbs
- Paprika for topping
- Cook or microwave the potatoes in their skins until done. When cool enough to handle, peel them. Dice four of them and mash the other four coarsely. Set aside until needed.
- Preheat the oven to 350º F.
- Heat the oil in a large skillet. Add the onion and sauté over medium heat until golden. Add the vegetables of your choice, layering quicker-cooking vegetables like peas, corn, and zucchini over longer cooking ones like cauliflower, broccoli, and leeks. Add a bit of water; cover and cook until the vegetables are tender but not overdone, about 5 minutes.
- Sprinkle the flour into the skillet, then pour in the stock. Add the optional nutritional yeast. Cook for a minute or two, stirring constantly. until the liquid thickens.
- Stir in both the diced and mashed potatoes. Heat through gently. Stir in the seasoning blend, thyme, and parsley. Season with salt and pepper. Pour the mixture into the pie crust and pat in.
- Sprinkle the bread crumbs evenly over each pie, then top with a sprinkling of paprika.
- Bake for 35 to 40 minutes, or until the crust is golden. Let the pies stand at room temperature for 10 minutes or so, then cut into wedges and serve.
Nutrition Information:
Per Serving (with broccoli, mushrooms, and carrots): 179 calories; 3.4g fat; 34.5g carbs; 4.8g fiber; 4.5g protein; 107mg sodium
- Browse more of VegKitchen's vegan casserole recipes.
- Explore A Colorful Vegan Christmas (Vegetarians Welcome!) for a complete listing of holiday dinner recipes, menus, and desserts.
- Here are more recipes for plannin a Vegetarian and Vegan Friendly Thanksgiving.
75 comments on "Hearty Vegetable Pot Pie"
Mahima Hi. 5 stars for this recipe!! I wanted to know how to reheat this pie if it was made about 5-6 hours before the meal. Do reply. Thanks a lot!
Kelly This was simply delicious! I used mushrooms, broccoli, peas and zucchini, and it was one of the best vegan main dishes I have made since going vegan almost a year ago. I am going to try to freeze the second pie!
Brenda Anderton I want to make individual pot pies. What would you recommend for the dough?
Chrysla Made this tonight. It was wonderful! My veggie combination was celery, purple onion, leaks, broccoli, and mushrooms. I did use the mashed potatoes but I left out the cubed ones as I did not want an overly starchy pot pie. I also used a generous amount of herbs de Provence instead of Mrs. Dash. I used another pie crust to top instead of bread crumbs. I made a little too much for one pie so I was happy to eat this for dinner while the pie cooked. My non-vegan husband kept nibbling out of my bowl until he finally declared that this dish be put in the regular rotation for the boys and himself. Yet another score on my list of vegan tasty dishes for our family. Thank you!
Heather Hi, I wanted to make this for vegan guests for the holidays. I was wondering if you had suggestions on how to make this gluten free?
Ellen I'm thinking you could add some cooked lentils or black beans to up the protein and fiber. Or some tofu or tempeh. Maybe halve the amount of potatoes to make room? Going to have to try this!
Chen Hi Nava. If using frozen pie crusts, do I need to blind bake them first? TIA!
Gwendolyn Lewis Hi Nava! These pot pies are always a winner! Thanks again for sharing the recipe.
Eileen This was the best vegetable pot pie recipe I have tried.
Patty church I need to freeze and bake another day. Has anyone done this? Do I bake then freeze or freeze it first and then bake? Thanks!
Danielle When using frozen vegetables, do i need to precook them on the stove still? Using frozen peas and carrots. Also, using potatoes and broccoli, if i cut them small enough, can i avoid boiling first?
Mary Does this recipe make one pie or two pies? My daughter is vegetarian, and I wanted to make this for her for her Christmas dinner. If it makes 2, then I could cut the recipe in half to make one, correct?
Nicole I made this for Christmas today. It was a fail 🙁 there was way too much potato and there whole thing was too bland and starchy. I would not recommend this recipe.
Yarm I made this with Christmas dinner! There is NONE left! You will love it! Thank you for the recipe! I did freeze one for another time!
Krysta Wanting to make this for 25 people. Can it be used in large dishes for a single pan?


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Brussels 'march against fear' cancelled due to fear of terrorist attack
A planned solidarity march to be held through the street of Brussels to show solidarity for terror victims has been
Read moreA planned solidarity march to be held through the street of Brussels to show solidarity for terror victims has been
Read moreIslamic State continued their recruitment efforts via social media, this time latching onto the Justin Bieber hashtag to show an
Read moreProsecutors say a French teacher who claimed he was stabbed in the neck by a man shouting "Islamic State!" made
Read moreTwo men are appearing in court for allegedly supplying IS with funds after the mother of a third man found
Read moreNo, not Islamic State in Iraq and Syria. Nutella have denied a 5-year-old girl a personalised jar because her name
Read moreHungover? No worries, just get your mate to phone your work and tell them it's Islamic State and you've put
Read moreGiovanni Gambino, the son of an infamous New York mobster has warned ISIS that the city is under their protection
Read moreReports in the British press suggest that hundreds of ISIS fighters are deserting the caliphate in a dispute over wages.
Read moreOmar Hussein, after leaving Morrisons to become Jihadist Abu Sa'eed Al Britani, has admitted that he misses UK food. He
Read moreDespite beheading prisoners, oppressing women, executing homosexuals and imprisoning those that speak out against them, Saudi Arabia has been named
Read moreFormer Morrisons security guard turned Islamic State Jihadist, Omar Hussain has written a blog post complaining his fellow terrorists are severely
Read more


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...I have thought a lot about the mechanism of developing a concept, presentation, talk and then how to deliver so everyone in the audience understands. Growing up I hated public speaking so I have a good sense of what it takes to get to a place where you would rather be in front of a crowd talking about a topic you care about than anywhere else. I would love to help others find that place too!
...At Harvard I was a Teaching Assistant at the Harvard extension school, working with local residents in a variety of math subjects. And in graduate school, I was a Teaching Assistant in college psychology courses, which made heavy use of math and statistics. After that, my 35-year career has inc...
...I enjoy tutoring and believe that no student should develop a fear of math or science. The material should be taught in a way that is engaging, fun, and student-centered. I am currently working as an AmeriCorps member at the Providence Children's Museum.
...I understand the multiple stresses students are under, especially as they endure the college application process with all its demands. I am eager to help students attain their goals in math. I graduated with honors from Fairfield University with a math degree and education minor.
...I will teach you/your child creatively, find the right approach for you and help you overcome your challenges. My students regularly achieve higher grades and build increased confidence. I have more than 8 years of experience teaching in high schools and community colleges, as well as working with private students.


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Hot springs are natural habitats for thermophilic Archaea and Bacteria. In this paper, we present the metagenomic analysis of eight globally distributed terrestrial hot springs from China, Iceland, Italy, Russia, and the USA with a temperature range between 61 and 92 ∘C and pH between 1.8 and 7. A comparison of the biodiversity and community composition… (More)
Microbes affect nutrient and energy transformations throughout the world's ecosystems, yet they do so under viral constraints. In complex communities, viral metagenome (virome) sequencing is transforming our ability to quantify viral diversity and impacts. Although some bottlenecks, for example, few reference genomes and nonquantitative viromics, have been… (More)
f W e welcome the attention of Stedman et al. to the novel RNA viruses that we recently identified in an archaea-dominated community (1) and their analysis of viral and cellular codon usage as an approach to assist in assigning potential hosts. However, we are not convinced that their analysis of such a limited number of RNA viruses leads to any compelling… (More)
A new type of viral-induced lysis system has recently been discovered for two unrelated archaeal viruses, STIV and SIRV2. Prior to the lysis of the infected host cell, unique pyramid-like lysis structures are formed on the cell surface by the protrusion of the underlying cell membrane through the overlying external S-layer. It is through these pyramid… (More)
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Mayhem # 1805429
About MeJette here. I am back after a few year hiatus and am now located in NYC and I am ready to work. Current hair color: Pink.I want to model with a purpose! I believe model status is something that should be earned. I'm chill to work with. I show up to WORK. My hair constantly changes!!! Modeling for me is my art. No matter what the style, the pose, the goal of the photo, I can make myself FEEL it. I want to be used for different types of projects. I can pull off any style and be comfortable. I want to be pushed to my creative limit. I hate being late. I love traveling. I wanna get out there and do my thing. I am a "tiny little thing". I know that can be against me, but I am DETERMINED to prove that being under 5'7'' isn't a career killer. This is work and I take it seriously. Don't forget to practice your inner beauty! :) Resume: Bosocov's Model, Fall Fashion Show '05 (runway) Joico Model, Hershey Hair Show '10 (runway) Keen Modeling Agency Syber Cyrens Donatelli Modeling Agency CreditsHave you worked with Jette 22? Add Credits for Jette 22! >>Credit NotesPhotographers:1387266 (BCR Greg) 21 (EA Photography) 457992 (Crow Photography) 1321761 (C. Everett Geist) 457992 (Jeff Michaels Photography) Hair Stylists: 441871 (Lady Wop) 1472753 (Green Bunny) Makeup Artist: 441871 Friends


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The former West Ham midfielder has been hit with the penalty after being found guilty of using foul and abusive language towards referee Iain Brines in the 1-1 draw with Celtic
The former West Ham captain was furious with referee Iain Brines in the 1-1 draw with Celtic at Parkhead earlier in November, when the official allowed Bhoys striker Tony Watt back on to the field in what he felt was a dangerous position after receiving treatment for an injury.
Despite Lomas apologising to Brines in person after the match, the SFA disciplinary committee found him guilty of using foul and abusive language towards an official, resulting in a six-match ban from the dugout.
However, Lomas also acquired a suspended two-match ban following another dismissal in the match against Ross County in September.
A statement on St Johnstone website read: "The SFA have handed Steve Lomas an eight-match touchline suspension following his dismissal from the dugout in the match against Celtic on November 11 for foul and abusive language towards a match official.
"This ban consists of six games for the offence committed at Celtic Park plus the suspended two-game ban which was already hanging over him following the game against Ross County in Dingwall in September."
Lomas faces yet more disciplinary action after being sent to the stands again towards the end of his side's 1-0 defeat by Hibernian on Wednesday evening.


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Man Charged In Nearly Year Old Deadly DUI CrashA Kendall man accused in a deadly DUI accident in Coral Springs has been charged nearly a year after the crash.
Principal Maimed In Accident Meets Those Who Saved His LifeA South Florida high school principal who survived a devastating accident that took his legs met with the group that saved his life.
Principal Maimed By Alleged Drunk Driver Throws Out 1st Pitch At Marlins GameJavier Perez showed off his throwing arm at Marlins Park on Friday night. He also showed off his considerable emotional strength.
Principal Who Lost Legs In DUI Accident Wants 'To Run A Marathon Again'It was the story that gripped the community – a horrific accident on April 26.
Son Tried To Keep Mother From Driving Before Alleged DUI CrashOn April 26th, a silver GMC SUV plowed through a fence at Tamiami Park by Southwest 117th Avenue, onto a ball field, running over and maiming Javier Perez, the beloved principal of South Dade Senior High. The husband and father was at the park coaching his 5-year-old son's baseball game.
Live Feed Of Graduation Helps Injured Principal Join Ceremony In SpiritThere was a loud ovation Monday night at the Bank United Center at the University of Miami for South Dade High School Principal Javier Perez.
Principal Injured In DUI Crash Off VentilatorA beloved high school principal run over by an alleged drunk driver is closer to recovery.
Accused DUI Driver Fitted With GPS After Release Mix-UpThe woman charged in a DUI crash that injured a South Dade High principal was fitted with monitor after being let out without it.
Woman Connected To DUI Crash That Injured Principal Should Have Never Bonded OutThe woman arrested for a drunk driving crash that injured a high school principal should have never bonded out.
Woman Hurts Cabbie In DUI Rollover On Miami BeachAt least one person was hurt in an early morning crash on Miami Beach that police said was caused by a drunk driver.
Driver Arrested For DUI After Causing 6-Car Crash Including MDPD CruiserA multi-car crash, which included a marked Miami-Dade police car, shut down a stretch of Calle Ocho Tuesday night.
Woman Who Texted "Driving Drunk Woo" Before Deadly Crash Accepts Plea DealA woman charged with DUI manslaughter for a 2013 crash which killed her friend in downtown Miami accepted a plea deal Friday.


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Genesis Volare 953 Di2 Frameset - 2015 MAD-VOLARE-953-DI2-FS-2015-GROUP |
£0.00 |
Key Features
About Genesis
Road Bike Size Guide
The Volare 953 Di2 Frameset The Volare Di2 frameset is constructed from Reynolds 953 Chromoly and is heat-treated and air-hardened to provide a superior strength to weigth ration. The Volare range is Genesis' top line, premium steel framed road range and it leaves nothing but a sweet taste in the mouth of the rider. All Volares are lightweight, lively on the road and extremely comfortable over long distances which makes this bike perfect for the sportive rider or weekend racer! The steel frame gives a unique and characterful ride which is augmented with the inclusion of a 44mm headtube and a carbon bladed fork which keep the bike feeling lightweight and adds vibration damping and a stiff and rigid front end. NB: Image for illustration purposes only, Frame, Fork and Headset Only |


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About Platinum
Platinum Properties is a full-service family-owned boutique brokerage in New York City. Our network of international and domestic investors, developers and landlords has positioned Platinum Properties since 2005 as the smart choice for luxury real estate transactions in New York City Real Estate—sometimes they even make history:
- Platinum Properties sold one unit for $2996 per square foot, the second highest price per square foot in Tribeca in 2011.
- Platinum Properties sold the three remaining penthouses at Trump World Tower for $33.7 million after they languished on the market for six years.
- Platinum Properties sold the penthouse at The Continuum South Tower in Miami Beach in a record 36 hours and then resold it three years later for the highest price on the East Coast outside of New York City at a record ($25MM).
Platinum Properties was founded by Khashy Eyn. Alongside his sister Dezireh, a licensed attorney, co-founder and Platinum's COO; and lifetime family friend Daniel Hedaya, Platinum's President, this tight-knit family environment attracts clients from around the globe as well as landlords and developers in New York City.
With headquarters on Wall Street, and an office on Paris' Champs Elysees—Platinum Properties is positioned as a turnkey solution for international investors and locals alike. "When the world thinks about New York, these are the places they think of and we're the people doing business there every day. Whether it's sales or leasing, residential or commercial, it's our backyard and we're happy to talk to you about opportunities here," says Khashy. All Platinum principals are at their desks in business suits from 7 AM to field calls from European clientele, and until after 10 PM to service investors in Asia.


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Multi-channel hard disk recording capability is combined with 20 MB class wavetable, multiple effects busses, 7 independent 24 bit effect processors, channelized EQs and more right on a single PCI card. Any application can control over a million parameters for all these facilities via MIDI, including digital audio mixing with digital effects through the new multi-channel internal bus system.
20 MB Wave ROM
1,267 plus 256 (if PLG100-VL is fitted) voices, and 46 drum kits in total.
32 Parts Multi Timbral and 64 + 1 Max Polyphony
32 parts internal plus 16 parts external MIDI. Maximum of 64 notes simultaneous polyphony and plus 1 (if PLG100-VL is fitted).
Synthesis Capabilities
The most advanced digital filter with LPF and HPF, ADR type envelope generator, channelized EQ with hi/low and gain and more. Drum Setup parameters such as pitch, filter, EG and even reverb/chorus/variation sends for each instrument of a drum kit can be controlled.
Digital Audio
6 stereo / 12 mono tracks for playback - 16/32 bit, 48/44.1/22/11/8 kHz with rate converter; 4 tracks for recording - 16/32 bit, 44.1 kHz.
24 Bit Resolution Effect Processors
Up to 7 independent effects blocks, each with many effect programs and tons of program-dependent parameters. 12 types of Reverb, 14 Chorus, 70 Variation, 43 Insertion 1, 43 Insertion 2 effects, 5-Band Total EQ and 4 types of vocal harmony effect (when upgraded with PLG100-VH) can be used simultaneously for the wavetable synthesizer and the digiital audio.
2 A/D Inputs
The digital effects can be applied to the sound source from the Analog MIC/LINE Input.
Modular Synthesis Plug In System Expansion
The SW1000XG can be expanded with cards from the Modular Synthesis Plug-in system. There are 6 cards available: PLG100VH, PLG100XG, PLG150AN, PLG150PF, PLG150DX, and PLG150VL. Any 100 Series board will work with any SW1000XG. Any boards will work with the SW1000XG-P shipped from August 1999. To use PLG150 series boards with older SW1000XGs, please contact your dealer to have it updated at no charge.
A special version of Yamaha's XGworks MIDI/audio sequencer and tone generator editor will be bundled. SW1000XG is fully functional in many leading software programs such as Steinberg's Cubase VST, emagic's Logic Audio, Twelve Tone Systems' Cakewalk Pro Audio, Sonic Foundry's SoundForge, IQS's SAW.
Music / Sound Production Suite with DSP Factory
Internal multipin bus interface for connection to DSP Factory card system, Yamaha's ultimate digital mixing / processing PCI card which was recently announced.
Architecture PCI Bus Master (PCI version 2.1)
OC Compatibility Windows 95 (95 Plug and Play compatible)
Audio Converter Stereo 20 bit resolution ADC; Stereo 18 bit resolution DAC; Stereo 24 bit resolution internal processing
Digital Audio Full duplex playback and recording
SNR (A-weighted) less than 94dB
Recording Tracks 1 stereo / 2 mono (External analog in); 1 stereo/ 2 mono (Internal loopback)
Playback Tracks 6 stereo / 12 mono
Recording Rate 44.1 KHz
Playback Rate 48 / 44.1 / 22 / 11 / 8 KHz
Recording Resolution 16 / 32 bit
Playback Resolution 16 / 32 bit
MIDI Features Tone Generation Method: AWM2; Maximum Simultaneous Polyphony: 64 notes / 32 parts; Wave ROM size: 20 MB; Sound Module Mode: XG, TG300B
Internal Voices/Program Structure Normal Programs; Total Voices: 1,267; XG mode: 1,074; TG300B mode: 614
Drum Programs Total Programs: 46; XG mode: 36; TG300B mode: 10; C/M mode: 1
Effects 6 independent effects: Reverb (12 types); Chorus (14 types); Variation (70 types); Insertion 1, 2 (43 types each), 5-band EQ (4 types)
Jacks & Terminals A/D input (stereo-mini); Analog output R, L (RCA-pin); Digital output (S/PDIF); MIDI interface (DIN 6 pins)
Digital Interface DSPF: for DSP Factory DS2416; XG Plug-in Board: for PLG100-VL/VH
System Requirements PC Windows 95 installed; Pentium 166Mhz or upper; 32 MB RAM or upper; PCI Slot
Dimensions ( D x W x H) 198 x 127 x 21.6 mm
Weight 140 g
Trademarks and Registered Trademarks *All trademarks and registered trademarks shown are the property of their respective owners.
Additional Information
Manufacturer | Yamaha |
Price | $519.95 |
Product Condition | New - On Clearance |


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"A Cultural History of the Banana" may have it beat...
Here's one fun fact: In this country, in the time it takes to watch one episode of Glee, 138 students drop out of high school.
In dollars spent on federal lobbying efforts last year, BU comes out the big winner.
Italian scientists say they have have developed a cold fusion energy device capable of producing 12,400 W of heat power with an input of just 400 W.
Breaking down the reasons behind the astronomical cost of education in today's world
The national default rate on student loans hit 7%, the Department of Education reported.
In a few weeks, for the first time in their lives, many Americans will kick off the fall in a cubicle rather than a classroom.


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Any way to remove home screens..I don't use them all just wondering if u can remove them Sent from my DROID2 using DroidForums App
Well you can download a homescreen replacement from the marketplace like adw or launcher pro. They have options to use fewer homescreens. Sent from my DROID2 using DroidForums App
Yeah. I use adw, and it works great. Tried launcher pro, but didn't like it so much. Sent from my DROID2 using DroidForums App
Anyone finding that with 2.3.4 the settings are getting reset for: Home screen default FB contact sync It has happened to be twice in the last three days. Not a major deal but I wonder what else is getting reset. Sent from my Droid using DroidForums


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Not only does Autosport Plus in Canton, Ohio offer sales and installation of Jeep lift kits but we also offer Northeast Ohio's largest selection of custom wheels and tires. Stop in today and let us work out a rim and tire package for your Jeep. Financing and Nationwide shipping is available. 330.454.5705.
Autosport Plus is located on Rt62 in Canton, Ohio sells and installs more Jeep lift kits than anyone else in the Canon and Akron area..PERIOD! Stop by our award-winning location on any given day and it looks like a Jeep event is being held! Our vehicles have been featured on Mfg's website's, publications, and more. If you're looking for a Jeep specialist, look no further than Autosport Plus! Call or stop in today. 330.454.5705
Autosport Plus
2136 Columbus Rd NE
Canton, Ohio 44705
Monday - Friday 10-7
Saturday 10-5


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Monday, September 2, 2013
Just arrived. Mission 727s.
I've owned 700s, 717s and 770s, but this is the first pair I've seen available in my neck of the woods. Second from top of the line 770 in it's debut model year, I've been dying to hear it for a long, long time.
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If a stranger offered to give you a big bag of money on the street, would you take it?
Free e-mail watchdog
Answer this question
If a stranger offered to give you a big bag of money on the street, would you take it?
Answer for question:
Your name:
Answer #1
| 04/07 2008 00:40
I'll take it as long as I get their business card and check it to their ID. What kind of a stupid f***ed up question is this?
73 %
Answer #2
| 04/07 2008 01:02
Yes, I would take it
67 %
Answer #3
| 04/07 2008 01:45
of course i would
47 %
Answer #4
| 04/07 2008 02:36
I would try to ask him why he was giving it away first, cuss lots of OPRAH people out there giving money away, and if he was one and I past it up and then saw OPRAH on TV saying she was trying to give money away in that area I be out of luck.
15 %
Answer #5
| 04/07 2008 03:20
take it then have it checked out by police
10 %
Answer #6
| 04/07 2008 04:39
this is wut u want to know. r u that bored. zzzzzzzzzzzzz!!!
10 %
Answer #7
| 04/07 2008 06:38
Only if he insisted !!
10 %
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The Whipper Snappers Studio experience begins by providing an atmosphere that makes everyone feel like smiling. Then they help those smiles turn into grins, giggles and guffaws with interesting props just begging to be investigated. What better way than to capture the magic of the moment than giving kids ways to laugh, play, discover -- just be kids. The company specializes in children photography offering parents a means of recording their pleasant memories with their kids without compromising on quality or poor pictures.
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Apple Cider Pie
Recipe by Derf
Made this for a bake sale, they got $6.00 for it!! Hope the buyer liked it, have no idea what it tastes like! but it sounds good. Will try it on the family at next family dinner and let you know.
Top Review by woodland hues
A really good apple pie! Made to take to a Thanksgiving dinner at the neighbour's. Easy to prepare, baked for 45 min. The thin-sliced apples were done perfectly. Thanks, Derf!
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1 ReviewIngredients Nutrition
- 3⁄4 cup apple cider or 3⁄4 cup apple juice
- 1⁄2 cup chopped pitted dates or 1⁄2 cup raisins
- 1 tablespoon cornstarch
- 1⁄2 teaspoon ground ginger
- 1⁄2 teaspoon grated fresh lemon rind
- 2 store bought or homemade pie crusts
- 1⁄2 cup sugar
- 1 tablespoon all-purpose flour
- 5 cups thinly sliced, peeled tart cooking apples
- Preheat oven to 375 degrees.
- In a small saucepan, stir together the apple cider, dates, cornstarch, ginger, and the lemon rind.
- Bring to a boil over moderate heat, stirring constantly.
- Cook for 2 minutes or until mixture is thickened, stirring constantly.
- Remove from heat.
- Line a 9 inch pie plate with one of the pie crusts.
- Trim crust even with edge of pie plate.
- In a large bowl, stir together the sugar and flour.
- Add the apples, toss until apples are coated.
- Spoon the apple mixture into crust.
- Spoon the cider mixture over apple mixture.
- Cut slits in the remaining pie crust, place on top of cider mixture.
- Trim top crust to 1/2 inch beyond edge of pie plate.
- Fold top crust under bottom crust and crimp edge.
- Sprinkle with a little additional sugar.
- Bake for 40 to 45 minutes or until apples are tender and crust is golden.
- If the edge of the crust seems to be browning too quickly, cover with foil.
- Cool on wire rack for 2 hours before serving.


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Review of existing data on potential contributors to the development of obesity in at-risk populations has provided insights for the development of community-based interventions in the African-American community. Black women in the United States are disproportionately affected by obesity, with almost two-thirds considered obese based on body mass index. Obesity has been directly linked to cardiovascular morbidity and mortality in black women. Therefore, understanding contributors to the genesis of obesity in black women is imperative. While biologic differences likely result in varying obesity prevalence across racial/ethnic groups, behaviors such as post-partum weight retention and limited leisure-time physical activity, may especially contribute to obesity in black women. Black women also appear to be particularly susceptible to cultural, psychosocial, and environmental factors that can promote weight gain. Therapeutic interventions are being tailored to specifically address these social determinants of health and to foster lifestyle modification. However, more work is needed to evaluate specific interventional tools that can counteract barriers to behavior change for black women. The current literature suggests that successful community-based interventions focused on obesity in black women should engage faith-based organizations, community organizations advocating for black women's health issues, and community advisory boards to design interventions promoting social support and environmental changes for lifestyle modification.
Leon, Benjamin; Miller 3rd, Bernard V; Zalos, Gloria et al. (2014) Weight Loss Programs May Have Beneficial or Adverse Effects on Fat Mass and Insulin Sensitivity in Overweight and Obese Black Women. J Racial Ethn Health Disparities 1:140-147 |
Powell-Wiley, Tiffany M; Miller, Paige E; Agyemang, Priscilla et al. (2014) Perceived and objective diet quality in US adults: a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES). Public Health Nutr 17:2641-9 |


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Dougal and Jaikie set off travelling the world, but first they must visit Mr Craw and his Castle Gay fully of mysterious characters.
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Sadhana (Paperback)
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Written by Nobel Prize Laureate Rabindranath Tagore and available in paperback for the first time, "Sadhana is a profound, highly accessible introduction to India's ancient spiritual heritage.
Few figures in history have been as important as Rabindranath Tagore in bringing Indian philosophy and spiritual teachings to the West. Although he was known primarily as a poet, his work is deeply religious, imbued with his belief that God can be found through personal purity and service to others. "Sadhana (sometimes translated from the Sanskrit as "spiritual practice" or "spiritual discipline") is a beautifully written, concise distillation of the great resources of Indian philosophy. With the surge of interest in Indian spirituality, it will be welcomed with enthusiasm by readers everywhere.


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