text:
|United States Patent
|Patent No.:
|Date of Patent:
|Feb. 10, 2009
|Altered insulin-like growth factor binding proteins
|Briony Forbes, Adelaide (AU)
|University of Adelaide, Adelaide (AU), Type: Foreign Company
|Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.
|Appl. No.:
|PCT Filed:
|Jul. 11, 2003
|PCT No.:
|§ 371 (c)(1), (2), (4) Date:
|Dec. 29, 2004
|PCT Pub. No.:
|PCT Pub. Date:
|Jan. 22, 2004
|Prior Publication Data
|US 2006/0153853 A1 Jul. 13, 2006
|Foreign Application Priority Data
|Jul. 12, 2002
|Int. Cl.
|U.S. Cl.
|530/350; 536/23.1
|Field of Search
|References Cited
|Yamano et al.
|Wells, 1990, Biochemistry 29:8509-8517. *
|Ngo et al., 1994, The Protein Folding Problem and Tertiary Structure Prediction, pp. 492-495. *
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|Doerks et al., 1998, Trends in Genetics 14:248-250. *
|Smith et al., 1997, Nature Biotechnology 15:1222-1223. *
|Brenner, 1999, Trends in Genetics 15:132-133. *
|Bork et al., 1996, Trends in Genetics 12: 425-427. *
|Richardson et al. 2003. Virchows Arch. 442: 329-335. *
|Ngo et al., 1994, The Protein Folding Problem and Tetiary Structure Prediction, pp. 492-495. *
|Skolnick et al., 2000, Trends in Biotech. 18(1):34-39. *
|Bork et al., 1996, Trends in Genetics 12:425-427. *
|Eiseman et al., Clin Cancer Res. 2007; 13: 2121-2127. *
|Rajaram et al., Endocr Rev 1997;18: 801-31. *
|Boulle et al. J Clin Endocrinol Metab 1998; 83: 1713-20. *
|Moore et al. Int J Cancer. 2003: 105: 14-19. *
|Kibbey et al. Mol Pharmacol 2006;69: 833-45. *
|elmhurst.edu/˜chm/vchembook/561aminostructure.html; downloaded on Feb. 15, 2008 at 3:55:54pm; 3 pages. *
|Imai, Y. et al., "Protease-resistance form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-1 actions on porcine smooth muscle cells in culture.", Journal of Clinical Investigation, vol. 100, No. 10, 1997, pp. 2596-2605.
|Lucic, M. et al., "Secretion in Escherichia coli and phage display of recombinant insulin-like growth factor binding protein-2.", Journal of Biotechnology, vol. 61, 1998, pp. 95-108.
|Clemmons, David R.; "Use of mutagenesis to probe IGF-binding protein structure/function relationships"; Endocrine Reviews (2001), vol. 22 (6); pp. 800-817.
|Qin Xuezhong et al.; "Studies on the role of human insulin-like growth factor-II (IGF-II)-dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts using protease-resistant IGBP-4 analogs"; Journal of Bone and Mineral Research (1999), vol. 14(12), pp. 2079-2088.
|Miyakoshi, Naohisa et al.; Systemic administration of insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) increases bone formation parameters in mice by increasing IGF bioavailability via an IGFBP-4 protease-dependent mechanism; Endocrinology (2001); vol. 142(6); pp. 2641-2648.
|Canover, C. A. et al.; Cleavage analysis of insulin-like growth-factor (IGF) dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants; Journal of Biological Chemistry, American Society of Biolochemical Biologist, Birmingham, US (1995); vol., 270 (9), pp. 4395-4400.
|Clemmons, D. et al.; "Role of insulin-like growth factor binding protein in the control of IGF actions"; Progress of Growth Factor Research; vol. 6, No. 2-4; 1995; pp. 357-366.
|Bramani, S. et al.; "Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I"; Journal of Molecular Endocrinology; vol. 23, 1999, pp. 117-123.
|* cited by examiner
|Primary Examiner —Elizabeth C. Kemmerer
|Assistant Examiner —Christina Borgeest
|Art Unit — 1649
|Exemplary claim number — 1
|Attorney, Agent, or Firm — Foley & Lardner LLP
Altered IGFBPs are able to bind IGF, but the release is inhibited by resistance to protease cleavage and/or reduced binding to extracellular matrix (ECM). Alterations have been made in IGFBP-2 to the linker domain in particular and to two amino acid motifs found to be important for ECM binding. IGF-1 mediated proliferation of cancer cells have been inhibited by use of the altered IGFBPs.
|24 Claims, 6 Drawing Sheets, and 7 Figures
[0001] This invention relates to an altered Insulin-like Growth Factor Binding Protein (IGFBP) with reduced IGF (Insulin-like Growth Factor) release characteristics. The IGFBP of the invention is proposed to be useful for therapeutic purposes, such as treatment of certain cancers. A specific form of the invention relates to an altered IGFBP-2.
[0002] Insulin-like growth factors (IGF-I and IGF-II) are small, highly-related proteins (˜7.5 kilodaltons) which mediate anabolic, mitogenic and anti-apoptotic activities in a wide variety of cell types. These actions result from IGF interaction with and subsequent activation of the type 1 IGF receptor (IGF1R) (Sepp-Lorenzino, (1998), Baserga, 1999). A second unrelated receptor (the type 2 IGF receptor or IGF2R) has the major function of regulation of IGF-II levels by internalisation and degradation (Wang et al., 1994) and current evidence suggests that the IGF2R acts as a tumour suppressor of IGF-II-dependent tumours (Braulke, 1999).
[0003] IGFs are produced by the liver, providing circulating IGF, and are also secreted locally in most tissues. A family of 6 high-affinity IGF binding proteins (IGFBP-1 to -6) act to increase the half-life of IGFs in circulation (predominantly as the IGFBP-3·ALS·IGF complex) and also to transport IGFs to target tissues. Within target tissues IGFBPs can either enhance or inhibit IGF action. IGFBPs can inhibit the interaction of IGF by blocking binding to the IGF1R. However, under certain circumstances IGFBPs can release IGF, thereby making IGF available for binding to the IGF1R. This results in an enhancing effect on IGF action. Release mechanisms include 1) proteolysis of the IGFBPs and 2) IGFBP binding to the extracellular matrix (ECM), both of which lower their affinity for IGF. Extracellular matrix binding is also believed to assist the localisation of IGF close to the cell surface and therefore near IGF1Rs. The outcome of IGFBP action is controlled by a balance between local proteolytic activity and the binding of IGFBPs to the ECM.
[0004] Substantial evidence (in vivo and in vitro) implicates insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in cancer. Many tumour cells (including prostate and breast) secrete more IGF-II and IGFBP-2 than their normal counterparts and their serum levels commonly rise as cancers progress (Cohen et al., (1994); Thrasher et al.,(1996); Ho et al., (1997); Chan et al., 1998). IGF secreted by tumour cells binds to the Type 1 IGF receptor potentiating tumourigenesis and metastasis (DiGiovanni et al., 2000).
[0005] The proteolysis of IGFBP-2 has been detected under a number of normal and abnormal physiological conditions. For example, IGFBP-2 fragments have been detected in human milk and cleavage occurred predominantly in the linker region between the N- and C-domains and including sites at residues 168 and 180/181 of hIGFBP-2 (Ho and Baxter, 1997; Elminger et al., 1999). Proteolysed IGFBP-2 is also found in serum during pregnancy. IGFBP-2 is also cleaved by proteases produced by cancer cells (Michell et al., 1997). The specific cancer cell proteases have not been well characterised although Cathepsin D produced in vitro by prostate epithelial cells has been shown to degrade IGFBP-2 (Kanety et al., 1993; Nunn et al., 1997). Preferential proteolysis of IGFBP-2 has been demonstrated in colonic cancers (Michell et al., 1997) and neuroblastoma cells (Menouny et al., 1997). Specific cleavage sites have not been described for proteolytic products generated by cancer proteases.
[0006] Protease cleavage sites have been identified in the IGFBP-3, -4 and -5 sequences. Proteolysis is generally within the linker regions of these proteins although it can be in the C-domain. Protease resistant IGFBP-4 and IGFBP-5 (Imai et al., 1997) have been generated by mutating specific residues at cleavage sites or by deletion of some linker region residues (deletion of 121-141 of IGFBP-4 rendered it resistant to a protease in pregnancy serum (Byun et al., 2000).
[0007] IGFBP-2 binds to human fibroblast extracellular membrane preparations (Arai et al., 1996) and glycosaminoglycans (Russo et al 1997, Arai et al., 1996). There are 2 potential matrix binding sites within the IGFBP-2 sequence. Current evidence suggests that the basic region of hIGFBP-2 (residues 227-244), corresponding to residues 201-218 of hIGFBP-5, may act as a site for matrix binding (Arai et al., 1996). Using a synthetic peptide based on residues 201-218 of hIGFBP-5 (residues known to be important for matrix binding) Arai et al., 1996, inhibited IGFBP-2 binding to heparin-Sepharose. Hodgkinson et al., (1994) predicted a glycosaminoglycan (GAG) binding site in IGFBP-2 based on a short GAG-binding consensus sequence described by Cardin and Weintraub (1989). This XBBXBX (B=basic, X=undefined) motif at residues 179-184 of hIGFBP-2 lies in the central domain. There is no published evidence that this motif plays a role in GAG binding.
[0008] For the purposes of this specification the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning. Also a reference within this specification to a document is not to be taken as an admission that the disclosure therein constitutes common general knowledge in Australia.
[0009] This invention arises from the alteration of IGFBP-2 in a manner that inhibits the release of IGF-I and IGF-II. The inhibition of release results from the introduction of changes to the IGFBP-2 resulting in reduced binding to extracellular matrix (ECM) and reduced susceptibility to proteolysis by one or more proteases. This altered IGFBP-2 is proposed to be useful for its ability to inhibit growth of IGF-dependent tumours such as colon, prostate and breast cancers. This is to the inventor's knowledge the first time that an altered IGFBP has been constructed to contain both reduced ECM binding and reduced proteolytic susceptibility and the first time that this combination has been shown to be effective at inhibiting the release of IGF-I and IGF-II. It is proposed that this approach will be effective not only for IGFBP-2 but also for other IGFBPs. Additionally this is to the inventor's knowledge the first time that there have been functional data to show the location of the two ECM binding sites for IGFBP-2, and a demonstration that these together with alteration of proteolysis sites result in inhibition of the release of IGF-I and IGF-II. Additionally this is the first time to the inventor's knowledge that an overlapping IGF and ECM binding site has been altered in a manner that still allows IGF binding but not ECM binding.
[0010] The invention might in a first broad aspect be said therefore to reside in an altered IGFBP-2 molecule able to effect binding of IGF-I or IGF-II with high affinity characterised in an inhibited release of IGF on contact with extracellular matrix or exposure to a protease.
[0011] In a second form of the first aspect of the invention might be said to reside in an altered IGFBP molecule able to effect binding of IGF-I or IGF-II with high affinity characterised in an inhibited release of IGF on contact with extracellular matrix and exposure to a protease.
[0012] Cleaved IGFBP-2 has greater than 10 fold weaker affinity for IGF (Carrick, 2001). It is desirable for the altered IGFBP-2 to have at least to have an affinity for IGF-I equivalent to the IGF type 1 receptor which is 10 fold lower than that of native IGFBP-2 for IGF-I. This allows effective competition with the receptor for IGF binding and the term high affinity binding of IGF should be understood in that context.
[0013] Regions required for IGF binding have not been fully elucidated however considerable effort has been made to define these regions. Thus for example we (Hobba et al., 1998) and others (Zeslawski et al., 2001) have identified residues in the distal part of the N-domain involved in IGF binding. Similarly, the minimum C-domain length of IGFBP-2 required for IGF binding has been described (Forbes et al., 1998). WO 00/23469 also describes the IGF binding domain which document is incorporated herein in its entirety by reference. Methods for assaying IGF binding are known in the art and can be readily employed to ascertain whether the altered IGFBP is able to bind IGF or not.
[0014] The inventor has identified two ECM sites for IGFBP-2 and shown that mutations in these lead to reduced heparin binding. The first ECM site for IGFBP-2 spans sequence 179-184 and consists of the sequence PKKLRP [SEQ ID No 1] and was originally proposed on the basis of homology with the consensus sequence for matrix binding proteins (namely XBBXBX, Hodgkinson et al., 1994). The second ECM binding sequence of IGFBP-2 identified is at 227-244 by homology with IGFBP-3 and IGFBP-5 and consists of sequence KHGLYNLKQCKMSLNGQR [SEQ ID No 2]. Suitable reference sequence is available from sequence databases. Thus the nucleotide sequence of cDNA of human IGFBP-2 might be accessed from the NCBI sequence database as accession number NM000597. Suitable amino acid sequence predictions can be made from the nucleotide sequence.
[0015] The invention might also in a third form of the first aspect be said to reside in an altered IGFBP-2 molecule able to effect binding of IGF-I or IGF-II with high affinity said IGFBP-2 molecule having alterations in any one or more amino acids in both ECM binding sites, a first ECM binding site being located at sequence 179 to 185 and a second ECM binding site being located at sequences 227 through 244, the alterations separately and together inhibiting the binding of the IGFBP-2 to ECM.
[0016] Preferably residues between 227 and 236 are substitution mutations because it is thought that at least part of this site may also be important for IGF binding. Whereas the 179-185 alteration may be achieved by deletion, inversion, substitution or other gross alteration, however preferably this is altered by amino acid substitution.
[0017] Generally substitution of a basic amino acid for an amino acid of different character, that is either neutral, or acidic is found generally to have a disrupting effect on matrix binding motifs.
[0018] It is found by the inventor that utilising the mutations so far introduced into IGFBP-2 that alteration of one of the two ECM sites alone is not sufficient to totally abolish ECM binding. Whilst the present invention preferably provides for the alteration of both ECM sites, the invention might also encompass the provision of alteration in one only of the ECM binding sites, and perhaps in combination with an altered protease cleavage and/or binding site.
[0019] Set out below are sequences shown or proposed to be the ECM binding sites for all six IGFBPs.
[0020] ECM binding sites (in the C-domain between the 14th and 16th cysteine residues)
[0022] Sites for IGFBP 3 and IGFBP 5 have been published previously the present data establishes the binding site for IGFBP-2 and the sites for IGFBP1, 4, and 6 are suggested by reason of amino acid alignment.
[0023] The invention might also encompass an altered IGFBP-1, -4, or -6 having an alteration in the ECM binding sites.
[0024] Alternatively in this first aspect of the invention it might be that a satisfactory decreased release of IGF is achieved solely by inhibition of ECM binding by amino acid substitution at the second ECM binding site. Such decreased release might be the result of the close association between the IGF binding site and the second ECM binding site, and therefore the first aspect of the invention might reside in an altered IGFBP molecule and perhaps preferably an IGFBP-2 molecule that has one or more amino acid substitutions in the ECM binding site that still allows binding of IGF, whilst still inhibiting binding of ECM and thereby also inhibiting IGF release.
[0025] It is thought that the positively charged residues are important for binding and thus substitution of these are likely to result in inhibition of binding. The substitution might be a non conservative substitution such as the following alanine (A) substitutions for lysine (K).
[0027] Substitutions of the lysine residues by other amino acids might also be contemplated.
[0028] The altered IGFBP-2 of the first aspect of the invention preferrably also has an alteration at any one or more proteolytic cleavage sites whereby to inhibit the release of an IGF when subjected to a protease specific for the proteolytic cleavage site.
[0029] A preferred form of the first aspect of the invention comprises substitution mutations at the first and second ECM binding sites, perhaps those referred to above, together with any one or more deletions in the linker domain that remove one or more proteolytic cleavage sites.
[0030] As indicated above in certain aspects the invention encompasses alterations that inhibit IGF release on exposure to one or more proteolytic enzymes. Many proteolytic enzymes to which IGFBPs are susceptible have as their target the linker domain. The proteolytic enzyme that the altered IGFBP is exposed to on delivery for therapeutic purposes may differ depending on the conditions to be treated. It is known that tumour cell lines vary in the proteolytic enzymes they produce. It has been shown by the inventor that a substantial deletion in the linker domain of IGFBP-2 leads to resistance to proteolytic cleavage whilst at the same time maintaining protein stability and maintaining IGF binding at high affinity. A preferred alteration of the IGFBP that leads to protease resistance is therefore one or more deletions within the linker domain. The size of the deletion or deletions may be varied. It has been found by the inventor that a deletion of substantially all of the linker region still led to IGF binding. With a deletion of substantially all of the linker domain it is preferred that amino acids from about 180 through to 191 are maintained.
[0031] Smaller deletions may also be suitable for removal of sites that make the IGFBP susceptible to proteolysis, thus deletions of amino acids about 110 through to about 170 has resulted in a decrease susceptibility to proteolysis.
[0032] Resistance to proteolysis may also be achieved by more targetted alteration to sites important for proteolytic cleavage or binding.
[0033] Substitution of residues other than those involved in proteolysis or ECM binding might also be contemplated, and these might be conservative substitutions or non-conservative substitutions, deletions, duplications, inversions and other rearrangements, provided that the altered IGFBP is still capable of high affinity IGF binding. Additionally the altered IGFBP might have additional changes such as glycosylations or other chemical modifications.
[0034] The proteins may be a fusion protein which assists in the purification, thus the protein may include a C terminal 6 histidine tag which provides for nickel affinity purification. Other fusion purification systems are also known and may be used. For bacterial systems a fusion protein may include a signal sequence, such as one derived from ompA, adapted to have the protein secreted through the surface of the cell.
[0035] In a second aspect the invention might be said to reside in a nucleic acid encoding one or more of the proteins or amino acid sequences of the first aspect of the invention. In a preferred form the nucleic acid is carried by a vector, the vector having nucleic acid operably linked with a control sequence including a promoter for transcription leading to expression of the protein or amino acid. Any one of the very many known vectors for that purpose may be used. Alternatively the vector may be used simply to introduce the nucleic acid encoding the altered IGFBP into a host cell for integration into a chromosome host cell, and might therefore be a retroviral expression vector.
[0036] In a third form the invention might be said to reside in a recombinant cell carrying a vector or the nucleic acid of the second aspect of the invention. The host cell might be intended for expression of the altered IGFBP which can thus be produced and purified such that the purified protein may be then separately used or administered as desired. The host cell might be bacterial, yeast, plant or mammalian. Alternatively the host cell may be intended for introduction into a treated animal such as a human for gene therapy purposes.
[0037] In a fourth form the invention might be said to reside in a pharmaceutical composition. The composition including the altered IGFBP-2 of the first aspect of the invention as an active component. The pharmaceutical composition may be formulated in accordance with an approved method, and may include a carrier which may or may not be fused to the altered IGFBP, or conjugated with the altered IGFBP. The composition may additionally include other medicinal agents, pharmaceutical agents, adjuvants, diluent, excipients and the like.
[0038] The invention might in a fifth form also be said to reside in a method for decreasing serum and/or tissue levels of biologically active IGF-I or IGF-II by administering to a mammal an amount of altered IGFBP of the first aspect of this invention effective to inhibit the release of IGF-I or IGF-II from the altered IGFBP and thereby decrease serum and tissue levels of biologically active IGF-I or IGF-II.
[0039] The fifth aspect of the invention contemplates the expression of exogenous nucleic acid encoding the altered IGFBP-2 in one or more cells of the animal of human patient, as a so called gene therapy approach.
[0040] Generally, gene therapy is used to increase (or overexpress) altered IGFBP-2 levels in the mammal. Nucleic acids which encode IGFBP-2 be used for this purpose. Several nucleic acid molecules may be generated using the degeneracy of the genetic code that encodes the desired amino acid sequence.
[0041] There are two principal approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells for purposes of gene therapy: in vivo and ex vivo. For in vivo delivery, the nucleic acid is injected directly into the patient, usually at the site where the viperine is required. Preferably this might be in the liver. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187.
[0042] There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retrovirus.
[0043] In vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990). For a review of known gene marking and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). See also WO 93/25673 and the references cited therein.
[0044] Alternatively purified altered IGFBP can be administered to the mammal in a suitable carrier the altered IGFBP may be administered orally parenterally, topically transdermally. It might be preferred to provide the altered IGFBP in slow release from. Determination of appropriate dosages and formulation may be achieve by one of ordinary skill in the art using only routine experimentation. See for example Remington's Pharmaceutical Sciences (Martin E. E. ed, latest edition), Mack Publishing Co, Easton Pa.
[0045] Further, included within the scope of the present invention is the co-administration of an altered IGFBP-2 together with a cytotoxic or anticancer agent. Such agents suitably include antimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g. cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines (e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin, idarubicin, adriamycin, daunomycin), topoisomerase inhibitiors (e.g., etoposide, camptothecins) or any other antineoplastic agents. (estramustine phosphate, prednimustine).
[0046] It is anticipated that altered IGFBP-2 used in combination with various anticancer drugs can give rise to a significantly enhanced cytotoxic effect on cancerous cells, thus providing an increased therapeutic effect. Specifically, as a significantly increased growth-inhibitory effect is obtained with the above disclosed combinations utilizing lower concentrations of the anticancer drugs compared to the treatment regimes in which the drugs are used alone, there is the potential to provide therapy wherein adverse side effects associated with the anticancer drugs are considerably reduced than normally observed with the anticancer drugs used alone in larger doses.
[0047] Compounds of the present invention can be administered in a variety of ways including enteral, parenteral and topical routes of administration. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, vaginal, and the like.
[0048] Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or move thereof. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., N.J. (1991), which is incorporated herein by reference.
[0049] In a sixth aspect the invention might be said to reside in a method of reducing IGF mediated proliferation of a population of cancerous cell in the presence of IGF, by contacting the population of cells with an altered IGFBP according to this invention.
[0050] Conditions that might be amenable to treatment in this way include cancers that are IGF dependant and these may include certain cancers of the breast, prostate, colorectal, lung, thyroid, ovaries, and brain as well as in childhood leukaemias, glioblastomas, and neuroblastomas.
[0051] It is contemplated that treatment of say prostate cancer according to the present invention may not necessarily be used on its own but as an adjunct to other methods.
[0052] By way of a shorthand notation the following three and one letter abbreviations for amino acid residues are used in the specification as defined in Table 1.
[0053] Where a specific amino acid residue is referred to by its position in the polypeptide of an protein, the amino acid abbreviation is used with the residue number given in superscript (i.e. Xaan)
[0055] FIG. 1. Schematic diagram of the hIGFBP-2 expression vector constructed from the mammalian expression vector pXMT2 (Rathjen et al., (1990); Whyatt et al., (1993)). A colicin E1 origin of replication (ori) and the β-lactamase ampicillin resistance gene (Amp) enable selection and propagation in E. coli. Mammalian expression of the IGFBP-2 cDNA is driven by the adenovirus major late promoter (MLP). The dihydrofolate reductase (DHFR) gene is present for selection. A 6 Histidine tag is encoded at the 3′ end of the IGFBP-2 cDNA sequence.
[0056] FIG. 2. Purified hIGFBP-2 and mutants were separated on a 12% tricine SDS polyacrylamide gel. Proteins were stained with Coomassie blue (except for Des(114-170)His hIGFBP-2 which was stained with sypro ruby). All mutants migrated with the expected size as estimated by comparison with molecular size standards (Novex Broad range). A minor breakdown product was detected in this particular K234AHis preparation.
[0057] FIG. 3. Schematic of hIGFBP-2 and truncated hIGFBP-2 showing the conserved N- and C-cysteine domains connected by the linker domain. The know disulphide bonds (shown as transverse white stripes) and residue numbers are indicated above. Potential cleavage and ECM sites are also identified.
[0058] FIG. 4. BIAcore analysis of hIGFBP-2 and mutants binding to IGF-I.
[0059] Human IGFBP-2 (at concentrations of 3.1, 6.25, 12.5, 25 and 50 nM) was allowed to associate with the Biosensor surface (70 resonance units of hIGFBP-2) for 300 s (from t=150 s) and then dissociated for 900 s. Real time binding is measured in response units. Kinetic studies were performed at a flow rate of 30 μl/min to minimize mass transfer effects. The IGF-I-coated biosensor surface was regenerated with 10 mM HCl between binding cycles. For each binding curve the response obtained using control surfaces (no protein coupled) was subtracted.
[0060] FIG. 5A Analysis of susceptibility of truncated hIGFBP-2 to proteolysis in culture media conditioned by cancer cells. Truncated hIGFBP-2 and hIGFBP-2 were exposed to culture media of a) T84 colon cancer and b) PC3 prostate cancer for 0, 3 or 24 hours at 37 degrees. Samples were separated by SDS polyacrylamide gel electrophoresis on 10% tricine gels and transferred to nitrocellulose. hIGFBP-2 and cleavage products were detected with an anti-IGFBP-2 polyclonal antibody (left). hIGFBP-2 migrates at 34 kDa whereas Des(114-170)His migrates at 20 kDa. A dimer is present in hIGFBP-2 preparations. Proteolytic fragments range in size between 14 to 20 kDa. Densities of bands indicated on the blots were quantitated using the NIH image program and amounts of uncleaved or cleaved hIGFBP-2 graphed (right).
[0061] FIG. 5B Analysis of susceptibility of truncated hIGFBP-2 to proteolysis in culture media conditioned by cancer cells. Truncated hIGFBP-2 and hIGFBP-2 were exposed to culture media of c) DU145 and d) LNCaP prostate cancer cell lines for 0, 3 or 24 hours at 37 degrees. Samples were separated by SDS polyacrylamide gel electrophoresis on 10% tricine gels and transferred to nitrocellulose. hIGFBP-2 and cleavage products were detected with an anti-IGFBP-2 polyclonal antibody (left). hIGFBP-2 migrates at 34 kDa whereas Des(114-170)His migrates at 20 kDa. A dimer is present in hIGFBP-2 preparations. Proteolytic fragments range in size between 14 to 20 kDa. Densities of bands indicated on the blots were quantitated using the NIH image program and amounts of uncleaved or cleaved hIGFBP-2 graphed (right), and
[0062] FIG. 6 Proliferation of HT-29 colorectal cells measured by Cell Titre Glo Assay (Promega). Apoptosis was induced with 5 mM butyrate and the ability of IGF-1 to promote cell survival was measured. Native IGFBP-2, DES(114-170) and DES (114-170) K180A K181A were added to cells treated with IGF-1 at various concentrations (0, 0.1, 0.3 and 0.65 nM) in the presence of butyrate. Results are expressed as a percentage of proliferation in the presence of culture medium alone. As Des (114-170) and Des (114-170) K180A K181A have two fold lower affinity for IGF-I and IGF-II respectively the percentage proliferation for these treatments was adjusted accordingly.
Materials and Methods
Mutagenesis and Subcloning
[0063] Mutations of cDNA encoding hIGFBP-2 in the pBluescript vector (Stratagene, La Jolla, Calif., USA) were introduced using the Quikchange mutagenesis method (Stratagene). The following oligonucleotides were used to introduce the lysine (K) to alanine (A) mutations and the deletion mutant Des(114-170)His:
[0065] The deletion mutant Des(114-170)His was generated by sequentially introducing two NaeI restriction sites in the cDNA encoding residues 114 and 170 respectively. The new clone was then digested with NaeI and religated to delete out the sequence between these sites.
[0066] The cDNA clones encoding resultant mutant IGFBPs were transformed into DH5α E. coli. Clones were sequenced to confirm correct introduction of mutations. They were subsequently subcloned into the pXMT-2 vector using XhoI and EcoRI restriction sites and transformed into DH5α E. coli (see FIG. 1; Rathjen P. D. et al., (1990); Whyatt L. M. et al., (1993)). Mutant IGFBP-2 proteins were expressed upon transient transfection of COS-1 monkey kidney cells (ATCC:CRL 1650) with the mutant IGFBP-2 cDNAs. Cos-1 cells are cultured in DMEM (GIBCO)+10% FCS. The method of culture and purification is described in Forbes et al., (1998)
Purification and Analysis of Purity
[0067] Proteins were purified using standard Nickel affinity purification techniques taking advantage of a 6 histidine tag at the C-terminus of each protein. Purification is from culture medium as the IGFBP is secreted (Forbes et al, 1998). Following elusion from the nickel column, proteins were further purified using reverse phase high performance liquid chromatography (HPLC). Purity was analysed by rpHPLC, SDS PAGE and mass spectrometry. The mass of each mutant was determined by electrospray mass spectrometry (by Dr. Chris Bagley, Hanson Centre) and found to be correct (generally within the limits of the mass spec=1 mass unit/10,000 daltons).
Measurement of IGF Binding Affinities
[0068] IGF binding affinities of hIGFBP-2 and mutants were determined by surface plasmon resonance using the BIAcore with IGF-I or IGF-II coupled to the sensor surface (for details of method see Carrick et al., (2001). IGF-I or IGF-II (70RU) was coupled to a CM-5 biosensor chip (BIAcore Inc) via amine groups using standard coupling procedures (Lofas and Johnsson, 1990). Briefly, at 5 μl/min a CM5 chip was activated with 35 μl NHS (0.4 mg)/EDC (2.6 mg) and then 35 μl IGF (10 μg/ml) was coupled in 10 mM sodium acetate pH 4.5. Unreacted groups were inactivated with 35 μl 1M ethanolamine-HCl, pH 8.5. Kinetic studies with a range of hIGFBP-2 or mutant concentrations (50, 25, 12.5, 6.25 and 3.1 nM) were performed at 40 μl/min to minimize mass transport effects with 300 secs allowed for association and 900 secs for dissociation. The IGF surface was regenerated with 10 mM HCl.
Proteolysis Assay
[0069] The source of proteases for the proteolysis assays was conditioned medium of cancer cells. Cells were grown to confluence in the presence of foetal calf serum. (T84 cells are grown in DMEM: Ham's F12 (50:50 v:v) with 10% foetal bovine serum FBS; LNCaP were grown in RPMI+6% FBS; PC3 and DU145 were grown in DMEM+10% FBS, all media and FBS are from GIBCO). Cells were then washed 2×2 hours in serum free culture medium. Cells were then cultured for 3 days in serum free conditions and the medium was collected. Conditioned medium was concentrated approximately 10 fold using a centricon-10 (Millipore Corp, Mass. USA). Purified hIGFBP-2 or mutants thereof (250 ng in 2 μl) were mixed with conditioned medium for 24 hours at 37° C. to allow proteolysis. Proteins were separated on 12% tricine SDS polyacrylamide gels and transferred to nitrocellulose. Nitrocellulose filters were probed with a specific polyclonal anti-IGFBP-2 antibody (raised in our laboratory) to detect IGFBP-2 and IGFBP-2 fragments. A secondary goat anti-rabbit antibody conjugated to avidin alkaline phosphatase (Sigma) was used to detect the anti-IGFBP-2 antibody. Substrates for avidin alkaline phosphatase (nitroblue tetrazolium and 5 bromo 4 chloro 3-indolyl phosphate p-toluidine salt) were added and coloured bands indicated presence of IGFBP-2.
Matrix Binding Assay
[0070] Heparin was biotinylated using biocytin hydrazine (Pierce) using conditions recommended by the manufacturer. Following the reaction the biotinylated heparin was concentrated using a centricon-3 (Millipore Corp, Mass. USA) and was dialysed against H2O. Biotinylated heparin was coupled to a streptavidin biosensor chip in 0.3M NaCl and HBS (hepes buffered saline containing surfactant, BIAcore Inc.). hIGFBP-2 and mutants at different concentrations (6.25 nM to 300 nM) were injected at 10 μl/min. Regeneration of the surface was achieved with 2M NaCl.
Proliferation of HT-29 Colorectal Cells
[0071] Cells are plated at 12,000 cells per well in 96 well plates in RPMI (GIBCO)+10% FCS (foetal calf serum), grown for 2 days, washed in serum free RPMI for 3 hours and then treated with butyrate (5 mM, Sigma) or butyrate with IGF-I at various concentrations in RPMI+5% BSA. In this experiment different amounts of IGFBP-2 or mutant IGFBP-2 were added to the butyrate+IGF-1 treated cells. Proliferation is measured using the Cell titre Glo kit from Promega. This measures ATP levels basically. IGF rescues cells from apoptosis and the binding proteins (native or mutant) inhibit the ability of IGF to rescue cells from apoptosis (sequester IGF away from the receptor).
Results and Discussion
Cloning, Expression, Purity and IGF Binding Affinities
[0072] Five mutants have been designed for introduction of protease resistance or interruption of matrix binding (K180A K181AHis, K227AHis, K234AHis, K237AHis, Des(114-170)His). These were purified to homogeneity (FIG. 2) and subjected to mass spectral analysis to confirm they had the expected mass. The residues K180 and K181 are potential sites of protease cleavage (Ho, J. P. & Baxter, R. C. (1997) and are also possibly involved in matrix binding (Hodgkinson, et al (1994)). K227, K234 and K237 are residues in the analogous regions of IGFBP-3 and -5 corresponding to matrix binding motifs. The potential sites of proteolytic cleavage and matrix binding are highlighted in FIG. 3.
[0073] The purified mutants were tested for their ability to bind IGF-I by BIAcore analysis (FIG. 4). All mutants have similar affinities to native hIGFBP-2. Mutant K237AHis has about 2 fold higher affinity for both IGF-I and IGF-II and Des(114-170)His has a 5 fold or 2 fold decrease in affinity for IGF-I and IGF-II respectively (Tables 2 and 3).
[0074] Table 2 shows the kinetic constants obtained from BIAcore analysis of hIGFBP-2 and mutant hIGFBP-2 binding to IGF-I. Data was analysed using BIAevaluation software 3.0 and fit to a Langmuir 1:1 binding model. The dissociation constant (KD) was determined from the calculation of kd/ka, where ka is the association rate and kd is the dissociation rate. Relative KD is equal to KD of hIGFBP-2/KD of hIGFBP-2 mutant.
[0076] Table 3. Kinetic constants obtained from BIAcore analysis of hIGFBP-2 and mutant hIGFBP-2 binding to IGF-II. Data was analysed using BIAevaluation software 3.0 and fit to a Langmuir 1:1 binding model. The dissociation constant (KD) was determined from the calculation of kd/ka, where ka is the association rate and kd is the dissociation rate. Relative KD is equal to KD of hIGFBP-2/KD of hIGFBP-2 mutant.
Protease Assays
[0078] The mutant binding proteins were tested for protease sensitivity in the assay described in materials and methods. We first analysed the truncation mutant Des(114-170)His and observed protease resistance in a number of conditioned media including T84, HT29, CaCO (all colon cancer cells) and PC3 (prostate cancer cell line). Table 4 outlines which cell lines were used, the relative amount of protease activity (qualitative from observations of gels) and which conditioned media contained proteases which are no longer able to cleave the truncation mutant.
[0079] Table 4. List of cell lines used as sources of conditioned media containing proteolytic activity. Cell lines are grouped in to cancer types. A qualitative score is given to each cell line indicating the amount of proteolytic activity evident upon incubation with hIGFBP-2 (central column). If the truncation of hIGFBP-2 (Des(114-170)His hIGFBP-2) resulted in protection against cleavage by conditioned media compared with hIGFBP-2 then this is indicated in the column on the right.
[0081] Importantly, there was a significant amount of easily detectable protease activity in the T84 cell line conditioned medium (FIG. 5A and 5B). The truncation mutant was clearly resistant to proteolysis in this medium. Proteolysis of hIGFBP-2 was also easily detected in PC3 conditioned medium and the truncation mutant was also resistant to proteolysis by this medium (FIG. 5A). In other cell lines there was less proteolytic activity making detection of protease resistance more difficult (eg LIM1215). In other cell lines the truncation mutant was clearly proteolysed (FIG. 5B).
[0082] These results highlight the fact that each cell line produces a different array of proteases. It is not known which protease is cleaving hIGFBP-2 in the T84 and PC3 media. The cleavage product in T84 medium corresponds to a C-terminal fragment of hIGFBP-2 as detected by an antibody specific for C-terminal residues. We have tested the other mutants in the T84 conditioned medium for protease resistance. As these residues lie outside the 114-170 truncation it was not surprising that none were resistant to proteolysis, suggesting that cleavage does not occur at K180, K181, K227, K234, K237.
Heparin Binding
[0083] We analyzed matrix binding using the commonly used model system of heparin binding. We analyzed heparin binding using the BIAcore. Preliminary data show that the K234A mutation reduces heparin binding 5 fold (FIG. 8) and the K180A, K181A mutation has a great effect on heparin binding. This data indicates that there are probably 2 heparin binding sites on IGFBP-2.
[0084] Table 5. Heparin binding affinities of hIGFBP-2 and K234AHis IGFBP-2 were measured by surface plasmon resonance. Data was analysed using BIA evaluation software 3.0 and fit to a Langmuir 1:1 binding model. The dissociation constant (KD) was determined by the calculation of kd/ka, where ka is the association rate and kd is the dissociation rate.
Proliferation of HT-29 Colorectal Cells
[0086] The assay shows that HT-29 colorectal cancer cells undergo apoptosis in 5 mM Butyrate. Addition of IGF-I rescues cells from butyrate induced apoptosis in a dose dependent manner. Additional IGFBP-2 inhibits the ability of IGF-I to rescue cells from butyrate induced apoptosis by sequestering the IGF away from the IGF receptor. The mutants Des (114-170) and Des (114-170) K180A K181A are more effective at inhibiting the action of IGF-I. This assay shows little difference between Des (114-170) and Des (114-170) K180A K181A suggesting the greatest benefit is the resistance to proteolysis conferred to the molecule by removal of protease cleavage sites. Mutation of positions K180A and K181 A could also protect from further proteolysis or inhibit interaction with the extracellular matrix. However, under the conditions of this assay it is not possible to detect a significant difference between Des (114-170) and Des (114-170) K180A K181A.
[0087] Arai et al.,. Endcrinology 137, 4571-4575 (1996)
[0088] Arai et al., J. Biol. Chem. 269, 20388-20393 (1996)
[0089] Baserga, Exp. Cell Res. 253(1),1-6 (1999)
[0090] Braulke, Horm. Metab. Res. 31(2-3), 242-246 (1999)
[0091] Byun et al., J Clin Endocrinol Metab 85(1):373-81 (2000).
[0092] Chan et al., Science 279, 563-566 (1998)
[0093] Cardin. & Weintraub Arteriosclerosis 9, 21-32 (1989).
[0094] Carrick et al., J. Biol. Chem. 276, 27120-27128 (2001)
[0095] Cohen et al., J. Clin. Endocrinol. Metab. 79, 1410-1415(1994)
[0096] DiGiovanni et al., PNAS 97, 3455-3460 (2000)
[0097] Elminger et al., Mol. Cell. Endocrinol. 175(1-2), 211-218 (1999)
[0098] Firth et al., J. Biol. Chem. 273, 2631-2638 (1998)
[0099] Forbes et al., J. Biol. Chem. 273, 4647-4652 (1998)
[0100] Ho & Baxter, Clin. Endocrinol. 46, 333-342 (1997)
[0101] Ho & Baxter Endocrinology 138, 3811-3818 (1997).
[0102] Hobba et al., J. Biol. Chem. 273:19691-19698 (1998).
[0103] Hodgkinson et al. J. Mol. Endocrinol. 13, 105-112 (1994);
[0104] Imai et al., J. Clin. Invest. 100(10), 2596-2605 (1997)
[0105] Kanety, et al. J. Clin. Endocrinol. Metab. 77, 229-233 (1993).
[0106] Lofas and Johnsson, J. Chem. Soc. Chem. Commun. 21, 1526-1528 (1990)
[0107] Menouny et al., Endocrinology 138, 683-690 (1997).
[0108] Michell et al., Br. J. Cancer 76, 60-66 (1997).
[0109] Nunn et al., J Cell. Physiol. 171, 196-204 (1997).
[0110] Rathjen et al., Cell 62(6),1105-1114 (1990)
[0111] Russo et al. Endocrinology 138, 4858-4867 (1997)
[0112] Sepp-Lorenzino, Breast Cancer Res. Treat. 47(3), 235-253 (1998)
[0113] Thrasher et al., J. Urol. 155, 999-1003 (1996)
[0114] Wang et al., Nature 372, 464-467 (1994)
[0115] Whyatt et al., Mol. Cell. Biol. 13, 7971-7976 (1993)
[0116] Zeslawski et al., EMBO J. 20, 3638-44 (2001)
1. An altered human IGFBP-2 molecule able to bind IGF-I or IGF-II with high affinity, which differs from a human IGFBP-2 molecule by one or more of the following substitutions or deletions:
(i) the lysine at one or more of positions 180, 181, 227, 234 and 237 of the human IGFBP-2 molecule has been replaced with a neutral or acidic amino acid; and/or
(ii) amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
2. The altered human IGFBP-2 molecule of claim 1, wherein the lysine at one or more of positions 180, 181, 227, 234 and 237 has been replaced with alanine.
3. The altered human IGFBP-2 molecule of claim 1, wherein the lysine at position 180 has been replaced with alanine.
4. The altered human IGFBP-2 molecule of claim 1, wherein the lysine at position 181 has been replaced with alanine.
5. The altered human IGFBP-2 molecule of claim 1, wherein the lysines at positions 180 and 181 have been replaced with alanines.
6. The altered human IGFBP-2 molecule of claim 5, wherein amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
7. The altered human IGFBP-2 molecule of claim 1, wherein the lysine at position 234 has been replaced with alamine.
8. The altered human IGFBP-2 molecule of claim 7, wherein amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
9. The altered human IGFBP-2 molecule of claim 1, wherein the lysines at positions 180, 181 and 234 have been replaced with alanines.
10. The altered human IGFBP-2 molecule of claim 9, wherein amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
11. The altered human IGFBP-2 molecule of claim 1, wherein amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
12. The altered human IGFBP-2 molecule of claim 1, which differs from a human IGFBP-2 molecule by the following substitutions and deletions:
(i) the lysine at one or more of positions 180, 181, 227, 234 and 237 of the human IGFBP-2 molecule has been replaced with a neutral or acidic amino acid; and
(ii) amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
13. An isolated nucleic acid molecule encoding an altered human IGFBP-2 molecule able to bind IGF-I or IGF-II with high affinity, which differs from a human IGFBP-2 molecule by one or more of the following substitutions or deletions:
(i) the lysine at one or more of positions 180, 181, 227, 234 and 237 of the human IGFBP-2 molecule has been replaced with a neutral or acidic amino acid; and/or
(ii) amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
14. The isolated nucleic acid molecule of claim 13, wherein the lysine at one or more of positions 180, 181, 227, 234 and 237 has been replaced with alanine.
15. The isolated nucleic acid molecule of claim 13, wherein the lysine at position 180 has been replaced with alanine.
16. The isolated nucleic acid molecule of claim 13, wherein the lysine at position 181 has been replaced with alanine.
17. The isolated nucleic acid molecule of claim 13, wherein the lysines at positions 180 and 181 have been replaced with alanines.
18. The isolated nucleic acid molecule of claim 13, wherein the lysines at positions 180 and 181 have been replaced with alanines and amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
19. The isolated nucleic acid molecule of claim 13, wherein the lysine at position 234 has been replaced with alanine.
20. The isolated nucleic acid molecule of claim 13, wherein the lysine at position 234 has been replaced with alanine and amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
21. The isolated nucleic acid molecule of claim 13, wherein the lysines at positions 180, 181 and 234 have been replaced with alanines.
22. The isolated nucleic acid molecule of claim 13, wherein the lysines at positions 180, 181 and 234 have been replaced with alanines and amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
23. The isolated nucleic acid molecule of claim 13, wherein amino acids 114-170 of the human IGFBP-2 molecule have been deleted.
24. The isolated nucleic acid molecule of claim 13, encoding an altered human IGFBP-2 molecule able to bind IGF-I or IGF-II with high affinity, which differs from a human IGFBP-2 molecule by the following substitutions and deletions:
(i) the lysine at one or more of positions 180, 181, 227, 234 and 237 of the human IGFBP-2 molecule has been replaced with a neutral or acidic amino acid; and
(ii) amino acids 114-170 of the human IGFBP-2 molecule have been deleted.


text:
Jul 4, 2012The corpse of Rambhai, an Indian fisherman who died in a Karachi prison, is likely to be repatriated to the country Monday. Activist Jatin Desai says Rambhai died way back on May 25, and since then, his family that lives in Gujarat as well as voluntary organisations and individuals in Mumbai have been ''working desperately'' to get his body back. The month-long delay has compounded their trauma. -- .
Jul 4, 2012Hansaben Parmar, the widowed mother of young Bhavesh who is languishing in Lahore's Kot Lakhpat jail since nearly eight years, has expressed relief at hearing the campaign to release him has gone international. Unaware of himself, the mentally ill youth had crossed over to Pakistan aboard the Samjhauta Express and was arrested upon arrival. --
Jul 4, 2012Awais Sheikh, counsel for Sarabjit Singh as well as other Indian prisoners in Pakistan, is preparing to file a writ petition in a Pakistani court seeking the release of Bhavesh Parmar, the Mumbai-based software engineer who is in Lahore Central Prison since almost eight years. Mumbai activist Jatin Desai has helped secure power of attorney for the lawyer in this case. --
May 22, 2012Siddhivinayak temple will conduct a day-long auction of gold offerings received by the deity on May 27. Around 120 items comprising finger rings, golden chains, coins, modaks and durva grass have been displayed at the temple for public viewing. --
Apr 26, 2012Amar Chitra Katha Thursday published a comic book on `Salim Ali: The Bird Man of India' based on India's best known ornithologist. The title was launched in Hornbill House, the premises of the Bombay Natural History Society (BNHS) by director Dr Asad Rahmani in the presence of other wildlife enthusiasts. --
Feb 23, 2012With the tenth anniversary of the Godhra train fire and the subsequent Gujarat riots coming up on Monday, Feb 27, activists in Mumbai will relay a live webcast of the anniversary commemoration taking place at Gulbarg Society. This is the colony in Ahmedabad where former MP Ehsan Jafri was killed. Godhra train victims' families not to attend. -- India's largest all women's car drive is back on Sunday, 26th February 2012. For more details log on to lavasawomensdrive.com
Jul 13, 2011Horror stories of dismembered bodies and pieces of burnt flesh emanated from the blast site at Zaveri Bazar. Residents and shopkeepers were at their wits' end trying to rush survivors who were missing an arm or a leg to hospital even as they tried to piece together the puzzle before doing so.
Jul 13, 2011The white tiles of the casualty ward of Hurkissondas Hospital were marked with bloodied footprints. Attendants kept busy mopping up pieces of torn clothing and cotton swabs, all soaked in blood.
Jul 13, 2011Shocked shopowners pointed to the remains of the scooter upon which the explosive device had been placed in Zaveri Bazar. They reported seeing an umbrella wrapped in wires upon the vehicle that did not belong to any of the local residents.
Jul 13, 2011A group of diamond brokers was the first to react as soon as the bomb went off outside Panchratna Building in Opera House. They bundled bodies of bloodied men into taxis to take them to hospital without waiting to see if they were dead or alive. Cab drivers did not charge a penny.
Copyright © 2019 Bennett, Coleman & Co. Ltd. All rights reserved. For reprint rights: Times Syndication Service

subset: commoncrawl


text:
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text:
"I found out I can see aura (wow!) but I can't do it at will very well. Do you know any tips (or websites) on how to develop this pstchic ability?"
Merry Meet Seeker!
Wow! Yes, that is most definitely how one feels when for the first time the colors pop up spontaneously! And you are very lucky indeed, I might add, because most people have to work incredibly hard to develop their auric sight.
As for tips, here goes my penny's worth:
You need to practice. I have two exercises in mind for you. When doing these exercises, practice it against a white or cream backdrop. I also prefer good light. Why? Because light is needed to see color. Ask anyone who scuba dives. The deeper one goes, the darker the water gets and the less color one sees. As for the backdrop, colors manifesting against a light background are clearer. (As you study the topic you will find that psychics all have their own ideas on this one:))
The first exercise you can perform alone. Place your hand against a white wall, with your body the full arm length away. Relax your eyes and "unfocus" or "soft focus" them. You should not be looking directly at your hand. If you are using your right hand, look slightly to its left. You will start seeing colors materializing gradually. The more practiced you get; the more layers of color will appear. Resist the temptation to look at the colors directly, because they will disappear! Also, try not to blink too much. Do this exercise daily for 5 minutes, but not more than twice a day. You can also do this exercise by placing yourself in front of a mirror against a white backdrop. You will quickly establish the distance you need from the mirror suited to your auric site. Find a spot about 3 to 4 inches to the left or the right of your neck. Relax and allow the colors to materialize.
The second exercise will require a partner. Let your partner stand against a wall. Ask him or her not to move about too much, especially at first. Stand 4 to 5 yards away from your partner. "Unfocus" or "Soft focus" your eyes. Find a spot about 6 to 8 inches to the left or to the right of your partner. Relax and allow the colors to materialize. At first only small sections may materialize. With practice, the entire aura will pop out in all its glory. Importantly, you can adjust the distances to suit your personal auric sight. It must work for YOU.
Finally, just a quick word on ethics. It is not really acceptable to view the auras of others intentionally without their permission. It can be like eavesdropping or prying. Sure, sometimes you will see the aura of another without trying, especially when it develops at first. You need to train yourself to switch it off though when not required.Â
Hope you have lots and lots of fun in developing your auric sight. A good book to buy is How to See and Read the Aura by Ted Andrews. Watch out when using web sites. There are quite a few charlatans out there.
Brightest Blessings!


text:
SpringerBriefs in Mathematical Physics
Book Series
There are 33 volumes in this series
There are 33 volumes in this series
Published 2014 - 2018
SpringerBriefs are characterized in general by their size (50-125 pages) and fast production time (2-3 months compared to 6 months for a monograph).
Briefs are available in print but are intended as a primarily electronic publication to be included in Springer's e-book package.
Typical works might include:
SpringerBriefs in Mathematical Physics showcase, in a compact format, topics of current relevance in the field of mathematical physics. Published titles will encompass all areas of theoretical and mathematical physics. This series is intended for mathematicians, physicists, and other scientists, as well as doctoral students in related areas.
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Springer Briefs in a nutshell
SpringerBriefs specifications vary depending on the title. In general, each Brief will have:


text:
If a physician has got disruptive behavior for many years, you won't alter the culture immediately. But it is possible to implement protocols and policies which will help safeguard your clinic.
Prohibit criminal harassment that comes with a clear statement your group strictly prohibits sexual harassment in addition to harassment on the grounds of race, color, sex, faith, disability and the rest of the categories protected by state and federal regulation.
The policy must define harassment and provide examples of improper behavior. This can be useful when addressing a scenario where a disruptive physician won't operate with a colleague due to gender, religion, ethnicity, etc.
Prohibit retaliation against any worker to make sure that if staff members or doctors make complaints regarding a tumultuous doctor they are protected from any retaliatory behavior. You can visit https://www.thedisruptivephysician.com/ to know more about disruptive physician behavior.
Many clinics require some effort and time to come up with behavior criteria. As soon as you've implemented behavior policies, make sure you train each doctor on what policies imply and how they'll be enforced, apply those policies regularly. It's a time-tested endeavor, but when the group is directed by the policies in your manual, your clinic direction will become simpler.


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The Last Moment รักสามเศร้า
Jul. 19, 2008Thailand107 Min.N/A
what going on?
The story of Payu, Fah and Nam, fresh college graduates from the Interior Design faculty. After college, they are faced with important transitions of their lives, which includes career, romance etc. Fah chooses to marry her boyfriend, leaving a career future in interior design. Nam chooses to end her affection for Payu and plans to get a Master degree in England. For Payu, Nam's and Fah's closest friend, chooses to end his crush with Fah and goes back to Chiang Rai to look after the family business.
ชื่อต้นฉบับ รัก สาม เศร้า
คะแนน IMDb 7.3 40 votes
คะแนน TMDb 42 votes


text:
Regrettably, this isn't how a lot of people address their bodies. They have an inclination to eat toxic foods, they don't physical exercise, they snooze below required, and do all kinds of other things that mess up with their wellbeing. Certainly, they don't accomplish that on reason.
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All in all, this arrangement is obviously designed to torpedo the countrywide economic system. It's not an accident. It's not an unintended consequence. The Globalists could be criminals, However they aren't Silly criminals.
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It has become the earth's strongest alliances. And still most people have almost certainly never ever heard about it, for the reason that its existence is a intently guarded governing administration magic formula.
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MooFree May
UK activists have resumed their attack on the dairy industry.
At the end of May, the winner of the Ahuwhenua Trophy for the top Māori sheep and beef farm will be announced at a gala dinner in Gisborne.
Good Māori farms shouldn't just be pigeon-holed as good Māori farms, they should just be recognised as top farms globally.
The East Coast of the North Island features prominently in this year's Ahuwhenua Young Māori Farmer Award for Sheep and Beef.
Two farms from the North Island east coast and one from Central Hawkes Bay are the finalists in this year's Ahuwhenua competition for the top Māori sheep and beef farm.
Landcorp is to sponsor the Ahuwhenua Trophy, which recognises excellence in Maori dairy and sheep and beef farming.
Massey University has signed as a sponsor of the Ahuwhenua Trophy, awarded annually to the country's premier Māori farmer.
About 700 people in late May attended a gala awards night in Whangerei, to see Omapere Rangihamama Trust farm, near Kaikohe, awarded the Ahuwhenua Trophy.
Prime Minister Bill English says in most regions Maori now have the potential to become the largest long-term investors.
On Friday the winner of the Ahuwhenua Trophy for the top Maori sheep and beef farm will be named at a gala awards dinner in Whangarei.
UK activists have resumed their attack on the dairy industry.
The shelf life of fresh milk can be extended up to two months by a technology developed by an Australian…


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Tuesday, November 20, 2018
Gratitude Tree - Oui Jar
6 comments:
I LOVE this!!! So unique and am always wondering how to repurpose things instead of throwing them away!!! This would work for any holiday!ReplyDelete
This is a really cute idea! Love it. But love mine more!!ReplyDelete
Love the recycling idea here .Thanks for sharing your work with us here at http://ttcrd.blogspot.com/ReplyDelete
Good luck with your entry hope we see more soon
Shirley-Anne DT
Super cute!!! Thank you for joining us at City Crafter, good luck!!! Amy DTReplyDelete
Thank you so much for entering the challenge at Through The Craft Room Door this week. Looking forward to seeing you again in the next challenge.ReplyDelete
Hugs & Craftiness,
AngelaThrough The Craft Room Door DT
Taste Of Craftiness {My Blog}
Through The Craft Room Door
OH! I LOVE this idea too!! The thankful tags are brilliant!!ReplyDelete
THANK YOU for taking the time to leave your comments. I hope I've inspired you to get a bit crafty today ;)


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In the interest of not publishing a book myself, I'm just going to list recent reads, and a few authors. I tend to skew heavily toward mystery, science fiction and fantasy, while staying far, far away from best sellers.
1. The Bible. Well, duh. Each rereading teaches you more.
2. Anything by Agatha Christie. Yes, by now the books are definitely dated, but Christie not only wrote well, she masterfully broke all the "rules" of mystery writing (see: The Murder of Roger Ackroyd, Murder on the Orient Express for starters). In the same vein, check out Ngaio Marsh (New Zealander who was a contemporary of Christie), Dorothy Sayers (who doesn't love Peter Wimsey?) and Margary Allingham.
3. The Lunar Chronicles by Marissa Meyer. The first book in the series is Cinder, a retelling of the classic Cinderella story, with several twists. Cinder, you see, is part cyborg. There are five main books in the series, each bringing in another fairytale character. These are technically young adult novels, but are written well enough to be enjoyed by all ages.
4. Ready Player One by Ernest Cline. I'm reading this for the third time to refresh my memory before the movie comes out next week. The book is a love letter to all things 1980s, and if you lived through that decade, the book is bound to bring a smile to your face. I've young friends who are already expressing disappointment in the movie, just based on the trailers. ::Sniff:: Their opinions don't hold much water, given they were barely toddlers in the '80s. The book is well worth the read.
5. Monster Hunter International by Larry Correia. Vampires, werewolves, things that go bump in the night and much worse are real, and could be your neighbor. Good things MHI has a secret government contract to hunt down and take care of the dangerous ones. If you are a gun control nut, stay away from this series; Correia is a gun nut who shoots competitively and describes the arsenal in loving detail. The books are a well-written hoot. Bonus points because the main character is an accountant (well, until he stays late to do some work and ends up having to kill his boss, who has gone all werewolf on him).
6. The School of Essential Ingredients by Erica Bauermeister. There are three books in this loose series, all centered around food, though really about relationships. If it tells you anything, I see I read all three in under a week two summers ago. It should tell you something, as these are outside my usual mystery/scifi/fantasy zone.
So, so many more...
Cleo Coyle - actually a writing team, putting out mysteries revolving around a Greenwich Village coffeehouse. Bonus, books include an appendix with fun coffee facts and beverage and dessert recipes
Jennifer Chiaverini - Sylvia opens her home as a quilting retreat, and the books tell the stories of the women who attend. Bonus, Ms. Chiaverini is a Wisconsin resident.
Jim C. Hines - the Magic ex Libris series. Libriomancers are able to pull items from books into reality, using them to fight those bent on destruction. Bonus, the arguements between Gutenberg and Ponce De Leon. Really.
These are just the things that popped out at me as I looked down the list of titles in my kindle library. I'm close to 500 titles there; truly, there is quite a bit of nonfiction as well.
If nothing else, I'm hoping this list might inspire you to say, "Ugh! Horrible titles! I beat I can read better books than that!" That won't bother me, as long as you are reading.


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