text:
|Publication number
|Publication type
|Application number
|PCT number
|Publication date
|Oct 1, 2002
|Filing date
|Feb 12, 1997
|Priority date
|Feb 13, 1996
|Fee status
|Also published as
|Publication number
|Andrej Sourovoi, Guenther Jung
|Original Assignee
|Andrej Sourovoi
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (18), Non-Patent Citations (24), Referenced by (40), Classifications (26), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a 371 of PCT/EP97/00629 filed in Feb. 12, 1997.
This invention relates to lipid compounds and their use, for example for the transport of biologically active substances or molecules in cells.
Liposomes are spherical, self-closed structures composed of lipid bilayers which entrap in their interior a portion of the solvent, in which they float. They may consist of one or more concentric membranes, and their size ranges from several nanometers to several dozens of micrometers.
Liposomes are mostly made from amphiphilic molecules which can be characterized by having a hydrophilic (often named the polar head) and a hydrophobic group (nonpolar tail) on the same molecule. In most cases, liposome-forming molecules are not soluble in water. However, under certain circumstances, they form colloidal dispersions.
Liposomes can be large or small and may be composed from one to several hundred of concentric bilayers. With respect to the size and the nature of the layer (lamellae), they can be classified as multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles LUVs).
SUVs have a diameter from 20 to 600 nm and consist of a single lipid bilayer which surrounds the interior aqueous compartment. LUVs have a diameter from 600 to 30000 nm. MLVs vary greatly in size for up to 10000 nm and contain more than one lipid bilayer, therefore they are multi-compartmental in their structure.
Liposomes can be produced in a number of ways. The so-called "thin-film hydration" method results in the formation of heterogeneous dispersions of predominantly MLVs. By using charged lipid compositions rather high fractions of LUVs can be produced. Said dispersions can be further treated (mechanically, electrostatically or chemically) in order to produce solutions of SUVs. Most frequently these methods include extrusion through filters with pores of different diameter, or sonication.
Alternatively, liposomes can be prepared by lyophilization, where the lipid-film is then dissolved in a volatile solvent (for example tert-butyl alcohol), frozen and lyophilized.
A variety of methods for preparing liposomes have been described in the periodical and patent literature: Szoka and Papahadjopoulos in: Ann. Rev. Biophys. Bioeng. 9, 467-508 (1980) as well as U.S. Pat. Nos. 4,229,360, 4,241,046, 4,235,871.
The most important liposome feature is their ability to dissolve, protect and carry hydrophilic or hydrophobic molecules. For negatively charged drugs, including some proteins, positively charged liposomes can be used. Improvements in therapy were observed, despite the known fact that positively charged liposomes can be toxic.
Various DNA transfection methods have been developed in the past twenty years. These methods include the calcium phosphate precipitation method, DEAE-dextran method, electroporation method, microinjection, receptor mediated endocytosis, liposomes and viral vectors. However, most of these methods posess some significant drawbacks: they are either too inefficient, or too toxic, or too complicated and tedious to be effectively adapted to biological and therapeutical protocols both in vitro and in vivo. For instance, the most frequently used in vitro calcium phosphate precipitation method is too inefficient (average transfection frequency of 1 in 104 cells). Electroporation is much more efficient than the calcium phosphate method. However, this method is too aggressive (maximum efficiency is obtained at about 50% of cell death) and, in addition, this method requires a special apparatus. Microinjection is efficient, but it is too tedious and not practical. All these methods cannot be used in vivo.
A receptor mediated endocytosis method involves polylysine as a basic polymer for interacting and packaging of DNA. Polylysine has been modified with different ligands (transferrin, insulin, asialoorosomukoid, or galactose) in order to target modified protein-DNA complexes to cell surface receptors: Wu, G. Y. et al. in: J. Biol. Chem. (1987) 262:4429-4432, Cotten, M. et al. in: Proc. Natl. Acad. Sci. (USA) (1990) 87:4033-4037, Huckett, B. et al. in: Biochem. Pharmacol. (1990) 40:253-263, Plank, C. et al. in: Bioconjugate Chem. (1992), 533-539. The method has been dramatically improved by use of inactivated adenovirus to facilitate exit of DNA from endosomes, see: Wagner, E. et al. in: Proc. Natl. Acad. Sci. (USA) (1992) 89:6099-6103, Christiano, R. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:2122-2126. The major disadvantage of the described approach includes an inherent inability to control the protein conjugation chemistry and to prepare such conjugates in a reproducible fashion.
At present the best transfection efficiencies both in vitro and in vivo are obtained with retrovirus, adenovirus, and some others, see for example: Kerr, W. G. and Mule, J. J. in: J. Leucocyte Biol. (1994) 56:210-214, Hwu, P. and Rosenberg, S. A. in: Cancer Detect. Prevent. (1994) 18:43-50, Rosenfeld, M. A. et al. in: Cell (1992) 68:143-155. Nevertheless, the use of viral vectors poses several considerations including the requirement of extensive cell culture manipulations, low titers for certain virus systems and the cell tropism of the virus. In addition, immune reactivity against viral vectors may cause problems. Most importantly, the safety issues related to the use of viral vectors are not completely resolved to date.
Liposomes have also been used to introduce DNA into cells both in vitro and in vivo. The most successful liposome systems use different cationic lipids like dioleyloxypropyl-trimethylammonium (DOTMA, which forms a reagent in combination with phosphatidylethanolamine (PE)), dioleoyloxypropyl-trimethylammoniummethyl sulfate (DOTAP), dimethylaminoethane-carbamoyl cholesterol, dioctadecylamidoglycylspermine, 2,3-dioleyloxy-N-(2(sperminecarboxamido)ethyl)-N,N-dimethyl-1 propanamine (DOSPA), which in combination with PE forms a reagent, see: Felgner, P. L. in: Proc. Natl. Acad. Sci. (USA) (1987) 84:7413-7417, U.S. Pat. No. 5,208,036, Leventis, R. and Silvius, J. R. in: Biochimica et Biophysica Acta (1990), 124-132, Gao, X. and Huang, L. in: Biochem. Biophys. Res. Commun. (1991) 179:280-285, Behr, J.-P. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6982-6986.
The advantage of using the above mentioned compounds is that the cationic liposome is simply mixed with DNA and added to the cell. Transfection efficiency is usually high when compared to other physical methods of DNA transfer. Besides for delivery of DNA, a specific compound has been used to deliver mRNA and proteins into cultured cells, see: Malone, R. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6077-6081, and Debs, R. et al. in: J. Biol. Chem. (1990) 265, 10189-10193. Some of the above mentioned compounds have been used to transfect reporter or therapeutically utile genes in vivo, see: Nabel, G. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:11307-11311, Zhu, N. et al. in: Science (1993) 261:209-211. Finally, a DNA transfection protocol has been developed that makes use of the cyclic cationic peptide gramicidin S and PE, see: Legendre, J.-Y. and Szoka, F. C. in: Proc. Natl. Acad. Sci. (USA) (1993), 90:893-897. The above mentioned system takes advantage of the DNA binding ability and the membrane destabilization properties of gramicidin S.
The main disadvantage of cationic liposomes includes their relatively high cytotoxicity. In addition, most of the above mentioned compounds are not active or show highly reduced activity in the presence of serum. Most of them need the use of PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion. Studies on the mechanism responsible for transfection using the cationic lipids have not been fully addressed to date. The need exists, therefore, for a less toxic, non-infectious and more efficient delivery of biological molecules into the cytoplasm and nuclei of living cells.
The object of the present invention is to overcome the above mentioned and other drawbacks of the state of the art. According to a first aspect there are lipid compounds to be provided to allow an improvement of the transport of biologically active agents or molecules through membranes and thus in cells or cell organelles.
This object is solved primarily by the compounds according to claim 1. Preferred compounds are claimed in the subclaims 2 to 11. Derivated products and applications of the novel compounds according to the invention are mentioned in claims 12 to 24 (complexes), claims 25 and 26 (methods for the preparation of complexes), claim 27 (liposome), claims 28 to 32 (liposome formulations) and claims 33 to 42 (methods for the transport of substances and agents through membranes). The wording of all the claims is included into the description by reference.
The compounds according to the invention are illustrated by formula I as follows:
R1 and R2 are the same or different and are an alkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms;
R3, R4, and R5 are the same or different and are hydrogen, alkyl, or alkylamine, having from 1 to 8 carbon atoms, or an amino acid, an amino acid derivative, a peptide, or a peptide derivative;
W is hydrogen, a carboxyl group, or a side chain group of amino acids, amino acid derivatives, peptides or peptide derivatives;
Y is a linking group having at least one atom other than hydrogen, particularly —CO—, —(CH2)mCO—, —(CH2—)m, —(CHOHCH2—)m, wherein m is from 1 to 20, —CH2—S—CH2—, —CH2—SO—CH2—, —CH2—SO2—CH2—, —CH2—SO2— or —SO2—;
Z is an ester, ether or amide bond;
n is from 1 to 8; and
X is an anion, particularly a pharmaceutically acceptable anion.
The compounds according to the invention comprise lipids (detergents, surfactants) the main characteristics of which are well-known. The compounds comprised by formula I can be present in the form of their optical isomers (R- or S-configuration) or in the form of mixtures thereof.
According to formula I the lipid compounds are prepared in the form of ionic salts. This is due to the fact that per se neutral compounds are present as ions in (aqueous) solution. Of course the invention is also to include the corresponding neutral compounds. The compounds according to formula I can be provided with further charges by appropriate selection of substituents, for example in the substituent W by appropriate selection of the side chain group of an amino acid.
For a better understanding of the claims and description are given the following definitions:
Alkyl refers to a fully saturated branched or unbranched carbon chain radical.
Alkenyl refers to a branched or unbranched unsaturated carbon chain radical having one or more double bonds.
Alkynyl refers to a branched or unbranched unsaturated carbon chain radical having one or more triple bonds.
Amino acids refer to a monomeric unit of a peptide, polypeptide or protein. The twenty protein amino acids (L-isomers) are: alanine ("AA"), arginine ("R"), asparagine ("N"), aspartic acid ("D"), cysteine ("C"), glutamine ("Q"), glutamic acid ("E"), glycine ("G"), histidine ("H"), isoleucine ("I"), leucine ("L"), lysine ("K"), methionine ("M"), phenylalanine ("F"), proline ("P"), serine ("S"), threonine ("T"), tryptophane ("W"), tyrosine ("Y"), and valine ("V"). The term amino acid, as used herein, also includes analogues of the protein amino acids, D-isomers of the protein amino acids, β-, y- and other amino acids, unnatural amino acids, and their analogues.
Biologically active substance refers to any molecule or mixture or complex of molecules that exerts biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, steroids, vitamins, polyanions, nucleosides, nucleotides, polynucleotides, etc.
Buffers referred to in this disclosure include: "Hepes" which is N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and used here as a buffer at about pH 7; "PBS" is a phosphate buffer saline, and is 10 mM phosphate and 0.9% wt. NaCl, used here as an isotonic physiological buffer at pH 7.4; "Transfection buffer" is 10 mM Hepes and 0.9% wt. NaCl and used here as a buffer at about pH 7.4; "Tris" is tris(hydroxymethyl)amino methane, also used here as a buffer at about pH 7.
Cell-targeted complexes or liposomes applied to the cells include further molecules, e. g. on their surfaces, capable of recognizing a component on the surface of said targeted cell. Cell recognition components include: ligands for cell surface receptors, antibodies to cell surface antigens etc.
Charge-masked complexes or liposomes are to be understood as positively charged, comprising a compound of formula I and a bound polymer which covers the surface of the liposome, for example. A polymer can be covalently linked to any lipid forming the liposome, or be adsorbed on their surface.
A complex is defined as the product made by mixing of two or more components. Such a complex is characterized by a non-covalent interaction (ionic, hydrophilic, hydrophobic etc.) between two or more components.
DNA represents deoxyribonucleic acid which may comprise unnatural nucleotides. DNA may be single-stranded or double-stranded.
Drug refers to any prophylactic or therapeutic compound which is used in the prevention, diagnosis, alleviation, treatment, or cure of disease in a human or an animal.
A liposome formulation is a composition of substances including a liposome which includes entrapped material for diagnostic, biological, therapeutic or other use.
An optional co-lipid is to be understood as a compound capable of producing a stable liposome, either alone, or in combination with other lipid components. Examples of optional co-lipids are phospholipid-related materials, such as lecithin, phosphatidylcholine, dioleylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatic acid, cerebrosides, dicetylphosphate, etc., non-phosphorous lipids like steroids and terpenes. Additional non-phosphorous lipids are, e. g. stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, dioctadecylammonium bromide, amphoteric polymers, triethanolamine lauryl sulfate, cationic lipids described before and the like.
A pharmaceutically acceptable ion is an ion which itself is non-toxic.
A polyanion is a polymeric structure, where more than one unit of the polymer bears a negative charge and the net charge of the polymer is negative.
A polycation is a polymeric structure, where more than one unit of the polymer bears a positive charge and the net charge of the polymer is positive.
A polynucleotide is DNA or RNA containing more than one nucleotide. Polynucleotides are intended to include cyclic polynucleotides and unnatural nucleotides, and can be made by chemical methods, or by use of recombinant technology, or by both.
A polypeptide is to be understood as a series of two or more amino acids coupled via covalent linkage.
RNA represents ribonucleic acid which may comprise unnatural nucleotides. RNA may be single-stranded or double-stranded.
Preferably the above described compounds according to formula I comprise Y that is carbonyl, i. e. —CO—. This results in a linkage of the peptide bonding type —CO—N<.
With the lipid compounds according to formula I the group Z preferably includes an ester linkage, i. e. the group —O—CO—.
In further preferred embodiments R1 and R2 include an alkyl or alkenyl group of 10 to 20 carbon atoms, preferably 12 to 18 carbon atoms. The groups R1 and R2 are preferably the same. Of the described preferred groups the alkenyl groups are preferred. Accordingly, the groups R1 and R2 comprise, besides palmityl or stearyl groups, preferably the oleyl group or moieties of the linolic or linoleic acid.
In formula I preferably n is 2. Further, the group W preferably is a side chain group of a basic amino acid, in particular a side chain group of lysine or ornithine.
The groups R3, R4 and R5 may be alkyl groups having 1 to 8 carbon atoms, in particular the methyl group. Preferably the three groups R3, R4 and R5 are the same. In a particularly preferred embodiment R3, R4 and R5 are hydrogen.
Generally, all conceivable anions may be used as counterion X, with pharmaceutically acceptable anions being preferred for most applications of the present invention. Preferably X may be a halide anion, in particular a chloride anion.
Preferred compounds according to formula I are given in claim 10, to which particular reference is made. A particularly preferred compound is L-lysine-bis-(O,O′-cis-9-octadecenoyl-β-hydroxyethyl)amide-dihydrochloride or an optical isomer thereof.
Compounds covered by the definition of formula I and preferred according to the invention can be prepared by preparing the corresponding compound using the following coupling:
amino acid or peptide-----dialkanol amine-----fatty acid or fatty alcohol
Compounds preparable in this manner are particularly adapted to the applications which will be discussed below. Said compounds are characterized in particular by a considerably minor toxicity than so far known lipid compounds used for similar applications.
Besides the described lipid compounds the invention includes complexes which will be prepared by means of said novel compound. The expression "complex" is to be understood in the sense of the above definition. It is not necessarily a completely formed liposome. However, as the exact mechanisms of the complex formation are not known, the case that primarily liposomes are formed from lipides and these in turn will complex with polyanions, polycations or complexes thereof, is not excluded according to the invention.
The characteristics of the complexes according to the invention are claimed and described in claims 12 to 17, and 18 to 24, respectively. Explicit reference is made to these claims. The complex formation relies essentially on the fact that the compounds according to the invention have a positive charge.
The ratio between lipide compound and the other constituents of the complex, that is in the (binary) polyanion-lipid complexes the ratio polyanion and lipid compound, may be varied considerably upon the desired application. Preferably, in favour of the lipid compound this ratio is larger than 1:1 (considering charge), so that a positive total or netto charge is resulting for the complex to allow an interaction with the negatively charged surface of biological membranes in a simple manner. If necessary a particularly advantageous ratio will be determined experimentally. Thus for example, in a DNA transfection that ratio of DNA and lipid compound will be determined by experiment which results in an optimized expression of the transfected DNA.
Generally the complexes according to the invention can be realized with all molecules having a negative charge. Preferred are such complexes that use a polynucleotide as the polyanion.
In the ternary complexes given in claims 18 to 24 it is preferred that the polycation is a polypeptide. Generally it is possible to use neutral polypeptides for the formation of ternary polynucleotide-polypeptide-lipid complexes which are also to be covered by the present invention.
Surprisingly, during the formation of ternary complexes was found, for example, that by using polypeptides, the transfection of polynucleotides will be increased significantly as compared to the use of polynucleotide-lipid complexes. The peptide sequences mentioned in claim 23, to which explicit reference is made, can be used preferably. The first sequence represents the C-terminal portion of the nucleoprotein of the hepatitis B virus (HBV). Although a final scientific explanation is not available at present, there seems to be a possible correlation of the positive effect of these peptide sequences in the ternary complexes and the comparably high portion of basic amino acids in the sequences.
The described complexes for particular applications can optionally be charge-masked or cell-targeted for interaction with particular cells or cell organelles.
Further the invention comprises the methods of claims 25 and 26 for the preparation of the complexes according to the invention. The preparation and the contacting involved therein are realized by common methods as well-known to those skilled in the art. Thus for example, in the preparation of binary complexes are combined buffer solutions including the polyanion and the lipid compound, respectively. In a corresponding manner is the preparation of ternary complexes, wherein first a polyanion-polycation complex is obtained in a buffer solution and subsequently the buffer solution is combined with a buffer solution containing the lipid compound.
Further the invention comprises liposomes, which are preparable or prepared from at least one of the lipid compounds according to the invention. The preparation of the liposomes is performed in conventional ways well-known in the state of the art.
Further the invention comprises liposome formulations in aqueous solution including at least one biologically active substance (material, molecule etc.) and one lipid component. Therein the lipid component comprises at least one of the lipid compounds according to the invention. Besides the biologically active substance and the lipid component the liposome formulation includes conventional solution constituents of an aqueous solution, which may be a solution in pure water or preferably conventional buffer solutions.
As is given in the wording of claim 28, a single compound of formula I or a mixture of such compounds may be applied as a lipid component. Furthermore it is possible, to use the compound or compounds according to the invention with additional co-lipids as defined above, for example PE, POPE in mixture.
As long as liposome formation is possible, the amount of biologically active substance is generally not critical. Commonly the substance will be present in amount of up to 10% by weight in the liposome formulation. A value of, for example, 0.01% by weight is to be mentioned as a lower limit. A concentration range of 1 to 5% by weight of biologically active substance is preferred.
The compound according to the invention can preferably amount to 1% to 100% of the lipid component. In preferred embodiments of the liposome formulation there will be one or more co-lipids present, wherein such co-lipids amount to 30 to 70% of the lipid component.
In case the biologically active substance is a drug, the amount thereof is conventionally selected according to the desired therapy. Preferably there will be 1 to 5% by weight of the drug present in the liposome formulation. In case of drugs applied as biologically active substance, there can pharmaceutically acceptable excipients be present in the liposome formulation.
As mentioned above with the complexes according to the invention, also the liposomes or liposome formulations according to the invention will optionally be charge-masked or cell-targeted for interaction with certain cells.
Finally, the invention comprises methods for the transfer of polyanions or polycations or for the transfer of biologically active substances in general through biological membranes, in particular for introduction in cells or cell organelles. In this context particular reference is made to claims 33 to 38, and 39 to 42, respectively. For the methods according to the invention can be used either the compounds according to the invention themselves or compositions including such compounds.
During the performance of the method by incubation in vivo there can additional stabilizers be present, like for example polyethylene glycol.
Further details of the method according to the invention will be described below.
The compounds and other parts of the present invention have various advantages. One of the advantages of the compounds described here are that they allow up to 100% inclusion of polyanionic materials in a convenient protocol. On the other hand, the incubation of positively charged complexes with negatively charged cell surfaces results in a rapid and improved uptake, in particular of polyanionic substances and other biologically active compounds in general. The latter allows the introduction of complexed or included polyanionic substances like for example DNA, in an amount not known so far with such cells.
The particular advantages of the compounds disclosed herein are as follows. First, these compounds represent the novel liposome forming lipids. The geometry of both the aliphatic chains in the compounds of formula I allows their organization in stable double layer structures. The polar head (e. g. amino acid) can be varied as a function of the application. This will allow the easy introduction of different modifications to the amino group, the side chain or the bonding, for example. The cytotoxicity of most of the cationic compounds disclosed here are favourable as compared to that reported with other cationic amphiphiles. All the bondings illustrated in formula I will readily be hydrolized in the cells which results in the formation of non-toxic compounds.
Further, the positively charged lipids of formula I have an improved transfection efficiency in the presence of serum, in contrast to other cationic lipids of the state of the art. The ability to transfect cells in the constant presence of serum is advantageous for various reasons: transfection occurs more easily and is less time consuming, the requirements as to media and serum are reduced, the cells are not depleted of serum which could deteriorate cell functions and viability.
The second specific advantage over the disclosed state of the art is derived from the novel method for the introduction of polynucleotide (in particular DNA) into ternary complexes. Said complex is in particular formed from positively charged lipids of formula I and a complex made of polynucleotide and cationic polypeptide. According to the method there will be formed a first complex made of polynucleotide-cationic polypeptide which in the following step will be complexed with a positively charged lipid of formula I. An exact regulation of the composition will determine the biological activity of the final complex.
The advantage of this procedure in relation to the transfection steps known in the state of the art is for example the fact that the novel method results in an up to 300 times increased transfection efficiency. Furthermore, by using the novel method, a detection of transfection (e. g. detection of the expressed protein) will easily be registered after less than two hours from the start of transfection. Further, the novel procedure allows working in "microscale" for cell transfection (for example 96-well size) which is desirable for the screening of a large number of samples and allows automation of the process in total.
The compounds of formula I are particularly useful in the preparation of liposomes, however, they can be used in other cases where cationic lipids find application. They may be used in industrial applications, for example. Of particular interest is the application of said compounds in combination with cationic lipids which are acceptable for pharmaceutical formulations (creams, pastes, gels, colloidal dispersions and the like) and/or cosmetic compositions (makeups, lipsticks, polishes, body lotions, moisturizing creams, shampoos and the like).
Formulations comprising the compounds of formula I are advantageous for achieving desirable intracellular delivery of biologically active substances, such as polynucleotides, peptides, proteins, steroids and other natural or synthetic compounds. The intracellular delivery can be into the cytoplasm, into the nucleus, or both. Such intracellular delivery can be achieved in tissue culture (in vitro) and may be used, for example, for transfecting cells with desired polynucleotides (e. g. DNA), or delivery of proteins and the like.
Formulations comprising the compounds of formula I can also be used for ex vivo therapy, where cells isolated from the organism are transfected in vitro and then implanted to the organism. An example of this application is to transfect bone marrow cells.
Intracellular delivery can also be achieved in the whole organism (in vivo) and thus may be useful in several applications like gene therapy, antisense and antigene therapy. Intracellular delivery in vivo can also be used for DNA vaccination with an aim to induce immune response (humoral and/or cellular) to the desired protein.
Intracellular delivery utilizing compounds of formula I is also useful for delivery of anticancer and antiviral compounds, antibiotics and the like.
Cell selectivity can be achieved by incorporating cell recognition compounds, for example on the surface of the vesicle such as antibodies, ligands for cell-surface receptors and the like. Increased stability and further selectivity can be achieved by coating the liposome vesicle with an appropriate charge-masked natural or synthetic compound such as polymers and neutral, or negatively charged lipids.
Liposome vesicles comprising compounds of formula I can be used for the induction of a specific immune response to an antigene of interest which is incorporated in the liposome. Additional components, like N-palmitoyl-S-(2,3-bis(palmitoyl-oxy)-(2RS)-propyl)-R-cystein (Pam3Cys) or N-acetyl-myramyl-L-threonyl-D-isoglutamine (MDP) and derivatives thereof, may be particularly useful.
Compounds of formula I are of interest for the introduction of a lipophilic moiety into polymers, and particulary in peptides in order to increase their uptake by a cell, or to increase their incorporation in the lipid-containing vesicles and the like. Activated compounds of formula I can also be used for modification of proteins.
Further details of the so far presented parts of the invention will be apparent from the following description of preparation procedures, lipid compounds and examples for application thereof. In the description of each example reference will be made to the accompanying drawings.
Method for the Preparation of Selected Compounds of Formula I
Using well-known techniques, a person skilled in the art can readily make additions, deletions or substitutions, increase or decrease the preferred polypeptide amino acid sequences, or simply use another sequence encoding for different cationic polypeptides. It should be understood, however, that such variations are within the scope of this invention. It should also be understood that the examples provided below are for illustrative purposes only and are not to be construed as limiting this invention in any way.
This reaction scheme is applicable to the sythesis of preferred compounds of formula I, wherein Z is an ester bond and n is 2. In this reaction scheme P1 and P2 are protecting groups, and are the same or different; W is a side chain of amino acid, R1 and R2 are the same and are alkyl or alkenyl having 6 to 24 carbon atoms.
Compounds of formula (A) are commercially available in the optically pure form. To effect the formation of compounds of formula (B), the carboxyl group of the amino acid of formula (A) is activated with an appropriate agent, and is then allowed to react with the imino group of diethanol amine. Methods for activation of amino acids are known in the art. For example, dicyclohexylcarbodiimide/N-hydroxysuccinimide method can be used for epimerization-free amidation of protected amino acids.
The amide of formula (B) is prepared by dissolving the amino acid of formula (A) and a preformed salt of N-hydroxysuccinimide and diethanolamine in an appropriate polar solvent such as dimethylformamide. The mixture is cooled to 0� C. and then a solution of dicyclohexylcarbodiimide is added. This reaction is effected by stirring the solution for one hour at 0� C. and eight hours at room temperature. The resulting amide of formula (B) is then recovered by some standard separatory means.
To effect the formation of compounds of formula (C), the amide of formula (B) is dissolved in an appropriate solvent (e. g. dichloromethane). To this is added an appropriate tertiary amine, such as, for example, triethylamine in an excess molar amount. The mixture is cooled to about 0� C. The alkylating agent of the desired chain length and degree of unsaturation is added in an excess molar amount, preferably about 3 to about 4 times the amount of the amide of formula (B). For example, oleoylchloride can be used to effect the addition of 9-octadecenoyl groups. This mixture is then stirred for approximately 3 hours under N2 atmosphere at room temperature. The product of formula (C) is then extracted. The compound of formula (C) is then deprotected in an appropriate way, depending on the used protecting groups, extracted and further purified by chromatographic means.
Boc-Lys(Boc)-OH *DCHA (2.63 g, 5 mmol) was suspended in ethylacetate and converted to the free acid using ice cold 2 M H2SO4. Boc-Lys(Boc)-OH in ethylacetate was dried with MgSO4 and evaporated to dryness. The residual oil was dissolved in about 15 ml of dimethylformamide (DMF) containing 1.1 g (5 mmol) of a preformed salt of N-hydroxysuccinimide and diethanolamide (HOSu*N(EtOH)2) and was cooled at 0� C. with ice-water. While stirring, a solution of 1.13 g (5.5 mmol) of dicyclohexylcarbodiimide (DCC) in 5 ml of DMF was added to the mixture which was stirred for one hour at 0� C. and then continuously at room temperature for another 8 hours. The mixture was concentrated to dryness in vacuum. After the addition of about 50 ml of ethylacetate to the residue, most of the dicyclohexylurea (DCU) precipitated and was filtered off. The ethylacetate phase was washed with aqueous solutions of NaHCO3 (5%) and citric acid (5%) in 0.1 M of NaCl, dried with MgSO4 and evaporated to dryness to give 1.7 g of the crude product as a colorless oil.
A solution of 1.7 g (3.92 mmol) of Boc-Lys(Boc)-N(EtOH)2, 1.64 ml of triethylamine and 3.93 mol of oleoylchloride (11.7 mmol each) in 50 ml of dichloromethane (DCM) was stirred for 30 min at 0� C. and 4 hours under N2 atmosphere in the dark at room temperature. The mixture was quenched with methanol, concentrated in vacuum, redissolved in hexane and washed three times with 0.1 M KOH in methanol/water (1:1 vol.) at 0� C., then once with 0.1 M aqueous NaCl. The hexane phase was concentrated in vacuum and the residual was treated with 50 ml of a mixture of trifluoroacetic acid (TFA)/DCM (1:1 vol.) for 40 min at room temperature. The mixture was repeatedly mixed with toluene and reconcentrated in vacuum, and then applied in chloroform to a column of Silica Gel 60. The column was eluted with an ascending gradient of methanol in chloroform, and the pure compound (2.3 g, 62% yield) eluted at about 20% methanol. The hydrochloride was prepared by dissolving the product in dry ethylacetate saturated with HCl gas and evaporating to dryness. (Analysis: ES-MS: Mol. mass determined=762 (calculated=762); TLC: Rf=0.69 in ethylacetate:acetic acid:water=(4:1:1); HPLC: Rt=14.19 min; column Nucleosil C2 4.0�100 mm, gradient 30 to 90% of acetonitrile in 0.1% of TFA in water within 20 min).
In a similar manner, but substituting the appropriate starting material, the following compounds were prepared:
L-Lysine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (2)
L-Lysine-bis-(O,O′-myristoyl-β-hydroxyethyl)amide dihydrochloride (3)
L-Ornithine-bis-(O,O′-myristoyl-β-hydroxyethyl)amide dihydrochloride (4)
L-Ornithine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride (5)
L-Ornithine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (6)
L-Arginine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride (7)
L-Arginine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (8)
L-Serine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride (9)
Glycine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (10)
Sarcosine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (11)
L-Histidine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (12)
L-Glutamine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride (13)
The compound Boc-Asp(OBzl)-N(EtOH)2 was prepared in the same manner as described above for preparing compound Boc-Lys (Boc)-N(EtOH)2. The compound Boc-Asp(OBzl)-N(EtOPalm)2 was prepared similar to the compound Boc-Lys(Boc)-N(EtOOL)2 with minor modifications. The reaction proceeded overnight, the mixture was diluted with ethylacetate and then poured into a saturated aqueous solution of NaHCO3 to destroy residual palmitoylchloride and precipitate sodium palmitate. The precipitate was filtered off and the organic phase was evaporated to dryness and redissolved in hot methanol. The product was precipitated in cold methanol and collected by filtration with the yield of 55% (TLC: Rf=0.64 in chloroform:methanol=100:2). The benzyl-protecting group was removed from the product (1.5 g) by treating it overnight with NH4COOH (0.65 g) in the presence of freshly prepared Pd black (about 0.1 g) in 15 ml of DMF. Pd black was filtered off and the mixture was evaporated in vacuo. The residual was precipitated from methanol/water solution to give the desired product Boc-Asp-N (EtOPalm)2 (14) with the yield of 85%. (Analysis: ES-MS: Mol. mass determined=796,5 (calculated=796); TLC: Rf=0.4 in chloroform:ethyl acetate:methanol=9:3:1).
0.5 g (0.62 mmol) of Boc-Asp-N(EtOPalm)2 was treated with a mixture TFA/DCM=1:1 for 30 min at room temperature. The mixture was repeatedly evaporated with toluene. The residual was dissolved in 10 ml of DMF, neutralized with diisopropylethylamine (DIPEA) and was reacted for two hours with 1.2 equivalents of 9-fluorenylmethyl-succinimidyl-carbonate (Fmoc-OSu). The reaction mixture was evaporated and redissolved in methanol. The desired compound Fmoc-Asp-N(EtOPalm)2 (15) was precipitated to afford 0.44 g of the compound with a yield of 77%. (Analysis: TLC: Rf=0.4 in chloroform:ethyl acetate:methanol=9:3:1).
Using similar procedures, but substituting the appropriate starting material, the following compounds were prepared:
N-α-tert-butoxycarbonyl-L-glutamic acid-γ-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide Boc-Glu(N(EtOPalm)2)-OH (16)
N-α-fluorenylmethyloxycarbonyl-L-glutamic acid-γ-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide Fmoc-Glu(N(EtOPalm)2)-OH (17)
N-α-tert-butoxycarbonyl-L-aspartic acid-β-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide Boc-Asp(N(EtOPalm)2)-OH (18)
In addition, intermediates of their synthesis, or of the above described synthesis, can be used for the preparation of liposomes:
L-glutamic acid-γ-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide H-Glu(N(EtOPalm)2)-OH (19)
L-aspartic acid-β-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide H-Asp(N(EtOPalm)2)-OH (20)
L-aspartic acid-α-N′-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide H-Asp(N(EtOPalm)2) (21)
Preparation and Characteristics of a Complex Made of DNA and Compound (1)
Fluorescence studies. Fluorescence measurements were carried out on an Aminco SPF-1000Sc spectrophotometer, using a 1 cm light path cell with a slit width for excitation and emission of 10 nm. The binding of (1) to nucleic acids was observed from the displacement of ethidium bromide, which upon intercalation in between the DNA base pairs acts as a fluorescence probe. Fluorescence was measured immediately following addition of increasing amounts of (1) to DNA complexed with ethidium bromide (5 μg/ml calf thymus DNA, 8�10−6 M bp (base pairs); ethidium bromide 1:50 bp in 10 mM Hepes, 150 mM NaCl, pH 7.4). Ethidium bromide displacement was monitored by the decrease of the ethidium bromide fluorescence (excitation=540 nm, emission=600 nm) that occurs when it is released from DNA (FIG. 1). Fluorescence intensity is gradually decreased with increasing concentration of the cationic lipid. This is an indication for compaction and/or aggregation occurring in DNA. The ratio between (1) and DNA at which the fluorescence quenching does not change any more, corresponds to about 6:1 (w/w). The obtained ratio corresponds to the charge ratio of about 3.5:1.
Transfection of Cells
Cell cultures and plasmids
HeLa (CCL2, epitheloid carcinoma, human), COS-7 (CRL 1651, kidney, SV-40 transformed, African green monkey), HT-29 (HTB 38, adenocarcinoma, colon, human), CV-1 (CCL 70, kidney, African green monkey), 818-4 (adenocarcinoma, pancreas, human), H4IIE (CRL 1548; hepatoma, rat), K562 (CCL 243; chronic myelogenous leukemia, human), HL-60 (CCL 240; promyelotic leukemia, human) were grown in 5% CO2 at 37� C. on plastic tissue culture flasks in RPMI 1640 or DMEM with 10% fetal calf serum and supplemented with penicillin at 100 units per ml, streptomycin at 100 μg per ml, 2 mM glutamine and 0.1 μM dexamethasone for H4IIE cells.
Plasmids pCMVL and pCMVμ-gal encode luciferase and β-galactosidase genes, respectively, and their expression is under control of the cytomegalovirus (CMV) immediate-early promoter. Plasmids pZeoSV and pZeoSVLacZ encode the Sh-ble resistant protein to Zeocin™ (registered trademark) which allows for selection both in prokaryotic and eukariotic cells. Eukaryotic expression is under control of the CMV promoter. pZeoSVLacZ in addition encodes β-galactosidase gene under control of the SV40 early enhancer-promoter. Plasmid DNAs were purified on a QIAGEN™ (registered trademark) column using the QIAGEN™ plasmid purification procedure.
Transfection of tissue culture cells. Preparation of cells, transfection protocol and assays for transfection efficiency. The details of the individual transfections are given in the result section.
a) Adherent Cells
At about 24 hours before transfection nearly confluent cell monolayers were trypsinized. The cells were resuspended in fresh medium and placed either in 24-well plates (5�104 cells per well) or in 6-well plates (2-2.5�104 cells per well). Just before transfection, the cells were placed in fresh medium with or without 10% FCS. In general, cells were transfected at 50 to 70% confluence. 24 hours after transfection, cells were washed three times with 2 ml of PBS and lysed in 100 μl (24-well plates) or 200 μl (6-well plates) of lysis buffer (77 mM K2HPO4,23 mM KH2PO4,0.2% Triton X-100, 1 mM dithiotreitol, pH 7.8). Cell debris was removed by centrifugation (14000 rpm for 2 min). Usually, enzyme activity (see below) was determined directly after cell lysis. However, lysates can be stored at −20� C. without loss of activity.
b) Suspension Cells
At 24 hours before transfection, the cells were placed in fresh complete growth medium. Just before transfection, the cells were collected, resuspended in fresh medium with or without 10% FCS at 0.25�106 cells per ml, and placed in 24-well plates, 2 ml per well.
The cells were normally harvested 24 hours after transfection (the time of harvesting is an important variable and should be optimized), collected by centrifugation and resuspended in 10 ml of PBS, again centrifuged, and the cell pellet was transferred into a 1.5 ml Eppendorf tube with 1 ml PBS. The cells were centrifuged in the Eppendorf centrifuge (14000 rpm for 20 sec), The resulting cells were lysed in 100 μl of lysis buffer (described above). The sample was then centrifuged (14000 rpm for 2 min) and the supernatant was carefully transfered to a fresh centrifuge tube.
c) Transfection Protocols
Transfection of cells using compound (1). Plasmid DNA (from a stock solution of a concentration of about 1 mg/ml) and cationic lipids (from a stock solution of a concentration 1 to 2 mg/ml) were diluted separately in 1.5 ml Eppendorf tubes with 10 mM Hepes, 0.9% NaCl buffer, pH 7.4, to the final volume of 100 μl. The amount of DNA in typical transfections varied from 1 to 2 μg per 100 μl. The amount of a cationic lipid varied from 0 to 20 μg per 100 μl. Two solutions were mixed together to form a DNA-lipid solution. After incubation for 10 min at room temperature the resultant DNA-lipid solution was added to the cell cultures, growing as described before, and gently mixed. Alternatively, the DNA-lipid solution was diluted with 800 μl (for 24- or 6-well plates) of the appropriate medium (+/−FCS) and mixed. The diluted solution was then overlayed onto the cells, rinsed with the appropriate medium. The complexes were incubated with the cells for 1 to 24 hours. A typical incubation time in the absence of serum was 4 hours. The transfection medium was then substituted with a complete growth medium. In the presence of serum, the transfection medium was usually not replaced and the cells continued to grow in the presence of complexes until the end of experiments (one step transfection). After 24 hours cells were worked up as described above.
Transfection of cells using ternary complexes of (1) Plasmid DNA was diluted with 10 mM Hepes, 0.9% NaCl buffer (pH 7.4) containing the appropriate peptide (for example according to claim 23) to the final volume of 100 μl. The amount of a peptide varied between 0 to 20 μg per 100 μl of buffer. A typical concentration was 2 to 5 μg peptide per 100 μl of buffer. After incubation for 10 min, the resultant solution was mixed with 100 μl of a cationic lipid solution, and the whole procedure was continued as described above.
d) Enzyme Assays
Luciferase assay. The luciferase activity in cell extracts was assayed using a Berthold Lumat instrument (LB 9501). The luciferase assay was performed by adding 5 to 10 μl of cell extract. The sample was placed in the LB 9501 and the instrument automatically injected 100μl of the injection buffer (20 mM tricine, 1.07 mM (MgCO3)4Mg(OH)2, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin, 530 μM ATP, pH 7.8) into the sample, measured light emission and displayed it as an integrated value for the first 10 sec of light production.
β-Galactosidase assay. β-Galactosidase activity in cell extracts was measured on the 96-well microtiter plates. 10 μl of cell extracts were diluted with 70 μl of "β-Gal" buffer (33 mM NaH2PO4, 66 MM Na2HPO4, 2 mM MgSO4, 40 mM β-mercaptoethanol) and 25 μl of ONPG solution (o-nitrophenyl-β-D-galactopyranoside 4 mg/ml in B-Gal buffer). The mixture was kept at 37� C. for 30 min to 1 hour and the reaction was stopped by adding 150 μl of 1 M sodium bicarbonate. The absorbance was measured at 405 nm.
Staining of cells with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). The rinsed cells were fixed with 1% glutaraldehyde solution in 0.1 M sodium phosphate, 1 mM MgCl2 buffer (pH 7.0) for 15 min at room temperature. The fixing solution was removed, cells were washed three times with PBS, and staining solution (0.2% X-Gal, 10 mM sodium phosphate, 150 mM NaCl, 1 MM MgCl2, 3.3 MM K4Fe(CN)6 and 3.3 mM K3Fe(CN)6, pH 7.0) was overlayed onto the cells and incubated for 1 to 8 h at 37� C. The stained cells on culture dishes or cover slides were visualized using inverted or phase contrast microscopes, respectively.
Protein determination. Protein content in the supernatant was assayed using the technique of Lowry (Bio-Rad protein determination kit).
Determination of cytotoxicity is done using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay.
Adherent Cells
a) Compound (1). The transfection efficiency of compound (1) on HeLa cells in the presence or absence of serum was compared to DOTAP. 5�104 cells per well (24-well plate) in 1 ml of complete RPMI 1640 were plated one day before transfection. Just before transfection, the medium was removed and cells were supplied with 1 ml fresh medium with or without serum. To assess the optimum ratio between cationic lipids and DNA, various quantities of both lipids in the transfection buffer (final volume 100 μl) were mixed with 100 μl of DNA solution containing 1 μg of pCMVL. After incubation for 10 min at room temperature 100 μl of this mixture containing 0.5 μg of the plasmid and half of the indicated amount of lipid was added to each well. In the presence of serum, plates were incubated for 24 hours without exchange of medium. In the absence of serum, the transfection medium was removed after 4 hours, and the cells were supplied with 1 ml fresh serum supplemented medium.
The cells were harvested for luciferase assay 24 hours after transfection as described above. The results are plotted in FIG. 2 as the total amount of a cationic lipid per 1 μg of PCMVL. In the case where cells were transfected in the presence of serum, the optimum expression of luciferase activity was obtained using DOTAP/DNA ratio of 10:1 (w/w) and a ratio of compound (1)/DNA of 5:1 (w/w), respectively. In the absence of serum, the optimum expression of luciferase activity of both lipids was obtained using a lipid/DNA ratio of 5:1 (w/w). Compound (1) mediated transfection, in comparison to DOTAP transfection, was 12-fold and 6-fold higher in presence and in absence of serum, respectively.
b) Ternary complex of compound (1). 5�104 cells per well in 1 ml of complete RPMI 1640 were plated one day before transfection. Just before transfection, the medium was removed and cells were supplied with 1 ml fresh medium with or without serum. To assess the influence of the HBV peptide on transfection of HeLa cells, various quantities of the peptide were mixed with 1 μg of pCMVL to make the final volume 100 μl, and incubated for 10 min. Then 100 μl of a solution containing 7.5 μg of compound (1) was added with mixing. After additional incubation for 10 min 100 μl of the transfection solution (containing 0.5 μg of DNA and 3.75 μg of compound (1)) was applied to the cells. After 4 hours transfection medium has been replaced with 1 ml of fresh complete growth medium.
24 hours after transfection cells were lysed and cell extracts were analysed for gene expression as described above. Transfection levels (with the transfection in the presence of serum) are plotted in FIG. 3 as a percentage of the value obtained with DOTAP transfection. The results show that the optimum amount of the peptide in these experiments was 5 μg, and that the expression of the luciferase activity was 14-fold and 200-fold higher than those achieved with compound (1) and DOTAP transfections, respectively. In the case of the transfection performed under serum free conditions, the optimum amount of the peptide was the same and the expression of the luciferase activity was about 10-fold higher than those achieved with compound (1) and 150-fold higher than with DOTAP transfections, respectively (data not shown).
c) Time dependence. 5�104 HeLa cells per well (24-well plate) in 1 ml of complete RPMI 1640 were plated one day before transfection. Just before transfection, the medium was removed and cells were supplied with 1 ml fresh medium with serum. 50 μl of the transfection solution containing 0.25 μg of pCMVLuc, 0.5 μg of HBV-peptide and 1.87 μg of compound (1) were added to each well with mixing. After 2 hours transfection medium has been replaced with 1 ml of fresh complete growth medium. The cells were harvested for luciferase assay at the indicated time as described above. The results are plotted in FIG. 2c as the total luciferase activity per well. As can be seen, luciferase activity rapidly increases, reaching maximum at 12 to 16 hours. Already after 2 hours, 4.5�104 light units were obtained. The same luciferase activity was observed after 3 hours with compound (1) mediated transfection, and only after 5 to 6 hours after DOTAP mediated transfection.
Suspension Cells
α) Compound (1)
The transfection efficiency of compound (1) on K562 cells in the presence of serum was compared to DOTAP. To assess the optimum ratio between cationic lipids and the DNA, various quantities of both lipids were added to a given amount of pCMVL (1 μg per well, or per 0.5�106 cells in 2 ml of the complete RPMI 1640, 24-well plates). 200 μl of the transfection mixture was added into each well and the plate was incubated for 24 h at 37� C. (one-step transfection). Cells were lysed as described above. The results are plotted in FIG. 4 as the total luciferase activity per well versus the amount of cationic lipid per 1 μg DNA. The optimum expression of luciferase activity was obtained using a DOTAP/DNA ratio of 5:1 (w/w) and compound (1)/DNA ratio of 7.5:1 (w/w). The obtained ratio corresponds to the charge ratio of lipid to DNA about 2:1 and 5:1 for DOTAP and compound (1), respectively (with the assumption that 1 μg of DNA contains 3.1 nm phosphate anionic charges). Compound (1) mediated transfection was more than 10-fold higher than those obtained with DOTAP.
B) Ternary Complex of Compound (1)
To assess the influence of peptide concentration on the transfection of K562 cells, various quantities of HBV-peptide were mixed with 1 μg of pCMVL to make the final volume 100 μl, and incubated for 10 min. Then 100 μl of a solution containing 10 μg of compound (1) was added with mixing. After additional incubation for 10 min the transfection solution (200 μl) was applied to the cells. All other conditions were exactly the same as described in the previous experiment. The results are shown in FIG. 5 as the total luciferase activity per well versus the amount of added peptide (per 1 μg DNA). The results show that the optimum amount of the peptide was 2 μg, and that the expression of the luciferase activity was 3- to 4-fold higher than those achieved with compound (1) and 30- to 40-fold higher than with DOTAP transfections, respectively.
γ) Dose Dependency
A "stock" solution was made from 1 μg of PCMVL, 2 μg HBV-peptide and 10 μg compound (1) in the final volume of 200 μl. The DNA dose dependency was checked in one-step transfection by adding from 5 to 200 μl of this solution to the cells. Results from this experiment are plotted in FIG. 6. The expression of the luciferase activity was dependent on the amount of added DNA and was linear in the range from 0 to 0.25 μg of DNA. In principal, under these conditions as little as 0.5 ng of reporter DNA (without carrier DNA) could be detected. The obtained results also indicate that once formed, these ternary complexes are very stable and do not dissociate with dilution.
Intracellular Introduction of Oligonucleotides
Fluorescein labelled phosphotioate oligonucleotide (5′-ACT TGG ATA CGC ACG-fluorescein-3) (SEQ ID NO.1) was used to access the intracellular delivery and distribution of oligonucleotide in the presence and absence of compound (1).
HeLa cells (plated one day before the experiment, 2�105 cells per well, 6-well plate) were grown on glass microscope slides in 2 ml of RPMI 1640 supplemented with 10% FCS. Before the experiment, the medium was replaced with RPMI 1640 without serum.
Formation of oligonucleotide-compound (1) complexes was done as follows. 10 μg of phosphothioate oligonucleotide was dissolved in 90 μl of transfection buffer. 16 μg and 50 μg of compound (1) were diluted with transfection buffer to the final volume of 100 μl. The two solutions were mixed together and incubated for 10 min. Oligonucleotide solution without compound (1) was diluted with 100 μl of transfection buffer. The resultant solutions (200 μl per well) were added to the cell cultures (final concentration of oligonucleotide was about 1 μM, compound (1) 8 μM and 25 μM). The complexes were incubated with the cells for 4 hours. The medium was then substituted with a complete growth medium.
At 4 and 24 hours, cells were washed three times with PBS and fixed with 1% glutaraldehyde solution for 15 min. After fixation, cells were washed three times with PBS and mounted in glycerol mounting solution (10 mM phosphate, 150 mM NaCl, 70% glycerol, pH 7.5). The localization of fluorescein labelled phosphothioate oligonucleotide was determined by fluorescent microscopy, using a Zeiss fluorescent microscope.
Incubation of cells with 1 μM fluorescein labelled oligonucleotide resulted in a faint fluorescence both after 4 and 24 hours. In contrast, all cells incubated with 1 μM fluorescein labelled oligonucleotide in the presence of compound (1) exhibited very bright punktate fluorescence in the cytoplasm. After 24 hours the cells show very clear punktate fluorescence in the cytoplasm. The fluorescence intensity was higher in the presence of 25 μM compound (1).
Experiments performed in the presence of serum did not change the overall fluorescence and distribution of fluorescein labelled oligonucleotide.
Intracellular Introduction of Anionic Polypeptides
Fluorescein labelled anionic polypeptide (fluoresceinpoly(Glu)21) (SEQ ID NO:2) was used to access the intracellular delivery and distribution of anionic polypeptide in the presence and absence of compound (1). Conditions of the experiment were exactly the same as described in the previous example.
Incubation of cells with 1 μM fluorescein labelled poly-(Glu)21 did not result in fluorescence neither after 4 nor 24 hours. In contrast, all cells incubated with 1 μM fluorescein labelled oligonucleotide in the presence of 25 μM compound (1) exhibited bright cytoplasmic punktate fluorescence already after 45 min. The localization of the fluorescence vesicles was somehow different form those observed with fluorescein labelled oligonucleotide. After 24 hours cells exhibited very bright punktate fluorescence in the cytoplasm, most likely localized within the same structures.
Liposome Formulations
The following compositions illustrate the use of compounds according to the present invention in formulations comprising biologically active agents:
7.1 A topical formulation was prepared by dissolving 0.25 mg of prednisolone acetate (21-acetoxy-1,4-pregnadien-11β, 17a-diol-3,20-dion) and 50 mg of L-lysine-bis-(O,O′-oleoyl-β-hydroxyethyl) amide dihydrochloride in 2 ml of dichloromethane:ethanol (1:1) solution. The solvent was evaporated under a stream of nitrogen. The film mixture was placed under vacuum overnight to vaporate off the residual solvent completely. The dry film was then suspended in 2 ml of 0.9% NaCl solution. The solution was sonicated until visually clear.
7.2 (+)-α-tocopherol (5,7,8-trimethyltocol, vitamin E) and 100 mg of L-lysine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride were dissolved in 5 ml of dichloromethane:ethanol (1:1) solution. The solvent was evaporated under a stream of nitrogen and the residual was evaporated off under vacuum. The dry film was then suspended in 4 ml of 10 mM Hepes, 0.9% NaCl, pH 7.4, and sonicated until visually clear.
7.3 20 mg retinol (3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclo-hexenyl)-2,4,6,8-nonatetraen-1-ol) and 80 mg of L-lysine-bis-(O,O′-myristoyl-β-hydroxyethyl)amide dihydrochloride were dissolved in 5 ml of dichloromethane:ethanol (1:1) solution. The solvent was evaporated and the dry film was placed under vacuum overnight. The film was then suspended in 5 ml of 10 mM Tris, 0.9% NaCl, pH 7.4.
7.4 3 mg N-palmitoyl-S(2,3-bis(palmitoyl-oxy)-(2RS)-propyl-R-cystein and 25 mg of L-lysine-bis-(O,O′-palmitoyl-β-hydro-xyethyl)amide dihydrochloride were dissolved in 5 ml of dichloromethane:ethanol (1:1) solution and evaporated under vacuum until dryness. The resulting film was suspended in 1 ml of distilled water and treated with ultrasound until clear.
Besides the portions described so far, the present invention also comprises the use of lipid compounds according to the invention for the introduction of lipophilic portions into peptides as well as the lipopeptides produced in this manner. Further details will become apparent from the following:
Peptide Synthesis
General Procedure
the synthesis of peptides having lipophilic modifications at their C- or N-terminus, or both, or within the chain, was accomplished by all-stepwise solid phase Fmoc-based synthetic methods using automated peptide synthesizer ABI 350 or multiple peptide synthesizer.
Peptides were usually assembled on a Rink amide MBHA resin (4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamido-norleucyl-methylbenzhydrylamin resin, Novabiochem) or on a trityl resin (2-chlorotritylchloride resin, Novabiochem).
The couplings of the Fmoc-amino acids (10 molar excess) were performed with N-N′-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in DMF usually for about one hour. The couplings of the lipophilic amino acid-bis-hydroxyethyl amides were achieved using their three molar excess, the same DIC/HOBt activation method and a coupling time for about 3 hours. The completeness of coupling was monitored with a ninhydrin test. The Fmoc group was removed with piperidine:DMF=1:2 within 15 min.
Cleavage and final deprotection was performed with a mixture of n-cresol:dimethylsulfide:ethanediol:TFA=3:3:3:91 for two hours at room temperature. After filtration, peptides were precipitated from the mixture and washed several times with diethyl ether. The crude peptides were purified on a semipreparative Nucleosil C2 column (8�250 mm) and an ascending gradient of acetonitrile in water, each containing 0.1% of TFA. Purified peptides were lyophilized from the mixture of tert-butyl alcohol:water=4:1 and stored at −4� C. Their homogeneitiy was verified by amino acid analysis, analytical HPLC and electrospray-ionization mass spectrometry.
According to this procedure the following lipopeptides were prepared:
GLFGAIAAGFIENGWEGLIDG-D(N(EtOPalm)2)-NH2 (SEQ ID NO: 3)
E(N(EtOPalm)2)-MQRGNFRNQRKMVKGGRAPRKKG (SEQ ID NO: 4)
E(N(EtOMyr)2)-MQRGNFRNQRKMVKGGRAPRKKG (SEQ ID NO: 5)
GRGDSPGSG-D(N(EtOPalm)2)-NH2 (SEQ ID NO: 6)
Acetyl-GRGDSPGSG-D(N(EtOPalm)2)-NH2 (SEQ ID NO: 7)
YNRNAVPNLRGDLQVLAQKVARTL-E(N(EtOPalm)2-NH2 (SEQ ID NO:8)
E(N(EtOPalm)2-YNRNAVPNLRGDLQVLAQKVARTL-E(N(EtOPalm)2-NH2 (SEQ ID NO:9)
YPS-E(N(EtOPalm)2-PDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2 (SEQ ID NO:10)
Further the invention comprises the use of lipid compounds according to the invention for the introduction of glycolipophilic portions into peptides as well as glyco-lipopeptides produced in this manner. Further details will become apparent from the following:
Synthesis of Glyco-Lipopeptides
1.8 g (5 mmol) of D(+)-lactose monohydrate was reacted with 40 ml of saturated NH4HCO3 for 6 days at 30� C. To remove the excess of NH4HCO3, the reaction mixture was diluted with water (20 ml) and concentrated in vacuo to half of its original volume. This procedure was repeated 6 times. Finally, the water was removed by lyophilization. The resulting crude amino sugar was used without further purifiaction for the synthesis of Fmoc-Asn(lactose)-OtBu derivatives. 480 mg (1.17 mmol) of Fmoc-Asp-OtBu and 228 mg (1.52 mmol) of HOBt were idssolved in DMF (5 ml) and after cooling to 4� C., 205 mg (1.63 mmol) of DIC was added. After stirring at 4� C. for 15 min and at 25� C. for 20 min, 5.85 mmol crude 1-amino lactose was added to the active ester in 6 ml of DMF:H2O=2:1. After stirring for 6 hours the solvent was evaporated in vacuo and diethylether was added. The precipitated product was filtered, washed with cold ether and cold water. The tBu-protecting group was cleaved by 70% TFA in water (20 min at room temperature). After evaporating of the TFA/water mixture in vacuo, the product was dissolved in tert-butyl alcohol:water=4:1 and lyophilized. Crude Fmoc-Asn(lactose)-OH was purified by reversed-phase chromatography (Lichroprep C18 column 25�310 mm, isocratic elution at 30% acetonitrile/-water/0.1% TFA) to give 280 mg of HPLC pure Fmoc-Asn(lactose)-OH (yield 35% based on starting Fmoc-Asp-AtBu. Analysis: +FAB-MS (MH+)=679 (calculated=679), Rt=6.59 min on Nucleosil C18 4�150 mm, gradient 30 to 100% of acetonitrile/0.1% TFA in water/0.1% TFA in 30 min).
32.5 mg (0.048 mmol) of Fmoc-Asn(lactose)-OH and 7.25 mg (0.05 mmol) HOBt were dissolved in 200 μl of DMF and 6.31 mg (0.05 mmol) DIC was added. After stirring for 30 min the formed active ester was coupled overnight to 30 mg (0.016 mmol) of Boc-(Lys(NH2))4-Lys2-Lys-Acx-bAla-Acx-E(N(EtOPalm)2)-peptidyl resin. The resin was carefully washed and the Fmoc group was removed with 20% piperidine treatment. The glycopeptide was cleaved of, deprotected and worked up under identical conditions as described above. Finally, it was purified by semipreparative HPLC to give 10.6 mg of N-lactosylated lipopeptide.
FIG. 1 of the accompanying illustrations shows the fluorescence intensity as a function of the amount of compound (1) added.
FIG. 2 shows the effect of the ratio of DOTAP/DNA and compound (1)/DNA on the transfection of HeLa cells. The transfection of compound (1) is illustrated in the presence (▪) and in the absence (□) of serum. The DOTAP transfection is illustrated in the presence () and in the absence (◯) of serum. Transfection levels are given as total light units per 50000 cells. Each point is to represent the mean�SD of triplicate transfection.
FIG. 3 illustrates peptide enhanced gene transfer to HeLa cells. Transfection levels are given as percentages of the value obtained with DOTAP transfection and show the mean of triplicate transfection.
FIG. 4 illustrates the effect of the ratios DOTAP ()/DNA and compound (1) (▪)/DNA on transfection of K562 cells. Transfection levels are given as total light units per 500000 cells. Each point is to represent the mean�SD of triplicate transfection.
FIG. 5 illustrates peptide enhanced gene transfer to K562 cells. Transfection levels are given as total light units per 500000 cells and show the mean�SD of triplicate transfection.
FIG. 6 illustrates transfection levels versus the amount of added DNA. Transfection levels are given as total light units per 500000 cells and show the mean�SD of triplicate transfection.
|Cited Patent
|Filing date
|Publication date
|Aug 4, 1978
|Oct 21, 1980
|Battelle Memorial Institute
|Process for the dehydration of a colloidal dispersion of lipsomes
|Feb 24, 1978
|Nov 25, 1980
|Papahadjopoulos Demetrios P
|Method of encapsulating biologically active materials in lipid vesicles
|Nov 30, 1978
|Dec 23, 1980
|Papahadjopoulos Demetrios P
|Method of encapsulating biologically active materials in lipid vesicles
|Nov 16, 1990
|May 4, 1993
|Syntex (U.S.A.) Inc.
|N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
|Jan 6, 1986
|Jul 16, 1986
|Syntex (U.S.A.) Inc.
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|The Upjohn Company
|Lipophilic polyamines useful for treating hypercholesterolemia
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|Citing Patent
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|Compositions and methods of use of targeting peptides for diagnosis and therapy of human cancer
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|May 2, 2008
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|Amino acid lipids and uses thereof
|Sep 30, 2008
|May 31, 2011
|Board Of Regents, The University Of Texas System
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|May 29, 2007
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|Jul 17, 2002
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|Feb 23, 2004
|Jan 6, 2005
|Wadih Arap
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|Sep 7, 2001
|Apr 7, 2005
|Wadih Arap
|Biopanning and rapid analysis of selective interactive ligands (brasil)
|Aug 30, 2002
|May 4, 2006
|Renata Pasqualini
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|Oct 19, 2004
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|Mar 17, 2005
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|Sep 30, 2008
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|Renata Pasqualini
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|U.S. Classification
|424/450, 564/291
|International Classification
|Cooperative Classification
|European Classification
|Jan 8, 1999
Owner name: SOUROVOI, ANDREJ, RUSSIAN FEDERATION
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:JUNG, GUENTHER;REEL/FRAME:009684/0567
Effective date: 19981018
|May 20, 2003
|Certificate of correction
|Mar 13, 2006
|Fee payment
Year of fee payment: 4
|Mar 25, 2010
|Fee payment
Year of fee payment: 8
|Mar 27, 2014
|Fee payment
Year of fee payment: 12


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IWD 2017: We won't have true equality until women have the housing and support they need
This International Women's Day we're using hard data to shatter community perceptions about homelessness.
Contrary to the stereotype of the older male rough sleeper, the person mostly likely to seek help from a homeless service is a woman aged 25-34 years. Last financial year, 62% of Victorians needing homeless help were female. That's nearly 2-in-3 clients.
Homelessness is on the rise in every city, town and suburb. But worryingly, the rate at which women are seeking help is growing much faster than men. Over the last 3 years, there has been a 14% increase in the number of women needing help from services, compared to a 10% rise in men.
Women's homelessness can be easier to ignore, because they're less likely to be sleeping on the street, but the effects of homelessness are damaging and lasting, no matter what form it takes.
Women are more likely to be at risk of homelessness because of inherent financial disadvantage, a lack of housing that women on low incomes can afford, and the increasing reporting of family violence, which continues to be a major driver of homelessness. A mother on single parenting payments looking for a two-bedroom rental in Melbourne, has just 2-in-100 properties which she could afford (DHS, Rent Report, 2016).
Number of women seeking assistance from Victorian homelessness services
Rate of growth
(2012-13 vs 2015-16)
Source: Specialist Homelessness Services data (Australian Institute of Health and Welfare)
The enormous demand on homelessness services combined with limited places to house clients means that agencies have to turn away too many people in need. Last year, twice as many women were turned away from homelessness services every day, when compared to men. Critically, CHP has called for our sector to be better resourced to focus on preventing homelessness in women through housing and support programs that help women and children stay housed.
Justice Connect Homeless Law has released a two-year report on its Women's Homelessness Prevention Project. The report pulls together 10 women's stories and 10 calls for change.
- Strengthen safeguards to make evictions into homelessness a last resort
- Invest in services proven to keep women in housing and resolve legal issues stemming from family violence
- Improve the legal framework for victims of family violence to keep their housing
- Improve legal mechanisms for exiting leases due to family violence
- Prevent victims of family violence being penalised for damage or arrears caused by perpetrators
- Strengthen policies and oversight to avoid inappropriate debts for public housing tenants (for more on this recommendation see our recent position paper, Through the Roof: Improving the Office of Housing's policies and processes for dealing with housing debts)
- Promote the ability of tenants to retrieve and keep their belongings when their tenancy ends
- Make human rights meaningful and accessible
- Support the private rental sector to avoid unnecessary evictions
- Plan for – and invest in – significant growth in affordable housing.
No. of people turned away from Vic homelessness services each day (AIHW, 2015-16)
Number of Victorian clients turned away each day
What's on
21st August
The International Mental Health Conference
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subset: commoncrawl


text:
At Hanson Landscape & Design, our goal is to give you a lush lawn that adds beauty to your home or business. We have different fertilizer and weed control options available to you. When it comes to fertilizing your lawn, there are options for early spring, spring, summer, early fall and late fall so that you have a lawn that will be in good health.
When you want a thick lawn that looks fantastic and is well cared for, Hanson Landscape & Design can help you. We offer affordable solutions, making the investment worth the time that you will save and the beautiful lawn that you will have. Because we are a highly experienced Minneapolis lawn care company, you know that you are getting the best.
Customized Solutions For Your Lawn
Not every lawn requires the same number of fertilizer or weed control applications. In other words, every lawn is different. The lawn type, the soil type, and other factors specify the fertilizer mix and the rate in which it is to be applied. Only a professional lawn care service will be able to determine the perfect fertilizer and weed control program for your lawn. At Hanson Landscape & Design, we have that experience, so you can rely on us to present you with the proper program that will give you the beautiful lawn that you want and need.
Lawn Treatment That Is Safe For The Environment
The fertilizer and weed control systems that we use are safe for the environment. We don't use fertilizers that contain phosphorus because it is not safe for the environment and because Minnesota law prohibits the use of it in fertilizers. The reason is because phosphorous can encourage algae blooms in many of Minnesota's lakes and ponds. Furthermore, homes that are located around lakes require a great deal of care because it is important that the lake plants and aquatic animals are not exposed to anything that can hurt them. We treat the environment with the respect that it deserves. This also means our treatments are safe for you, your family, and your pets.
We take pride in the fact that we only offer treatment options that are safe for the environment while also being highly effective. This also helps you reduce your carbon footprint. Many customers throughout the Twin Cities opt for our fertilizer and weed control services because they want to ensure that they can achieve what they want for their lawn without hurting the ecosystem.
Contact A Minneapolis Lawn Care Service
Two components of a healthy lawn are fertilizer and weed control. You want the good things to grow and the bad things to stay away. At Hanson Landscape & Design, we fertilize the good and we eliminate the bad so that your lawn can look beautiful. Not only does this result in curb appeal, but a healthy lawn. To learn more about our fertilizer, weed control, and other services, call us at 952-393-6385 to schedule a free estimate.


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Cannot find your local Phoenix Contact site? Use our selector
Network cable, PROFINET CAT5 (100 Mbps), 4-position, PVC/PVC, green RAL 6018, shielded, Plug straight M12 SPEEDCON / IP67, Coding: D, on Plug straight M12 SPEEDCON / IP67, Coding: D, Cable length: 70 m
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text:
Autumn Matai Black Tea
- Imprimer
- Agrandir
Automne 2015; vieux jardins; thé noir en feuilles entières
Aucun nouveau produit à l'heure actuelle
Automne 2015; vieux jardins; thé noir en feuilles entières
The Yunnan black tea made by Mrs Pu Jin Jing in Matai is my favorite one. Matai is located in Bangdong area, in Lincang County. Because of their old tree gardens origin, the leaves have plenty of power to deliver. It has a thick mouthfeeling, complex fragrances and long lasting Huigan. Perfect for brewing in a glass during a cold winter day, excellent when enjoyed in a Gaiwan at the end of a Pu-erh marathon.
The Autumn version can be enjoyed over five or six infusions.


text:
posted by kristie .
Could you show me step by step how to find the inverse of this problem:
log with base of 2 (x+1)
It is not clear what the problem is. You are given a function of x. Let's calli it y. Are you trying to come up with an equation for x in terms of y?
If log(base2)of (x+1) = y,
then 2^y = x+1 and
x = 2^y - 1
the exact problem was :
find f^-1(x).
f(x) = log(base2)(x+1)
"find f^(-1)(x)" means find the inverse function of f(x).
I believe that my previously derived fomula for x in terms of y IS the inverse function. Just relabel it as
f^-1 (x) = 2^x -1
f^(-1) of (f(x)) = x
give mats formula
i could answer that but can you tell me what this sign^ stands for please!!!!!


text:
|This room looks so much better!! The colour is called Apium. The kids love it !! And I can wash the walls without paint peeling off!!
|The collages look awesome on the feature wall.. The kids have promised to keep their new room clean!! hmmmm let's see how long that lasts !!


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Pacman Gobbles Up a Felony Arrest Warrant in Las Vegas
Breaking Adam "Pacman" Jones news this a.m., courtesy the Las Vegas Sun, which is reporting that the Clark County District Attorney's Office has issued a felony arrest warrant for the Dallas Cowboys' latest and greatest. The warrant was issued late last night, as Jones hasn't paid $20,000 in gambling debts to Caesars Palace. Notes the story:
Chief Deputy District Attorney Bernie Zadrowski, who runs the bad check unit, forwarded the request, along with a two-count criminal complaint charging Jones with fraud and theft, to Justice of the Peace Joe Bonaventure on Thursday.
In the warrant request, Zadrowski alleged that Jones has not paid back three markers, two for $5,000 each and one for $10,000, that Caesars Palace gave Jones on Sept. 3.
Links props courtesy ... TMZ.com, which offers the story with this headline: "Caesars to Pacman: Et Tu Deadbeat?" Brilliant. --Robert Wilonsky
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The five year old: "Mummy I am not sure if I am happy because I am not sure I am here because I think I might just be a dream."
Me "Gosh. Ok. So, how do we decide if this is real or not I wonder?"
"I don't know Mummy. And Mummy the other thing I think is that when I am asleep, the person who is dreaming me wakes up."
I am proud.
I am a little afraid.
I am wondering why my parents forked out for my philosophy degree – the five year old pretty much has it nailed.


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Trendy and modern, this beach front bar and lounge is where the sophisticated set go to sip on cocktails while listening to live music or a DJ and dance the night away.
Located toward the center of Trois Ilets, Le Kano Bar is a good choice if you'll be staying in this town or an adjacent neighborhood.
Location: Trois Ilets, Martinique
Help us improve! We welcome your corrections and suggestions.


text:
Kobe Bryant News
Kobe Bryant news continually updated from thousands of sources around the net.
American Terrorism: Plain and Simple
The Bachelorette Couples - Who Broke Up, Who Stayed Tog...
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Kobe Bryant news is powered by NewsRank ®
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Updated: Mon Jun 26, 2017 08:02 am
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text:
Cop Shoot Cop
Cop Shoot Cop is ranked number 4,455 in the overall artist rankings with a total rank score of 162.
Listen to Cop Shoot Cop on YouTube
Cop Shoot Cop best albums
The following albums by Cop Shoot Cop are ranked highest in the greatest album charts:
This may not be a complete discography for Cop Shoot Cop. This listing only shows those albums by this artist that appear in at least one chart on this site. If an album is 'missing' that you think deserves to be here, you can include it in your own chart from the My Charts page!
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Heymama is the premier content destination and curated membership community of kickass lady bosses who embody and give voice to modern motherhood.
#SocialBoss: This is How The Brooklyn Blonde Balances Motherhood
Office Boss: 5 Kate Spade Women Share This One Key to Success
Sun Seeker: Bree Hannemann Hikes Utah's Wilderness
14 Things Successful Working Moms Have Stopped Doing
What Does Your Aura Say? Mama Medicine Knows.
A 'SMART' Way To Beat Post Partum Depression
Sun Seeker: Fallon Santiago's Tips for Fun in the Sun
Secrets to a Successful Divorce
Sparking Creativity with Your Kids
Sleep Secrets from the Baby Sleep Expert
Dear Daughter: Actress Denise Vasi's Wishes for Her Little Girl


text:
got long on some 62.5 OCT
Will oil prices stabalize above $50, leading to SLB bulls?
Will SLB break $81 and head higher? Or is it forming a top, only to be crushed by oil market once again?
WFM looks like a head a shoulder play....will probably be following the weekly's for possible in opportunity's, should it not cross 2/3 fibonaci
Made 20% in two weeks on AMBA as it hits all-time highs. Placing a stop at 30.68 which seems to be just below the current projection to retrace in coming days/weeks. If the stock continues to break upwards, I will close the gap and probably enlist a trailing-stop.


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Role of earthmoving equipment in managing the soil erosion
One of the leading scientific magazines has recently published an interesting article on the problems of soil erosion in Bangkok. According to the article, because of soil erosion, by 2030, parts of Bangkok will be submerged in water. This publication is based on a scientific study conducted by some of the researchers on the effects of global warming, soil erosion particularly in the coastal region, rising sea level and such other related issues. The erosion control specialists warn that immediate preventive steps should be taken so as to avert the tragedy looming to happen by 2030.
Uses of earth moving equipment:
Soil erosion has become a global threat, and many countries across the globe stare at the disasters caused by the erosion. Thanks to the modern earth moving equipment like the trenching machine, JCBs, directional drilling machines which have helped in effectively managing the problems of soil erosion. According to the erosion control specialists, these earthmoving equipment can be effectively employed to prevent soil erosion which in turn helps in improving the fertility of the soil.
Effects of soil erosion:
Experts point out that soil erosion can cause several disasters. Apart from affecting the fertility of the soil, it can also affect the delicate ecological balance of the region. Soil erosion can also affect the structural safety of bridges and water reservoirs. They further point out that soil erosion can turn the vast grasslands into barren lands. However, you can remedy this situation by digging trenches along the course of the river or by constructing Gabion retainer walls. For this, you will have to employ a proper trenching machine. You can avail the trenching machine hire services at reasonable rentals.
Backhoe loader:
Alternatively, the erosion control specialists point out that you can effectively prevent soil erosion by employing backhoe loader. This earthmoving equipment is similar to the excavator. The machine can be used to dig trenches and move the soil to a different predetermined location. You can also get different services of backhoe hire Melbourne firms provide at reasonable rentals. Experts point out that employing a backhoe for purposes of digging trenches would be cost effective. The equipment accurately digs the trench, and it can also be effectively employed to backfill the trench. See more at State Plant Hire
Grader machine:
Apart from these, erosion control experts also normally employ grader machine also called as road grader for soil erosion prevention activities. There are many firms, which offer services of grader hire Melbourne wide, which are available with varying capabilities and features. The type of grader to be used depends on the nature of work that is to be accomplished. The grader machine is normally used for leveling and also for preparing base course for asphalting of roads. The graders are effectively used to level areas with uneven terrain.
Cost effective procedure:
These earth moving equipment are normally operated by trained operators. If you have undergone training in operating the earth moving equipment, then you may hire only the machine and dispense with the services of the operator. Many of the construction companies now hire these earthmoving equipment because these are cost-effective and, the work can be completed within a short span of time and with a greater degree of accuracy.


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Tag: Jimmy Kimmel
Max Ehrich and His Band, Infinity, To Perform First Concert
"The Young and the Restless'" Max Ehrich (Fenmore Baldwin) has gone from soap star to rock star seemingly overnight. He and his boy band, Infinity, just recently released a music video of their version of Rihanna's Stay, and now the group is prepping for their first ever live performance!
ABC Announces Hosts of 'Emmys Red Carpet Live'
Sunday, September 23 marks ABC's turn to host the "The Primetime Emmy Awards" (now in its 64th year) and in honored tradition the network will also be airing the pre-show, "Emmys Red Carpet Live." This year the red carpet festivities will be hosted by the network's Josh Elliott and Lara Spencer ("Good Morning America" and "Good Afternoon America"), Chris Harrison ("The Bachelor" and "The Bachelorette") and Clinton Kelly ("The Chew").


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Security National Life Insurance Co. takes over Wichita Executive Centre
Security National Life Insurance Co. has taken over ownership of the Wichita Executive Centre downtown, the company announced Monday.
The office tower, formerly known as the SC Telcom building and the Kansas State Bank & Trust building, had been owned and operated by Real Development Corp., informally known in Wichita as "the Minnesota Guys." The transfer was effective July 1, Security National said.
"We are happy to take the reins of Wichita Executive Centre and intend to return it to its former glory as a Class A office building in Wichita," Bryce Baker, vice president of real estate at Security National, said in the news release.
The building is 40 percent occupied. Current tenants include New York Life, The Research Partnership, Woolsey Petroleum, Vincent Oil, Edmiston Oil and the Trust Company of Kansas.
Security National said it plans to invest $3 million to $5 million in the 19-story building over the next two years.
"Our commitment to the tenants and to the city is to own and manage this building for the long-term," Baker said in the release. "As a life insurance company, we have a different outlook on this property than a bank lender may have. For our company, the Wichita Executive Centre is a long-term investment, not a 'buy-and-flip' proposition. We intend to do everything we can to make this building better for the long term because that is good business for the tenants, for the city and for us as the investor."
Security National Life Insurance is a wholly owned subsidiary of Security National Financial Corp. (NASDAQ: SNFCA), based in Salt Lake City.
Security National staff has met with various tenants during the month in hopes of re-establishing confidence in the building's management. In 2011, the building's tenants went public with their issues with the building's management under Real Development, including neglected maintenance and crumbling infrastructure.
And Real Development has had a series of issues beyond the Wichita Executive Centre, including an investigation by the Kansas Securities Commission, which has alleged that Real Development's principals used fraudulent collateral to secure a bank loan. Last year, Real Development auctioned off three buildings it owned in downtown Wichita, saying it wanted to concentrate on its larger properties, like Wichita Executive Centre.
Security National has hired Delton Sandefer of Essential Property Management as the local property manager, and Sandefer has relocated his company into the building, the release says.
"Past issues with the building have been widely reported but that is definitely in the past," Baker said in the release. "Going forward, our goal is to demonstrate solid performance, steady improvements, tenant follow-through and total transparency. We are a conservative company and we believe that consistent, quality performance is what really matters."
Space ranging from 1,000 to 16,000 square feet is available, including one full floor with panoramic views of the city.
Tony Utter of Utter Commercial Real Estate Inc. is the leasing broker.


text:
Documents Required with Application Form
Applicants must attach with application form attested photocopies of the following certificates and documents.
The documents & certificates must be attested by a gazetted Government servant. The stamp of the officer must bear his/her full name, designation and current place of duty. Selected candidates shall have to submit all original certificates at the time of interviews/admission.
The original documents of the selected candidates will be retained by the College concerned and will be returned on leaving the Institution.
(The application forms and photocopies of the documents submitted by the candidates not selected will not be returned to them).
1. Two Copies of Detailed Marks Certificate of the examination on the basis of which admission is sought.
2. An equivalence certificate from the Inter Board Committee of Chairmen, Islamabad, if the qualifying certificate is from an Institute abroad.
3. Hafiz-e-Quran certificate from a Registered Madrassa if the candidate claims to be Hafiz-e-Quran.
4. Matirc/Secondary School pass Certificate (Science/Equivalent).
5. Migration Certificate from the respective board (only from admitted candidates).
(a) Disability registration certificate from social and Welfare Department, Government of Khyber Pakhtunkhwa.
(b) Disability medical certificate on prescribed proforma from concerned certified specialist if applying against seats reserved for the disabled.
7. Two copies of Domicile certificate of the candidate. (Domicile certificate submitted with the Entrance test form will not be changed at a later stage of admission process).
8. A copy of Domicile certificate of the father of the applicant; if the father is not alive, then the death certificate of the father along with the domicile certificate of the mother have to be submitted.
9. A copy of Computerized National Identity Card of the candidate or Computerized Registration Form.
10. A copy of Computerized National Identity Card of the father of the applicant; if the father is not alive, a copy of the National Identity card of the mother or guardian.
11. A copy of parent's valid OPF Membership card and Foreign Exchange Remittance Card (Valid on the closing date of application form for admission) in case of candidates applying against Overseas Pakistani's Seats.
12. A copy of attempt certificate form the concerned BISE if the period between SSC and F.Sc. is more than two academic years.
13. Original Undertaking on judicial stamp paper of Rs.20/- duly attested by Notary Public/Political Agent as per specimen given in the prospectus/admission from (only for admitted candidates).
14. Three passport size colored photographs of the applicant attested on the back.
15. Certificate of location of educational institutions from principals/Head Masters of the concerned institutions have to be submitted by the candidates of FATA and backward areas.
16. The certificate must be countersigned by the EDO and DCO of the concerned district and Political Agent of the agency.
17. Certificate from the Institution last attended with duration of stay.
18. In case, the candidate has obtained F.Sc part-I and part-II education from different institutions he/she has to produce a certificate indicating the institution from where F.Sc. Part-I and Part-II have been obtained.