Improvements in or relating to diagnostic/therapeutic agents

专利摘要:
PURPOSE: Diagnostic and/or therapeutically active agents, more particularly to diagnostic and/or therapeutically active agents incorporating moieties having affinity for sites and/or structures within the body so that diagnostic imaging and/or therapy of particular locations within the body may be enhanced. CONSTITUTION: Targetable diagnostic and/or therapeutically active agents, e.g. ultrasound contrast agents, comprises a suspension in an aqueous carrier liquid of a reporter comprising gas-containing or gas-generating material, the agent being capable of forming at least two types of binding pairs with a target.
公开号:KR20000052830A
申请号:KR1019990703659
申请日:1997-10-28
公开日:2000-08-25
发明作者:요 클라베네스；폴 롱베트；안데르스 회그세트；헬게 톨레샤우그；알란 쿠트베르트슨；아슬락 고달；라르스 호프；가이르 고그스타트；클라우스 브린；안네 네베스타트；다그핀 뢰프하우그；할디스 헬레부스트；마그네 솔바켄
申请人:조오지 디빈센조, 토브 아스 헬지, 에바 요한손；니코메드 이메이징 에이에스；
IPC主号:

专利说明:

Diagnostics / therapies or related improvements {Improvements in or Relating to Diagnostic / Therapeutic Agents}
The present invention relates to diagnostic and / or therapeutically active agents, and more particularly to diagnostic and / or therapeutically active agents in admixture with residues that interact with or have affinity with sites and / or structures in the body. Diagnostic imaging and / or treatment of can be improved. Of particular interest are diagnostic agents for using ultrasound imaging, referred to below as target ultrasound contrast agents.
Ultrasound imaging is known to constitute an effective and valuable diagnostic tool, for example in the study of the vascular system, in particular cardiacography and tissue microvascular system. Various contrast agents have been proposed to increase the resulting acoustic image, including solid particles, emulsion droplets, gas bubbles and suspensions of encapsulated gas or liquid. In general, low-density contrast agents that are easily compressible are recognized to be particularly effective in the backscattering surface of the acoustics they produce, which has given considerable interest to the manufacture of gas-containing and gas-generating systems.
Gas-containing contrast agents are also known to be effective in magnetic resonance (MR) imaging, for example, as magnetizing contrast agents that act to reduce MR signal strength. Oxygen-containing contrast agents also represent useful paramagnetic MR contrast agents.
It has also been observed that gases such as carbon dioxide can be used as negative oral or intravascular contrast agents in the field of X-ray imaging.
Radioactive isotopes of radioactive gases, such as inert gases such as xenon, are also used for scintogram imaging, for example blood imaging.
Targeted ultrasound contrast agents include (i) reporter residues that can interact with ultrasound irradiation to generate a detectable signal; (ii) one or more vectors, in particular, having affinity for a target site and / or structure in the body, for example a particular cell or region of pathology; And (iii) one or more binders linking the reporter and vector (s), consequently not directly binding them.
The molecules and / or structures to which the diagnostic / therapeutic agent is intended to bind are referred to as targets below. Targets must be present and useful in this area / structure in order to obtain a specific burn or therapeutic effect in a selected area / structure in the body. Ideally, they are present only in the region of interest, but may also be present in other locations in the body that can cause posterior problems. This target can be defined as a molecular species (eg, a target molecule) or an unknown molecule or more complex structure present in the region to be imaged and / or treated, and can specifically or selectively bind to a given vector molecule. have.
Such vectors are attached or bound to reporter residues to bind these residues to the regions / structures to be imaged and / or treated. The vector may specifically bind to the selected target, or may only selectively bind, again having affinity for a limited number of other molecules / structures, again causing back problems.
There is a limited prior art related to target ultrasound contrast agents. Thus, for example, US-A-5531980 discloses one or more film-forming surfactants in which the reporter is at least partially present in a layered or layered form (the surfactant comprises a "living species designed for specific target purposes"). And a reporter comprising an aqueous suspension of air or gas microbubbles stabilized by one or more vectors). It is mentioned that the microbubbles are not directly encapsulated by the surfactant material, but are mixed in liquid filled liposomes that stabilize the microbubbles. Layered or layered surface-active substances such as phospholipids present in these liposomes inevitably comprise a lipophilic tail "back-to-back" and one or more lipid bilayers with hydrophilic heads both inside and outside. In form (see, eg, Schneider, M. on "Liposomes as drug carriers: 10 years of research" in Drug targeting, Nyon, Switzerland, 3-5 October 1984, Buri, P. and Gumma, A. (Ed), Elsevier, Amsterdam 1984).
EP-A-0727225 describes a target ultrasound contrast agent in which the reporter comprises a compound having sufficient vapor pressure, the proportion of which is gaseous at the body temperature of the subject. This compound binds a surfactant or albumin carrier comprising a protein-, peptide- or carbohydrate-based cell adhesion molecule ligand as a vector. Reporter residues in such contrast agents correspond to the phase transfer colloidal system described in WO-A-9416739; It is currently recognized that the administration of such phase transfer colloids may grow unregulated, possibly leading to the generation of microbubbles which, for example, cause dangerous color shifts of the myocardial vascular system and the brain (eg, literature See Schwarz, Advances in Echo-Contrast [1994 (3)], pp 48-49.
WO-A-9320802 suggested that tissue-specific ultrasound imaging improvements can be achieved using acoustically reflective oligolayered liposomes bound to tissue-specific ligands such as antibodies, peptides, lectins and the like. These liposomes are carefully selected to degas and do not have the favorable echo of gas-based ultrasound contrast agents. Also, for example, references to this technique in targets for fibrin, tromby and atherosclerotic regions are described in Alkanonyuksel, H. et al. Pharm. Sci. (1996) 85 (5), 486-490; J. Am. Coll. Cardiol. (1996) 27 (2) Suppl A, 298A; and Circulation, 68 Sci. Sessions, Anaheim 13-16 November 1995].
In addition, there are a number of publications relating to ultrasound contrast agents referred to during reporter passage, including the possible use of monoclonal antibodies and / or substances that can be dissolved by the reticuloendothelial system as vectors that do not give important practical details. Enables burn improvement of organs such as the liver-for example, WO-A-9300933, WO-A-9401140, WO-A-9408627, WO-A-9428874, US-A-5088499, US-A-5348016 And US-A-5469854. Typically, to focus on target cells, prior art targeted contrast agents tend to increase contrast at certain locations in the body, eg, tumor cells, by using one vector that strongly contends with one target. In contrast to this principle of using one vector to bind one vector with high affinity, the present invention provides for a wide variety of vector-target interactions (e.g., affinity for different targets on the same or different cell types). Partly based on the discovery that diagnostic and / or therapeutically active agents having more advantageous properties can be obtained by using a plurality of different vectors having and / or agents associated with one or more vectors). In this way, the binding of the gas-containing and gas-generating diagnostics and / or therapeutic agents is, for example, with low or high affinity, between one vector having specificity for one or more receptors or for one or more target types. This can be accomplished by forming multiple bond pairs between one or more vectors with Mars. Such multiple binding of an agent vector-bound to one or more target molecules / structures, for example, increases target specificity and / or fundamental interactions with lower levels of similar molecules / structures to targets expressed elsewhere in the body. It is possible to produce advantageous target properties by remarkable interaction at the target site of interest.
It is well known to use one vector which binds with high affinity to one target. However, the present invention is directed to forming a plurality of binding pairs with low affinity between one type of vector and one type of target or a plurality of types between one or more types of vector and one or more types of targets with low or high affinity. It is based on the discovery that the desired combination of gas-containing and gas-generating diagnostic and / or therapeutic agents can be obtained by forming a binding pair of. Thus, multiple binding of a vector bound agent to one or more target molecules / structures may, for example, increase target specificity and / or fundamental interaction with lower levels of molecules / structures for other expressed targets in the body. It may also have advantageous target properties by making the interaction at the target site desired.
Thus, according to one aspect of the invention, a reporter containing a gas-containing or gas-generating substance in a targetable diagnostic and / or therapeutically active agent, such as an aqueous carrier liquid (eg, an injectable carrier liquid) Ultrasound comprising a suspension, capable of forming two or more types of binding pairs, eg, coupled to one or more than two vectors capable of binding to two or more binding sites Contrast agent is provided.
One advantageous embodiment of the present invention is that a limited attachment to a target is a very useful property of a diagnostic and / or therapeutically active agent, which property can be achieved using a vector that yields temporary retention over fixed attachment to the target. It is based on the further finding that it can. Thus such diagnostic and / or therapeutically active agents may be effectively present in the form of a delayed flow with the vascular endothelial, for example by transient interaction with endothelial cells, rather than being held fixed at a particular site. Thus, such diagnostic and / or therapeutically active agents may be concentrated on the vessel wall when the ultrasound contrast agent provides improved echo over the bulk of the bloodstream lacking anatomical features. Thus, these diagnostic and / or therapeutically active agents may allow for improved burns of capillary systems including microvessels, for example to facilitate differentiation between normal and improperly spread tissue in the heart and It may be useful for visualizing structures such as, Cooper cells, tromby and atherosclerotic damage or for visualization of neo-vascular and inflammatory tissue regions. The present invention is particularly suitable for burn changes occurring in normal blood vessels located in the area of tissue necrosis.
Binding affinity is thought to be dependent on the number of interactions and their strength. Thus, the density of the vector molecules at the surface of the receptor unit may be selected to suit the degree of interaction between the specific agent and the target.
The term multi-specificity also refers to a gas consisting of one or more vectors comprising specific components for one or more cell surface receptors and at the same time a second component having specificity for a substrate or receptor-based binding that elicits a therapeutic response. Used to refer to an injectable carrier solution of a containing or gas-generating substance. Thus, targeting vectors such as the anti-fibrin antibodies described in Lanza et al., Circulation (1996) 94 (12), pp 3334, annexin V athero platelet binding peptides such as YRALVDTLK or fibrin coagulation Multi-specific imaging agents, including any other vector known to be known, have local antithrombobolic activity by having fibrinogen activity, such as streptokinase, plasminogen activator (tRA), urokinase (uPA), or prourokinase (scuPA). It is included within the scope of the present invention in conjunction with drugs or enzymes having a therapeutic effect. The invention also extends to the inclusion of vectors with increased specificity for tumor cells in combination with vectors or drug molecules that act as chemotherapeutic agents capable of inhibiting tumor growth.
Although not all, it is known that many target molecules are not expressed alone at the target site; Such molecules are overexpressed by the target cell in the co-position, but at lower levels in other places in the body in the target construct. In such a situation it may be advantageous to use a reporter having a multiplicity of a vector having a relatively low affinity for the target, since a site of high target density at which the reporter allows for multiple (and also strong) binding to the reporter. (Eg, gas-containing agents that introduce vector folic acid and glutathione that bind to overexpressed hydrochloric acid receptors and glutathione-S-trasperase receptors, respectively, as tumor cells). On the other hand, low target density sites will not provide sufficient interaction with such low affinity vectors that bind the target. In this embodiment of the invention, the low affinity vector is capable of interacting with a target molecule or structure of less than 10 ⁸ M ⁻¹ , eg, less than 10 ⁷ M ⁻¹ , preferably less than 10 ⁶ M ⁻¹ . Can be considered to have _a binding constant K _a . Thus, a further embodiment of the present invention provides that the desired binding of gas-containing and gas-generating diagnostic and / or therapeutic agents can be obtained by forming a binding pair with low affinity between one or more vector types and one or more targets. Based on discovery. Therefore, multiple vectors can be used to increase specificity, and reporters will only bind to target cells or structures that exhibit specific binding of the target molecule.
In addition, to create increased binding force, it would be useful to select a plurality of vectors that bind to other portions of the target structure, for example, an epitope. This will be particularly advantageous when the target density is low.
Products containing two or more vectors with different specificities, ie, binding to different target molecules on different cells, are advantageous as "general purpose" agents for the detection of a range of diseases, such as, for example, other forms of cancer. Can be used. Thus, for example, the use of such agents enables the detection of metastatases and is often heterogeneous with respect to the expression of target molecules (eg antigens).
In the scope of the present invention, reporter units will typically be present in combination with the vector. Often, in another type of targeting process called pre-targeting, a vector (often a monoclonal antibody) is administered alone, followed by a reporter that binds to a residue capable of specifically coupling the vector molecule. (If the vector is an antibody, the reporter may bind to an immunoglobulin-binding molecule such as Protein A or an anti-immunoglobulin antibody). The advantage of this protocol is that time is allowed for the removal of vector molecules that do not bind their targets, thereby substantially reducing the underlying problem associated with the presence of excess reporter-vector conjugates. In the present invention, there may be mentioned preliminary targeting of one specific vector, a reporter unit coupled to another vector, and a residue binding to the first vector. In the present invention it is important to determine the rate at which the ultrasound contrast agent bound to the target or detached from the target in some cases and in particular in the assessment of the blood reflux rate at certain sites such as myocardium. This can be achieved in a controlled manner by subsequent administration of a vector or other agent that replaces or leaves the contrast agent from the target.
Useful vectors according to the present invention include cell attachment proteins themselves with ligands for cell adhesion proteins and corresponding ligands on endothelial cell surfaces. Examples of cell adhesion proteins are integrins, most of which bind to the Arg-Gly-Asp (RGD) amino acid sequence. If desired, the vector may be primarily targeted to specific cell adhesion proteins expressed on activated endothelial cells, such as those found at or near inflammation or other pathological response sites. Other vectors that can be used include proteins and peptides that bind to cell-surface proteoglycans, which are complexes of proteins and sulfated polysaccharides found in most cells. Such proteoglycans cause all nucleated cells from vertebrates to have a negative surface charge. Also, in the present invention, such charges can be used to electrostatically interact with the endothelial surface, for example by using vectors of positive charges containing cationic lipids.
A further aspect of the present invention is that the vector (s) are included in the reporter non-covalently, for example, to the reporter in such a way that they are not easily exposed to the target or receptor. Therefore, increased tissue specificity can be achieved by applying additional processes of exposing the vector, such as exposing the agent to external ultrasound after administration to change the diffusion capacity of the residue containing the vector, for example.
The reporter may be, for example, any simple form of any suitable gas-containing or gas-generating ultrasound contrast formulation. Representative examples of such formulations include agglutinated surface membranes (eg, gelatin as described in WO-A-8002365), pimogen proteins (eg, US-A-4718433, US-A-4774958, US-A Albumin, such as human serum albumin, as described in -4844882, EP-A-0359246, WO-A-9112823, WO-A-9205806, WO-A-9217213, WO-A-9406477 or WO-A-9501187), Polymeric materials (e.g., synthetic biodegradable polymers as described in EP-A-0398935, elastic interfacial synthetic polymer membranes as described in EP-A-0458745, microparticle biodegradable polys as described in EP-A-0441468) Aldehydes, microparticle N-dicarboxylic acid derivatives-polycyclic imides of polyamino acids such as those described in WO-A-9317718 and WO-A-9507434, non-polymeric and non-polymeric wall-producing materials (eg, As described in WO-A-9521631 or a surfactant (e.g., polyoxyethylene-polyoxy such as Pluronic) Lohylene block copolymer surfactant, polymeric surfactant as described in WO-A-9506518, or WO-A-9211873, WO-A-9217212, WO-A-9222247, WO-A-9428780 or WO-A- Polymer surfactants, such as those described in 9503835) (eg, at least partially encapsulated) microbubbles.
Other useful gas-containing contrast agent formulations include gas-containing solids such as microparticles (especially microparticle masses) that contain gas or otherwise associated therewith (eg, EP-A-0122624, EP-A). Contained within voids, cavities or voids as described in -0123235, EP-A-0365467, WO-A-9221382, WO-A-9300930, WO-A-9313802, WO-A-9313808 or WO-A-9313809 (Or) attached to its surface). It is believed that the reverberation of such microparticle contrast agents can be derived directly from gases (eg, microbubbles) and / or gaseous / related gases liberated from solid materials (eg, upon dissolution of the microparticle structure). do.
The above-mentioned documents relating to gas-containing contrast agent formulations are incorporated herein by reference.
Other gas-containing materials such as gas microbubbles and microparticles are preferably initially no more than 10 μm (eg, 7 μm or less) in order to be able to pass freely through the pulmonary artery after administration such as intravenous injection. Have an average size.
When the phospholipid-containing composition is used in the present invention, for example in the form of phospholipid-stabilized gas microbubbles, representative examples of useful phospholipids are lecithin (ie phosphatidylcholine), eg egg yolk lecidine or soy lecithin. Natural lecithin such as, or synthetic or semisynthetic lecithin, for example dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; Phosphatidic acid; Phosphatidylethanolamine; Phosphatidylserine; Phosphatidylglycerol; Sphingomyelin; Fluorinated optional homologs; Mixtures of the foregoing and mixtures with other lipids such as cholesterol. For example, naturally occurring (eg, induced egg yoke or soybean), semisynthetic (eg, partially or wholly hydrogenated) and synthetic phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and (or It may be particularly advantageous to use large amounts (at least about 75%) of phospholipids, including molecules with an overall charge, such as cardiolipin).
Other examples of lipids that can be used to prepare gas-containing contrast agents include fatty acids, stearic acid, palmitic acid, 2-n-hexadecylstearic acid, oleic acid, and other acids containing lipid structures. These lipid structures are particularly important when coupling with one or more amino acids. Attachment of the functionalized spacer component resulting from a lipid altered amino acid (eg dipalmitolysine, distearoyl-2,3-diaminopropionic acid) characterizing the coupling position for binding of one or more vector molecules It is thought to be a useful precursor for.
Another aspect of the invention relates to the synthesis of lipopeptide constructs comprising a lipid reporter attached to a linker moiety (eg, PEG, polyamino acids, alkyl halides, etc.), the linker coupling one or more vector molecules. To be functionalized appropriately. It is particularly preferred to include a charged linker component (eg two or more lysine residues) for the fixation of the reporter component in the microbubble through electrostatic interaction with the negatively charged membrane. Multi-specific targeting can be achieved by mixing and 'doping' the phospholipid gas containing construct with one or more targeted lipopeptide sequences. In addition, Tam et al., Proc. Natl. Acad. Sci. As described in USA, 1989, 86, 9084, multi-specificity can be obtained by assembling one or more vectors on a branched lysine core structure. Lipopeptides or phospholipids, including combinatorial libraries synthesized by chemical synthesis, are also described, as described in Lowe et al., Combination Chemistry, Chemical Society Reviews, 1995, 309-317. Can be used to achieve multi-specificity.
Also within the scope of the present invention are functionalized microbubbles having at least one reactive group for non-specific reactions with receptor molecules located on the cell surface. Microbubbles comprising thiol residues can bind to cell surface receptors, for example, via disulfide exchange reactions. The reversible nature of this covalent bond means that bubble flow is adjustable by changing the redox environment. Similarly, 'activated' microbubbles of membranes comprising active esters such as N-hydroxysuccinimide esters can be used to modify amino acids found to be multiplicity of cell surface molecules.
Representative examples of gas-containing microparticle materials that may be useful in accordance with the present invention include carbohydrates (eg hexoses such as glucose, fructose or galactose; disaccharides such as shoe Cross, lactose or maltose; pentose, for example aribinose, xylose or ribose; α-, β- and γ-cyclodextrin; polysaccharides such as starch, hydroxyethyl starch, amylose, Amylopectin, glycogen, inulin, pullulan, dextran, carboxymethyl dextran, dextran phosphate, ketodextran, aminoethyldextran, alginate, chitin, chitosan, hyaluronic acid or heparin; and sugar alcohols, alditol, eg Mannitol or sorbitol), inorganic salts (e.g. sodium chloride), organic salts (sodium citrate, sodium acetate or sodium tartrate), x-ray contrast agents (e.g. Trizoic acid, ditrizoic acid, iotalamic acid, ioxaglic acid, iohexel, iopentol, iopamidol, iodixanol, iopromide, methamide, iodipamide, meglumine, iodipamide, meth Carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, in the 3- and / or 5-positions, as in glutamine acetioate and meclomine ditrizoate; Any commercially available carboxylic acid and nonionic amide contrast agent containing one or more 2,4,6-triiodophenyl groups with substituents such as acylamino, N-alkylacylamino or acylaminomethyl and polypeptides and proteins (eg For example, gelatin or albumin such as human serum albumin).
According to the present invention, any biocompatible gas may be present in the reporter of the contrast agent, and the term "gas" as used herein refers to any substance in the form of substantially almost gas (including steam) at normal human temperature of 37 ° C. Mixtures). Thus, for example, the gas may be air; nitrogen; Oxygen; carbon dioxide; Hydrogen; Inert gases such as helium, argon, xenon or krypton; Sulfur fluorides such as sulfur hexafluoride, disgadisulfide or pentafluoride trifluoromethylsulfur; Selenium hexafluoride; Optionally halogenated silanes such as methylsilane or dimethylsilane; Low molecular weight hydrocarbons (for example containing up to 7 carbon atoms), for example, alkanes such as methane, ethane, propane, butane or pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, ethylene, pro Alkenes such as phen, propadiene or butene, or alkynes such as acetylene or propine; Ethers such as dimethyl ether; Ketones; ester; Halogenated low molecular weight hydrocarbons (eg containing up to 7 carbon atoms); Or mixtures of any of the foregoing. Advantageously, at least some of the halogenated atoms in the halogenated gas are fluorine atoms such that the biocompatible halogenated hydrocarbon gas is, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoro Romethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethylfluoride, 1,1-difluoroethane and perfluorocarbons, eg Mixtures with other isomers such as, for example, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (eg, perfluoro-n-butane, optionally perfluoro-iso-butane) ), Perfluoroalkanes such as perfluoropentane, perfluorohexane and perfluoroheptane; Perfluoroalkenes such as perfluoropropene, perfluorobutene (eg, perfluorobut-2-ene) and perfluorobutadiene; Perfluoroalkynes such as perfluorobut-2-yne; And perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluoro Perfluorocycloalkanes such as rocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane. Other halogenated gases include fluorinated (eg perfluorinated) ethers such as methyl chloride, fluorinated (eg perfluorinated) ketones such as perfluoroacetone and perfluorodiethyl ether. The use of perfluorinated gases, such as sulfur hexafluoride and perfluoropropane, perfluorobutane and perfluoropentane, has been found to be highly stable in the blood flow of microbubbles containing such gases. It may be particularly advantageous at
For example, the reporter may be prepared by any simple process by preparing a gas-containing or gas-generating formulation. Representative examples include contacting a gas with a surfactant and mixing them in the presence of an aqueous carrier, as described in WO 9115244; Or by spraying a solution or suspension of the wall-generating material in the presence of a gas to obtain co-microcapsules, as described in EP 512693A1; Preparation of solid microspheres by a double emulsion process as described in US 5648095; As described in EP 681843A2, a process for preparing hollow microcapsules by a spray drying process; Gas microbubble suspensions are prepared by shaking a aqueous solution containing lipids in the presence of a gas as described in US Pat. No. 5,469,854 to prepare a gas-filled liposome.
Suitable attachment processes of the desired vector to the reporter include surface modification of the reporter performed with a suitable linker using a reactor on the surface of both the reporter and the vector. It may be particularly advantageous to physically mix the vector containing material and the reporter material at any stage of the process. Such a process will produce the formulation or attach the vector to the reporter. Any process step may remove excess vector not bound to the reporter after separation, for example by washing the gas-containing molecules by suspension. A preferred aspect is to use lipopeptide constructs that introduce functional groups such as thiols, maleimide biotin, etc., which can be premixed with other reporter molecules, if desired, prior to generation of the gas-containing formulation. Attachment of the vector can be performed using the linker reagents listed below.
Combination of reporter units to the desired vector can generally be accomplished by covalent or non-covalent means involving interaction with the reporter and / or one or more functional groups located in the vector. Examples of chemically active functional groups that can be used for this purpose include carbohydrate groups, bisinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyls in addition to amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups , Imidazolyl and phenolic groups.
Thus, covalent coupling of reporters and vectors can be done using linking agents comprising reactive moieties that can react with such functional groups. Examples of reactive moieties that can react with sulfhydryl groups show particular reactivity with sulfhydryl groups, but are described in Gurd, FRN in Methods Enzymol. (1967) 11, 532 of the form X-CH ₂ CO-, wherein X = Br, Cl or I, which may also be used to modify imidazolyl, thioether, phenol and amino groups α-haloacetyl compounds. N-maleimide derivatives are also considered selective to sulfhydryl groups, but may be further useful in coupling to amino groups under certain conditions. N-maleimide is described by Kitagawa, T. et al. in Chem. Pharm. Bull. (1981) 29, 1130, incorporated into a linking system for reporter-vector binding or as described in Kovacic, P. et al. in J. Am. Chem. Soc. (1959) 81, 1887, as polymer crosslinkers for bubble stabilization. See, eg, Traut, T. et al. in Biochemistry (1973) 12, 3266, a reagent such as 2-iminothiolane that introduces thiol groups via the conversion of amino groups to be considered sulfhydryl reagents when the linkage occurs through the formation of disulfide bridges. Can be. Thus, reagents that introduce reactive disulfide bonds into the reporter or vector may be useful because a linkage may occur due to disulfide changes between the vector and the reporter; Examples of such reagents include Elman reagent (DTNB), 4,4'-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide and methyl-2-pyridyl disulfide (Kimura, T et al. in Analyt. Biochem. (1982) 122, 271).
Examples of reactive moieties that can react with amino groups include alkylating agents and acylating agents. Examples of representative alkylating agents include:
i) an α-haloacetyl compound exhibiting specificity for an amino group in the presence of a reactive thiol group, for example, see Wong, YH.H. in Biochemistry (1979) 24, 5337, in the form of X-CH ₂ CO-, wherein X = Cl, Br or I;
ii) Michael-type reactions or as described in Smith, D.G. et al. in J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964) 91, 589, N-maleimide derivatives capable of reacting with amino groups via acylation by addition to cyclic carbonyl groups;
iii) aryl halides such as reactive nitrohaloaromatic compounds;
iv) McKenzie, J.A. et al. in J. Protein Chem. (1988) 7, 581] alkyl halides;
v) aldehydes and ketones capable of forming a Schiff base with amino groups, wherein the adducts formed are generally stabilized through reduction to yield stable amines;
vi) epoxide derivatives capable of reacting with amino, sulfhydryl or phenolic hydroxyl groups such as epichlorohydrin and bisoxirane;
vii) chlorine-containing derivatives of s-triazine which are highly reactive against nucleophiles such as amino, sulfhydryl and hydroxy groups;
viii) s-triazine compounds based aziridine capable of reacting with nucleophiles such as amino groups by ring opening (see, eg, Ross, WCJ in Adv. Cancer Res. (1954) 2, 1) ;
ix) squaric acid diethyl ester (Tietze, L. F. in Chem. Ber. (1991) 124, 1215);
x) α-haloalkyl ethers which are more reactive alkylating agents than ordinary alkyl halides by activation with ether oxygen atoms (Benneche, T. et al. in Eur. J. Med. Chem. (1993) 28, 463 ]).
Representative amino-reactive acylating agents include:
i) forming stable urea and thiourea derivatives, respectively, and described in Schick, A.F. et al. in J. Biol. Chem. (1961) 236, 2477; isocyanates and isothiocyanates, in particular aromatic derivatives, used for protein crosslinking;
ii) Herzig, D.J. et al. sulfonyl chlorides described in in Biopolymers (1964) 2, 349, which may be useful for the introduction of fluorescent reporter groups into linkers;
iii) acid halides;
iv) active esters such as nitrophenyl esters or N-hydroxysuccinimidyl esters;
v) acid anhydrides such as mixed, symmetric or N-carboxy anhydrides;
vi) Bodansky, M. et al. other reagents useful for the formation of amide bonds as described in 'Principles of Peptide Synthesis' (1984) Springer-Verlag;
vii) acyl azide, wherein the azide group is a hydrazide derivative preformed using sodium nitrate, as described, for example, in Wetz, K. et al. in Anal. Biochem. (1974) 58, 347. Occurs from);
viii) for example, Rasmussen, J.K. azlactone attached to a polymer such as bisacrylamide as described in Reactive Polymers (1991) 16, 199; And
ix) Imidoesters that form stable amidines upon reaction with amino groups (see, eg, Hunter, M. J. and Ludwig, M. L. in J. Am. Chem. Soc. (1962) 84, 3491).
Carbonyl groups, such as aldehyde functional groups, can react with weak protein bases at pH at which the nucleophilic protein side chain functional groups are protonated. Weak bases include 1,2-aminothiol, which can be found in the N-terminal cysteine residue, which is described, for example, in Ratner, S. et al. in J. Am. Chem. Soc. (1937) 59, 200, optionally forming a stable 5-membered thiazolidine ring with an aldehyde group. Other weak bases such as phenyl hydrazone are described, for example, in Heitzman, H. et al. in Proc. Natl. Acad. Sci. USA (1974) 71, 3537.
Aldehydes and ketones can also react with amines to form Schiff bases, and can advantageously be stabilized through reductive amination. Alkoxyamino residues can readily react with ketones and aldehydes to produce stable alkoxamines (see, eg, Webb, R. et al. In Bioconjugate Chem. (1990) 1, 96).
Examples of reactive moieties that can react with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups (eg, Herriot RM in Adv). Protein Chem. (1947) 3, 169]. Carboxylic acid modifiers such as carbodiimides that react through O-acylurea formation and amide bond formation may also be usefully employed; Linking can be facilitated through the addition of amines or results in direct vector-reporter coupling. Useful water-soluble carbodiimides include 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide (CMC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (See, eg, Zot, HG and Puett, D. in J. Biol. Chem. (1989) 264, 15552). Other useful carboxylic acid modifiers include isoxazolium derivatives such as Woodwards Reagent K; chloroformates such as p-nitrophenylchloroformate; Carbonyldiimidazole, such as 1,1'-carbonyldiimidazole; And N-carvalkoxydihydroquinolines such as N- (ethoxycarbonyl) -2-ethoxy-1,2-dihydroquinoline.
Other useful reactive moieties are bisinal diones such as p-phenylenediglyoxal, which include guanidinyl groups (Wagner et al. In Nucleic acid Res. (1978) 5, 4065); And diazonium salts capable of undergoing an electrophilic substitution reaction (Ishizaka, K. and Ishizaka T. in J. Immunol. (1960) 85, 163). Bis-diazonium compounds are readily prepared by treating aryl diamine with sodium nitrate in acidic solution. If functional groups in the reporter and / or vector are desired, they may be converted to other functional groups, for example, prior to the reaction to impart further reactivity or selectivity. Examples of methods useful for this purpose include the conversion of amines to carboxylic acids using reagents such as dicarboxylic acid anhydrides; Conversion of amines to thiols using reagents such as N-acetylhomocysteine thiollactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane or thiol-containing succinimidyl derivatives; conversion of thiols to carboxylic acids using reagents such as α-haloacetate; Conversion of thiols to amines using reagents such as ethyleneimine or 2-broboethylamine; Carbodiimide followed by conversion of the carboxylic acid to the amine using a reagent such as diamine; And conversion of the alcohol to thiol using a reagent such as tosyl chloride, followed by transesterification with thioacetate and hydrolysis of the thiol with sodium acetate.
Vector-reporter coupling can also be performed using enzymes such as zero-length linkers; Thus, for example, transglutaminase, peroxidase and xanthine oxidase can be used to prepare linked products. Reverse proteolysis can also be used for linkage through amide bond formation.
Non-covalent vector-reporter coupling is, for example, between a polylysinyl-functionalized reporter and a polyglutamyl-functionalized reporter through high affinity binding interactions such as chelation or avidin / biotin bonds in the form of stable metal complexes. By electrostatic charge interaction. Polylysine coated noncovalently on the negatively charged membrane surface can also nonspecifically increase the affinity of the microbubbles to cells through charge interactions.
Vectors can also be coupled to known protein or peptide sequences to bind phospholipids. In many cases other proteins are attached primarily to surfaces consisting of phospholipid head groups, while a single molecule of phospholipid can attach to proteins such as translocases and thus can be used to attach vectors to phospholipid microspheres; One example of such a protein is β2-glycoprotein I (Chonn, A., Semple, S.C. and Cullis, P.R., Journal of Biological Chemistry (1995) 270, 25845-25849). Phosphatidylserine-binding proteins are described in Igarashi, K. et al. in Journal of Biological Chemistry 270 (49), 29075-29078. Annexin is a type of phospholipid binding protein, many of which specifically bind to phosphatidyl-serine [Raynal, P and H.B. Pollard. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins ", Biochim. Biophys.Acta 1197: 63-93] .The combination of these phosphatidylserine-binding proteins with phosphatidylserine- If the amino acid sequence of the binding protein is known, the phospholipid-binding protein is synthesized or isolated, used for binding to the vector, and can be located anywhere in the molecule. Can be avoided.
It is also possible to obtain molecules that specifically bind to the surface (or “membrane”) of the microspheres by direct screening of a molecular library for microsphere-binding molecules. For example, phage libraries that display small proteins can be used for this selection. This selection can be made by simply mixing the microspheres with the phage display library and releasing the phage bonds to the suspended microspheres. If desired, such selection may be made in "physiological conditions" (eg, in the blood) to remove peptides that cross-react with blood components. The advantage of this type of selection process is that only the binding molecules attached to the fully suspended microspheres rise to the top so that only binding molecules that do not destabilize the microspheres should be selected. It may also be possible to introduce some kind of “stress” (eg, pressure) during the selection process to ensure that destable binding moieties are not selected. This selection can also be carried out by shearing conditions, for example by first reacting phage with microspheres and passing the microspheres through a surface coated with an anti-phage antibody under flow conditions. In this way it may be possible to select a binder that is capable of inhibiting shear conditions present in vivo. The binding moieties identified in this way can be coupled to a vector molecule consisting of general means for attaching any vector molecule to microspheres (via chemical binding or peptide synthesis, or at the DNA-level for recombinant vectors).
Lipopeptide linkers containing vectors coupled to or coupled to peptides, elements capable of modulating membrane insertion, may also be useful. An example is described in Lynnhouts, J.M. et al. in Febs Letters (1995) 370 (3), 189-192. Bioinert molecules composed of known membrane insertion anchors / signals can be used as vectors for specific applications and examples are described in Xie, Y. and Morimoto, T. in J. Biol. Chem. (1995) 270 (20), 11985-11991, H1 hydrophobic segment from Na, K-ATPase α-subunit. This anchor group may also be fatty acid (s) or cholesterol.
Coupling can also be performed using avidin or streptavidin with four high affinity binding sites for biotin. Thus, avidin can be used to bind the vector to the reporter when both the vector and the reporter are biotinylated. Such examples are described in Bayer, E.A. and Wilchek, M. in Methods Biochem. Anal. (1980) 26, 1). The method can also be extended to a process that can facilitate the linking of the reporter to the reporter, bubble association and possibly possibly increased echo.
Non-covalent coupling can also take advantage of the bifunctional nature of bispecific immunoglobulins. Such molecules can specifically bind two antibodies and link them. For example, bispecific IgG or chemically treated bispecific F (ab) ' ₂ segments can be used as the linking agent. Heterobifunctional bispecific antibodies have also been reported to link two different antigens (Bode, C. et al. In J. Biol. Chem. (1989) 264, 944 and Statrz, UD et al. in Proc. Natl. Acad. Sci. USA (1986) 83, 1453]. Similarly, any reporter and / or vector having two or more antigenic determinants (described in Chen, Aa et al. In Am. J. Pathol. (1988) 130, 216) is an antibody. It can be crosslinked by the molecule, forming a potentially increased echo of multi-bubble cross-linked assembly.
So-called zero-length linkers which induce direct covalent bonds of two reactive chemical groups without introducing additional linking materials (eg using carbodiimide or upon enzymatically induced amide bond formation) are preferred. And a therapeutic agent such as a biotin / avidin system that induces a non-covalent reporter-vector linkage and a therapeutic agent that induces hydrophobic or electrostatic interactions.
However, most commonly the linking agent comprises two or more reactive moieties linked by spacer elements, as described above. The presence of such spacers allows difunctional linkers to react with specific functional groups in one molecule or two different molecules to form a bond between these two components and introduce an external linker-derived material into the reporter-vector bond. The reactive moieties in the linking agent may be identical (homofunctional reagents) or different (heterofunctional reagents or heteropolyfunctional reagents when several dissimilar reactive moieties are present) and may be molecules or molecules between any chemical species. Can cause covalent bonds.
The nature of the foreign material introduced by the linking agent can have important implications for the target strength and general stability of the final product. Thus, for example, it is desirable to introduce labile bonds containing spacer arms that mix biodegradable or chemically sensitive or enzymatic cleavage sites. Such spacers may also contain polymeric components, for example, which act as surfactants and increase bubble stability. Such spacers may include reactive moieties as described above, for example, to increase surface crosslinking, or may include tracer elements such as fluorescent probes, spin labels or radioactive materials.
The spacer element may generally consist of aliphatic chains that effectively separate the reactive moieties of the linker at a distance of 5 to 30 mm 3. They may also include macromolecular structures such as poly (ethylene glycol). Such polymeric structures, referred to herein as PEG, are simple neutral polyesters, which are used in biotechnological and biomedical applications (eg, Milton Harris, J. (ed) "Poly (ethylene glycol) chemistry, biotechnical and biomedical applications "Plenum Press, New York, 1992). PEG is highly hydrated with two or three water molecules soluble in most solvents, including water, and bound to each ethylene glycol segment in an aqueous environment; This has the effect of preventing the uptake of proteins on other polymers or PEG-modified surfaces. PEG is known to be non-toxic and not harmful to active proteins or cells, but covalently linked PEG is known to be non-immune and non-antigenic. In addition, PEG is easily modified and can bind to other molecules that have little effect on its chemistry. Their advantageous solubility and biological properties are evident from the many possible uses of PEG and copolymers of PEG, including block copolymers such as PEG-polyurethane and PEG-polypropylene.
Suitable molecular weights for the PEG spacer used according to the invention are, for example, from 120 daltons to 20 kilodaltons.
The main mechanism for uptake of particles by cells of the reticuloendothelial system (RES) is opsonism by plasma proteins in the blood; These mark the foreign particles absorbed by the RES. The biological properties of the PEG spacer elements used in accordance with the present invention can serve to increase contrast cycle time in a manner similar to that observed for PEGylated liposomes (Klibanov, AL et al. In FEBS Letters (1990) 268, 235-237 and Blume, G. and Cevc, G. in Biochim. Biophys. Acta (1990) 1029, 91-97).
Other useful protein modifications include partial or complete deglycosidation by nuramidase, endoglycosidase, or periodate because deglycosidation is often less absorbed by liver, spleen, macrophages, etc. Glycosidation often results in increased uptake by the liver and macrophages; Preparation of the truncated form by proteolytic cleavage includes reduced size and shorter half-life in circulation; And cationization (see, eg, Kumagi et al. In J. Biol. Chem. (1987) 262, 15214-15219; Triguero et al. In Proc. Natl. Acad. Sci. USA (1989) 86, 4761-4765; Pardridge et al. In J. Pharmacol.Exp. Therap. (1989) 251, 821-826 and Pardrige and Boado, Febs Lett. (1991) 288, 30-32.
Increased coupling efficiency for the region of interest can also be achieved using antibodies bound to PEG spacer ends (Maruyama, K. et al. In Biochim. Biophys. Acta (1995) 1234, 74-80). And Hansen, CB et al. In Biochim. Biophys. Acta (1995) 1239, 133-144.
In some cases it is believed to include the PEG component as a stabilizer upon binding directly to the reporter in the same molecule in which the vector (s) is bound or PEG does not act as a spacer.
Other representative spacer elements include structural polysaccharides such as polygalacturonic acid, glycosaminoglycans, heparinoids, cellulose and marine polysaccharides such as alginates, chitosan and carrageenan; Storage polysaccharides such as starch, glycogen, dextran and aminodextran; Polyamino acids and their methyl and ethyl esters such as lysine, glutamic acid and aspartic acid alone or in copolymers; And polypeptides, oligosaccharides and oligonucleotides which may or may not bind to enzyme cleavage sites.
In general, the spacer element may comprise cleavable groups such as bisinal glycol, azo, sulfone, ester, thioester or disulfide groups. Chemical formula
-(Z) mYXC (R ¹ R ² ) .XY (Z) n- where X and Z are -O-, -S- and -NR- where R is hydrogen or an organic group Y is a carbonyl, thiocarbonyl, sulfonyl, phosphoryl or similar acid-forming group, respectively; m and n are each 0 or 1; R ¹ and R ² are each hydrogen, an organic group, or -XY (Z); spacers comprising a biodegradable methylene diester or diamide group of m-group or together with divalent organic groups) may be useful; For example, as described in WO-A-9217436, such groups are readily biodegradable, for example in the presence of estase in vivo, but are stable in the absence of such enzymes. Thus, they can be advantageously linked to a therapeutic agent to allow slow release of the therapeutic agent.
Poly [N- (2-hydroxyethyl) methacrylamide] is a useful spacer material by low interaction with cells and tissues (Volfova, I., Rihova, B. and VR and Vetvicka, P. in J. Bioact.Comp. Polymers (1992) 7, 175-190). Experiments with similar polymers consisting primarily of closely related 2-hydroxypropyl derivatives have shown that cells are only absorbed to a lesser extent by the mononuclear phagocyte system (Goddard, P., Williamson, I., Bron, J., Hutchkinson). , LE, Nicholls, J. and Petrak, K. in J. Bioct.Compat.Polym. (1991) 6, 4-24).
Other useful polymeric spacer materials include:
i) copolymers of methyl methacrylate and methacrylic acid; They may be eroded (see Lee, P.I. in Pharm. Res. (1993) 10, 980), and carboxylate substituents may cause higher swelling than neutral polymers;
ii) block copolymers of polymethacrylate with biodegradable polyesters (see San Roman, J. and Guillen-Garcia, P. in Biomaterials (1991) 12, 236-241);
iii) polymers of cyanoacrylates, ie esters of 2-cyanoacrylic acid, which are biodegradable and have been used in the form of nanoparticles for selective drug delivery (Forestier, F., Gerrier, P., Chaumard, C., Quero, AM, Couvreur, P. and Labarre, C. in J. Antimicrob. Chemoter. (1992) 30, 173-179);
iv) polyvinyl alcohols which are water soluble and generally considered biocompatible (see Langer, R. in J. Control. Release (1991) 16, 53-60);
v) copolymers of vinyl methyl ether and maleic anhydride mentioned as bioeroded (Finne, U., Hannus, M. and Urtti, A. in Int. J. Pharm. (1992) 78. 237-241 ] Reference);
vi) polyvinylpyrrolidone, for example, rapidly filtered by elongation with a molecular weight of less than about 25,000 (Hespe, W., Meier, AM and Blankwater, YM in Arzeim.-Forsch./Drug Res. (1977) 27, 1158-1162);
vii) polymers and copolymers of short-chain aliphatic hydroxy acids such as glycolic acid, lactic acid, butyric acid, valeric acid and caproic acid (see Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9) ) And copolymers which mix aromatic hydroxy acids to increase their degradation rate (Imasaki, K., Yoshida, M., Fukuzaki, H., Asano, M., Kumakura, M.). , Mashimo, T., Yamanaka, H. and Nagai.T. In Int. J. Pharm. (1992) 81, 31-38);
viii) polyesters composed of alternating units of ethylene glycol and terephthalic acid, for example Dacron ^R as non-degradable but very biocompatible;
ix) for example polyurethanes (Kobayashi, H., Hyon, SH and Ikada, Y. in "Water-curable and biodegradable prepolymer" -J. Biomed. Mater. Res. (1991) 25, 1481-1494 ] Aliphatic hydroxy acid polymers when mixed with (Younes, H., Nataf, PR, Cohn, D., Appelbaum, YJ, Pizov, G. and Uretzky, G. in Biomater.Artif.Cells Artif.Organs (1988) 16, 705-719), the block copolymer comprising a biodegradable segment;
x) flexible “soft” segments known to be resistant to transplanted tissue, including, for example, poly (tetramethylene glycol), poly (propylene glycol) or poly (ethylene glycol) and for example 4,4 Polyurethanes that can be mixed with aromatic “hard” segments including '-methylenebis (phenylene isocyanate) (Ratner, BD, Johnston, AB and Lenk, TJ in J. Biomed. Mater. Res: Applied Biomaterials ( 1987) 21, 59-90; Sa Da Costa, V. et al. In J. Coll.Interface Sci. (1981) 80, 445-452 and Affrossman, S. et al. In Clinical Materials (1991) 8, 25 -31);
xi) hydrolyzable ester linkages (see Song, CX, Cui, XM and Schindler, A. in Med. Biol. Eng. Comput. (1993) 31, S147-150). And poly (1,4-dioxan-2-one) which may include glycolide units to improve absorbency (Bezwada, RS, Shalaby, SW and Newman, HDJ in Agricultural and synthetic polymers: Biodegradability and utilization (1990) (ed Glass, JE and Swift, G.), 167-174-ACS symposium Series, # 433, Washington DC, USA-American Chemical Society.
xii) Rabbit studies (Brem, H., Kader, A., Epstein, JI, Tamargo, RJ, Domb, A., Langer, R. and Leong, KW in Sel. Cancer Ther. (1989) 5, 55 And rat studies (Tamargo, RJ, Epstein, JI, Reinhard, CS, Chasin, M. and Brem, H. in J. Biomed. Mater. Res. (1989) 23, 253-266). Polyanhydrides, such as copolymers of sebacic acid (octanedioic acid) and bis (4-carboxy-phenoxy) propane, which have been shown to be useful for controlled release of drugs in the brain without toxic effects;
xiii) biodegradable polymers containing ortho-ester groups used for controlled release in vivo (see Maa, Y. F. and Heller, J. in J. Control. Release (1990) 14, 21-28); And
xiv) polyphosphazenes, alternately inorganic polymers composed of phosphorus and nitrogen atoms (see Chrommen, J. H., Vandorpe, J. and Schacht, E. H. in J. Control. Release (1993) 24, 167-180).
The following table lists linking agents and reagents for protein modification that may be useful for preparing the target traceable therapeutic agent according to the present invention.
Heterodifunctional coupling agents
ConnectionReactivity 1Reactivity 2Explanation ABHcarbohydratePhotoreactivityANB-NOS-NH ₂ PhotoreactivityAPDP (1)-SHPhotoreactivityIodide Disulfide Linkers APG-NH ₂ PhotoreactivityReacts selectively with Arg at pH 7-8 ASIB (1)-SHPhotoreactivityIodinable ASBA (1)-COOHPhotoreactivityIodinable EDC-NH ₂ -COOHZero-length coupling agent GMBS-NH ₂ -SHSulfo-GMBS-NH ₂ -SHreceptivity HSAB-NH ₂ Photoreactivity
Sulfo-HSAB-NH ₂ Photoreactivityreceptivity MBS-NH ₂ -SHSulfo-MBS-NH ₂ -SHreceptivity M ₂ C ₂ Hcarbohydrate-SHMPBHcarbohydrate-SHNHS-ASA (1)-NH ₂ PhotoreactivityIodinable Sulfo-NHS-ASA (1)-NH ₂ PhotoreactivityWater Soluble, Iodideable Sulfo-NHS-LC-ASA (1)-NH ₂ PhotoreactivityWater Soluble, Iodideable PDPHcarbohydrate-SHDisulfide Linker PNP-DTP-NH ₂ PhotoreactivitySADP-NH ₂ PhotoreactivityDisulfide Linker Sulfo-SADP-NH ₂ PhotoreactivityWater Soluble Disulfide Linker SAED-NH ₂ PhotoreactivityDisulfide Linker SAND-NH ₂ PhotoreactivityWater Soluble Disulfide Linker SANPAH-NH ₂ PhotoreactivitySulfo-SANPAH-NH ₂ Photoreactivityreceptivity SASD (1)-NH ₂ PhotoreactivityWater Soluble Iodide Disulfide Linker SIAB-NH ₂ -SHSulfo-SIAB-NH ₂ -SHreceptivity SMCC-NH ₂ -SHSulfo-SMCC-NH ₂ -SHreceptivity SMPB-NH ₂ -SHSulfo-SMPB-NH ₂ -SHreceptivity SMPT-NH ₂ -SHSulfo-LC-SMPT-NH ₂ -SHreceptivity SPDP-NH ₂ -SHSulfo-SPDP-NH ₂ -SHreceptivity Sulfo-LC-SPDP-NH ₂ -SHreceptivity Sulfo-SAMCA (2)-NH ₂ PhotoreactivitySulfo-SAPB-NH ₂ Photoreactivityreceptivity (1) = iodizable; (2) = fluorescence
Homo-functional coupling agent
ConnectionResponsiveExplanation 0-NH ₂ BMH-SHBASED (1)PhotoreactivityIodide Disulfide Linker BSCOES-NH ₂ Sulfo-BSCOES-NH ₂ receptivity DFDNB-NH ₂ DMA-NH ₂ DMP-NH ₂ DMS-NH ₂ DPDPB-NH ₂ Disulfide linker DSG-NH ₂ DSP-NH ₂ Disulfide linker DSS-NH ₂ DST-NH ₂ Sulfo-DST-NH ₂ receptivity DTBP-NH ₂ Disulfide linker DTSSP-NH ₂ Disulfide linker EGS-NH ₂ Sulfo-EGS-NH ₂ receptivity SPBP-NH ₂
Biotin
Diagnosis / TreatmentResponsiveExplanation Biotin-BMCC-SHBiotin-DPPE * Preparation of Biotinylated Liposomes Biotin-LC-DPPE * Preparation of Biotinylated Liposomes Biotin-HPDP-SHDisulfide linker Biotin-hydrazidecarbohydrateBiotin-LC-HydrazidecarbohydrateIodoacetyl-LC-Biotin-NH ₂ NHS-Iminobiotin-NH ₂ Reduced affinity for avidin NHS-SS-Biotin-NH ₂ Disulfide linker Photoreactive BiotinNucleic acidSulfo-NHS-Biotin-NH ₂ receptivity Sulfo-NHS-LC-Biotin-NH ₂ DPPE = dipalmitoylphosphatidylethanolamine; LC = long chain
Protein Modification Diagnostics / Therapies
Diagnosis / TreatmentResponsivefunction Elman reagent-SHQuantification / Detection / Protection DTT-S.S-restoration 2-mercaptoethanol-S.S-restoration 2-metcaptylamine-S.S-restoration Traut reagent-NH ₂ -SH introduced SATA-NH ₂ Introduced protected -SH AMCA-NHS-NH ₂ Fluorescent marker AMCA-hydrazidecarbohydrateFluorescent marker AMCA-HPDP-S.S-Fluorescent marker SBF-chloride-S.S-Fluorescence Detection of -SH N-ethylmaleimide-S.S--SH block NHS-acetate-NH ₂ -NH ₂ block and acetylation Citraconic Anhydride-NH ₂ Reversibly block and introduce negative charge DTPA-NH ₂ Introducing the chelator BNPS-ScartolTryptophanTryptophan residue cleavage Bolton-Hunter-NH ₂ Introducing iodinable groups
The linking agent used in the present invention can typically be linked to the reporter (s) with some degree of specificity, and can also be used to attach one or more therapeutically active agents.
Ultrasound imaging modalities that can be used in accordance with the present invention are based on B-mode imaging (e.g., the sum or difference of the fundamental frequencies of the emitted ultrasonic pulses, their low or high harmonics or the pulses emitted and the frequencies derived from these harmonics). Using the time-varying magnitude of the generated signal envelope, two-dimensional and three-dimensional image techniques such as images from fundamental frequencies or second harmonics are preferred, color Doppler images and Doppler size images, and the last two and the foregoing Combinations of any of the forms. Surprisingly good second harmonic signal was obtained from the target monolayer-stabilized microspheres according to the present invention. Continuous images of tissue, such as the heart or kidney, can be collected with the aid of appropriate synchronization techniques (eg gating or respiratory movement of the subject) to reduce the effects of migration. Measurement of changes in resonant frequency or frequency absorption involving stationary or delayed microbubbles can also be usefully made to detect contrast agents.
The present invention provides a tool for therapeutic drug delivery in cooperation with the vector-mediated direction of the product at the desired site. "Treatment" or "drug" means a therapeutic agent that has an effective effect on a particular disease in a living person or a non-human animal. Combinations of drugs and ultrasound contrast agents are described, for example, in WO-A-9428873 and WO-A-9507072, but such products lack a vector having affinity for a particular site and thus are preferred sites before or during drug release. Shows a relatively poor specific delay.
The therapeutic compound used according to the invention may be encapsulated inside the microbubble or attached or mixed to the stabilizing membrane. Thus, the therapeutic compound of the present invention is linked to a part of the membrane or physically mixed into a stabilizing material, for example, via covalent or ionic bonds, and especially before the drug acts in the body when the drug has similar polarity or solubility to the membrane material. To prevent leakage from the product. Release of the drug may be initiated only by wet contact with the blood and administered or by a catalyzed dissolution process by other internal or external influences, such as the use of enzymes or ultrasound. The destruction of gas-containing microparticles using external ultrasound is known as an example in WO-A-9325241 as a phenomenon in ultrasound contrast agents; The rate of drug release can vary depending on the type of therapeutic use using a particular amount of ultrasound energy from the transducer.
The therapeutic agents of the present invention can be covalently bound to the encapsulated membrane surface using, for example, suitable linking agents as described above. Thus, for example, a phospholipid or lipopeptide derivative, to which a drug is bound via a biodegradable bond or linker, may be prepared first, and then the derivative may be mixed with this material to prepare a reporter as described above.
Representative therapeutic agents suitable for use in the drug delivery compositions of the present invention include any known therapeutic drug or active homologue thereof containing a thiol group capable of coupling with a thiol-containing microbubble under oxidative conditions producing a disulfide group. In combination with the vector (s) such drug / vector-modified microbubbles can accumulate in the target tissue; Administration of a reducing agent such as reduced glutathione can release the drug molecule from the target microbubbles in the vicinity of the target cell, thereby increasing the local concentration of the drug and increasing the therapeutic effect. In addition, the compositions of the present invention are first prepared without a therapeutic agent and then coupled or coated onto the microbubble immediately before use, such as by adding the therapeutic agent to a suspension of microbubbles in an aqueous medium to adhere or adhere the therapeutic agent to the microbubble. can do.
Other drug delivery systems include vector-modified phospholipid membranes doped with lipopeptide structures comprising poly-L-lysine or poly-D-lysine chains along with target vectors. For application to gene therapy / antisense techniques of particular importance on reporter-mediated drug delivery, microbubble carriers are concentrated with DNA or RNA through electrostatic interaction with cationic polylysine. This method has the advantage that the vector (s) used for targeted delivery are not directly attached to the polylysine carrier residue. This polylysine chain is also more tightly anchored to the microbubble membrane by the presence of lipid chains. The use of ultrasound to increase the delivery effect is also considered useful.
In addition, the free polylysine chain is first modified with drug or vector molecules and then concentrated on the opposite surface of the target microbubbles.
Representative and non-limiting examples useful in accordance with the present invention include vincristine, vinblastine, vindesine, busulfan, chlorambucil, spiroplatin, cisplatin, carboplatin, methotrezeate, adriamycin, mitomycin, Bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopurine, mitotan, procarbazine, darktinomycin (antimycin D), daunorubicin, doxorubicin, hydrochloride, taxol, plicamycin, aminogluglucin Tethymides, estrasmustin, flutamide, lutrolide, megestrol acetate, tamozifene, testosterone, trilostane, amsacrine (m-AMSA), asparaginase (L-asparaginase), Blood products such as etoposide, anti-neoplastic agents such as interferon a-2a and 2b, hematoporphyrin or derivatives of the foregoing; Biological response modifiers such as muram peptides; Antifungal agents such as ketoconazole, nystatin, griseofulvin, flucitocin, myconazole or amphotericin B; Growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, cortisone acetate, dexamethasone, flunisolidide, hydrocortisone, methylprednisolone, paramethaneson acetate, prednisolone, prednison, triamcinolone or pulluled Hormones such as locortisone acetate; Vitamins such as cyanocobalamine or retinoids; Enzymes such as alkaline phosphatase or manganese superoxide dismutase; Anti-allergic agents such as amelezanox; Inhibitors of tissue factors such as monoclonal antibodies and compounds that downregulate the expression of Fab fragments, synthetic peptides, non-peptides and tissue factors thereof; Platelet inhibitors such as GPIa, GPIb and GPIIb-IIIa, ADP reporter, thrombin reporter, Von Willebrand factor, prostaglandin, aspirin, ticlopidine, clopigogrel and leopro; Inhibitors of aggregate protein targets such as FIIa, FVa, FVIIa, FVIIIA, FIXa, FXa, tissue factor, heparin, hirudin, pyrullog, argatroban, DEGR-rFVIIa and Annexin V: inhibitors of fibrin formation and t- Fibrin degradation promoters such as PA, urokinase, plasmin, streptokinase, rt-plasminogen activator and r-stapillokinase; Anti-angiogenic factors such as methoxyprogesterone, pentosan polysulfate, suramin, taxol, thalidomide, angiostatin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondine; Circulating drugs such as propranolol; Metabolic enhancers such as glutathione; anti-nodal agents such as p-aminosalicylic acid, isoniazid, capreomycin, sulfate, cyclocezin, etabutol, ethionamide, pyrazineamide, rifampin or streptomycin sulfate; Antiviral agents such as acylclevir, amantadine, azidomidine, ribavirin or vidarabine; Vasodilators such as diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin or pentaerythritol tetranitrate; Daphsone, Chloramphenicol, Neomycin, Sephachlor, Sepaderoxyl, Sephalexin, Sepradin, Erythromycin, Clindamycin, Lincomycin, Amoxicillin, Ampicillin, Bacampicillin, Carbenicillin, Diclozacillin, Cyclacillin Antibiotics such as picclozacillin, hetacillin, methicillin, naphcillin, penicillin, polymizin or tetracycline; Anti-inflammatory agents such as diflunisal, ibuprofen, indomethacin, meclefenamate, mefenamic acid, naprogen, phenylbutazone, pyroxicam, tolmethine, aspirin or salicylate; Antiprotozoal agents such as chloroquine, metronidazole, quinine or meglumine antimonate; Antirheumatic agents such as penicylamine; Anesthetics such as paregory; Opiates such as codeine, morphine or opiates; Cardiac glycosides such as deslanside, digitoxin, digosine, digitalin or digitalis; Neuromuscular blockers such as atraccurium mesylate, galamine triethoxide, hexafluorenium bromide, metocurin iodide, pancuronium bromide, succinylcholine chloride, tubocurin chloride or becuronium bromide; Amobarbital, Amobarbital Sodium, Aprobarbital, Butabarbital Sodium, Chloral Hydrate, Etchlorbinol, Etynamate, Flulazepam Hydrochloride, Glutetimide, Metotrimethrazine Hydrochloride, Methi Sedatives such as plylon, midazolam hydrochloride, paraldehyde, pentobarbital, secobarbital sodium, debutal, temazepam or triazolam; Local anesthetics such as bupivacaine, chloroprocaine, ethidocaine, lidocaine, mepivacaine, procaine or tetracaine; Acid additions such as dropperidol, etomidate, fentanyl citrate and dropperidol, ketamine hydrochloride, methohexyl sodium or thiopental and pharmaceutically acceptable salts (eg hydrochloride or hydrobromide) Salts or basic salts such as sodium, calcium or magnesium salts) or derivatives thereof (for example acetate). Other examples of therapeutic agents include genetic material such as nucleic acids, RNA, and DNA of natural or synthetic origin, including recombinant RNA and DNA. DNA encoding specific proteins can be used to treat many different forms of disease. For example, tumor necrosis factor or interleukin-2 gene can be provided to treat advanced cancer; Thymidine kinase gene can be provided to treat ovarian cancer or brain tumor; The interleukin-2 gene can be provided to treat neuroblastoma, malignant melanoma or kidney cancer; Interleukin-4 gene may be provided to treat cancer.
The lipophilic derivative of the drug linked to the microbubble membrane via hydrophobic interaction may have a therapeutic effect as part of the microbubble or after release from the microbubble, for example by the use of ultrasound. If the drug does not have desirable physical properties, lipophilic groups can be introduced to fix the drug to the membrane. Preferably, the lipophilic group should be introduced in a manner that does not affect the efficacy of the molecule in vivo or that the lipophilic group is cleaved to release the active drug. The lipophilic group can be introduced by various chemical means depending on the functional group available to the drug molecule. Covalent coupling can be accomplished using functional groups in drug molecules that can react with suitably functionalized lipophilic compounds. Examples of lipophilic moieties include branched and unbranched alkyl chains, cyclic compounds, aromatic moieties and fused aromatic and nonaromatic cyclic systems. In some cases the lipophilic moiety consists of a suitably functionalized steroid such as cholesterol or related compounds. Examples of particularly suitable functional groups for derivatization include nucleophilic groups such as amino, hydroxy and sulfhydryl groups. Suitable methods for lipophilic derivatization of any drug containing sulfhydryl groups, such as captopril, include the formation of thiol esters by direct alkylation, for example by reaction with an alkyl halide under basic conditions and with an active carboxylic acid. It may include. Representative examples of derivatization of any drug having carboxyl functionality, such as athenol or chlorambucil, include amide and ester formation by coupling, respectively, with amines and alcohols containing suitable physical properties. Preferred embodiments include cholesterol attachment to the therapeutic compound by forming a degradable ester bond.
A preferred use of the present invention relates to angiogenesis in which new blood vessels are produced by branching from existing blood vessels. The primary stimulus for this method may be an inadequate supply of nutrients and oxygen to the cells of the tissue (hypoxia). These cells can respond by secreting many angiogenic factors; One example is vascular endothelial growth factor. These factors initiate the secretion of proteases that degrade proteins in the basement membrane as well as inhibitors that limit the action of these possible harmful enzymes. The combined effect of the signal from the reporter for loss of adhesion and angiogenic factors migrates, proliferates and rearranges endothelial cells and finally synthesizes a new perivascular basal membrane.
A tumor must initiate angiogenesis to maintain its growth rate when it reaches a millimeter in length. This method is a promising target for therapeutic adjustment because angiogenesis involves characteristic changes in endothelial cells and their environment. Transformations involving angiogenesis are also very promising for diagnosis and a preferred embodiment is a malignant disease, but this concept also shows great promise in inflammation and various inflammation-related diseases. This factor is also involved in revascularization of the infarcted part of the myocardial layer and occurs when stenosis occurs in a short time.
Many known reporters / targets involved in angiogenesis are shown in the table below. Using the targeting principles described herein, angiogenesis can be detected by most imaging modalities when used in medicaments. Contrast-enhanced ultrasound may have additional advantages, and the contrast agent is microspheres that are limited inside the blood vessels. Even if the target antigen is found in many cell types, these microspheres attach exclusively to endothelial cells.
So-called prodrugs can also be used as therapeutic agents according to the invention. Thus, drugs can be induced to alter their physicochemical properties and applied for inclusion into the reporter; The drug so induced may be considered a prodrug and is generally inactive until cleavage of the inducer regenerates the active form of the drug.
Targeting a gas-filled microbubble containing a prodrug-activated enzyme against a region of the pathology can be an image that targets the enzyme and visualized when the microbubble is properly targeted to the region of the pathology and at the same time disappears from the non-target region It is possible. In this way it is possible to determine a suitable time for infusion of the prodrug to an individual patient.
Another alternative is to mix prodrugs, prodrug-activating enzymes and vectors in the same microbubble in a system where the prodrug is activated only after some external stimulation. Such stimulation may be, for example, a rupture of the microbubble by external ultrasound after the tumor-specific protease as described above or the desired target has been achieved.
Therapeutic agents can be readily delivered according to the invention, for example, to diseases or necrotic areas in the heart, general blood vessels and other sites such as the liver, spleen, kidney and lymphatic system, body cavity or gastrointestinal system.
The product according to the invention can be used for targeted therapeutic delivery in vivo or in vitro. Later in the specification such products may be useful in in vitro systems such as diagnostic kits or the characterization of different components of different diseases in blood or tissue samples. A technique similar to attaching them to in vitro polymer particles (eg, monodisperse magnetic particles) to separate certain blood components or cells from a sample is described herein for separation of gas-containing materials by suspension and repeated washing. It can be used using a low density of reporter units in the treatment of.
Vectors that can be usefully used to generate multi-specific target traceable contrast agents according to the present invention include:
i) An antibody that can be used as a vector for a wide range of targets and has advantageous properties such as very high specificity, high affinity (preferably), and possibility of modification affinity as desired. Whether the antibody is bioactive or not depends on the specific vector / target combination. Both conventional and genetically treated antibodies can be used, and genetically treated antibodies allow for the accepted treatment of the antibody, for example, for specific needs regarding affinity and specificity. The use of human antibodies may be desirable to avoid possible immune responses against vector molecules. A further useful class of antibodies is the so-called bispecific antibodies that have specificity for two different target molecules in one antibody molecule. Such antibodies are useful for, for example, enhancing the formation of bubble clusters, and can also be used for various therapeutic purposes, for example, transport of toxic residues to a target. Various characteristics of bispecific antibodies are described in McGuinness, B.T. et al. in Nat. Biotechnol. (1996) 14, 1149-1154; by George, A.J. et al. in J. Immunol. (1994) 152, 1802-1811; by Bonardi et al. in Cancer Res. (1993) 53, 3015-3021; and by French, R. R. et al. in Cancer Res. (1991) 51, 2353-2361.
ii) cell adhesion molecules, their receptors, cytokines, growth factors, peptide hormones and fragments thereof. Such vectors rely on normal biological protein-protein interactions with target molecular receptors and in many cases generate biological responses upon binding to the target and are therefore bioactive; These may be of relatively insignificant relationship with the vector targeting the proteoglycan.
iii) non-peptide agonists / antagonists or inactive binders of receptors for cell adhesion molecules, cytokines, growth factors and peptide hormones. This category is not agonist or antagonist, but may include non-living vectors that may exhibit useful targets.
iv) Oligonucleotides and modified oligonucleotides that bind DNA or RNA through Watson-Crick or other forms of base-pairing. Such oligonucleotides, which are generally in vivo in the extracellular space as a result of cellular damage and are generally non-living, can be useful, for example, for targeting of necrotic regions associated with many different pathological conditions. Oligonucleotides can be designed to bind to specific DNA- or RNA-binding proteins such as transcription factors that are very often highly overexpressed or activated in tumor cells or activated immune or endothelial cells. Combination libraries can be used to select oligonucleotides that specifically bind to any possible target molecule and thus can be used as target vectors.
v) DNA-binding drugs may behave similarly to oligonucleotides but may exhibit biologically active and / or toxic effects when taken up by cells.
vi) various small molecules, including bioactive compounds known to bind to biological receptors. Such vectors or their vectors can be used to generate non-living compounds that bind to the same target.
vii) Vector molecules can be generated from combinatorial libraries without necessarily knowing the exact molecular targets by functionally selecting molecules (in vitro, ex vivo or in vivo) that bind to the region / structure to be imaged.
viii) A variety of small molecules, including known bioactive compounds for binding to various types of biological receptors. Such vectors or targets thereof can be used to generate non-living compound binding to the same target.
ix) a protein or peptide that binds to the glucosamioglycan side chain, eg, heparan sulphate comprising a larger molecule's glucosaminoglycan-binding moiety, has a biological response upon binding to the glucosaminoglycan side chain Does not cause Proteoglycans are not found in red blood cells and eliminate the undesirable uptake of these cells.
Other peptide vectors and lipopeptides thereof of particular interest for targeted ultrasound imaging are as follows: atherosclerotic plaque binding peptides such as YRALVDTLK, YAKFRETLEDTRDRMY and RALVDTEFKVKQEAGAK; Thrombus binding peptides such as NDGDFEEIPEEYLQ and GPRG, platelet binding peptides such as PLYKKIIKKLLES; And cholecystokinin, α-melanosite-stimulating hormone, heat stable enterotoxin 1, vasoactive enteric peptide, synthetic alpha-M2 peptide from the third heavy chain complementarity-determining region and homologs thereof for tumor application.
The following table shows the various vectors that can be targeted to specific types of targets and the indicated regions used for the target traceable diagnostic and / or therapeutic agents according to the invention comprising such vectors.
Protein and Peptide Vectors-Antibodies
Vector formTargetDescription / Use AreaRef Antibodies (Common)CD34Common vascular diseases, normal blood vessel walls (eg myocardial layer), activated endothelial cells, immune cells〃ICAM-1〃〃ICAM-2〃〃ICAM-3〃〃E-selectin〃〃P-selectin〃〃PECAM〃〃Integrins such as VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, β ₁ α ₇ , β ₁ α ₈ , β ₁ α _v , LFA-1, Mac -1, CD41a, etc.〃〃GlyCAMVascular wall in lymph nodes (highly specific for lymph nodes)〃MadCam 1〃〃FibrinTromby〃Tissue factorActivated endothelial, tumor〃MyosinNecrosis, myocardial infarction〃CEA (cancer antigen)tumor〃Mucintumor〃Multiple Drug Resistance Proteinstumor〃Prostate Specific AntibodyProstate cancer〃Cathepsin BTumors (various species of proteases often overexpress somewhat specific in various tumors-cathepsin B is such a protease)〃Transferrin receptorTumor, blood vessel wallMoAb 9.2.27 Antibodies Upregulated in Tumor Cell Growth〃VAP-1Adhesion molecule Band 3 proteinUpregulated during phagocytic activity CD44Tumor cells β2-micro-globulinNormally MHC species 1NormallyAntibody Antibody AntibodyIntegrinα vβ 3 CD44 β2-micro-globulin MHC species 1tumor; Angiogenic tumor cellscabb
a) Heider, KH, M. Sproll, S. Susani, E. Patzelt, P. Beaumier, E. Ostermann, H. Ahorn, and GR Adolf, 1996. "Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas ". Cancer Immunology Immunotheraphy 43: 245-253.
b) I. Roitt, J. Brostoff, and D. Male. 1985. Immunology, London: Gower Medical Publishing, p. 4.7
c) Stromblad, S., and D. A. Cheresh. 1996. "Integrins, angiogenesis and vascular cell survival". Chemistry & Biology 3: 881-885.
Protein and Peptide Vectors-Cell Adhesion Molecules, etc.
Vector formReceptorDescription / Use AreaRef L-selectinCD4MadCAM1GlyCam 1Common vascular diseases, normal vascular wall (eg myocardial layer), activated endothelial, lymph nodesOther SelectinCarbohydrate Ligands (Sialyl Lewis x) Heparan SulfateCommon vascular diseases, normal vascular wall (eg myocardial layer), activated endothelialRGD-peptideIntegreen〃PECAMPECAM, and othersEndothelial, cells in the immune systemIntegrins such as VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, β ₁ α ₇ , β ₁ α ₈ , β ₁ α _v , LFA-1, Mac -1, CD41a, etc.Laminin, collagen, fibronectin, VCAM-1, thrombospodine, vitronectin, etc.Endothelium, blood vessel wall, etc.Integrin receptors such as laminin, collagen, fibronectin, VCAM-1, thrombospodine, vitronectin and the likeIntegrins such as VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, β ₁ α ₇ , β ₁ α ₈ , β ₁ α _v , LFA-1, Mac -1, CD41a, etc.Cells, blood vessel walls, etc. in the immune systemNerve cell adhesion molecule (N-CAM) RGD-peptideProteoglycan N-CAM (homologous affinity) integrinAngiogenesisc
Vector containing cytokine / growth factor / peptide hormones and segments thereof
Vector formReceptorDescription / Use AreaRef Epidermal growth factorEGF-receptor or related receptortumorNerve growth factorNGF-receptortumorSomatostatinST-receptortumorEndothelinEndothelin-ReceptorVessel wallInterleukin-1IL-1-receptorInflammation, different types of activated cellsInterleukin-2IL-2-receptor〃Chemokines (receptors in which approximately 20 different cytokines are partially distributed)Chemokine receptors, proteoglycansInflammationTumor necrosis factorTNF-receptorInflammationParathyroid hormonePTH-receptorBone diseaseBone Morphogenic ProteinBMP-receptorBone diseaseCalcitoninCT-receptorBone diseaseColony Stimulating Factors (G-CSF, GM-CSF, M-CSF, IL-3)Corresponding specific receptor, proteoglycanEndothelialInsulin-like growth factor IIGF-I ReceptorTumors, other growth tissuesAtria sodium diuretic factorANF-receptorKidney, blood vessel wallVasopressinVasopressin receptorKidney, blood vessel wallVEGFVEGF-receptorsEndothelial area, angiogenesisFibroblast growth factorFGF-receptor, proteoglycanEndothelium, angiogenesisSchwann cell growth factorProteoglycan Specific Receptors
Various protein and peptide vectors
Vector formReceptorDescription / Use AreaRef StreptavidinkidneyKidney diseaseBacterial Fibronectin-Binding ProteinsFibronectinVessel wallFc-Parts of AntibodiesFc-receptorMonocyte-macrophageTransferrinTransferrin-receptorTumor vessel wallStreptokinase / Tissue Plasminogen ActivatorTrombyTrombyPlasminogen, plasminFibrinTromby, tumorMast cell proteinasesProteoglycan ElastaseProteoglycan Lipoprotein LipaseProteoglycan AgglutinaseProteoglycan Extracellular Superoxide DismutaseProteoglycan Heparin Joiner IIProteoglycan Retinal survival factorProteoglycan Specific Receptors Heparin-bound brain mitosisProteoglycan Specific Receptors Apolipoproteins such as apolypoprotein BProteoglycan specific receptors (eg, LDL receptors) Apolipoprotein ELDL Receptor Proteoglycans Adhesion-promoting proteins such as furfurin ProteoglycanViral coat proteins such as HIV, herpes ProteoglycanMicrobial AdhesinComplex of "antigen 85" mycobacteriaFibronectin, collagen, fibrinogen, vitronectin, heparan sulfateβ-amyloid precursorProteoglycanΒ-amyloid Accumulation in Alzheimer's DiseaseTenasin, for example tenasin CHeparan Sulfate, Integrin
Vector containing non-peptide agonist / cytokinin antagonist / growth factor / peptide hormone / cell adhesion molecule
Vector formReceptorDescription / Use AreaRef Endothelial antagonistsEndothelial receptorsVessel wallDesmopressin (vasopressin homologue)Vasopressin receptorRenal vessel wallDemoxitoxin (oxytocin homologue)Oxytocin receptorReproductive system, mammary gland, brainAngiotensin II Receptor Antagonists C-11974, TCV-116Angiotensin II ReceptorVascular wallNonpeptide RGD-HomologsIntegreenCell walls in the immune system
Vectors containing antiangiogenic factors
Vector formTargetDescription / Use AreaRef AngiostatinEC of tumorPlasminogen SegmentK Cartilage-induced inhibitorsEC of tumor J β-cyclodextrin tetradecasulfateTumor, inflammation C Pumagiline and HomologsTumor, inflammation E Interferon-αEC of tumor K Interferon-γEC of tumor E Interleukin-12EC of tumor E LinomideTumor, inflammation A MedroxyprogesteroneEC of tumor K Metalloproteinase inhibitorsEC of tumor K Pentosan polysulfateEC of tumor K Platelet factor 4EC of tumor M SomatostatinEC of tumor K SuraminEC of tumor K TexolEC of tumor K ThalidomideEC of tumor K ThrombospondinEC of tumor K
Vector containing angiogenic factors
Vector formTargetDescription / Use AreaRef Acid Fibroblast Growth FactorEC of tumor K AdenosineEC of tumor K AngiogeninEC of tumor K Angiotensin IIEC of tumor K Basement membrane componentstumorFor example, tenasin, collagen IVM Basic fibroblast growth factorEC of tumor K BradykininEC of tumor K Calcitonin gene-related peptideEC of tumor K Epidermal growth factorEC of tumor K Fibrintumor K Fibrinogentumor K HeparinEC of tumor K HistamineEC of tumor K Hyaluronic acid or its segmentEC of tumor K Interleukin-1αEC of tumor K Laminin, laminin segmentEC of tumor K NicotinamideEC of tumor K Platelet activatorEC of tumor K Platelet-induced endothelial growth factorEC of tumor K Prostaglandins E1, E2EC of tumor K SpermineEC of tumor K SpermineEC of tumor K Substance PEC of tumor K Transformation Growth Factor-αEC of tumor K Transformation Growth Factor-βEC of tumor K Tumor Necrosis Factor-αEC of tumor K Vascular Endothelial Growth Factor / Vascular Permeability FactorEC of tumor K Vitronectin A
Vector molecules other than angiogenic factors recognized to have a known affinity for receptors involved in angiogenesis
Vector formTargetDescription / Use AreaRef AngiopoietinTumor, inflammation B α ₂ -antiplasminTumor, inflammation Combinatorial libraries, compoundsTumor, inflammationFor example: compounds that bind to the basement membrane after degradationEndoglinTumor, inflammation D EndocrineTumor, inflammation D Endostatin [collagen segments]Tumor, inflammation M Factor VII-associated antigenTumor, inflammation D FibrinopeptidesTumor, inflammation ZC Fibroblast Growth Factor, BasicTumor, inflammation E Hepatosite growth factorTumor, inflammation I Insulin-like growth factorTumor, inflammation R InterleukinTumor, inflammationFor example: IL-8I Leukemia inhibitory factorTumor, inflammation A Metalloproteinase inhibitorsTumor, inflammationFor example, BatimastadE Monoclonal antibodiesTumor, inflammationFor example: for angiogenic factors or their receptors or components of a fibrinolytic systemPeptides such as cyclic RGD _D FVTumor, inflammation B, Q Placental growth factorTumor, inflammation J Placenta Proliperin-Related ProteinsTumor, inflammation E PlasminogenTumor, inflammation M Plasminogen activatorTumor, inflammation D Plasminogen Activator InhibitorTumor, inflammation U, V Platelet activator antagonistTumor, inflammationInhibitors of angiogenesisA Platelet-induced growth factorTumor, inflammation E PlayotropinTumor, inflammation ZA ProliperinTumor, inflammation E Proliperin Related ProteinsTumor, inflammation E SelectinTumor, inflammationFor example, E-selectinD SPARCTumor, inflammation M Snake venom (RGD-containing)Tumor, inflammation Q Tissue Inhibitors of MetalloproteinasesTumor, inflammationFor example, TIMP-2U ThrombinTumor, inflammation H Thrombin-receptor-active tetradecapeptideTumor, inflammation H Thymidine phosphorylaseTumor, inflammation D Tumor growth factorTumor, inflammation ZA
Receptors / targets associated with angiogenesis
Vector formTargetDescription / Use AreaRef BigglycanTumor, inflammationDermatan Sulfate ProteoglycanX CD34Tumor, inflammation L CD44Tumor, inflammation F Collagen Types I, IV, VI, VIIITumor, inflammation A DecorinTumor, inflammationDermatan Sulfate ProteoglycanY Dermatan Sulfate ProteoglycanTumor, inflammation X EndothelinTumor, inflammation G Endothelin receptorTumor, inflammation G Fibronectintumor P Flk-1 / KDR, Flt-4Tumor, inflammationVEGF receptorD FLT-1 (fms sheep tyrosine kinase)Tumor, inflammationVEGF-A receptorO Heparan SulfateTumor, inflammation P Hepatosite growth factor receptor (c-met)Tumor, inflammation I Insulin-like growth factor / mannose-6-phosphate receptorTumor, inflammation R Integrin: β ₃ and β ₅ , integrin α _V β ₃ , integrin α ₆ β ₁ , integrin α ₆ , integrin β ₁ , integrin α ₂ β ₁ , integrin α _V β ₃ , integrin α ₅ integrin α _V β ₅ , fibrin ReceptorTumor, inflammationSubunit of Laminin Receptor Fibronectin ReceptorD, P Intracellular adhesion molecules-1 and -2Tumor, inflammation P
Serrated gene productTumor, inflammation T Ly-6Tumor, inflammationLymphocyte active proteinN Matrix metalloproteinaseTumor, inflammation D MHC Species IITumor, inflammation V-shaped gene productTumor, inflammation T Osteopontintumor Z PECAMTumor, inflammationAlias CD31P Plasminogen activator receptorTumor, inflammation ZC Platelet-induced growth factor receptorTumor, inflammation E Selectin: E-, P-Tumor, inflammation D Sialyl Lewis-XTumor, inflammationBlood group antibodiesM Stress Proteins: Glucose Regulated, Heat Shock Family and OthersTumor, inflammationMolecular ChaperoneCindecanTumor, inflammation T ThrombospondinTumor, inflammation M TIE receptorTumor, inflammationTyrosine kinases with Ig- and EGF positive domainsE Tissue factorTumor, inflammation Z Tissue Inhibitors of MetalloproteinasesTumor, inflammationFor example, TIMP-2U Transformed growth factor receptorTumor, inflammation E Urokinase type plasminogen activator receptorTumor, inflammation D Vascular Cellular Adhesion Molecule (VCAM)Tumor, inflammation D Vascular Endothelial Growth Factor-related ProteinTumor, inflammation Vascular Endothelial Growth Factor-A ReceptorTumor, inflammation K Von willebrand factor-associated antibodiesTumor, inflammation L
Oligonucleotide vector
Vector formReceptorDescription / Use AreaRef Oligonucleotides complementary to genes for repeat sequences such as ribosomal RNA, Alu-sequences,DNA that can be produced by necrosisAll other diseases, including tumor myocardial infarction necrosisOligonucleotides complementary to disease-specific mutations (eg, mutated oncogenes)DNA preparable by necrosis in areas of related diseasetumorOligonucleotides Complementary to Infectious DNAInfectious DNAViral or bacterial infectionTriple or quadruple-helix forming oligonucleotidesSame as above exampleSame as above exampleOligonucleotides with recognition sequences for DNA- or RNA-binding proteinsDNA-binding proteins such as transcription factors (often overexpressed / activated in tumor or activated endothelial / immune cells)Tumor activated endothelial activated immune cells
Modified nucleotide vector
Vector formReceptorDescription / Use AreaRef Phosphorothioate oligosOn unmodified oligosOn unmodified oligos2'-O-methyl substituted oligos〃〃Illusion oligos〃〃Oligos with hairpin structure to increase degradation〃〃Oligos with terminal phosphorothioate〃〃2'-fluoro oligos〃〃2'-amino oligos〃〃DNA-binding drugs bound to oligos (see, eg, below)〃Increased binding affinity compared to pure oligosPeptide nucleic acids (PNAs, oligonucleotides having a peptide backbone)〃Increased binding affinity and stability compared to standard oligos
Nucleoside and Nucleotide Vectors
Vector formReceptorDescription / Use AreaRef Adenosine or homologueAdenosine receptorsVascular wall heartADP, UDP, UTP and moreVarious nucleotide receptorsMany tissues, such as the brain, spinal cord, kidneys, spleen
Receptors Including DNA-binding Drugs
Vector formReceptorDescription / Use AreaRef Acridine derivatives distamycin netroplopsin actinomycin D echinomycin bleomycinDNA prepared by necrosisAll other diseases related to tumors, myocardial infarction and necrosis or other methods of releasing DNA from cells
Receptor containing protease substrate
Vector formTargetDescription / Use AreaRef Peptide or Nonpeptidic SubstrateCathepsin BTumors, various types, for example, various variants capable of somewhat overexpressing the protease of cathepsin B
Receptors Including Protease Inhibitors
Vector formTargetDescription / Use AreaRef Peptide or non-peptidic inhibitors such as N-acetyl-Leu-Leu-norrousinCathepsin BTumors, various types, for example, various variants capable of somewhat overexpressing the protease of cathepsin BBestin ([(2S, 3R) -3-amino-2-hydroxy-4-phenyl-butanoyl] -L-leucine hydrochloride)AminopeptidaseTumors, for example on the cell surfacePepablock (4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride)Serine ProteaseTumor, vascular wall, etc.Commercially available inhibitors, such as captopril enalapril ricinooprilAngiotensin converting enzymeEndothelial cellsLow Specific Nonpeptidic CompoundsCoagulation factorVascular wall injury, tumor, etc.Protease Nexin (Extracellular Protease Inhibitor)Proteoglycan AntithrombinProteoglycan, aggregation factor
Vector from Combination Library
Vector formReceptorDescription / Use AreaRef Antibodies with Structures Determined During DevelopmentMay be unaware in preparing the functional selection of any of the aforementioned targets-or vectors that bind to selected diseased structuresAny diseased or normal structure of interest, for example, the walls of tromby, tumor or myocardial vesselsPeptides with Sequences Determined During Development〃〃Oligonucleotides with Sequences Determined During Development〃〃Variations of the Oligos Above〃〃Other compounds with structures determined during development〃〃
Carbohydrate vector
Vector formReceptorDescription / Use AreaRef Neo-GlycoproteinMacrophageGeneral Activation / InflammationOligosaccharides With Terminal GalactoseAsialo-glycoprotein receptorliverHyaluronanAggrecan (proteoglycan) "link protein" cell-surface receptor: CD44 Mannose Blood brain barrier, brain tumors and other diseases causing changes in BBBBacterial glycopeptides 〃
Geological vector
Vector formReceptorDescription / Use AreaRef LDL-like lipidsLDL-receptorArteriosclerosisPlatelet activator (PAF) antagonistPAF receptorDiagnosis of inflammationProstaglandin antagonists of inflammationProstograndine receptorDiagnosis of inflammation
Small molecule vector
Vector formTargetDescription / Use AreaRef AdrenalineCorresponding receptor Beta blockerAdrenergic beta-receptorsMyocardial Layer for Beta-1 BlockersAlpha-BlockerAdrenergic alpha-receptorsVessel wallBenzodiazepines Serotonin-HomologSerotonin-receptor Anti-histamineHistamine-receptorsVessel wallAcetyl-choline Receptor AntagonistACh-receptor VerapamilCa ²⁺ -Channel BlockerHeart muscleNifedipineCa ²⁺ -Channel BlockerHeart muscleAmylorideNa ⁺ / H ⁺ -exchangerIt blocks this exchange in the kidney and is generally upregulated in cells stimulated by growth factors.Digitalis GlycosideNa ⁺ / K ⁺ -ATP-asesMyocardial terminal vascular system, central nervous systemThromboses / prostaglandin receptor antagonists or agonistsThromboxane / prostaglandin receptorVascular wall, endothelialGlutathioneGlutathione-receptorrucotriene-receptorLungs, brainBiotinBiotin Transport Protein on Cell Surface FolateFolate Transport Protein on Cell SurfacetumorRiboflavinRiboflavin Transport Protein on Cell Surface MethotrexateFolate Transport Protein on Cell Surface ChloramCommon Delivery Mechanisms
<References to the previous table>

The following non-limiting examples illustrate the concept of multiple receptor specificities. Other combinations of vectors, spacers and reporters and binding techniques that lead to multiple vector introductions are also considered important in the present invention. As described in WO-A-9607434, confirmation of microparticle properties of the product was carried out using a microscopy microscope. Ultrasonic delivery measurements were performed using a wide band transducer on a suspension of the product showing increased sound beam attenuation compared to the standard. Specifically binds to expressing cells using microscopy and / or flow chambers containing immobilized cells, for example, by using cell colonies expressing target structures and additional cell colonies not expressing targets. The activity of the targeted agent can be studied. Radioactive, fluorescent or enzyme-labeled streptoavidine / avidin can be used to analyze biotin binding.
<Example 1>
Preparation and Biological Evaluation of Multi-Specific Gas-Containing Microbubbles of DSPS 'Doped' with Lipopeptides Containing Heparin Sulfate Binding Peptides (KRKR) and Bibronectin Peptides (WOPPRARI).
This example relates to the preparation of a target microbubble comprising multiple peptide vectors arranged in a straight sequence.
a) Synthesis of lipopeptides consisting of heparin sulphate binding peptide (KRKR) and bibronectin peptide (WOPPRARI)
Lipopeptides were synthesized on an ABI 433A automated peptide synthesizer starting with Fmoc-Ile-Wang resin (Novabiochem) on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids and palmitic acid prior to coupling. Simultaneous removal of peptides and side chain protecting groups from the resin was carried out in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O for 2 hours to give the crude product in 150 mg yield. . A 40 mg aliquot of the crude product was purified over 40 minutes by purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 70-100% (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate. . 16 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, where B = MeOH, A = 0.1% TFA / water, column Vydac 218TP1022: detection UV 260 and fluorescence, Ex ₂₈₀ , Em ₃₅₀ -product retention time = 19.44 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2198, found 2199
b) Preparation of gas-containing microbubbles of DSPS 'doped' with multi-specific lipopeptides consisting of heparin sulphate binding peptide (KRKR) and bibronectin peptide (WOPPRARI)
Lipopeptides of DSPS (Avanti, 4.5 mg) and a) were weighed into two vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming). The sample was cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were rolled overnight. The bubbles were washed several times with deionized water and analyzed with Coulter counters (size: 1-3 microns (87%), 3-5 microns (11.5%)) and wave attenuation (maximum attenuation: 3.5 MHz). Microbubbles were stable at 120 mmHg.
MALDI mass spectrometry was used to confirm the introduction into the DSPS microbubble as follows. About 0.05-0.1 ml of the microbubble suspension was transferred into a clean vial and 0.05-0.1 ml of methanol was added. The suspension was sonicated for 30 seconds and the solution analyzed by MALDI MS. The positive mode for lipopeptides showed M + H at 2200, with a predicted 2198.
c) In vitro studies of gas-containing microbubbles of DSPS 'doped' with multi-specific lipopeptides consisting of heparin sulphate binding peptide (KRKR) and bibronectin peptide (WOPPRARI):
Human endothelial cell line ECV 304 derived from normal umbilical cord (ATCC CRL-1998) on endothelial cells under flow conditions was treated with L-glutamine 200 mM, penicillin / stereptomycin (10.000 U / ml and 10.000 mcg / ml) and The cells were cultured in 260 ml Nunc culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whittaker) to which fetal serum (Hyclone Lot no.AFE 5183) was added. Upon reaching confluence, cells were secondary cultured at a split ratio of 1: 5 to 1: 7. 22 mm diameter cover glass (BDH, Cat no. 406/0189/40) was sterilized and placed on the bottom of a 12 well culture plate (Costar) before adding the cells in 0.5 ml complete medium with serum to the top. When the cells reached confluence, the coverslips were placed in the flow chamber normally prepared. The chamber consists of grooves engraved in a glass plate on which coverslips are placed with the cells, and the cells are in contact with the grooves forming the flow channels. The ultrasonic microbubbles of section b) were passed through a flow chamber from a reservoir maintained at 37 ° C. and sent back to the reservoir using a peristaltic pump. Flow rates were adjusted to promote physiologically appropriate shear rates. The flow chamber was placed under a microscope and the interaction between microspheres and cells was directly seen. The camera placed on the microscope was connected to a color video printer and a monitor. Gradual accumulation of microbubbles on the cells occurred, which was dependent on flow rate. As the flow rate increased, the cells began to detach from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
d) biological experiments
Case 1)
22 kg hybrid dogs were anesthetized with pentobarbital and mechanically ventilated. The chest was opened with a midline sternal incision, the anterior pericardium was removed, and a 30 mm gelled silicone rubber spacer was inserted between the heart and the P-5 transducer of the ATL HDI-3000 ultrasound scanner. The scanner was fixed to hepatic shortening once imaging in each end-cistol by delayed EGC response. The total volume of 2 ml microbubbles from b) was injected rapidly by intravenous injection. After 3 seconds, the imaged right ventricle was seen to contain contrast material, and after 3 seconds, the left ventricle was also filled, and a temporary attenuation shadow was observed with a faint part of the left ventricle. In addition, when the attenuation shading subsided, it was seen that the brightness of the myocardial layer increased substantially in the heart region away from the left ventricle. After the initial injection, the ultrasound scanner was fixed at continuous, high frame rate and high output power images using a known method of breaking ultrasound contrast bubbles at the imaged tissue site. After a few seconds, the scanner was adjusted back to its initial value. After that, the myocardial layer became darker and closer to the default. By moving the sliced image to a new position, the contrast effect appeared again, and by moving the slice back to its initial position, the tissue brightness near the default value appeared.
Case 2)
A total volume of 2 ml of microbubbles prepared in the same manner as b) above was injected using the same imaging method as above, except that lipopeptide was included in the preparation. Myocardial echo gain was much less severe and shorter duration than that observed in Case 1. At the end of the left ventricular attenuation phase, also myocardial contrast effect was almost lost, and myocardial echo increased in the anterior portion of the left ventricle, as was not observed in Case 1.
<Example 2>
Multi-Specific Gas-Containing Microbubbles of Lipopeptides Containing Heparin Sulfate Binding Peptides (KRKR) and Bibronectin Peptides (WOPPRARI) and DSPS 'Doped' with RGDC-Mal-PEG _2000- DSPE
This example relates to the preparation of a target microbubble comprising multiple peptide vectors.
Synthesis of one phosphatidyl ethanolamine (PE-PEG ₂₀₀₀ -Mal) acyl distearate - a) 3- maleimido propionyl amido -PEG ₂₀₀₀
Distearoyl phosphatidyl ethanolamine (DSPE) (37.40 mg, 0.005 mmol) in a solution of chloroform / methanol (3: 1), N-hydroxysuccinimido-PEG ₂₀₀₀ -maleimide, NHS-PEG-MAL (100 mg, 0.25 mmol) and triethylamine (35 μL, 0.25 mmol) were stirred at room temperature for 24 hours. After evaporation of the solvent under reduced pressure, the residue was purified by flash chromatography (chloroform / methanol, 8: 2). The product was obtained as 92 mg (66%) of white wax and demonstrated structure by NMR and maldi-MS.
b) synthesis of RGDC
RGDC peptides were synthesized on an ABI 433A automated peptide synthesizer (0.25 mmol scale, Fmol-Cyc (Trt) -Wang resin). HBTU was used to activate all amino acids. The crude peptide was removed from the resin and simultaneously deprotected in THF containing 5% EDT, 5% phenol and 5% water. After evaporation of excess degradation solution, the peptide was precipitated and polished several times with diethyl ether before air drying. The crude peptide was purified by purified Hplc and the fractions containing pure product were combined and lyophilized. Analytical hplc and MALDI-MS final characterizations were performed.
c) multi-specific gas-filled 'doped', phosphatidylserine-encapsulated with lipopeptide and RGDC-Mal-PEG _3400- DSPE containing heparin sulfate binding peptide (KRKR) and bibronectin peptide (WOPPRARI) The prepared microbubbles
DSPS (Avanti, 4.5 mg), lipopeptides from Example 1 a) (0.5 mg) and PE-PEG-MAL (0.5 mg) from section a) were weighed and placed in clean vials, 1.4% propylene glycol / 2.4 1.0 ml of% glycerol solution was added. The mixture was sonicated for 3 to 5 minutes, warmed to 80 ° C. for 5 minutes and then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were centrifuged at 1000 rpm for 3 minutes. The fluid was exchanged with 1 ml of PBS containing 1 mg of peptide RGDC and the pH was adjusted to 8. The binding reaction proceeded for 2 hours. Bubbles were washed in PBS until all unreacted RGDC was removed from the fluid as observed in MALDI-MS and then with water.
d) in vitro binding assays
Using the in vitro test described in Example 2 c), binding of microbubbles to endothelial cells was performed under flow conditions. Gradual accumulation of microbubbles on the cells occurred, which was dependent on flow rate. Control bubbles without vector did not adhere to endothelial cells detaching from cells under minimal flow conditions.
<Example 3>
Multi-specific gas-containing microbubbles encapsulated with thiolated anti-CD62-Mal-PEG ₂₀₀₀ -PE and thiolated anti-ICAM-1-Mal-PEG ₂₀₀₀ -PE and DSPS
This example relates to the preparation of a target microbubble comprising multiple antibody vectors for targeted ultrasound.
a) Preparation of gas-containing microbubbles encapsulated with PE-PEG ₂₀₀₀ -Mal and DSPS
PE-PEG _2000- MAL (0.5 mg) from DSPS (Avanti, 4.5 mg) and Example 2 a) were weighed into clean vials and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added. The mixture was warmed to 80 ° C. for 5 minutes and then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer, microclimate and washed with water.
b) Thiolation of anti-CD62 and anti-ICAM-1 antibodies
To each 0.3 mg of anti-CD62 and anti-ICAM-1 antibody dissolved in PBS buffer (pH 7, 0.5 ml), Traut reagent was added and the solution was stirred at room temperature for 1 hour. Excess reagent was separated from the modified protein on a NAP-5 column (Pharmacia).
b) Binding of thiolated anti-CD62 and anti-ICAM-1 antibodies to multi-specific gas-containing microbubbles encapsulated with DSPE-PEG _2000- MAL and DSPS
0.5 mg preparation of mixed thiolated antibody from b) was added to the microbubble aliquot from a) and the binding reaction was performed for 30 minutes on a roller table. After centrifugation at 2000 rpm for 5 minutes, the fluid was removed. The microbubbles were washed three times with water.
In addition, PEG spacer lengths may vary to include, for example, longer PEG ₃₄₀₀ and PEG ₅₀₀₀ , or shorter PEG ₆₀₀ and PEG ₈₀₀ . Also included is the addition of a third antibody, such as thiolated anti-CD34.
<Example 4>
Multi-Specific Gas-Containing Microbubbles of DSPS Covalently Coated with Fusion Peptides and Polylysine Containing Fibronectin Peptide Sequence NH ₂ FNFRLKAGOKIRFGGGGWOPP.RAIOH and PS Binding Components
a) Synthesis of PS binding component / fibronectin peptide sequence NH ₂ FNFRLKAGOKIRFGGGGWOPP.RAIOH
Peptides were synthesized on an ABI 433A automated peptide synthesizer starting with Fmoc-Ile-Wang resin on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids prior to coupling. Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% phenol, 5% EDT and 5% H ₂ O to afford the crude product in 302 mg yield. Crude product 25 over 40 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 20-40% (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at 9 mL / min flow rate. The mg aliquot was purified. 10 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 20-50% B, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water, column Vydac 218TP1022: detection UV 214 and 214 nm-product retention time = 12.4 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2856, found 2866
b) Preparation of microbubbles of DSPS noncovalently coated with PS binding component / fibronectin fragment fusion peptide NH ₂ FNFRLKAGOKIRFGGGGWOPP.RAIOH and polylysine
DSPS (Avanti, 5 mg) was weighed and placed in clean vials with poly-L-lysine (Sigma, 0.2 mg) and peptides from a) (0.2 mg). To this vial was added 1.0 ml of a 1.4% propylene glycol / 2.4% glycerol solution. This mixture was warmed to 80 ° C. for 5 minutes. This sample was cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were centrifuged at 1000 rpm for 3 minutes. After washing well with water, PBS and water, the final solution was tested for polylysine and peptide using MALDI-MS. No polypeptide material was observed in the final wash solution. Acetonitrile (0.5 ml) was then added and the microbubbles were broken by sonication. The resulting solution analysis was then performed on polylysine and PS-binding / fibronectin fusion peptides using MALDI-MS. The results are shown below.
MALDI EstimatesMALDI found Poly-L-lysine786, 914, 1042, 1170790, 919 1048, 1177 DSPS-binding protein28562866
Spacer elements included within the PS binding / fibronectin fusion peptide (-GGG-) range can also be replaced with other spacers such as PEG ₃₄₀₀ or polyalanine (-AAA-). It is also possible to use a preliminary target form to allow DSPS binding / fibronectin fragment fusion peptides to be associated with the cells first through fibronectin peptide binding. This is done by binding to the PS binding peptide after administration of the PS microbubbles.
Example 5
Encapsulated with phosphatidylserine and biotin-PEG ₃₄₀₀ -alanyl-cholesterol, streptavidin / biotinyl-endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and biotinyl Multi-specific gas-containing microbubbles functionalized with fibrin-antipolymerant peptide (Biotin-GPRPPERHQS.NH ₂ )
This example relates to the preparation of targeted ultrasonic microbubbles by using streptavidin as a linker between a biotidinylated receptor and a vector.
a) Synthesis of Biotin-PEG _3400- β-Alanine Cholesterol
To a solution of cholesterol-β-alanine hydrogen chloride (15 mg, 0.03 mmol) in 3 ml of chloroform / methanol (2.6: 1) was added triethylamine (42 mL, 0.30 mmol). The mixture was stirred at rt for 10 min and biotin-PEG _3400- NHS (100 mg, 0.03 mmol) in 1.4-dioxane (1 ml) was added dropwise. After stirring at room temperature for 3 hours, the mixture was evaporated to dryness and the residue was purified by flash chromatography to give 102 mg (89% yield) of white crystals. The structure was verified by NMR and MALDI-MS.
b) Synthesis of Biotinylated Endothelin-1 Peptide (Biotin-D-Trp-Leu-Asp-Ile-Trp.OH)
Peptides were synthesized in an ABI 433A automated peptide synthesizer starting with Fmoc-Trp (Boc) -Wang resin on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids prior to coupling. Removal of the peptide and side chain protecting groups from the resin in TFA containing 5% anisole and 5% H ₂ O simultaneously was carried out to give the crude product in 75 mg yield. Crude product 20 over 40 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 30-80% (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at 9 mL / min flow rate. The mg aliquot was purified. 2 mg of pure material was obtained after dry freezing of the pure fractions (analytical HPLC; gradient, 30-80% B, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water, column Vydac 218TP1022: detection- UV 214 nm-product retention time = 12.6 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1077, found 1077
c) Synthesis of Biotinyl-Fibrin-Anti-Polymer Peptide (Biotin-GPRPPERHQS.NH ₂ )
This peptide was synthesized and purified using the same protocol as described in section b). Pure product was characterized using hplc and MALDI mass spectroscopy.
d) multi-specific gas-containing microbubbles encapsulated with phosphatidylserine and biotin-PEG _3400- β-alanyl-cholesterol
Biotin-PEG _3400- β-alanine cholesterol (0.5 mg) of DSPS (Avanti, 4.5 mg) and section a) were weighed into vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming). The sample was cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were rolled overnight. The microbubble suspension was washed several times with deionized water and analyzed with a Coulter counter and wave attenuation (maximum attenuation: 3.5 MHz).
e) binding of fluorescein labeled streptavidin and biotinylated peptides from sections b) and c)
To the microbubble preparation from d) was added fluorescein bound streptavidin (0.2 mg) dissolved in PBS (1 ml). The bubble was placed on a roller table for 3 hours at room temperature. After washing well with water and analyzing by fluorescence microscopy, the microbubble was 1 ml of PBS containing biotinyl-endothelin-1 peptide (0.5 mg) and biotinyl-fibrin-anti-polymeric peptide (0.5 mg). Incubated in the middle. Microbubbles were washed well to remove unbound peptides.
<Example 6>
Phosphatidylserine, and streptavidin / biotinyl-endothelin-1 peptide (Biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and Biotinyl-fibrin-anti polymerrant peptide (Biotin-GPRPPERHQS. Multi-specific gas-containing microbubbles encapsulated with biotinylated lipopeptides used to prepare streptavidin 'sandwiches' with a mixture of NH ₂ )
a) Synthesis of lipopeptide dipalmitoyl-lysinyl-tryptophanyl-lysinyl-lysinyl-lysinyl (biotinyl) -glycine
Lipopeptides were synthesized on an ABI 433A automated peptide synthesizer starting with Fmoc-Gly-Wang resin (Novabiochem) on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids and palmitic acid prior to coupling. Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O to afford the crude product in 150 mg yield. . A 40 mg aliquot of the crude product was purified over 40 minutes by purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 70-100% (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate. . 14 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, where B = MeOH, A = 0.1% TFA / water, column Vydac 218TP1022: detection UV 260 and fluorescence, Ex280, Em350 Product retention time = 22 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1478, found 1471
b) Preparation of gas-containing microbubbles of DSDP 'doped' with the biotinylated lipopeptide sequence from section a)
Lipopeptides (0.5 mg) of DSPS (Avanti, 4.5 mg) and a) were weighed into two vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming). The sample was cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were rolled overnight. The microbubble suspension was washed several times with deionized water and analyzed by Coulter counter and wave attenuation. The introduction into DSPS microbubbles was confirmed using MALDI mass spectrometry as described in Example 1 b).
c) Encapsulated with phosphatidylserine and biotinylated lipopeptides, streptavidin / biotinyl-endothelin-1 peptide (biotin-D-Trp-Leu-Asp-Ile-Trp.OH) / biotinyl- Preparation of Multi-Specific Gas-Containing Microbubbles Functionalized with Fibrin-Anti Polymeric Peptides (Biotin-GPRPPERHQS.NH ₂ )
The microbubble preparation from b) was processed in a similar manner as described in Example 5 section e).
<Example 7>
Phosphatidylserine, and streptavidin / biotinyl-endothelin-1 peptide (Biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and Biotinyl-fibrin-anti polymerrant peptide (Biotin-GPRPPERHQS. Multi-specific gas-filled microbubbles encapsulated with biotin-DPPE used to make streptavidin 'sandwich' with a mixture of NH ₂ )
a) Preparation of Biotin-Containing Microbubbles
To a mixture of phosphatidylserine (Avanti, 5 mg) and biotin-DPPE (0.6 mg, Pierce) in a clean vial was added 5% propyleneglycol-glycerol in water (1 ml). This dispersion was warmed to 80 ° C. for 5 minutes and then cooled to room temperature. The headspace was flushed with perfluorobutane gas and the vial was shaken for 45 seconds in a cap mixer. After centrifugation, the fluid was removed and the resulting microbubbles were washed well with water.
b) phosphatidylserine, and streptavidin and biotinyl-endothelin-1 peptide (Biotin-D-Trp-Leu-Asp-Ile-Trp.OH) and Biotinyl-fibrin-anti polymerant peptide (Biotin- Binding of gas-filled microbubbles encapsulated with biotin-DPPE with a mixture of GPRPPERHQS.NH ₂ )
<Example 8>
Multi-specific gas-filled microbubbles encapsulated with a mixture of phosphatidylserine, streptavidin-Succ-PEG-DSPE and biotinylated human endothelial IgG antibodies and biotinylated transferrin
a) Synthesis of Succ-PEG ₃₄₀₀ -DSPE
For example, Nayal, Al. And carboxyl NH ₂ -PEG ₃₄₀₀ -DSPE using succinic anhydride by a method analogous to that described in Nayar, R. and Schroit, Biochemistry (1985) 24.5967-71. It was a true story.
b) Preparation of gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG _3400- DSPE
To a mixture (5 mg) of phosphatidylserine (90-99.9 mol%) and Succ-PEG _3400- DSPE (10-0.1 mol%) was added 5% propylene glycol-glycerol in water (1 ml). The dispersion was heated to 80 ° C. or less for 5 minutes and then cooled to room temperature. The dispersion (0.8 ml) was transferred to a vial (0.1 ml) and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer. After centrifugation, the fluid was exchanged with water and the wash was repeated.
c) Coupling of streptavidin to gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG _3400- DSPE
Streptavidin was covalently bound to Succ-PEG _3400- DSPE of the membrane by standard coupling methods using water soluble capbodiimide. The sample was added 5% propylene glycol-glycerol in water (1 ml) to a mixture (5 mg) of (10-0.1 mol%). The dispersion was heated to 80 ° C. or less for 5 minutes and then cooled to room temperature. The dispersion (0.8 ml) was transferred to a vial (0.1 ml) and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer. After centrifugation, the fluid was exchanged with water and the wash was repeated. The sample was placed on a roller table during the reaction. After centrifugation the fluid was exchanged with water and washing was repeated. For example, the functionality of a streptavidin attached by binding to a fluorescently labeled biotin, a biotinylated antibody (detected as a fluorescently labeled second antibody) or a biotinylated, fluorescent or radioactively labeled oligonucleotide Was analyzed. Analysis was performed by fluorescence microscopy or scintillation counting.
d) Preparation of multi-specific gas-filled microbubbles encapsulated with phosphatidylserine and streptavidin-Succ-PEG ₃₄₀₀ -DSPE non-covalently functionalized with biotinylated transferrin and human endothelial IgG antibodies
Bayer et al., Meth, Enzymol,. 62, 308 were used to incubate the microbubbles from section c) in a solution containing biotinylated human transferrin and human endothelial IgG antibodies. Vector-coated microbubbles were washed as described above.
Example 9
Multi-Specific Gas-Filled Microbubbles Encapsulated with Phosphatidylserine / Streptavidin-Succ-PEG-DSPE and Oligonucleotides Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and Biotin-GGCGCTGATGATGTTGTTGATTCTT
a) Synthesis of Succ-PEG ₃₄₀₀ -DSPE
Example 8 As described in a).
b) Preparation of gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG _3400- DSPE
As described in Example 8 b).
c) Coupling of streptavidin to gas-filled microbubbles encapsulated with phosphatidylserine and Succ-PEG _3400- DSPE
As described in Example 8c).
d) Preparation of Multi-Specific Gas-Filled Microbubbles Encapsulated with Phosphatidylserine / Streptavidin-Succ-PEG-DSPE and Oligonucleotides Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and Biotin-GGCGCTGATGATGTTGTTGATTCTT
The microbubbles from section c) were incubated in a solution containing a mixture of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotin-GGCGCTGATGATGTTGTTGATTCTT. Oligonucleotide-coated microbubbles were washed as described above. Binding of oligonucleotides to bubbles was detected, for example, using fluorescently labeled oligonucleotides for bubble attachment or by hybridizing oligonucleotides attached to labeled (fluorescent or radioactive) complementary oligonucleotides. For example, the functionality of the oligonucleotide-containing microbubbles was analyzed by hybridizing bubbles with immobilized DNA-containing sequences complementary to the attached oligonucleotides.
Another useful example included oligonucleotides that are complementary to ribosomal DNA (many copies per halfloid genome) and used oligonucleotides that are complementary to cancer genes.
<Example 10>
Multi-specific gas-filled microbubbles encapsulated with phosphatidylserine and phosphatidylethanolamine covalently functionalized with fibronectin and transferrin proteins
a) microbubble manufacturing
DSPS (Avanti, 4.5 mg) and DSPS (Avanti, 1.0 mg) were weighed into clean vials and 1 ml of 1.4% propylene glycol / 2.4% glycerol solution was added. This mixture was warmed to 80 ° C. for 5 minutes and filtered through a 4.5 micron filter. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken in a cap mixer for 45 seconds and the microbubbles were washed twice with distilled water and then resuspended in 0.1 M sodium borate at pH 9.
b) modification of fibronectin / transferrin
DSPS (0.5 mg) and transferrin (1.3 mg) were mixed in PBS and a solution containing NHS-fluorescein in DMSO was added. The mixture was stirred at rt for 1 h and then the protein was purified on a Superdex 200 column. The fluorescein labeled protein mixture in phosphate buffer, pH 7.5, was lyophilized.
c) microbubble deformation
The lyophilized product from b) was redissolved in 0.5 ml of water and 0.1 mmol of crosslinker SDBP (Pierce) was added to the fluorescein labeled fibronectin / transferrin mixture. The solution was incubated for 2 hours on ice, loaded on a NAP-5 column and eluted with PBS. To this was added 1 ml of the microbubble suspension from a) and incubated on a roller table for 2 hours at room temperature. After removing the unreacted material by flowing the microbubbles, the buffer was replaced with water and this process was repeated three times.
<Example 11>
Of a multi-specific hollow polymer molecule that introduces avidin to the polymer wall associated with oligonucleotide biotin-GGCGCTGATGATGTTGTTGATTCTT and biotinyl-endothelin-1 peptide biotin-D-Trp-Leu-Asp-Ile-Trp.OH. Produce
This example relates to the preparation of polymeric ultrasound contrast agents comprising multiple vectors attached to non-surfactants for targeted / therapeutic use.
a) Preparation of polymer molecules that introduce avidin into the polymer wall
P73 co-polymer molecules (as described in patent WO 96/07434) comprising amibin were prepared by a method comprising oil freeze drying of an oil-in-water emulsion using the following method: 5 ml of camphor at 60 ° C. An oil solution was prepared by dissolving 0.25 g of the biodegradable polymer p73 [oly (ethylidene bis (16-hydroxyhexadecanoate) coco (adipic acid)] in a 20 ml of water at 60 ° C. An aqueous solution was prepared by dissolving 0.4 g of-(16-polymer hexadecanoyloxyhexadecanoyl) -w-methoxypolyoxyethylene ester polymer An oil solution (0.2 ml) was added to an aqueous solution (0.8 ml) in a Vibrio mixer (Capmix). ) To form an oil-in-water emulsion The emulsion was frozen in dry ice and methanol and dried at a pressure of 200 mTorr for 24 hours to remove excess solvent. That the by was reconstituted as a suspension of the joint molecules was confirmed ultrasound contrast agent that is generated by the resistance to the microscopic observation, Coulter size distribution, acoustic attenuation and external pressure.
b) Synthesis of Biotin-D-Trp-Leu-Asp-Ile-Trp.OH
As described in Example 5 b).
c) binding of polymer molecules to introduce avidin
Centrifuge the molecules from a) and 1 ml of PBS buffer at pH 7.5 containing 0.2 mg of Biotin-GGCGCTGATGATGTTGTTGATTCTT and 0.2 mg of Biotin-D-Trp-Leu-Asp-Ile-Trp.OH from b) above. Supernatant was replaced with. After incubation for 24 hours, the molecules were washed well with PBS and water.
<Example 12>
Functionalization of Biotin-Containing Gas-Filled Albumin Microspheres (GAM) for Multi-Specific Targets
a) Preparation of Biotinylated Albumin Microspheres
GAM homogeneous suspension (6 × 10 ⁶ molecules / ml) in albumin 5 mg / ml was used and all preparations were performed at room temperature. Centrifuge two 10 ml aliquots (170 xg, 5 minutes) to increase the microspheres float, carefully remove 8 ml of excess fluid by aspirating and remove the same amount of air-saturated phosphate buffered saline And the precipitate was spun for 15-20 minutes to resuspend the microspheres. This process was repeated twice, after which a trace amount of free microsphere related albumin is believed to remain. 50 μl NHS-Biotin (10 mM in dimethylsulfoxide) was added to one of the aliquots (final concentration 50 μl); The remaining (control) aliquot received 50 μl of dimethylsulfoxide. After the tube containing the sample was rotated for 1 hour, about 20 µl of 50% aqueous glutaraldehyde was added to each tube to crosslink the microspheres. After further rotation, the tube was placed vertically overnight to allow the microspheres to move. The following day, the suspension was washed twice with phosphate buffered saline (PBS / HSA) containing 1 ml of human serum albumin and resuspended in PBS / HSA after the last centrifugation.
To determine the presence of microsphere related biotin, streptavidin (strep-HRP) bound to horseradish peroxidase was added to both suspensions and the tube was allowed to react for 1 hour by spinning. The microspheres were then washed three times and resuspended in 100 ml citrate-phosphate buffer (pH 5) containing 0.1 mg / ml phenylenediamine dihydrochloride and 0.01% hydrogen peroxide and spun for 10 minutes. Yellow-green development is a marker of the presence of enzymes. The following results were obtained.
sampleColor development Biotinylated Sphere + Strep-HRP2 + Control + Strep-HRP+
b) multi-specific gas-containing micromolecules
Multi-specific target products were prepared using biotinylated microspheres in a similar manner as exemplified in Examples 5), 6) and 7).
Example 13
Multi-Specific Gas-Containing Microbubbles of DSPS Functionalized with Heparin Sulfate Binding Peptides / Vibronectin Peptides / RGD Peptides and Fluorescein
a) Synthesis of lipopeptides containing RGD peptides and fluorescein reporter group: dipalmitoyl-Lys-Lys-Lys-Lys [acetyl-Arg-Gly-Asp-Lys (fluorescein)] Gly.OH
Lipopeptides were synthesized as described in Example 1) using commercial amino acids and polymers. Lipopeptides were degraded from the resin in TFA containing 5% phenol, 5% EDT and 5% H ₂ O. After evaporation in vacuo, the crude product was precipitated and triturated with diethyl ether. The crude product 40 over 40 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 60-100% (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at 9 mL / min flow rate. The mg aliquot was purified. 10 mg of pure material was obtained after lyophilization (analytical HPLC; gradient, 60-100% B, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water, column-Vydac 218TP1022: detection-UV 260- Product retention time = 20-22 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1922, found 1920
b) Synthesis of lipopeptides containing heparin sulphate binding sequence and bibronectin peptide
Synthesis and Purification as described in Example 1 a)
c) Preparation of multi-specific gas-containing microbubbles of DSPS functionalized with heparin sulphate binding peptide, bibronectin peptide, acetyl-RGD peptide and fluorescein
Lipopeptide DSPS (0.5 mg, 0.2 mmol) from DSPS (Avanti, 4 mg) and a) was weighed into two vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming). The sample was cooled to room temperature and the top space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were rolled overnight. The bubble was washed several times with deionized water and analyzed using MALDI-MS described in Example 1b). After microscopic analysis, the microbubbles appeared to consist of a range of 1 to 5 microns. In addition, the microbubbles fluoresced.
<Example 14>
Multi-Specific Gas-Containing Microbubbles of DSPS 'Doped' with Lipopeptides Affinity to Endothelial Cells, CD71 FITC-labeled Anti-Transferrin Receptor Antibody Covalently Modified
This embodiment relates to the preparation of multiple vector targeted ultrasound agents.
a) Synthesis of endothelial cell binding lipopeptides: 2-n-hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys -Leu-Ala-NH ₂
Lipopeptides shown below were synthesized in an ABI 433A automated peptide synthesizer starting with Rink amide resin on a 0.1 mmole scale using a 1 mmole amino acid cartridge.
HBTU was used to preactivate all amino acids and 2-n-hexadecylstearyl acid prior to coupling. Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT and 5% H ₂ O to afford the crude product in 150 mg yield. Purified HPLC (Vydac 218TP1022 column) using 90-100% concentration gradient (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate purified a 40 mg aliquot of crude material over 50 minutes. . 10 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 90-100% B, where B = MeOH, A = 0.01% TFA / water, column Vydac 218TP1022: detection UV 214 nm product retention time =). 23 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2869, found 2373.
b) Preparation of gas-containing microbubbles of DSPS 'doped' with endothelial cell binding lipopeptides and PE-PEG _2000- Mal
Lipopeptide DSPS (0.5 mg) from DSPS (Avanti, 4.5 mg) and a) were weighed together with PE-PEG ₂₀₀₀ -maleimide (0.5 mg) from Example 2 and placed in clean vials, 1.4% propylene glycol / 1 ml of 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes and then filtered through a 4.5 micron filter. The mixture was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were washed three times with distilled water.
c) Thiolation of FITC-labeled anti-transferrin receptor antibody
0.7 ml of FITC-labeled CD71 anti-transferrin receptor Ab (100 mg / mL, Becton Dickinson) in PBS was modified with Trout reagent (0.9 mg, Pierce) for 1 hour at room temperature. Excess reagent was separated from the modified protein on a NAP-5 column (Pharmacia).
d) Binding of thiolated FITC-labeled anti-transferrin receptor antibodies to gas-containing microbubbles of endothelial cell binding lipopeptides and 'doped' DSPS with PE-PEG _2000- Mal
A 0.5 ml aliquot (total 2 ml) of protein fraction from c) was added to the microbubbles from b) and the binding reaction was carried out for 10 minutes on a roller table. After centrifugation at 1000 rpm for 3 minutes, the protein solution was transferred and the binding reaction was repeated twice with 1 ml and 0.5 ml aliquots of the protein solution, respectively. The bubbles were then washed four times with distilled water and the samples analyzed by flow cytometer and microscope in the presence of antibody. Fluorescent colonies were observed above 92%.
(Comparison of flow cytometry of negative control microbubbles of DSPS (left curve) bound bubbles and CD71 FITC-labeled anti-transferrin antibody (filled curve, right) showing 92% fluorescence of colonies)
The introduction of lipopeptides into the microbubbles was confirmed using MALDI mass spectrometry as described in Example 1 b).
<Example 15>
Preparation of Multi-Specific Transferrin / Avidin Coated Gas-Containing Microbubbles for Targeted Ultrasound Imaging
This example relates to the preparation of microbubbles containing multiple protein vectors for targeted ultrasound / therapy.
a) Synthesis of thiol functionalized fat molecule: dipalmitoyl-Lys-Lys-Lys-Aca-Cys.OH.
Lipid structures shown above were synthesized in a ABI 433A automated peptide synthesizer starting with Fmoc-Ile-Wang resin (Novabiochem) on a 0.25 mmol scale using a 1 mmol amino acid cartridge. HBTU was used to preactivate all amino acids and palmitic acid prior to coupling.
Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT and 5% H ₂ O to afford the crude product in 250 mg yield. Purified HPLC (Vydac 218TP1022 column) using 90-100% concentration gradient (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate purified the 40 mg aliquot of crude product over 50 minutes. . 24 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, where B = 0.1% TFA / acetonitrile, A = 0.1% TFA / water, column Vydac 218TP1022: detection UV 214 nm) --Product retention time = 23 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1096, found 1099.
b) Preparation of gas-containing microbubbles of DSPS 'doped' with thiol containing fatty structures
Lipopeptide DSPS (0.5 mg) from DSPS (Avanti, 4.5 mg) and a) was weighed into clean vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming) and filtered through a 40 micron filter while hot. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were placed on a roller table overnight. The bubble was washed several times with deionized water and analyzed for thiol group introduction using Elman reagent.
c) Modification of Transferrin and Avidin with Fluorescein-NHS and Sulfo-SMPB
2 mg of transferrin (Holo, human, Alpha terapultic) and 2 mg of avidin (Sigma) in PBS (1 ml) containing 0.5 mg of fluorescein-NHS (Pierce) and 1 mg of sulfo-SMPB (Pierce) 0.5 ml of DMSO solution was added. The mixture was stirred at room temperature for 45 minutes and then passed through a Sephadex 200 column using PBS as eluent. Protein fractions were collected and stored at 4 ° C. before use.
d) Microbubble Bonds with Modified Transferrin / Avidin
To a thiol containing microbubble from b) 1 ml of a modified transferrin / avidin protein solution c) was added. After adjusting the pH of the solution to 9, the binding reaction was carried out at room temperature for 2 hours. After washing well with deionized water, it was analyzed by Coulter counter (1-7 microns (81%)) and fluorescence microscopy (high fluorescence microbubbles were observed).
<Example 16>
Preparation of Functionalized Gas-Filled Microbubbles for Targeted Ultrasound Imaging
This example relates in principle to the preparation of microbubbles with on-surface reactors for non-specific targets using an sulfide exchange reaction that performs binding to multiple cellular targets.
Thiol-containing fat structures (1.0 mg) from DSPS (Avanti, 5.0 mg) and Example 15 a) were weighed into clean vials and 0.8 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to each vial. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming) and filtered through a 40 micron filter while hot. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were placed on a roller table overnight. The bubble was washed several times with deionized water and analyzed for thiol group introduction using Elman reagent.
<Example 17>
Multi-Specific Gas-Containing Microbubbles of DSPS Including Lipopeptide and Captopril-Containing Molecules for Endothelial Cell Targets
This embodiment relates to the preparation of ultrasound agents for combined target and therapeutic use.
a) Synthesis of thiol functionalized fat molecule: dipalmitoyl-Lys-Lys-Lys-Aca-Cys.OH.
The structure was synthesized using a manual nitrogen foamer starting with a protected Rink Amide MBHA resin on a 0.125 mmol scale. All amino acids were purchased from Novabiochem and palmitic acid from Fluka. Coupling was performed using standard TBTU / HOBt / DIEA. Bromoacetic acid was coupled through the side chain of Lys as symmetric anhydride using DIC preactivation. Captopril (Sigma) dissolved in DMF was introduced onto the solid phase using DBU as the base.
Deprotection of the peptide and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT, 5% H ₂ O and 5% ethyl methyl sulfide to give the crude product in 250 mg yield. . The crude product over 60 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 70 to 100% B (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 mL / min. 10 mg aliquots were purified. 2 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, where B = 0.1% TFA / acetonitrile, A = 0.1% TFA / water, column Vydac 218TP1022: detection UV 214 nm) --Holding time = 26 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1265.
b) Synthesis of lipopeptides with affinity for endothelial cells: dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg-Arg-Val-Ala-Arg-Pro-Pro-Leu-NH ₂
Lipopeptides were synthesized on an ABI 433A automated peptide synthesizer starting with link amide resin (Novabiochem) on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids and palmitic acid prior to coupling. Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% phenol, 5% EDT and 5% H ₂ O to afford the crude product in a yield of 160 mg. A 35 mg aliquot of the crude product was purified over 40 minutes by purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 70-100% (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate. . 20 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 20-50% B, where B = MeOH, A = 0.01% TFA / water, column Vydac 218TP1022: detection UV 214 and 260 nm product retention). Time = 16 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2050, found 2055
c) Preparation of gas-containing microbubbles of DSPS containing captopril containing molecules for drug delivery and lipopeptides for targeting endothelial cells
DSPS (Avanti, 4.5 mg), product from a) (0.5 mg) and product from b) (0.5 mg) are weighed into vials and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution is added to each vial It was. The mixture was warmed to 80 ° C. for 5 minutes (stirring while warming). The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer, rolled for 1 hour, and washed well with deionized water. No starting material was observed in the final wash as evidenced by MALDI mass spectrometry. The introduction of the product from sections a) and b) into the microbubbles was confirmed using MALDI mass spectrometry as described in Example 1 b).
d) In vitro studies of gas-containing microbubbles of DSPS containing captopril containing molecules for therapeutic use and lipopeptides for targeting endothelial cells
Cell binding was examined under flow conditions using the in vitro assay described in Example 1 c). Gradual accumulation of cellular microbubbles occurred depending on the flow rate. As the flow rate increased, the cells began to deviate from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
Example 18
Preparation of Multi-Specific Gas-Containing Microbubbles of DSPS Loaded with Lipopeptides Containing Peptide Antibiotic Polymyxin B Sulfate and Helicoidal Peptides Having Affinity to Cell Membranes
A preparation of targeted microbubbles comprising multiple peptidic vectors with combined targets and therapeutic uses.
a) Synthesis of lipopeptides comprising helical peptides having affinity for cells: hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala- Ala-Leu-Lys-Leu-Ala-NH ₂
Example 14a) has been described.
b) Preparation of multi-specific gas-containing microbubbles
Lipopeptide (0.3 mg) from DSPS (Avanti, 5.0 mg), a) and product from polymyxin B sulfate b) (0.5 mg) were weighed and placed in clean vials, 1.4% propylene glycol / 2.4% glycerol solution 1.0 ml was added to the vial. The mixture was sonicated for 3 to 5 minutes, warmed to 80 ° C. for 5 minutes and then filtered through a 4.5 micron filter. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were centrifuged at 1000 rpm for 3 minutes. The microbubbles were washed in water until no polymyxin B sulfate or lipopeptides in the precipitate were detected by MALDI-MS. Microscopic observation showed that the size distribution of the bubble colonies was 1 to 8 microns as desired. To the washed bubble (about 0.2 ml) was added methanol (0.5 ml) and the mixture was placed in sonication for 2 minutes. It was found that the clear solution produced after analysis by MALDI-MS contained lipopeptides and polymyxin B sulfate (expected 1203, found 1207).
Example 19
Preparation of Multi-Specific Gas-Containing Microbubbles of DSPS Modified into Branched Structures Containing Drug Methotrexate and 'doped' with IL-1 Receptor Binding Sequence-Containing Lipopeptides
This example relates to the preparation of a targeted microbubble comprising a multiplex vector for target / therapeutic / drug release applications.
a) Synthesis of lipopeptides comprising interleukin-1 receptor binding peptides: dipalmitoyl-Lys-Gly-Asp-Trp-Asp-Gln-Phe-Gly-Leu-Trp-Arg-Gly-Ala-Ala.OH
Lipopeptides were synthesized on an ABI 433A automated peptide synthesizer starting with Fmoc-Ala-Wang resin (Novabiochem) on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids and palmitic acid prior to coupling. Removal of lipopeptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O to afford the crude product in 150 mg yield. . 30 mg aliquots of the crude product were purified over 40 minutes by purified HPLC (Vydac 218TP1022 column) using 90-100% concentration gradient (A = 0.1% TFA / water and B = MeOH) at 9 mL / min flow rate. . 4 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 90-100% B, where B = MeOH, A = 0.01% TFA / water, column Vydac 218TP1022: detection UV 214 nm product retention time =) 23 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2083, found 2088.
b) Synthesis of branched methotrexate corestructures comprising thiol residues:
Methotrexate was synthesized on an ABI 433A automated peptide synthesizer starting with Fmoc-Cys (Trt) Tentagel resin on a 0.1 mmol scale. Deprotection of the product and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT and 5% H ₂ O to afford the crude product in 160 mg yield. Crude product over 40 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 10-30% B (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at 9 mL / min flow rate. 30 mg aliquots were purified. 9 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 5-50% B, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water, column Vydac 218TP1022: detection UV 214 nm) Product retention time = 9.5 min). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 1523, found 1523.
c) preparation of multi-specific gas-containing microbubbles
DSPS (Avanti, 4.5 mg), thiol-containing lipopeptides from Example 15 a) (0.5 mg) and lipopeptides from a) (0.2 mg) were weighed and placed in clean vials, 1.4% propylene glycol / 2.4% glycerol 1.0 ml of solution was added to the vial. The mixture was sonicated for 3 to 5 minutes, warmed to 80 ° C. for 5 minutes and then filtered through a 4.5 micron filter. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer, the microbubbles were centrifuged at 1000 rpm for 3 minutes and the precipitate was discarded.
d) binding of methotrexate branched structures to thiolated microbubbles
The methotrexate structure preparation (0.5 mg) from b) was dissolved in PBS pH 8.0. This solution was added to the thiol containing bubble from c) and disulfide bond formation was carried out for 16 hours. After washing well with PBS and water, the bubbles were analyzed using microscopy and MALDI mass spectroscopy.
It is also believed that disulfide bonds that link the methotrexate structure to the microbubbles can reduce the free movement of free drug molecules in vivo. It is a drug delivery system with tumor specific vectors. Physiologically important reducing agents such as glutathione can be used to effect drug release.
Example 20
Preparation of microbubbles coated with poly-L-lysine in combination with fluorescein labeled DNA fragments from plasmid pBR322
This example relates to the manufacture of microbubbles for gene therapy / anti-sense applications. For example, specific targets can be obtained by further doping the microbubble membrane with vector modified lipid constructs as described in Example 1.
a) Preparation of DSPS Gas-Containing Microbubbles
DSPS (Avanti, 4.5 mg) was weighed and placed in clean vials. 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to the vial and the mixture was warmed to 80 ° C. for 5 minutes. The solution was then warmed up and filtered through a 4 micron filter. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer. The bubble was then washed with deionized water and discarded the precipitate. The microbubbles were then redissolved in 0.5 ml of water.
b) Preparation of poly-L-lysine / DNA complex and loading of DSPS microbubbles
To 1 mg of poly-L-lysine (70-150 kD) in a clean vial was added 0.1 ml of plasmid pBR322 (Biorad) fluorescein labeled digest dissolved in TE buffer (10 mM Tris-HCl, pH 8). This solution was made up to a total of 0.6 ml by adding water and adjusting the pH to 8. The complex reaction was carried out for 1 hour and 0.5 ml of polylysine-DNA solution was added to the microbubble suspension from a) above. After 1 hour, the bubble was observed to fluoresce under a microscope to confirm the presence of DNA.
Example 21
Preparation of Multi-Specific Gas-Filled Microbubbles Comprising Branched Core Peptides Composed of Dabsylated Atherom Platelet Binding Sequence and RGDS
This example relates to the preparation of microbubbles with thiol groups on the surface for transformation into thiol containing vectors for target / drug delivery and drug release.
a) Synthesis of Branched Peptides Davyl-Tyr-Arg-Ala-Leu-Val-Asp-Thr-leu-Lys-Lys- (NH2-Arg-Gly-Asp-Ser) -Gly-Cys.OH
Peptides were synthesized in an ABI 433A automated peptide synthesizer starting with Fmoc-Cys (Trt) -Tentagel resin on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids prior to coupling.
Removal of peptides and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% phenol, 5% EDT and 5% H ₂ O to afford the crude product in a yield of 160 mg. 30 mg aliquots of the crude product over 40 minutes with purified HPLC (Vydac 218TP1022 column) using a concentration gradient of 10-60% B (A = 0.1% TFA / water and B = acetonitrile) at 9 mL / min flow rate. Purified. 2.5 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 10-50% B, 20 min, where B = 0.1% TFA / acetonitrile, A = 0.01% TFA / water, column Vydac 218TP54: detection- UV 214 and 435 nm-product retention time = 21 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H expected 2070, found 2073.
b) Preparation of thiol containing gas-filled microbubbles
Examples 15 were described in a) and b).
c) Oxidative coupling of thiolated microbubbles with multi-specific peptides through disulfide bond formation
The precipitate of microbubbles from b) was discarded and replaced with dabsil-peptide solution (1 mg) in 0.7 ml of diluted ammonia solution (pH 8). To this was added 0.2 ml of a stock solution containing 6 mg of potassium ferricyanate dissolved in 2 ml of water. The vial was placed on a roller table and thiol oxidized for 2 hours. The bubbles were then washed well with water until the precipitate was free of dabsil-peptide, as evidenced by hplc and MALDI mass spectrometry.
Detection of microbubble bound peptides was performed by reducing disulfide bonds using the water soluble reducing agent Tris- (2-carboxyethyl) -phosphine. After reduction, as found by hplc and MALDI mass spectroscopy, the precipitate was found to contain free dabsil-peptide.
Other physiologically important reducing agents, such as reduced glutathione, are thought to be useful for initiating release.
<Example 22>
Gas-containing microbubbles comprising a polymer from ethylidene bis (16-hydroxyhexadecanoate) and adipoyl chloride and biotin-amidocaproate-Ala covalently bound to the polymer
a) Synthesis of Z-Ala-polymer (3-O- (carbenzyloxy-L-alanyl) -polymer)
As described in WO-A-9607434, polymers were prepared from ethylene bis (16-hydroxyhexadecanoate) and adipoyl chloride, and the polymer fraction with molecular weight 10000 was purified by gel permeation chromatography (GPC). 10 g of material (corresponding to 1 mmol OH group), Z-alanine (5 mmol) and dimethylaminopyridine (4 mmol) are dissolved in dry dimethyl formamide / tetrahydrofuran and then dicyclohexylcarbodiimide Was added. The reaction mixture was stirred at room temperature overnight. The dicyclohexylurea was filtered off and rotary evaporated to remove the solvent. The product was purified using chromatography, fractions containing the title compound were collected and solvent was removed using rotary evaporation. The structure of the product was confirmed by NMR.
b) Synthesis of Ala-polymer (3-O- (L-alanyl) -polymer)
Z-Ala-polymer (0.1 mmol) was stirred in toluene / tetrahydrofuran and glacial acetic acid (15% of total volume) and hydrogenated in the presence of 5% palladium on charcoal. The reaction mixture was filtered and concentrated in vacuo.
c) Synthesis of Biotinamidocaproate-Ala-Polymer
Biotinamidocaproate N-hydroxysuccinimide ester solution in tetrahydrofuran was added to H ₂ N-Ala-polymer dissolved in tetrahydrofuran and dimethylformamide and 0.1 M sodium phosphate buffer at pH 7.5. The reaction mixture was heated to 30 ° C. and stirred vigorously before TLC. The solvent was evaporated and the crude product was used without further purification.
d) gas-containing molecules comprising PEG 10000 methyl ether 16-hexadecanoyloxyhexadecanoate and biotin-amidocaproate-Ala-polymer
10 ml of a 5% w / w solution of biotin-amidocaproate-Ala polymer in (-)-camphor maintained at 60 ° C. was added to PEG 10000 methyl ether 16-hexadecanoyloxyhexadecanoate (WO-A-9607434). Prepared as described) to 30 ml of a 1% w / w aqueous solution. The rotor stator mixer (Ultra Turax® T25) was used at low speed for several minutes to emulsify the mixture, then frozen in a dry ice / methanol bath and lyophilized for 48 hours to obtain white powder. The title product was obtained.
e) acoustic characterization and microscopy of products
Confirmation of the microparticle characteristics of the product was carried out using an optical microscope as described in WO-A-9607434. Ultrasonic delivery measurements using a 3.5 NHz wideband transducer resulted in a particle suspension of less than 2 mg / mL sonic beam attenuation above 5 dB / cm.
f) multi-specific microparticles
Then, multi-specific target products were prepared using biotinylated microspheres similar to those illustrated in Examples 5), 6) and 7).
<Example 23>
Oligonucleotides Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and Biotinylated Fibrin-Anti-Polymeric Peptides (Biotin-GPRPPERHQS.NH ₂ ) and Streptavidin Functionalized and Multiplexed with Biotin-PEG ₃₄₀₀ -Acyl-Phosphatidylethanolamine and DSPS Preparation of Unusual Gas-Containing Microbubbles
a) Synthesis of Biotin-PEG ₃₄₀₀ -acyl-phosphatidylethanolamine
Dipalmitoyl phosphatidyl ethanolamine (21.00 mg, 0.03 mmol), biotin-PEG-CO ₂ -NHS (100 mg, 0.03 mmol) and triethylamine (42 μL, 0.30 mmol) in chloroform / methanol (3: 1) solution The mixture was stirred at rt for 2 h. After evaporation of the solvent under reduced pressure, the residue was subjected to flash chromatography (methylene chloride / methanol / water, 40: 8: 1). 112 mg (94%) of the product was obtained as a yellow gum and the structure was verified by NMR and MALDI-MS.
b) binding of fluorescein-linked streptavidin to gas-filled microbubbles
As described in Example 5 a), gas-containing microbubbles were prepared by mixing DSPS and biotin-PEG ₃₄₀₀ -acyl-phosphatidyl ethanolamine.
The microbubble suspension was divided into 0.2 ml aliquots and fluorescein bound streptavidin was added as shown in the table below. Samples were incubated at room temperature for 15-30 minutes on a roller table before removing excess protein by washing with PBS.
<Result>
Fraction numberAdded streptavidin (μg / 200: 1 sample)Incubation time (room temperature)% Fluorescein ParticlesParticle Average Diameter (microns) One0 2.0- 20 -12 (foam) 30.2 (3 x 10 ^-9 cm)30 minutes7.83.9 42 (3 x 10 ^-8 cm)30 minutes26.24.2 510 (3 x 10 ^-7 cm)15 mins30.5na 620 (3 x 10 ^-7 cm)30 minutes97.95.2 740 (3 x 10 ^-7 cm)15 mins96.75.1 8 DSPS contrast20 (3 x 10 ^-7 cm)15 mins0.63.7
Samples were analyzed by flow cytometer and microscope. The results are summarized in the table above.
c) Binding of oligonucleotide biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and biotinylated fibrin-anti-polymeric peptide biotin-GPRPPERHQS with streptavidin coded microbubbles
The particles of aliquot No. 6 were centrifuged and the supernatant was replaced with 1 ml of PBS buffer at pH 7.5 containing 0.2 mg of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and 0.2 mg of biotin-GPRPPERHQS (Example 5 c). After incubation for 24 hours, the particles were washed well with PBS and water.
Using this method, other biotinylated vectors or therapeutic agents can be bound to streptavidin or avidin coated microbubbles.
<Example 24>
Preparation of Microbubbles Functionalized with Thrombolytic Enzyme Tissue Plasminogen Activator and Thrombus-Targeted Lipopeptide and Encapsulated with DSPS
This example relates to the preparation of a thrombus target US agent containing a therapeutic thrombolytic agent.
a) Synthesis of lipopeptides having affinity for thrombi (dipalmitoyl-Lys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH ₂ )
The lipopeptides were synthesized on an ABI 433A automated peptide synthesizer starting with Rink amide resin (Novabiochem) on a 0.1 mmole scale using a 1 mmole amino acid cartridge. HBTU was used to preactivate all amino acids prior to coupling. Simultaneous removal of the peptide and side chain protecting groups from the resin in a TFA containing 5% phenol, 5% EDT, 5% anisole and 5% H ₂ O was carried out for 2 hours to give the crude product in 80 mg yield. . Purification HPLC (Vydac 218TP1022 column) purified 20 mg aliquots of the crude product. 6 mg of pure material was obtained after dry freezing. The product was characterized using MALDI mass spectroscopy and analytical HPLC.
b) modification of tissue plasminogen activator with sulfo-SMPB
A 0.1 ml solution of ammonium carbonate buffer containing 0.1 mg of t-PA (Sigma) was prepared by adding water to make 0.2 mL. To this solution was added 0.4 mg of sulfo-SMPB (Pierce) dissolved in 0.05 ml DMSO. The protein solution was allowed to stand for 45 minutes at room temperature and then purified on a Superdex 200 column. The product was eluted with PBS to collect modified protein fractions.
c) Preparation of Microbubbles Encapsulated with Thiol-Containing Lipopeptides and DSPS / Thromb binding Lipopeptides and Binding to Modified Tissue Platsminogen Activators
DSPS (Avanti, 5.0 mg) was weighed and lipopeptides from a) (0.5 mg) and thiol containing lipopeptides from Example 15 a) (0.5 mg) were placed in clean vials and 1.4% propylene glycol / 2.4% glycerol 1.0 ml of solution was added and the mixture was sonicated for 2 minutes and then warmed to 80 ° C. for 5 minutes. The solution was warmed and soon filtered through a 4 micron filter. The sample was cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and the microbubbles were washed twice with deionized water. The precipitate was discarded and replaced with 1 ml aliquot of the protein solution from b) above. The binding reaction was carried out for 1 hour. After the bubbles were centrifuged, the precipitate was exchanged with 1 mL of another protein solution. The incubation step was repeated until all protein solution was exhausted. The microbubbles were then washed well with water and analyzed with a Coulter counter. Microbubbles were tested by the flow chamber analysis described in Example 1 c). Microbubbles that have been modified with proteins that bind in greater numbers than those containing either lipopeptides / DSPS or DSPS itself have been found.
Targeting / therapeutic / ultrasonic activity of such microbubbles can be evaluated in in vitro and in vivo thrombus generation models.
<Example 25>
Multi-specific PFB gas filled microencapsulated encapsulated with DSPS and lipopeptides containing athenol for therapeutic use and heparin sulfate binding peptides (KRKR) and bibronectin peptides (WOPPRARI) for targets and DSPS bubble
a) Synthesis of lipopeptides consisting of heparin sulphate binding peptide (KRKR) and bibronectin peptide (WOPPRARI)
Synthesis and Purification as described in Example 1 a)
b) synthesis of protected athenol derivatives suitable for solid phase coupling
i) Synthesis of Methyl 4-[(2,3-epoxy) propoxy] phenyl acetate
A mixture of methyl 4-[(2,3-epoxy) propoxy] phenylacetate (4.98 g, 0.0 mol), epichlorohydrin (23.5 ml, 0.30 mmol) and pyridine (121 μl, 1.5 mmol) was added at 85 ° C. Stir for 2 hours. The reaction mixture was cooled down and excess epichlorohydrin was distilled off (rotor vapor). The residue was dissolved in ethyl acetate, washed with brine and dried (Na ₂ SO ₄ ). The solution was filtered and concentrated. The dark residue was chromatographed (silica, hexane / ethyl acetate 7: 3) to give 2.25 g (34%) of a colorless oil. The structure was observed in ¹ H (300 MHz) and ¹³ C NMR (75 MHz) spectra.
ii) Synthesis of methyl 4- [2-hydroxy-3-[(1-methylethyl) amino-propoxy] phenylacetate
Methyl 4-[(2,3-epoxy) propoxy] phenylacetate (2.00 g, 9.00 mol), isopropylamine (23 ml, 0.27 mmol) and water (1.35 ml, 74.7 mmol) were stirred overnight at room temperature. The reaction mixture was concentrated (rotor vapor) and the oil residue was dissolved in chloroform and dried (Na ₂ SO ₄ ). Filtration and concentration yielded a yellow oil in quantitative yield, which was used in the next step without further purification. The structure was verified by ¹ H and ¹³ C NMR analysis.
iii) Synthesis of 4- [2-hydroxy-3-[(1-methylethyl) aminol-propoxy] phenylacetic acid hydrochloride
Methyl 4-[(2-hydroxy-3-[(1-methylethyl) amino-propoxy] phenylacetate (563 mg, 2.00 moles) in 6 M hydrogen chloride (15 ml) was heated at 100 ° C. for 4 hours. The reaction mixture was concentrated (rotor vapor) and the residue was dissolved in water and freeze dried The structure was verified by ¹ H and ¹³ C NMR analysis MAL H Mass spectrometry gave an M + H estimate of 268.
iv) Synthesis of N-Boc-4- [2-hydroxy-3-[(1-methylethyl) aminol-propoxy] phenylacetic acid
A solution of 4- [2-hydroxy-3-[(1-methylethyl) aminol-propoxy] phenylacetic acid hydrochloride (2.0 mmol) in water (2.0 ml) was dissolved in water / dioxane (2: 1, 15 ml). ) Solution of sodium bicarbonate (0.60 g, 7.2 mmol). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in dioxane (5 ml) was added. The progress of the reaction was monitored by TLC analysis (silica, CHCl ₃ / MeOH / AcOH 85: 10: 5) and some di-tert-butyl dicarbonate was added until the conversion was complete. The reaction mixture was poured into water saturated with potassium hydrogensulfate and the organics extracted with ethyl acetate. The organic phase was washed with water and brine, dried (Na ₂ SO ₄ ) and filtered to give 0.6 g of crude material. The product was purified by chromatography (silica, CHCl ₃ / MeOH / AcOH 85: 10: 5). The solution was concentrated and the residue was dissolved in glacial acetic acid and freeze dried. Yield was 415 mg (56%) as a white solid. The structure was confirmed by ¹ H and ¹³ C NMR analysis.
c) Synthesis of Lipopeptides Functionalized with Athenol
The structures shown above start with a link amide MBHA resin (Novabiochem) on a 0.125 mmol scale using amino acids obtained from Nova Biochem, Palmitic Acid obtained from Fluka and compounds from a). Synthesis was carried out using a manual nitrogen foamer. Coupling was performed using standard TBTU / HOBt / DIEA protocol.
Deprotection of the peptide and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT and 5% H ₂ O. Crude material was precipitated from ether and purified liquid chromatography (Vydac) using a concentration gradient of 70-100% B (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 mL / min. 218TP1022 column) to purify crude material over 60 minutes. 38 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, 20 minutes, where B = 0.1% TFA / acetonitrile, A = 0.1% TFA / water, flow rate 1 mL / min, column Vydac 218TP54: detection UV 214 nm; retention time 25 minutes). Further characterization of the product was performed using MALDI mass spectroscopy (ACH matrix); M + H 1258, expected 1257.
d) Preparation of gas-filled microbubbles of DSPS comprising lipopeptides comprising heparin sulphate binding peptide (KRKR), bibronectin peptide (WOPPRARI) and lipopeptides containing athenol
DSPS (Avanti, 5.0 mg), product from a) (0.5 mg) and product from c) (0.5 mg) were weighed into vials and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to the vial. . The mixture was sonicated for 5 minutes and heated at 80 ° C. for 5 minutes (stirring while warming). This solution was filtered and cooled. The upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and washed well with deionized water.
The introduction of the athenol containing lipopeptides into the bubbles was confirmed using MALDI mass spectrometry as described in Example 1 b).
e) in vitro studies of multi-specific gas-filled microbubbles;
In vitro studies of microbubble suspensions were performed as described in Example 1 c). Gradual accumulation of microbubbles on the cells occurred, depending on the flow rate. As the flow rate increased, the cells began to detach from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
Example 26
PFB gas-filled microbubbles of DSPS containing cholesteryl esters of chlorambucil for diagnostic and therapeutic uses
This example relates to non-specific modification of multiplicity of cellular receptors on endothelial cells.
a) Synthesis of Cholesteryl 4- [4-bis (2-chloroethyl) amino] -phenyl] butanoate
DIC (170 μl, 1.10 mmol) was added to a solution of chlorambucil (sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15 ml). This mixture was stirred for 0.5 h at room temperature and added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction mixture was stirred overnight and poured into 5% sodium bicarbonate. This phase was separated and the organic phase was washed with brine and dried (MgSO ₄ ). The solution was filtered and concentrated, and the product was purified by column chromatography (silica, chlorolol) to give 560 mg of a colorless oil in 83% yield. The product was characterized using MALDI mass spectroscopy (ACH matrix) to obtain M + H at 674 as expected. In addition, ¹ H (500 MHz) and ¹³ C NMR (125 MHz) spectra were used to characterize and observe the structure.
b) Preparation of gas-containing microbubbles of DSPS comprising cholesteryl esters of chlorambucil for diagnostic and therapeutic uses
A mixture of DSPS (Avanti, 4.5 mg) and product from a) (0.5 mg) was placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to this vial. The mixture was sonicated for 5 minutes, heated at 80 ° C. for 5 minutes (stirring while warming) and cooled. The upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. No starting material was observed in the final wash as evidenced by MALDI mass spectrometry. The introduction of chlorambucil's cholesteryl ester into microbubbles was confirmed by the following MALDI mass spectrometry: About 50 μl of microbubbles were transferred to a clean vial containing about 100 μl of 90% methanol. The mixture was sonicated for 30 seconds and analyzed by MALDI mass spectrometry to yield M + H at 668, consistent with the structure from a).
Such microbubbles along with tumor specific vectors are believed to be useful as targeted drug delivery agents.
Example 27
Multi-Specific Gas-Filled Microbubbles of DSPS Including Cholesteryl Derivatives of Chlorambucil and Lipopeptides Containing Athenol
a) Synthesis of Protected Athenol Derivatives Suitable for Solid Phase Coupling
Example 25 As described in section b).
b) Synthesis of Lipopeptides Functionalized with Athenol
Example 25 As described in section c).
c) Synthesis of cholesteryl 4- [4- [bis (2-chloroethyl) amino] -phenyl] butanoate
Example 25 As described in section b).
d) Preparation of microbubbles of DSPS comprising cholesteryl esters of chlorambucil and athenol
A mixture of DSPS (Avanti, 5.0 mg), product from b) (0.5 mg) and product from c) (0.5 mg) is placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution is added to this vial. It was. The mixture was sonicated for 5 minutes and warmed at 80 ° C. for 5 minutes (stirring while warming). The solution was filtered cooled. The upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. As described in Example 1c), introduction of the athenol-containing lipopeptides into the microbubbles was confirmed by MALDI mass spectrometry.
e) In vitro study of multi-specific PFB gas-containing microbubbles of DSPS comprising lipopeptides containing cholesteryl derivatives of chlorambucil and athenol for diagnostic and therapeutic uses
The in vitro study described in Example 1 c) was used to assess cell binding under flow conditions. Gradual accumulation of microbubbles on the cells occurred, which was dependent on flow rate. As the flow rate increased, the cells began to detach from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
<Example 28>
Multi-specific gas-filled microbubbles of DSPS comprising lipopeptides containing lipophilic thiol esters of captopril for therapeutic use and atenols for cellular targets
a) Synthesis of Protected Athenol Derivatives Suitable for Solid Phase Coupling
Example 25 As described in section b).
b) Synthesis of Lipopeptides Functionalized with Athenol
Example 25 As described in section c).
c) Synthesis of cholanic acid thiol esters of captopril
A mixture of 5-β-cholanic acid (Sigma, 361 mg, 1.00 mmol) and DIC (77 μL, 0.50 mmol) in dichloromethane (5 ml) was stirred for 10 minutes, followed by captopril in dichloromethane (10 ml). (Sigma, 130 mg, 0.600 mmol) and DBU (180 μl, 1.20 mmol) solution. The reaction mixture was stirred overnight and poured into diluted hydrochloric acid. Chloroform (30 ml) was added. The phases were separated and the organic phase was washed with water and brine and dried (MgSO ₄ ). After filtration and concentration, the crude product was chromatographed (silica, chloroform / methanol / acetic acid, 95: 4: 1). The product was freeze dried from acetonitrile / water / ethanol mixture. Obtained as an off-white solid 137 mg, yield 49%. The structure was verified with ¹ H (500 MHz) and ¹³ C NMR (125 MHz) spectra. Further characterization of the product was performed using MALDI mass spectroscopy to obtain M + H peaks in the positive mode at m / z 584.
d) Preparation of gas-filled microbubbles of DSPS comprising lipopeptides containing lipophilic thiol esters of captopril for therapeutic use and atenols for cellular targets
A mixture of DSPS (Avanti, 5.0 mg), product from b) (0.5 mg) and product from c) (0.5 mg) is placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution is added to this vial. It was. The mixture was sonicated for 5 minutes, warmed at 80 ° C. for 5 minutes (stirring while warming) and cooled. The upper space was flushed with perfluorobutane gas and the vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. No starting material was observed in the final wash as evidenced by MALDI mass spectrometry. The introduction of the compounds from b) and c) into the microbubbles was confirmed by the following MALDI mass spectrometry: About 50 μl of microbubbles were transferred to a clean vial containing about 100 μl of 90% methanol. The mixture was sonicated for 30 seconds and analyzed by MALDI mass spectrometry (ACH-matrix) to yield peaks that matched the structures from b) and c), respectively.
e) in vitro studies of gas-containing microbubbles from d)
Cell binding was examined under flow conditions using the in vitro assay described in Example 1 c). Gradual accumulation of cellular microbubbles occurred depending on the flow rate. As the flow rate increased, the cells began to deviate from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
<Example 29>
Gas-filled microbubbles of phosphatidylserine containing biotinamide-PEG-β-Ala-cholesterol and cholesteryl derivatives of chlorambucil for diagnostic and therapeutic uses
a) Synthesis of N-Boc-β-alanineate
DIC (510 μl) was added to Boc-β-Ala-OH (1.25 g, 6.60 mmol) in dichloromethane (15 ml) under an inert atmosphere. The reaction mixture was stirred for 30 minutes and transferred to a flask containing a solution of cholesterol (1.16 g, 3.00 mmol) and DMAP (367 mg, 3.00 mmol) in dichloromethane (15 ml). The reaction mixture was stirred for 2 hours and then mixed with aqueous potassium hydrogensulfate solution. The phases were separated and the organic phase was extracted with chloroform. The combined organic phases were washed with aqueous potassium hydrogensulfate solution and water and dried (MgSO ₄ ). Filtration and concentration and the crude product was purified by column chromatography (silica, chlorolom / methanol 99: 1) to give 1.63 g of a white solid in 97% yield. ^The structure was confirmed by ¹ H NMR (500 MHz).
b) Synthesis of Cholesteryl β-alanine Hydrochloride
A solution of a) (279 mg, 0.500 mmol) from a) in 1 M hydrochloric acid in 1,4-dioxane (5 ml) was stirred at room temperature for 4 hours. The reaction mixture was concentrated to give a large amount of cholesteryl β-alanine hydrochloride. ^The structure was confirmed using ¹ H NMR (500 MHz) analysis and MALDI mass spectrometry to obtain an M + Ha peak at 482 (expected 481).
c) Biotin-PEG _3400- β-Ala-cholesterol
Triethylamine (42 μl, 0.30 mmol) was added to a solution of cholesteryl β-alanine hydrochloride (15 mg, 0.03 mmol) in chloroform / liquid methanol (2.6: 1, 3 ml). The mixture was stirred at rt for 10 min and a solution of biotin-PEG ₃₄₀₀ -NHS (100 mg, 0.03 mmol) in 1,4-dioxane (1 ml) was added dropwise. After stirring at room temperature for 3 hours, the mixture was evaporated to dryness and the residue was purified by flash chromatography to give 102 mg (89% yield) of white crystals. The structure was verified using NMR analysis and MALDI mass spectroscopy.
d) Synthesis of cholesteryl 4- [4- [bis (2-chloroethyl) amino] phenyl] butanoate
DIC (170 μl, 1.10 mmol) was added to a solution of chlorambucil (sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15 ml). This mixture was stirred for 0.5 h at room temperature and added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction mixture was stirred overnight and poured into 5% sodium bicarbonate. The phases were separated and the organic phase was washed with brine and dried (MgSO ₄ ). The solution was filtered and concentrated, and the product was purified by column chromatography (silica, chlorolol) to give 560 mg of a colorless oil in 83% yield. The product was characterized using MALDI mass spectroscopy to yield M + H at 674 as expected. In addition, ¹ H (500 MHz) and ¹³ C (125 MHz) NMR assays were used to characterize and verify the structure.
e) preparation of gas-filled microbubbles
A mixture of DSPS (Avanti, 5.0 mg), product from c) (0.5 mg) and product from d) (0.5 mg) is placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution is added to this vial. It was. The mixture was sonicated for 5 minutes, warmed at 80 ° C. for 5 minutes (stirring while warming) and cooled. The upper space was flushed with perfluorobutane gas and the vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. No starting material was observed in the final wash as evidenced by MALDI mass spectrometry.
The introduction of compounds from c) and d) into the bubbles was confirmed by MALDI mass spectrometry as described in Example 1 b).
<Example 30>
Gas-filled microbubbles of DSPS including lipopeptides containing chlorambucil for diagnostic and therapeutic uses
This example relates to the preparation of functionalized microbubbles with nonspecific affinity for the multiplicity of cell surface molecules.
a) Preparation of Lipopeptides Containing Chlorambucil
The structure was synthesized using a manual nitrogen foamer starting with Fmoc protected link amide MBHA resin (Nova Biochem) on the 0.125 mmol scale. Standard amino acids were purchased from Nova Biochem and palmitic acid were purchased from Fluka. Coupling was performed using standard TBTU / HOBt / DIEA protocol. Chlorambucil (Sigma) was coupled through the side chain of Lys as symmetric anhydride using DIC preactivation.
Deprotection of the peptide and side chain protecting groups from the resin was carried out simultaneously for 2 hours in TFA containing 5% EDT, 5% H ₂ O and 5% ethyl methyl sulfide. Crude liquid chromatography (Vydac 218TP1022 column) over 60 minutes using a gradient of 70 to 100% B (A = 0.1% TFA / water and B = 0.1% TFA / acetonitrile) at a flow rate of 10 mL / min. A 10 mg aliquot of the vaginal product was purified. 30 mg of pure material was obtained after dry freezing (analytical HPLC; gradient, 70-100% B, where B = 0.1% TFA / acetonitrile, A = 0.1% TFA / water, flow rate of 1 mL / min, column-Vydac 218TP54: detection-UV 214 nm-retention time = 26.5 minutes). Further characterization of the product was performed using MALDI mass spectroscopy; M + H 1295, expected 1294.
b) Preparation of gas-filled microbubbles comprising lipopeptides containing chlorampusyl for diagnostic and therapeutic uses
A mixture of DSPS (Avanti, 4.5 mg) and product from a) (0.5 mg) was placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to this vial. The mixture was sonicated for 5 minutes, warmed at 80 ° C. for 5 minutes (stirring while warming) and cooled. The upper space was flushed with perfluorobutane gas and the vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. No compound from a) was observed in the final wash in MALDI mass spectrometry.
The introduction of lipopeptide containing chlorambucil into microbubbles was confirmed by the following MALDI mass spectrometry: About 50 μl of microbubbles were transferred to a clean vial containing about 100 μl of 90% methanol. The mixture was sonicated for 30 seconds and analyzed by MALDI mass spectrometry (ACH-matrix) and found M + H peak, expected 1294 and M + Ha peak 1324, expected 1317 at 1300.
c) in vitro studies of gas-containing microbubbles of DSPS 'doped' with lipopeptides containing chlorambucil
Microbubbles were evaluated using the in vitro flow assay described in Example 1 c). Gradual accumulation of microbubbles on the cells occurred, depending on the flow rate. As the flow rate increased, the cells began to detach from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
<Example 31>
Gas-filled microbubbles of DSPS including lipopeptides containing athenol and lipophilic derivatives of captopril for diagnostic and therapeutic uses
a) Synthesis of Protected Athenol Derivatives Suitable for Solid Phase Coupling
As described in Example 25 b).
b) Synthesis of N-[(S) -3-hexadecylthio-2-methylpropionyl] proline
DIEA (188 μl, 1.10 mmol) was added to a solution of iodohexadecane (176 mg, 0.500 mmol), captopril (120 mg, 0.550 mmol) and DBU (165 μl, 1.10 mmol) in tetrahydrofuran (5 ml). Was added. This mixture was heated at 70 ° C. for 2 hours and concentrated. The residue was poured into water saturated with potassium hydrogensulfate and the organics were extracted with chloroform. The organic phase was washed with water and dried (MgSO ₄ ). The product was purified by chromatography (silica, CHCl ₃ / MeOH / AcOH 85: 10: 5) and lyophilized to give 105 mg (48%) of a white solid. Proven with ¹ H (500 MHz) and ¹³ C NMR (125 MHz) spectra and further characterized using MALDI mass spectroscopy to obtain MH in negative mode at m / z 440 as expected.
c) Preparation of gas-filled microbubbles of DSPS comprising lipopeptides containing athenol and lipophilic derivatives of captopril for diagnostic and therapeutic uses
A mixture of products from DSPS (Avanti, 4.5 mg), b) (0.5 mg) and c) was placed in a vial and 1.0 ml of 1.4% propylene glycol / 2.4% glycerol solution was added to this vial. The mixture was sonicated for 5 minutes, warmed at 80 ° C. for 5 minutes (stirring while warming) and cooled. The upper space was flushed with perfluorobutane gas and the vial was shaken for 45 seconds in a cap mixer and washed well with deionized water. No compound of b) or c) containing lipopeptides was observed in the final wash in MALDI mass spectrometry. As described in Example 1 b), the introduction of the compound of b) or c) containing lipopeptides into the bubble was confirmed by MALDI mass spectrometry.
d) In vitro studies of gas-containing microbubbles of DSPS comprising lipopeptides containing athenol and lipophilic derivatives of captopril for diagnostic and therapeutic uses
Microbubbles were evaluated using the in vitro flow assay described in Example 1 c). Gradual accumulation of microbubbles on the cells occurred, depending on the flow rate. As the flow rate increased, the cells began to detach from the coverslip and the microbubbles were still bound to the cells. Control bubbles without vector did not adhere to endothelial cells and disappeared from cells under minimal flow conditions.
<Example 32>
Floatation of endothelial cells by DSPS microbubbles containing multi-specific lipopeptides that bind to endothelial cells
This example was performed to show that the present invention can be used for cell isolation.
Human endothelial cell line ECV 304, derived from normal umbilical cord (ATCC CRL-1998), was treated with 200 mM L-glutamine, penicillin / streptomycin (10.000 U / ml and 10.000 mcg / ml) and 10% fetal bovine serum ( Hyclone Lot no.AFE 5183) was incubated in a Nunc culture flask (chutney 153732) in RPMI 1640 medium (Bio Whitaker). After trypsinization, cells were secondary cultured at a split ratio of 1: 5 to 1: 7. Two mills of cells from the trypsinated confluent culture were added to five sets of centrifuge tubes and DSPS doped with control microbubbles of DSPS, microbubbles of Example 1 or endothelial cell binding lipopeptides from Example 14 a) Microbubbles of 2, 4, 6, 8 or 10 mill bubbles were added per tube. After centrifugation at 400 g for 5 minutes, the cells were counted with a cell cooler counter at the bottom of the tube. It has been found that binding of four or more microbubbles to cells causes floating. In addition, all cells were suspended by endothelial cell binding lipopeptides bubbles, while about 50% were suspended with the microbubbles from Example 1.
<Example 33>
Gene Delivery of PFB Gas-Filled Microbubbles
This example relates to the preparation of targeted microbubbles for gene delivery.
a) Preparation of DSPS Lipopeptide Bubble / PFB Gas Coated with Poly-L-Lysine
Lipopeptides (0.5 ml) from DSPS (Avanti, 4.5 mg) and 17 b) were weighed and placed in two 2 ml vials. 0.8 ml of propylene glycol / glycerol (4%) in water was added to each vial. This solution was heated to 80 ° C. for 5 minutes and shaken. The solution was then cooled to room temperature and the upper space was flushed with perfluorobutane gas. The vial was shaken for 45 seconds in a Capmixer (Espe Capmix, 4450 vibrations / minute) and placed on a roller table for 5 minutes. The contents in the vial were mixed and the sample was centrifuged at 2000 rpm for 5 minutes and washed. The precipitate was removed and the same amount of distilled water was added. The washing process was repeated once.
Poly-L-lysine HBr (Sigma, 20.6 mg) was dissolved in 2 ml of water and aliquots (0.4 ml) were made up to 2 ml of water. 1.2 ml of diluted poly-L-lysine solution was added to 0.12 ml of DSPS-lipopeptide bubble suspension. After incubation, excess polylysine was removed by washing well with water.
b) transfection of cells
Endothelial cells (ECV 304) were incubated in a homogeneous matching layer in six wells. A 50 μl microbubble suspension from 5 μg DNA (green increased fluorescent protein vector from CLONTECH) and a) in RPMI medium was prepared with a final volume of 250 μl. The mixture was left at room temperature for 15 minutes, then 1 ml of complete RPMI medium was added. The medium was removed from the cell culture dish and the DNA-microbubble mixture was added to the cells.
c) ultrasonic treatment
After 15 min incubation, the selected wells were exposed to 1 MHz, 0.5 W / cm ² continuous wavelength ultrasound for 30 seconds.
Cells were cultured in a cell incubator (37 ° C.).
d) incubation and inspection
The cells were further incubated for about 4 hours 30 minutes in a cell culture incubator (37 ° C.). Thereafter, the medium containing the DNA-microbubbles was removed by suction, and 2 ml of complete RPMI medium was added. Cells were incubated for 40-70 hours before testing. Thereafter, most of the medium was removed and the cells examined by fluorescence microscopy. The results were compared with the results from control experiments in which DNA or DNA-polylysine was added to the cells.

权利要求:
Claims (38)
[1" claim-type="Currently amended] A target traceable diagnostic and / or therapeutically active agent comprising a suspension of a reporter consisting of a gas-containing or gas-generating material in an aqueous carrier liquid, wherein the target is capable of forming two or more types of binding pairs with the target.
[2" claim-type="Currently amended] The process of claim 1 wherein the gas comprises air, nitrogen, oxygen, carbon dioxide, hydrogen, inert gas, sulfur fluoride, hexafluoride, low molecular weight hydrocarbons, ketones, esters, halogenated low molecular weight hydrocarbons or any mixture thereof. Diagnostic and / or therapeutically active agent.
[3" claim-type="Currently amended] 3. The diagnostic and / or therapeutically active agent of claim 2, wherein the gas comprises a perfluorinated ketone, a perfluorinated ether or a perfluorocarbon.
[4" claim-type="Currently amended] The diagnostic and / or therapeutically active agent of claim 2, wherein the gas comprises sulfur hexafluoride or perfluoropropane, perfluorobutane or perfluoropentane.
[5" claim-type="Currently amended] The gas microbubble according to claim 1, comprising a gas microbubble stabilized with a coagulation resistant surface membrane, a filmogen protein, a polymeric material, a non-polymerizable and nonpolymerizable wall-forming material or a surfactant. Diagnostic and / or therapeutically active agents.
[6" claim-type="Currently amended] 6. The diagnostic and / or therapeutically active agent of claim 5, wherein the surfactant comprises one or more phospholipids.
[7" claim-type="Currently amended] 7. The diagnostic and / or therapeutically active agent according to claim 6, wherein at least 75% of the surfactant substance comprises phospholipid molecules individually carrying a total net charge.
[8" claim-type="Currently amended] The diagnostic and / or method according to claim 7, wherein at least 75% of the film-forming surfactant substance comprises at least one phospholipid selected from phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and cardiolipin. Therapeutically active agent.
[9" claim-type="Currently amended] 9. The diagnostic and / or therapeutically active agent of claim 8, wherein at least 80% of the phospholipids comprise phosphatidylserine.
[10" claim-type="Currently amended] 10. The diagnostic method according to any one of claims 1 to 9, wherein the gas-containing or gas-generating material is bound to two or more vectors or to one vector that is capable of binding to two or more binding sites. Or) therapeutically active agent.
[11" claim-type="Currently amended] The method of claim 1, wherein the gas-containing or gas-generating substance is bound to one or more target vectors having affinity for one or more cell surface receptors, and to induce a therapeutic response. A diagnostic and / or therapeutically active agent further comprising a moiety capable of binding to the receptor system.
[12" claim-type="Currently amended] The method of claim 1, wherein the vector (s) is an antibody; Cell adhesion molecules; Cell adhesion molecule receptors; Cytokines; Growth factor; Peptide hormones and fragments thereof; Non-peptide agonists / antagonists and non-living binders of receptors for cell adhesion molecules, cytokines, growth factors and peptide hormones; Oligonucleotides and modified oligonucleotides; DNA-binding drugs; Protease substrate / inhibitors; Molecules generated from combinatorial libraries; Biomolecules; And proteins and peptides that bind to cell-surface proteoglycans.
[13" claim-type="Currently amended] 13. The diagnostic and / or method of any one of claims 1 to 12, wherein the vector (s) have affinity for the target at a concentration at which the diagnostic / treatment agent interacts but does not bind statically to the target. Therapeutically active agent.
[14" claim-type="Currently amended] The diagnostic and / or therapeutically active agent of claim 13, wherein the vector (s) is selected from a ligand for cell adhesion protein and a cell adhesion protein having a corresponding ligand on the endothelial cell surface.
[15" claim-type="Currently amended] The diagnostic and / or therapeutically active agent of claim 1, wherein the vector (s) are positioned such that they are not easily exposed to the target.
[16" claim-type="Currently amended] The diagnostic and / or therapeutic according to any one of claims 1 to 15, wherein the vector is coupled or linked to the reporter by avidin-biotin and / or streptavidin-biotin interactions. Active agent.
[17" claim-type="Currently amended] 16. The diagnostic and / or therapeutically active agent according to any one of claims 1 to 15, wherein the vector (s) is covalently coupled or linked to the reporter.
[18" claim-type="Currently amended] 16. The diagnostic and / or therapeutically active agent according to any one of claims 1 to 15, wherein the vector (s) can be noncovalently coupled or linked to the reporter via electrostatic interaction.
[19" claim-type="Currently amended] 19. The diagnostic and / or therapeutically active agent of any one of claims 1 to 18, further comprising a moiety that is radioactive or effective as an X-ray contrast agent, photoimaging probe or spin label.
[20" claim-type="Currently amended] 20. The diagnostic and / or therapeutically active agent according to any one of claims 1 to 19, further comprising a therapeutic compound.
[21" claim-type="Currently amended] The method of claim 20, wherein the therapeutic compound is an antineoplastic agent, blood product, biological response modifier, antifungal agent, hormone or hormonal homologue, vitamin, enzyme, antiallergic agent, tissue factor inhibitor, platelet inhibitor, aggregate protein target inhibitor: fibrin formation Inhibitors, fibrin breakdown accelerators, anti-angiogenic agents, circulatory drugs, metabolic enhancers, anti-nodal agents, antiviral agents, vasodilators, antibiotics, anti-inflammatory agents, antiprotozoal agents, antirheumatic agents, anesthetics, opiates, cardiac glycosides, neuromuscular A diagnostic and / or therapeutically active agent characterized in that it is a growth blocker, a sedative, a local anesthetic, a general anesthetic or a genetic material.
[22" claim-type="Currently amended] 24. The diagnostic and / or therapeutically active agent of claim 20 or 23, wherein the therapeutic compound is covalently coupled or linked to the reporter via a disulfide group.
[23" claim-type="Currently amended] 22. A diagnostic and / or therapeutically active agent according to claim 20 or 21, wherein the lipophilic or lipophilic induction therapeutic compound is linked to the reporter via a hydrophobic interaction.
[24" claim-type="Currently amended] i) a first dosage composition comprising a pretargeting vector having affinity for the selected target; And
ii) comprising a second dosage composition comprising a diagnostic and / or therapeutically active agent as claimed in claim 1 comprising a vector having affinity for said pretargeting vector. Characterized in combination formulation.
[25" claim-type="Currently amended] The formulation of claim 24, wherein the pretargeting vector is a monoclonal antibody.
[26" claim-type="Currently amended] i) a first dosage composition comprising a diagnostic and / or therapeutically active agent as claimed in claim 1; And
ii) a second dosage composition comprising a substance capable of replacing or leaving said diagnostic and / or therapeutically active agent from its target.
[27" claim-type="Currently amended] i) a first dosage composition comprising the diagnostic and / or therapeutically active agent as claimed in claim 22; And
ii) a second dosage composition comprising a reducing agent capable of reductively cleaving a disulfide group that couples or links the therapeutic compound and reporter in the diagnostic and / or therapeutically active agent of the first dosage composition. Formulation formulation to be.
[28" claim-type="Currently amended] And a target traceable diagnosis as defined in claim 1, comprising coupling or connecting to a reporter comprising a gas-containing or gas-generating material and capable of forming two or more types of binding pairs with the target. (Or) a method of making a therapeutically active agent.
[29" claim-type="Currently amended] The method of claim 28, wherein the therapeutic compound is also combined with a reporter.
[30" claim-type="Currently amended] 24. Use of a diagnostic and / or therapeutically active agent as claimed in any one of claims 1 to 23 as a target traceable ultrasound contrast agent.
[31" claim-type="Currently amended] 24. A diagnostic and / or therapeutically active agent as claimed in any one of claims 1 to 23 is administered to a human or non-human animal and ultrasound, magnetic resonance, X-ray, radiation in at least a portion of the human or animal body. A method for generating an augmented image of a human or non-human animal, comprising generating a photographic or optical image.
[32" claim-type="Currently amended] The method of claim 31, wherein
i) administering a pretargeting vector having affinity for the selected target to the human or animal body; And
ii) administering the diagnostic and / or therapeutically active agent of any one of claims 1 to 23 comprising a vector having affinity for said pretargeting vector.
[33" claim-type="Currently amended] 33. The method of claim 32, wherein the pretargeting vector comprises a monoclonal antibody.
[34" claim-type="Currently amended] The method of claim 31, wherein
i) administering the diagnostic and / or therapeutically active agent of any one of claims 1 to 23 to the human or animal body; And
ii) administering a substance capable of replacing or leaving said diagnostic and / or therapeutically active agent from its target.
[35" claim-type="Currently amended] 35. The method of any one of claims 31-34, wherein the diagnostic and / or therapeutically active agent further comprises a therapeutic compound.
[36" claim-type="Currently amended] 36. The composition of claim 35, wherein the therapeutic compound is covalently coupled or linked to the reporter via a disulfide group, and subsequently a composition is administered comprising a reducing agent capable of reductively cleaving the disulfide group. Way.
[37" claim-type="Currently amended] A cell representing the target is fixedly positioned in the flow chamber, and a suspension of the diagnostic and / or therapeutically active agent as defined in any one of claims 1 to 23 in a carrier solution is passed through the chamber, A method for testing in vitro target traceability by a diagnostic and / or therapeutically active agent, comprising testing the binding of said diagnostic and / or therapeutically active agent to a diagnostic agent.
[38" claim-type="Currently amended] 38. The method of claim 37, wherein the flow rate of the carrier liquid is adjusted to indicate the shear rate occurring in vivo.

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同族专利:

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公开号 | 公开日

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CN1238700A|1999-12-15|

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WO1998018500A3|1998-07-23|

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NO991890L|1999-06-28|

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JP2001511765A|2001-08-14|

---

AU733477B2|2001-05-17|

---

EP1007101B1|2006-05-17|

---

NZ335799A|2000-11-24|

---

WO1998018500A8|1999-11-25|

---

BG103439A|2000-01-31|

---

AU4718297A|1998-05-22|

---

AT326242T|2006-06-15|

---

CA2269985A1|1998-05-07|

---

WO1998018500A2|1998-05-07|

---

EP1007101A2|2000-06-14|

---

DE69735901T2|2007-05-10|

---

DE69735901D1|2006-06-22|

---

BR9713978A|2000-05-02|

---

IL129445D0|2000-02-29|

---

HU0000357A2|2000-06-28|

---

NO991890D0|1999-04-21|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-10-28|Priority to GBGB9622366.4A

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1996-10-28|Priority to GBGB9622369.8A

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1996-10-28|Priority to GB9622366.4

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1996-10-28|Priority to GB9622369.8

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1997-02-04|Priority to GB9702195.0

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1997-02-04|Priority to GBGB9702195.0A

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1997-04-24|Priority to GBGB9708265.5A

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1997-04-24|Priority to GB9708265.5

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1997-06-06|Priority to GBGB9711839.2A

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1997-06-06|Priority to US4926497P

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1997-06-06|Priority to US4926697P

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1997-06-06|Priority to US4926397P

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1997-06-06|Priority to GBGB9711837.6A

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1997-06-06|Priority to GB9711837.6

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1997-06-06|Priority to GB9711839.2

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1997-10-28|Application filed by 조오지 디빈센조, 토브 아스 헬지, 에바 요한손, 니코메드 이메이징 에이에스

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1997-10-28|Priority to PCT/GB1997/002953

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2000-08-25|Publication of KR20000052830A

优先权:

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申请号 | 申请日 | 专利标题

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GBGB9622366.4A|GB9622366D0|1996-10-28|1996-10-28|Improvements in or relating to diagnostic/therapeutic agents|

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GBGB9622369.8A|GB9622369D0|1996-10-28|1996-10-28|Improvements in or relating to diagnostic/therapeutic agents|

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GB9622366.4|1996-10-28|

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GB9622369.8|1996-10-28|

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GBGB9702195.0A|GB9702195D0|1997-02-04|1997-02-04|Improvements in or relating to diagnostic/therapeutic agents|

---

GB9702195.0|1997-02-04|

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GB9708265.5|1997-04-24|

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GBGB9708265.5A|GB9708265D0|1997-04-24|1997-04-24|Contrast agents|

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US4926497P| true| 1997-06-06|1997-06-06|

---

US4926697P| true| 1997-06-06|1997-06-06|

---

US4926397P| true| 1997-06-06|1997-06-06|

---

GBGB9711837.6A|GB9711837D0|1997-06-06|1997-06-06|Improvements in or relating to diagnostic / therapeutic agents|

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GB9711837.6|1997-06-06|

---

GB9711839.2|1997-06-06|

---

GBGB9711839.2A|GB9711839D0|1997-06-06|1997-06-06|Improvements in or relating to diagnostic/therapeutic agents|

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PCT/GB1997/002953|WO1998018500A2|1996-10-28|1997-10-28|Improvements in or relating to diagnostic/therapeutic agents|