Patent Application
20140079635
This application relates to molecular probes and to methods of their use, and particularly relates to molecular probes that can bind to lipids and lipid matter and be used to detect and/or image lipids and lipid matter, such as myelin lipid and sphingolipid.
Myelin is a specialized membrane that ensheathes neuronal axons, promoting efficient nerve impulse transmission (Morell and Quarles (1999) Basic Neurochemistry: molecular, cellular, and medical aspects. In Siegel G J, ed. Myelin Formation, Structure, and Biochemistry. Lippincott-Raven Publishers, 79-83). Due to its important biological functions in the normal central nervous system (CNS) and its vulnerability in disease, several techniques have been developed to visualize and characterize myelin histopathology. These can be broadly divided into those based upon antibody immunohistochemistry (IHC) (Horton and Hocking (1997) Cereb. Cortex 7:166-177) and more traditional histochemical procedures. The classic histochemical stains include luxol fast blue MBN (Kluver and Barrera (1953) J Neurosci Methods 153: 135-146; Presnell and Schreibman (1997) Humanson's Animal Tissue Techniques, 5th ed.; Kiernan (1999) Histological and Histochemical Methods Theory and practice, 3rd ed.; Bancroft and Gamble (2002), Theory and Practice of Histological Techniques, 5 ed. and Sudan Black B (Lison and Dagnelie (1935) Bull. d'Histologie Appliquee 12: 85-91). Traditional chromogenic methods also include the Palweigert method ((Weigert (1884) Fortschr Deutsch Med 2: 190-192, (1885) Fortschr Deutsch Med 3:236-239; Clark and Ward (1934) Stain Technol 54:13-16), the Weil stain (Weil (1928) Arch Neurol Psychiatry 20:392-393; Berube et al. (1965) Stain Technol 40:53-62)), the Loyez method (Cook (1974) Manual of Histological Demonstration Methods, 5th ed.), and a method based on horse serum followed by subsequent reaction with diaminobenzidine (McNally and Peters (1998) J Histochem Cytochem 46:541-545). In addition, modified silver stains including the Gallyas method (Pistorio et al. (2005) J Neurosci Methods 153: 135-146) and Schmued's gold chloride technique (Schmued and Slikker (1999) Brain Res 837:289-297) have also been used as simple, high-resolution histochemical markers of myelin. More recently, fluoromyelin (Kanaan et al. (2005) Am J Physiol Regul Integr Comp Physiol 290:R1105-1114) and NIM (Xiang et al. (2005) J Histochem Cytochem 53:1511-1516) were introduced as novel myelin dyes, which enable quick and selective labeling of myelin in brain tissue sections. Although these myelin-staining techniques are widely used in vitro, none can be applied in vivo due to impermeability of the blood-brain barrier (BBB). The lack of in vivo molecular probes has limited the progress of myelin imaging and hindered efficacy evaluation of novel myelin repair therapies during their development.
Embodiments described herein relate to molecular probes that can selectively bind to lipids, including lipid matter, such as myelin, and be used in the detection and/or imaging of lipids or lipid matter, such as myelin, in a subject. The molecular probes can include a compound having the formula:
wherein X1 is a double bond, a triple bond, two or three conjugated double or triple bonds, or a combination of two or three conjugated double bonds and triple bonds; A1 and A2 are each independently C or N; each R1-R2 and R4-R13 is independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an aryloxy group, an alkaryl group, an aralkyl group, O, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group); wherein R10 and R11 and/or R12 and R13 may be linked to form a cyclic ring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic; wherein Z1-Z12, each independently, represent C, S, O, or N, but is not O or S if attached by a double bond to another such Z or if attached to another such Z which is O or S, and is not N if attached by a single bond to another such Z, which is N; or a pharmaceutically acceptable salt thereof.
The molecular probe can further include a radiolabel. The radiolabel can include at least one of 3H, 125I, 124I, 11C, or 18F. F. The molecular probe can optionally or additionally include a chelating group or a near infrared imaging group.
In some embodiments, the molecular probe can be used to detect lipids in a subject, associated with aberrant lipid accumulation (e.g., GL-3) in tissue. The aberrant lipid accumulation can be the result of a lysosomal storage disease, such as Fabry disease. In other embodiments, the molecular probe can be used in a method to screen agents that can treat aberrant lipid accumulation associated with lysosomal storage disease.
In other embodiments, the molecular probe can be used to detect and/or image lipid matter, such as myelin, in a subject. In one example, the molecular probe can readily enter the brain following systemic or parenteral administration and bind to lipids of myelin membranes. In other examples, the molecular probe can be administered systemically, locally, or topically to a subject to visualize myelin or myelinated nerves in a subject's peripheral nervous system.
The foregoing and other features and advantages of the present invention will become apparent to those skilled in the art to which the present invention relates upon reading the following description with reference to the accompanying drawings, in which:
The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups, such as cyclopentyl, cyclohexyl, and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 18 carbon atoms, preferably 1 to about 12 carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms. Substituents identified as “C1-C6 alkyl” or “lower alkyl” can contain 1 to 3 carbon atoms, and more particularly such substituents can contain 1 or 2 carbon atoms (i.e., methyl and ethyl). “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
The term “alkenyl” refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although again not necessarily, alkenyl groups can contain 2 to about 18 carbon atoms, and more particularly 2 to 12 carbon atoms. The term “lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, and the specific term “cycloalkenyl” intends a cyclic alkenyl group, preferably having 5 to 8 carbon atoms. The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkenyl” and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
The term “alkynyl” refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups can contain 2 to about 18 carbon atoms, and more particularly can contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms. The term “substituted alkynyl” refers to alkynyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkynyl” and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkynyl” and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Preferred substituents identified as “C1-C6 alkoxy” or “lower alkoxy” herein contain 1 to 3 carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).
The term “aryl” refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups can contain 5 to 20 carbon atoms, and particularly preferred aryl groups can contain 5 to 14 carbon atoms. Exemplary aryl groups contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the terms “heteroatom-containing aryl” and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
The term “aryloxy” as used herein refers to an aryl group bound through a single, terminal ether linkage, wherein “aryl” is as defined above. An “aryloxy” group may be represented as —O-aryl where aryl is as defined above. Preferred aryloxy groups contain 5 to 20 carbon atoms, and particularly preferred aryloxy groups contain 5 to 14 carbon atoms. Examples of aryloxy groups include, without limitation, phenoxy, o-halo-phenoxy, m-halo-phenoxy, p-halo-phenoxy, o-methoxy-phenoxy, m-methoxy-phenoxy, p-methoxy-phenoxy, 2,4-dimethoxy-phenoxy, 3,4,5-trimethoxy-phenoxy, and the like.
The term “alkaryl” refers to an aryl group with an alkyl substituent, and the term “aralkyl” refers to an alkyl group with an aryl substituent, wherein “aryl” and “alkyl” are as defined above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, and particularly preferred aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-1,4-diene, and the like.
As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
The term “cyclic” refers to alicyclic or aromatic substituents that may or may not be substituted and/or heteroatom containing, and that may be monocyclic, bicyclic, or polycyclic.
The terms “comprise,” “comprising,” “include,” “including,” “have,” and “having” are used in the inclusive, open sense, meaning that additional elements may be included. The terms “such as”, “e.g.”, as used herein are non-limiting and are for illustrative purposes only. “Including” and “including but not limited to” are used interchangeably.
As defined herein, the term “derivative”, refers to compounds that have a common core structure, and are substituted with various groups as described herein.
When referring to the terms “fluorescent trans-stilbene” or “fluorescent trans-stilbene derivative” or “fluorescent trans-stilbene compound” in the specification and the claims, it is intended that the terms encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates, active metabolites, and other such derivatives, analogs, and related compounds.
The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.
The phrase “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used.
The term “heteroatom-containing” as in a “heteroatom-containing alkyl group” (also termed a “heteroalkyl” group) or a “heteroatom-containing aryl group” (also termed a “heteroaryl” group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term “heteroalkyl” refers to an alkyl substituent that is heteroatom-containing, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, the terms “heteroaryl” and heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, etc.
The term “or” as used herein should be understood to mean “and/or”, unless the context clearly indicates otherwise.
The phrases “parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
By “substituted” as in “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.
When the term “substituted” appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase “substituted alkyl, alkenyl, and aryl” is to be interpreted as “substituted alkyl, substituted alkenyl, and substituted aryl.” Analogously, when the term “heteroatom-containing” appears prior to a list of possible heteroatom-containing groups, it is intended that the term apply to every member of that group. For example, the phrase “heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as “heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.”
“Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
The term “Fabry disease” refers to an X-linked inborn error of glycosphingolipid catabolism due to deficient lysosomal α-galactosidase A activity. This defect causes accumulation of globotriaosylceramide (ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes of the heart, kidneys, skin, and other tissues.
The term “atypical Fabry disease” refers to patients with primarily cardiac manifestations of the α-GAL deficiency, namely progressive globotriaosylceramide (GL-3) accumulation in myocardial cells that leads to significant enlargement of the heart, particularly the left ventricle.
A “carrier” is a female who has one X chromosome with a defective α-GAL gene and one X chromosome with the normal gene and in whom X chromosome inactivation of the normal allele is present in one or more cell types. A carrier is often afflicted with Fabry disease.
A “patient” or “subject” refers to a subject who has been diagnosed with a particular disease. The patient may be human or animal. A “Fabry disease patient” refers to an individual who has been diagnosed with Fabry disease and has a mutated α-GAL as defined further below. Characteristic markers of Fabry disease can occur in male hemizygotes and female carriers with the same prevalence, although females typically are less severely affected.
Human α-galactosidase A (α-GAL) refers to an enzyme encoded by the human Gla gene. The human α-GAL enzyme consists of 429 amino acids and is in GenBank Accession No. U78027.
As used herein in one embodiment, the term “mutant α-GAL” includes an α-GAL which has a mutation in the gene encoding α-GAL which results in the inability of the enzyme to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome. Such a mutation is sometimes called a “conformational mutant.”
Non-limiting, exemplary α-GAL mutations associated with Fabry disease which result in unstable α-GAL include L32P; N34S; T41I; M51K; E59K; E66Q; 191T; A97V; R100K; R112C; R112H; F113L; T141L; A143T; G144V; S148N; A156V; L166V; D170V; C172Y; G183D; P205T; Y207C; Y207S; N215S; A228P; S235C; D244N; P259R; N263S; N264A; G272S; S276G; Q279E; Q279K; Q279H; M284T; W287C; 1289F; M296I; M296V; L300P; R301Q; V316E; N320Y; G325D; G328A; R342Q; E358A; E358K; R363C; R363H; G370S; and P409A.
By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration. “Pharmacologically active” (or simply “active”) as in a “pharmacologically active” derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
As used herein, the term “pharmaceutically acceptable salts” or complexes refers to salts or complexes that retain the desired biological activity of the parent compound and exhibit minimal, if any, undesired toxicological effects. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, and polygalacturonic acid; (b) base addition salts formed with cations such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinc tannate salt or the like.
Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
All percentages and ratios used herein, unless otherwise indicated, are by weight.
This application related to molecular probes that can selectively bind to, detect, and/or image lipids or lipid matter, such as myelin, in a subject. In some embodiments, the molecular probes can bind to lipids that are associated with aberrant lipid accumulation in (or of) a cell, tissue, and/or organ. The molecular probes upon binding to the lipids in the cell, tissue, and/or organ can be detected or imaged to determine or quantify aberrant lipid accumulation in the cell, tissue, and/or organ of a subject. The aberrant lipid accumulation can be associated with a lysosomal or lipid storage disease or disorder, such as Gaucher disease, Niemann-Pick disease, Farber's disease, gangliosidoses, GM2 disorders, Krabbe disease, Metachromatic leukodystrophy, Wolman's disease, and Fabry disease. For example, the molecular probe can bind to globotriaosylceramide in tissue (kidneys, heart, skin, vasculature, etc.) of a subject that is formed as a result of a deficiency of the enzyme alpha galactosidase A, which is associated with Fabry disease.
The molecular probes can be administered in vitro, ex vivo, in vivo to a cell, tissue, and/or organ of the subject and upon binding to the lipid or lipid matter be readily visualized using conventional visualization techniques to indicate lipid presence and/or lipid accumulation in the cell, tissue, or organ of the subject including the heart, kidneys, skin, central and/or peripheral nervous system, and/or vasculature of the subject. In some aspects, the molecular probes can be used in a method of detecting aberrant lipid accumulation in vivo in a subject. In other aspects, the molecular probes can be used in a method of detecting aberrant lipid accumulation associated with a lysosomal storage disease (e.g., Fabry disease). In still other aspects, the molecular probes can be used in a method of screening agents for inhibiting aberrant lipid accumulation associated with a lysosomal storage disease. In yet other aspects, the molecular probes can be used for measuring the efficacy of an agent or therapy in inhibiting aberrant lipid accumulation in a subject.
In other embodiments, the molecular probes can target and selectively bind to lipid matter, such as myelin, and be used detect and/or image myelin in a subject. The molecular probes described herein upon administration to a mammal (e.g., systemic, parenteral, intravenous, topical, local administration) can readily and selectively localize to myelinated regions of the brain, central nervous system, and peripheral nervous system. The molecular probes can bind to myelin membrane and do not bind to a component of degenerating myelin fragments. The molecular probes can also be readily visualized using conventional visualization techniques to indicate myelinated regions of the brain, central nervous system, and peripheral nervous system. The molecular probes can be used in a method of detecting a level of myelination in vivo in a subject, a method of detecting a myelin related disorder in a subject, a method of monitoring the remyelination effects of an agent in an animal, and a method of screening the myelination effects of an agent in an animal.
The molecular probes can include a fluorescent stilbene derivative or a pharmacophore or analog thereof (e.g., coumarin pharmacophore) that is less than about 700 daltons and has a relatively high binding affinity (Kd) (e.g., at least about 1.0 nM) to isolated myelin fractions but a relatively low binding affinity (Kd) to isolated non-myelin fractions and/or has a relatively high binding affinity to lipids, such as GL-3, associated with a lysosomal storage disease.
In some embodiments, the molecular probe can include a fluorescent stilbene derivative having the following formula general formula:
wherein X1 is a double bond, a triple bond, two or three conjugated double or triple bonds, or a combination of two or three conjugated double bonds and triple bonds; A1 and A2 are each independently C or N, each R1-R2 and R4-R13 is independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an aryloxy group, an alkaryl group, an aralkyl group, O, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group); wherein R10 and R11 and/or R12 and R13 may be linked to form a cyclic ring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic; wherein Z1-Z12, each independently, represent C, S, O, or N, but is not O or S if attached by a double bond to another such Z or if attached to another such Z which is O or S, and is not N if attached by a single bond to another such Z, which is N; or a pharmaceutically acceptable salt thereof.
In other embodiments, the molecular probe can include a fluorescent trans-stilbene derivative having the following formula:
wherein X1 is a double bond, a triple bond, two or three conjugated double or triple bonds, or a combination of two or three conjugated double bonds and triple bonds; each R1-R2 and R4-R13 is independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an aryloxy group, an alkaryl group, an aralkyl group, O, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group); wherein R10 and R11 and/or R12 and R13 may be linked to form a cyclic ring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic; or a pharmaceutically acceptable salt thereof.
In some embodiment, R1 and/or R2 can be selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof, and each R4-R13 is H.
In one example, the molecular probe can include a fluorescent trans-stilbene derivative having the following formula:
wherein R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof, or a pharmaceutically acceptable salt thereof.
In a further aspect, the molecular probe can include a formula selected from the group consisting of:
and pharmaceutically acceptable salts thereof.
In other embodiments, the molecular probe can include a fluorescent trans-stilbene derivative selected from the group consisting of:
and pharmaceutically acceptable salts thereof.
In another aspect, the molecular probe can include a fluorescent coumarin derivative that is a pharmacophore of trans-stilbene. In an aspect of the invention, R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof, X1 is a double bond, and R10 and R11 are linked to form a heterocylic ring.
In one example, the fluorescent coumarin derivative can be a pharmacophore of trans-stilbene including the following formula:
wherein R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof; and pharmaceutically acceptable salts thereof.
In another example, the fluorescent coumarin derivative can be a pharmacophore of trans-stilbene including the following formula:
wherein R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof; and pharmaceutically salts thereof.
In a further example, the fluorescent coumarin derivative can be a pharmacophore of trans-stilbene including the following formula:
or a pharmaceutically acceptable salt thereof.
In a further example, the fluorescent coumarin derivative can include the formula:
or a pharmaceutically acceptable salt thereof.
In a further example, the fluorescent coumarin derivative can include the formula:
or a pharmaceutically acceptable salt thereof.
In other embodiments, the molecular probe can include the formula:
wherein X1 is a double bond, a triple bond, two or three conjugated double or triple bonds, or a combination of two or three conjugated double bonds and triple bonds; each R1-R2, R4-R8, and R10-R13 is independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an aryloxy group, an alkaryl group, an aralkyl group, O, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group); wherein R10 and R11 and/or R12 and R13 may be linked to form a cyclic ring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic; or a pharmaceutically acceptable salt thereof.
In some embodiment, R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof; and each R4-R8 and R10-R13 is H.
In one example, the molecular probe can include a fluorescent trans-stilbene derivative having the following formula:
wherein R1 and R2 are each independently selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, NO2, NH2, NHCH3, N(CH3)2, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2X, O—CH2—CH2—O—CH2—CH2—O—CH2—CH2X (wherein X═F, Cl, Br, or I), CN, C═O, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group, wherein R′ is H or a lower alkyl group), and alkyl derivatives thereof, alkoxy derivatives thereof; or a pharmaceutically acceptable salt thereof.
In one example, the fluorescent trans-stilbene derivative can include a formula selected from the group consisting of:
and a pharmaceutically acceptable salt thereof.
In other embodiments, the fluorescent stilbene derivative can include a formula selected from the group consisting of:
and
a pharmaceutically acceptable salt thereof.
In still embodiments, the molecular probe can include a formula selected from the group consisting of:
and pharmaceutically acceptable salts thereof.
The foregoing formulae represent the general structures of molecular probes found to be effective molecular probes for labeling lipids and lipid matter, such as myelin, in vivo as well as in vitro as described in the examples below. They are also characterized by their ability to be administered to a mammal or subject parenterally and selectively localize to aberrant lipid or lipid matter in tissues or organs. In some embodiments, the molecular probes can be administered to a mammal or subject parenterally and selectively localize to myelinated regions in the brain, central nervous system, and peripheral nervous system via direct binding to myelin membranes and not bind to degenerating myelin fragments.
The molecular probes are unique in that they exhibit negligible toxicities as demonstrated in both preclinical and clinical settings, making them suitable candidates for clinical imaging modalities and translational studies. In one example, once radiolabelled with positron-emitting radionuclide, they can be used for positron emission tomography to detect and quantify lipid or lipid matter, such myelin, contents in vivo.
Typically, the molecular probe can be formulated into solution prior to use. In one example, a molecular probe solution includes a 10 mM molecular probe solution. A molecular probe solution can also contain saline, DMSO, and HCL. One skilled in the art can utilize the molecular probe with pharmaceutical carriers and/or excipients in varying concentrations and formulations depending on the desired use.
In some embodiments, the molecular probe can be radiolabeled to aid in the detection of the molecular probe once it binds to myelin or a lipid. A ‘radiolabel’ as used herein is any compound that has been joined with a radioactive substance. Examples of radiolabels include positron emitting 3H, 125I, 124I, 11C, and 18F radiolabels.
In other embodiments, the molecular probe can be coupled to a chelating group (with or without a chelated metal group) to improve the MRI contrast properties of the molecular probe. In one example, as disclosed in U.S. Pat. No. 7,351,401, which is herein incorporated by reference in its entirety, the chelating group can be of the form W-L or V-W-L, wherein V is selected from the group consisting of —COO—, —CO—, —CH2O— and —CH2NH—; W is —(CH2)n where n=0, 1, 2, 3, 4, or 5; and L is:
wherein M is selected from the group consisting of Tc and Re; or
wherein each R3 is independently is selected from one of:
or a lipid binding, chelating compound (with or without a chelated metal group) or a water soluble, non-toxic salt thereof of the form:
wherein each R3 independently is selected from one of:
The chelating group can be coupled to at least one terminal benzene groups or the R1 or R2 groups. In one example, the chelating group can be coupled to terminal amino R1 and/or R2 group through a carbon chain link. The carbon chain link can comprise, for example about 2 to about 10 methylene groups and have a formula of, for example, (CH2)n, wherein n=2 to 10.
In one example, a molecular probe with the chelating group can have the following formula:
wherein X3 is a chelating group and n is 2 to 10; or a pharmaceutically acceptable salt thereof.
In another example, the molecular probe with the chelating group can have the following formula:
wherein X3 is a chelating group and n is 2 to 10; or a pharmaceutically acceptable salt thereof.
In another example, the molecular probe with the chelating group can have the following formula:
wherein X3 is a chelating group and n is 2 to 10; or a pharmaceutically acceptable salt thereof.
In another embodiment, the molecular probe can be coupled to a near infrared group to improve the near infrared imaging of the molecular probe. Examples of near infrared imaging groups that can be coupled to the molecular probe include:
These near infrared imaging groups are disclosed in, for example, Tetrahedron Letters 49 (2008) 3395-3399; Angew. Chem. Int. Ed. 2007, 46, 8998-9001; Anal. Chem. 2000, 72, 5907; Nature Biotechnology vol 23, 577-583; Eur Radiol (2003) 13: 195-208; and Cancer 67: 1991 2529-2537, which are herein incorporated by reference in their entirety.
The near infrared imaging group can be coupled to at least one terminal aryl or benzene group or R group. In one example, the near infrared imaging group can be coupled to at least one terminal benzene or aryl group.
In one example, the molecular probe with the near infrared imaging group can have the following formula:
wherein NIR is a near infrared imaging group; or a pharmaceutical salt thereof.
In another example, the molecular probe with the near infrared imaging group can have the following formula:
wherein NIR is a near infrared imaging group; or a pharmaceutical salt thereof.
By way of example, the molecular probe can include a compound having the following formula:
wherein n is 3 to 10; or a salt thereof.
In certain embodiments of the present invention, the molecular probes described herein can be administered to or contact an animal's brain tissue, central nervous system, and/or peripheral nervous system and be utilized for labeling and detecting myelinated regions of an animal's brain tissue, central nervous system, and/or peripheral nervous system. Myelinated regions of an animal's brain are typically found in the white matter of the brain in the myelin sheaths of neuronal axons. Myelin is an outgrowth of glial cells, more specifically oligodendrocytes, which serve as an electrically insulating phospholipid layer surrounding axons of many neurons. For purposes of the present invention, an animal's brain tissue is typically a mammal's brain tissue, such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
In some embodiments, the molecular probes described herein can be used for the in vivo detection and localization of myelinated regions of an animal's brain, central nervous system, and/or peripheral nervous system. The molecular probe can be administered to the animal as per the examples contained herein, but typically through intravenous injection. “Administered”, as used herein, means provision or delivery molecular probes in an amount(s) and for a period of time(s) effective to label myelin in an animal's brain central nervous system, and/or peripheral nervous system. The molecular probes can be administered to the animal can be enterally or parenterally in a solid or liquid. Enteral route includes oral, rectal, topical, buccal, and vaginal administration. Parenteral route includes intravenous, intramuscular, intraperitoneal, intrasternal, and subcutaneous injection or infusion.
An example of a dosing regimen is to administer about 40- about 50 mg/kg by weight to the animal. In one example at 5 min, the brain concentration of probe can range between about 4% to 24% ID/g to ensure sufficient visualization of the myelinated regions of the brain, central nervous system, and/or peripheral nervous system.
The molecular probes described herein can be used for neuroanatomical or neuropathological studies. Researchers studying normal brains can employ this method to examine the morphology and distribution of myelinated tissue in an animal. “Distribution” as used herein is the spatial property of being scattered about over an area or volume. In this case, the “distribution of myelinated tissue” is the spatial property of myelin being scattered about over an area or volume included in the animal's brain, central nervous system, or peripheral nervous system tissue. Researchers interested in neurotoxicology and neuropathology can also use this method in several ways. One way is to infer demyelination by the absence of the molecular probe labeling compared to normal control tissue (e.g., normal brain). A second way is to study morphological changes in the myelin such as a fragmented or beaded appearance of the myelin sheath. In yet another embodiment of the present invention, one skilled in the art can assess and quantify changes in myelin content in vivo.
In other aspects of the present invention, myelin in an animal's brain, central nervous system, and/or peripheral nervous system can be visualized and quantified using an in vivo imaging modality. The molecular probe may be visualized any time post administration depending on the application as typical molecular probes embodied in the present invention have a low clearance rate due to specific binding in the myelinated regions (e.g., at 60 min, the brain concentration of probe can be ≦50% of 5 min value to ensure that half time retention in normally myelinated brain is 60 min or longer).
An in vivo imaging modality as used herein is an imaging modality capable of visualizing molecular probes described herein in vivo (within a living organism). An example of an in vivo imaging modality is positron emission tomography (PET). PET is a functional imaging technique that can detect chemical and metabolic change at the molecular level. To function as a PET imaging molecular probe, embodiments of the present invention must meet a set of biological requirements known to the skilled artisan, some of which may include lipophilicity, binding affinity, binding specificity, brain uptake, retention, and metabolism. Another example of an in vivo imaging modality is MicroPET. MicroPET is a high resolution positron emission tomography scanner designed for imaging small laboratory animals. Other examples of imaging modalities that can be used include magnetic resonance imaging (MRI), near infrared (NIR) imaging, fluorescent microscopy, and mutiphoton microscopy.
For directly monitoring myelin changes in the white matter of a subject, molecular probes described herein can readily penetrate the blood-brain barrier (BBB) and directly bind to the myelinated white matter in proportion to the extent of myelination. Radiolabeled molecular probes described herein can be used in conjunction with PET as imaging markers to directly assess the extent of total lesion volumes associated with demyelination. This can provide a direct clinical efficacy endpoint measure of myelin changes and identify effective therapies aimed at protection and repair of axonal damages.
The molecular probes can also be used to diagnose a myelination related disorder in an animal through the use of in vivo myelin labeling. Thus, in certain embodiments, solutions containing the molecular probes describe herein can be used in the detection of myelin related disorders in an animal.
Methods of detecting a myelin related disorder include the steps of labeling myelin in vivo in the animal's brain tissue with a molecular probe described herein, visualizing a distribution of the molecular probe in the animal's brain tissue as described above and in the examples, and then correlating the distribution of the molecular probe with a myelin related disorder in the animal. In one example of detecting a myelin related disorder, the methods described herein can be used to compare myelinated axonal regions of the brain in the normal tissues of control populations to those of a suspect animal. If the suspect animal has a myelin related disorder, myelin may be virtually absent in lesioned areas thus indicating the presence of a myelin related disorder.
Myelination disorders can include any disease, condition (e.g., those occurring from traumatic spinal cord injury and cerebral infarction), or disorder related to demylination, remylination, or dysmyelination in a subject. A myelin related disorder as used herein can arise from a myelination related disorder or demyelination resulting from a variety of neurotoxic insults. Demyelination is the act of demyelinating, or the loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Bane Syndrome. Leukodystrophies are caused by inherited enzyme deficiencies, which cause abnormal formation, destruction, and/or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders share a poor prognosis leading to major disability. Thus, some embodiments of the present invention can include methods for the detection of neurodegenerative autoimmune diseases in an animal and more specifically the detection of multiple sclerosis in an animal.
Another embodiment includes a method of monitoring the efficacy of a remyelination therapy in an animal. Remyelination is the repair of damaged or replacement of absent myelin in an animal's brain tissue. The methods described include the steps of labeling myelin in vivo in the animal's brain tissue with a molecular probe described herein, then visualizing a distribution of the molecular probe in the animal's brain tissue (e.g., with a in vivo imaging modality as described herein), and then correlating the distribution of the molecular probe as visualized in the animal's brain with the efficacy of the remyelination therapy. It is contemplated that the labeling step can occur before, during, and after the course of a therapeutic regimen in order to determine the efficacy of the therapeutic regimen. One way to assess the efficacy of a remyelination therapy is to compare the distribution of the molecular probe before remyelination therapy with the distribution of the molecular probe after remyelination therapy has commenced or concluded.
Remyelination therapy as used herein refers to any therapy leading to a reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage related to demyelination. For example, a remyelination therapy can include administration of a therapeutic agent, therapies for the promotion of endogenous myelin repair, or a cell based therapy (e.g., a stem-cell based therapy).
In another embodiment of the present invention, methods are provided for screening for a myelination response in an animal's brain tissue to an agent. The method includes the initial step of administering an agent to the animal. Myelin in the animal's brain tissue is labeled in vivo with a molecular probe in accordance with the present invention. A distribution of the molecular probe in the animal's brain tissue is then visualized using a conventional visualization modality. Finally, the distribution of the molecular probe with the myelination response in the animal's brain tissue is correlated to the agent. One way to assess the myelination response in the animal's brain tissue is to compare the distribution of the molecular probe in an animal's brain tissue, which has been treated with a suspect agent with the distribution of the molecular probe in the brain tissue of a control population.
“Control Population” as used herein is defined as a population or a tissue sample not exposed to the agent under study but otherwise as close in all characteristics to the exposed group as possible.
The molecular probes described herein can be used to determine if an agent of interest has the potential to modulate demyelination, remyelination, or dysmyelination of axonal regions of an experimental animal's brain tissue.
In some embodiments, a molecular probe described above may be administered parenterally to a surgical subject prior to surgery such that the molecular probe binds to myelin and may be cleared from tissues that do not contain myelin. In another embodiment, a molecular probe may be applied directly to the surgical field during surgery, allowed to bind to myelin present, and the surgical site washed by lavage to clear unbound composition from the site. In some embodiments, during surgery, a light source tuned to the spectral excitation characteristics of the molecular probe may be applied to the surgical field. The molecular probe may be observed through an optical filter tuned to its spectral emission characteristics. It is contemplated that due to the specific binding of the molecular probes to nerves and other myelin-containing nervous tissue, that the myelin-containing nervous tissue are distinguishable from tissue not containing myelin. This enables the surgeon to avoid inadvertently cutting or damaging myelinated tissue by avoiding a visually detected peripheral nervous system tissue, or facilitates accurately administering treatment targeting a nerve or other myelin containing tissue, such as pharmaceutical or surgical nerve block.
In another embodiment, a molecular probe may be administered parenterally to a patient suspected of, or determined to be, suffering from a spinal pathology, such as but not limited to, spinal compression, spinal nerve root compression, or a bulging disc. For example, after binding to spinal myelin, and clearance from tissue that does not contain myelin without eliminating the specific myelin binding, the spine may be imaged for in vivo using radioisotope imaging such as PET, SPECT, or any combination thereof.
By inspection of the diagnostic images, the clinician may determine if, and where, the spinal cord, or associated nerve roots, are impinged, such as by the vertebral column Additional scans, such as CT or MRI, may also be conducted in conjunction with PET or SPECT scans, to provide additional information, such as the structure and relative positioning of elements of the vertebral column. In one embodiment, this method may be applied to a surgical procedure to image the spinal region intraoperatively.
In other embodiments, the molecular probes can be administered to an animal and utilized for labeling and detecting lipidated regions of the animal's kidneys, skin, heart and/or vasculature. In some embodiments, the molecular probes described herein can be used for the in vivo detection and localization of aberrant lipid accumulation of an animal's heart, kidneys, vasculature, and skin. The molecular probe can be administered to the animal as per the examples contained herein, but typically through intravenous injection.
The molecular probes described herein can be used for anatomical or pathological studies. Researchers studying aberrant lipid accumulation can employ this method to examine the morphology and distribution of lipid accumulation in tissue or organs of an animal. In this case the “distribution of lipid accumulation” is the spatial property of lipids being scattered about over an area or volume included in the animal's tissue or organs. Researchers interested in toxicology and pathology can also use this method in several ways. One way is to infer lipid accumulation by the presence of the molecular probe labeling compared to normal control tissue (e.g., normal kidneys). In yet another embodiment of the present invention, one skilled in the art can assess and quantify changes in aberrant lipid accumulation in vivo.
In other aspects, aberrant lipid accumulation in an animal's tissue or organs, such as kidneys, heart, or vasculature can be visualized and quantified using an in vivo imaging modality. The molecular probe may be visualized any time post administration depending on the application as typical molecular probes embodied in the present invention have a low clearance rate due to specific binding in the regions including aberrant lipid accumulation.
The molecular probes described herein can also be used to diagnose aberrant lipid accumulation associated with a lysosomal or lipid storage disease in an animal through the use of in vivo lipid labeling. Thus, in certain embodiments, solutions containing the molecular probes described herein can be used in the detection of lipid storage diseases in an animal.
Methods of detecting aberrant lipid accumulation include the steps of labeling lipids ex vivo or in vivo in the animal's tissue with a molecular probe described herein, visualizing a distribution of the molecular probe in the animal's tissue as described above and in the examples, and then correlating the distribution of the molecular probe with aberrant lipid accumulation in the animal. In one example, the methods described herein can be used to compare lipid accumulation in regions of the kidney in the normal tissues of control populations to those of a suspect animal. If the suspect animal has aberrant lipid accumulation, an increase quantity of lipids may be present in the tissue thus indicating the presence of an aberrant lipid accumulation.
Another embodiment of the application relates to a method of monitoring or measuring the efficacy of an agent or therapy in inhibiting aberrant lipid accumulation in a subject. The methods described include the steps of labeling lipids in vivo in the animal's tissue (e.g., kidneys) with a molecular probe described herein, then visualizing a distribution of the molecular probe in the animal's tissue (e.g., with a in vivo imaging modality as described herein), and then correlating the distribution of the molecular probe as visualized in the animal's tissue with the efficacy of the therapy or agent. It is contemplated that the labeling step can occur before, during, and after the course of a therapeutic regimen in order to determine the efficacy of the therapeutic regimen. One way to assess the efficacy of a therapy is to compare the distribution of the molecular probe before administration of the agent or therapy with the distribution of the molecular probe after therapy has commenced or concluded.
Another embodiment relates to a method for screening agents for inhibiting aberrant lipid accumulation associated with a lipid storage disease. The method includes the initial step of administering an agent to an experimental animal that has or is at risk of aberrant lipid accumulation, such as a transgenic mouse model of Fabry disease. Lipid accumulation in the animal's tissue is labeled in vivo or in vitro with a molecular probe as described herein. A distribution of the molecular probe in the animal's tissue can then visualized using a conventional visualization modality. Finally, the distribution of the molecular probe after the agents' response in the animal's tissue is correlated to the agent. One way to assess the agents' response in the animal's tissue is to compare the distribution of the molecular probe in an animal's tissue, which has been treated with a suspect agent with the distribution of the molecular probe in the tissue of a control population. “Control Population” as used herein is defined as a population or a tissue sample not exposed to the agent under study but otherwise as close in all characteristics to the exposed group as possible. The molecular probes described herein can be used to determine if an agent of interest has the potential to modulate lipid accumulation (e.g., G1-3 accumulation) of an experimental animal's tissue (e.g., kidneys) associated with a lipid storage disease.
We have identified a series of stilbene derivatives that displayed promising in vitro and in situ properties for imaging of myelinated white matter. Compared to previously reported myelin-imaging probes, these compounds showed improved solubility and binding affinity. The synthesis and biological evaluation of trans-stilbene derivatives is described below.
In this study, 4-nitrobenzaldehyde and 4-dimethylamino-benzaldehyde were employed to react with a Horner-Wadsworth-Emmons reagent, (p-nitrobenzyl)-phosphonic acid diethyl ester (1), to yield (E)-4,4′-dinitro-stilbene (2) and (E)-dimethyl-{4-[2-(4-nitro-phenyl)-vinyl]-phenyl}-amine (5). Further reduction of the nitro groups of 2 and 5 in the presence of SnCl2 in ethanol, furnished (E)-4,4′-diamino-trans-stilbene (3) and (E)-dimethyl-{4-[2-(4-amino-phenyl)-vinyl]-phenyl}-amine (6). Reduction of 2 also yielded a less polar, semi-reduced compound, 4-[2-(4-nitro-phenyl)-vinyl]-phenylamine (4) that was successfully separated and characterized by HNMR and HR-MS. Compound 4 was further protected with trifluoroacetic anhydride. Subsequently, methylation with iodomethane in the presence of potassium carbonate followed by hydrolysis and reduction yielded the monoalkylated compound, N-methyl-{4-[2-(4-amino-phenyl)-vinyl]-phenyl}-amine (7), which was purified by flash chromatography. In addition, an iodinated compound, 4-[2-(4-Iodo-phenyl)-vinyl]-phenylamine (9) was also synthesized through Horner-Wittig reaction (see Scheme 2).
4-Iodo-benzaldehyde readily reacted with 1 in DMF in the presence of NaH. Subsequent reduction with SnCl2 yielded Compound 9. Compounds 3, 6 and 7 are fluorescent compounds and soluble in EtOH, CH2Cl2, DMSO and other organic solvents. The excitation and emission spectra of 3, 6 and 7 (1 μM in DMSO), as recorded using a Cary Eclipse Fluorescent Spectrophotometer, are shown in
Compound 9 was selected for radiolabelling with 125I. The radiolabeling precursor, 4-[2-(4-tributylstannanyl-phenyl)-vinyl]-phenylamine (10), was first synthesized directly from the cold standard compound 9, in which the iodo group was replaced with a tributyltin group in the presence of Pd(PPh3)4. Iododestannylation reaction using no-carrier-added sodium [125I] iodide in the presence of hydrogen peroxide as the oxidant yielded [125I]9 (Scheme 2).
The radiochemical identity of [125I]9 was verified by co-injection with cold standard Compound 9. Following HPLC purification, [125I]9 was obtained in 70% radiochemical yield with a radiochemical purity of >98% and a specific activity of 80 TBq/mmol. As monitored by HPLC, [125I]9 was found stable to be kept at room temperature for up to 8 hrs and in the refrigerator for up to 2 months.
Binding affinities of newly synthesized compounds 6 and 7 were determined based on spectrophotometry. Myelin sheaths and non-myelin pellets were extracted from rat's brain homogenates according to subcellular fraction protocol (Martenson, R. E.; Deibler, G. E.; Kies, M. W. Extraction of rat myelin basic protein free of other basic proteins of whole central nervous system tissue. An analysis of its electrophoretic heterogeneity. J Biol Chem 1969, 244, 4268-4272). Briefly, the homogenates were successively mixed with different concentrations of sucrose and spun in a Beckman ultracentrifuge. Myelin sheaths and non-myelin containing pellets were well separated according to their different densities and located in different layers of sucrose. The proteins were then collected and washed thoroughly with Colman buffer (10 mM). The desired proteins were aliquoted and frozen at −80° C. for up to 6 months without noticeable change in its properties determined by electrophoresis (data not shown). Prior to binding assays, the protein fractions (myelin and pellet) were thawed and diluted with PBS (10 mM, pH 7.0). A series of concentrations of the protein fractions were incubated with tested compounds (6 and 7, 12.5 μM) for 1 h at room temperature. The free and bound tested compounds were then separated by centrifuging at 6000 rpm for 10 min and quantified.
As shown in
In vitro binding assay using radioligand is the most sensitive techniques available to quantitatively determine the binding affinities of compounds to certain proteins. Our previous studies have shown that BMB binds to myelin sheaths with high affinity and specifity (Stankoff, B.; Wang, Y.; Bottlaender, M.; Aigrot, M. S.; Dolle, F. et al. Imaging of CNS myelin by positron-emission tomography. Proc Nall Acad Sci USA 2006, 103, 9304-9309). For this reason, tritiated BMB was custom synthesized by American Radiolabeled Chemicals Inc. (St Louis, Mo.) and was used as the radioligand for binding assays. This allowed us to determine the binding affinities of the newly synthesized compounds using isolated rat myelin fractions. Saturation experiment was first conducted using [3H]BMB. As shown in
We then evaluated the myelin-binding properties of the newly synthesized compounds 3, 6, and 7 through in vitro staining of mouse brain tissue sections. Both myelinated corpus callosum and cerebellar regions were then examined by fluorescent microscopy. At 10 μM concentration, compounds 3, 6, and 7 selectively labeled both corpus callosum and cerebellum (
Following our in vitro tissue staining studies, we then evaluated the brain permeability and subsequent myelin-binding properties of 6 and 7 in the mouse brain. A dose of 6 or 7 (20-80 mg/kg) was administered via tail vein injection into wild-type mice. Three hours post injection, the mouse brains were perfused with saline followed by 4% paraformaldehyde (PFA) and removed. The fresh frozen brains were then sectioned. Fluorescent staining of myelinated regions such as the cerebellum were then directly examined under a microscope. As shown in
The partition coefficient (PC) is an important parameter of brain permeability. PC values ranging 1.0-3.5 often show good initial brain entry following i.v. injection (Wu, C.; Pike, V. W.; Wang, Y. Amyloid imaging: from benchtop to bedside. Curr Top Dev Biol 2005, 70, 171-213; Levin, V. A. Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J Med Chem 1980, 23, 682-684; Dishino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E. Relationship between lipophilicity and brain extraction of C-11-labeled radiopharmaceuticals. J Nucl Med 1983, 24, 1030-1038. For this reason, we radioiodinated Compound 9 and quantitatively determined the lipophilicity of [125I]9. Based on the conventional octanol-water partition measurement, the logPoct of [125I]9 was determined as 2.5±0.1, which falls in the range for optimal brain entry.
Encouraged by the aforementioned studies, we further evaluated the permeability of [125I]9 across the blood brain barrier. Following bolus tail vein injection of [125I]9 (0.2 ml, 0.185 MBq), the radioactivity concentration of [125I]9 in the brain was determined at 2, 30, and 60 min post injection. As shown in Table 1, [125I]9 displayed rapid brain entry at early time intervals. The initial brain entry was 2.29±0.66% ID/g at 2 min post injection. At 30 min post injection, the brain radioactivity concentration reached its peak level (2.65±0.27% ID/g). The radioactivity concentration slowly decreased to 1.12±0.23% ID/g at 120 min post injection. These results indicated that [125I]9 readily entered the brain. Retention of [125I] 9 at later time points was likely due to binding to myelin membranes as indicated by aforementioned in vitro and in situ staining studies.
To further evaluate the binding specificity of [125I]9 to myelin sheaths in the brain, in vitro autoradiography was carried out in mice. As shown in
Scheme 1, a) NaH, 4-nitrobenzaldehyde, DMF, MeOH, 83%; b) SnCl2, 1N HCl, THF; c) 4-Dimethylamino-benzaldehyde, DMF, EtOH, NaOCH3, 65%; d) SnCl2, EtOH, 64%; e) (CF3CO)2O, Et3N, THF; f) 1. NaH, MeI, DMF; 2. 1 N NaOH, MeOH; g) SnCl2, CH3COOH, reflux, 35% for 4 steps.
Under Ar, (4-nitro-benzyl)-phosphoric acid diethyl ester (1, 1.81 g, 6.6 mmol) and 4-nitrobenzaldehyde (1.00 g, 6.6 mmol) were dissolved in DMF (10 mL) and EtOH (10 mL). Then NaOCH3 (2.3 mL, 4.37 M) in MeOH was added and the suspension was stirred for another 3 hours. The solid was filtered and dried in vacuum to give 1.50 g (yield: 83%) of 4,4′-dinitro-trans-stilbene. 1H NMR (300 MHz, CDCl3): 8.28 (d, J=8.4 Hz, 4H), 7.94 (d, J=8.4 Hz, 4H), 7.69 (s, 2H).
To a solution of compound 2 (0.10 g, 0.4 mmol) in THF (20 mL) was added SnCl2 (1.50 g) dissolved in 1N HCl (10 mL). The reaction mixture was stirred overnight at room temperature. The acidic solution was then neutralized using 1N NaOH and extracted with ethyl acetate (3×20 mL). The combined organic phases were washed with water and brine, dried over Na2SO4, and concentrated. Purification with flash column (HE:EA=2:1 to 1:1) yielded 4,4′-diamino-trans-stilbene (3, 0.03 g, 40%) and 4-[2-(4-nitro-phenyl)-vinyl]-phenylamine (4). 1H NMR of 3 (300 MHz, CDCl3): 7.31 (d, J=8.4 Hz, 4H), 6.86 (s, 2H), 6.68 (d, J=8.4 Hz, 4H). HR-ESIMS of 3: m/z calcd for C14H14N2 (M+H+): 211.1230, found 211.1225. Melting point of 3: 206.1˜207.3° C. 1H NMR of 4 (300 MHz, CDCl3): 8.22 (d, J=8.0 Hz, 2H), 7.59 (d, J=6.86 Hz, 2H), 7.41 (d, J=8.57 Hz, 4H), 7.22 (d, J=17.14 Hz, 1H), 6.97 (d, J=12.57 Hz, 1H), 6.72 (d, J=10 Hz, 2H).
To a solution of 4-dimethylamino-benzaldehyde (2.24 g, 15 mmol) and (4-nitro-benzyl)-phosphoric acid diethyl ester (1, 4.10 g, 15 mmol) in DMF (20 ml) and EtOH (20 mL) was added to NaOCH3 (1.62 g, 30 mmol). The suspension was stirred and refluxed for 3 hrs. After cooled to room temperature, the precipitate was filtered and washed thoroughly with ethanol to give dimethyl-{4-[2-(4-nitro-phenyl)-vinyl]-phenyl}-amine (5, 2.55 g, 65%) as red solid, 5 was used without further purification.
To a solution of 5 (2.55 g, 9.5 mmol) in EtOH (100 ml) was added to SnCl2 (8.58 g, 38 mmol). The resulting mixture was refluxed for 4 hrs. The solvent was then removed under vacuum and NaOH (2 mol/L, 40 mL) was added to the residue. The crude solid was filtered and suspended in ethyl acetate (200 ml). The precipitates were then filtered to give dimethyl-{4-[2-(4-amino-phenyl)-vinyl]-phenyl}-amine (6, 1.45 g, 64%) as gray solid. 1H NMR (300 MHz, CDCl3): 7.40 (d, J=8.62 Hz, 2H), 7.33 (d, J=8.40 Hz, 2H), 6.86 (d, J=5.92 Hz, 2H), 6.76 (d, J=8.34 Hz, 2H), 6.69 (d, J=8.22 Hz, 2H), 2.99 (s, 6H). HR-ESIMS: m/z calcd for C16H18N2 (M+H+): 239.1543, found 239.1542. Melting point: 167.7˜168.5° C.
To a solution of 4 (50 mg, 2 mmol) dissolved in THF (5 mL) under argon was added to Et3N (1 mL). The solution was stirred for 4 hrs. The solvent was evaporated under vacuum and the protected product was then used without further purification.
To the solution of the above protected product dissolved in DMF (5 mL) were added NaH (0.10 g) and iodomethane (1 mL). The vial was sealed and stirred overnight. Then the solution was diluted with methanol (8 mL) and 1M NaOH solution (2 mL). After stirred for another 2 hours, the solution was extracted with ethyl acetate. The combined organic layer was washed with water and brine and dried over Na2SO4. Following concentration, the reduced product was then subsequently used without further purification.
To the suspension of the above compound in acetic acid (10 mL) was added tin (II) chloride (1.0 g). The suspension was heated to reflux for 2 hrs. After concentration, the residue was dissolved in ethyl acetate, washed with 2 N NaOH solution, water, and brine. Dried over Na2SO4, the solution was concentrated and purified by flash column (Hexanes:ethyl acetate=2:1 to 1:1) to give 15 mg of 7 (0.6 mmol, 35% yield for the above three steps). 1H NMR (400 MHz, CDCl3): 7.36 (d, J=8.3 Hz, 2H), 7.32 (d, J=8.2 Hz, 2H), 6.87 (AB, J=18.7 Hz, 16.5 Hz, 2H), 6.69 (d, J=8.2 Hz, 2H), 6.62 (d, J=8.3 Hz, 2H), 3.92 (br, 3H), 2.88 (s, 2H). HR-ESIMS: m/z calcd for C15H16N2 (M+H+): 225.1386, found 225.1385. Melting point: 143.7˜144.7° C.
To a solution of diethyl 4-nitrobenzylphosphate (0.44 g, 1.61 mmol) dissolved in DMF (10 mL) was added NaH (0.07 g, 1.75 mmol). The suspension was stirred for 1 hour followed by addition of 4-iodo-benzaldehyde (0.35 g, 1.51 mmol). The suspension was stirred for another 2 hours. Water was added and the solid was collected by filtration to give 8 (0.40 g, 1.14 mmol, yield: 75%). 1H NMR (400 MHz, CDCl3): 8.24 (d, J=8.65 Hz, 2H), 7.88 (d, J=8.68 Hz, 2H), 7.80 (d, J=8.19 Hz, 2H), 7.49 (m, J=8.2 Hz, 4H).
To a suspension of Compound 8 (0.20 g, 0.57 mmol) in ethanol (10 mL) was added Tin (II) chloride (1.00 g, 5 mmol) and heated to reflux for 4 hours under argon. The ethanol was evaporated under vacuum. The residue was dissolved in ethyl acetate, washed with 1 N NaOH, water, and brine. Dried over Na2SO4, the solution was concentrated and purified by flash column (Hexanes:ethyla acetate=2:1 to 1:1) to give 9 (0.18 g, quant. yield). 1H NMR (400 MHz, CDCl3): 7.66 (d, J=8.25 Hz, 2H), 7.35 (d, J=8.34 Hz, 2H), 7.04 (d, J=15.73 Hz, 1H), 6.71 (d, J=16.26 Hz, 1H), 6.70 (d, J=8.29 Hz, 2H). HR-ESIMS: m/z calcd for C14H12IN (M+H+): 322.0087, found 322.0084. Melting point: 213.4˜215.2° C.
Under Ar, the substrate 9 (0.05 g, 0.15 mmol) was mixed with (Bu3Sn)2 (1 mL), Pd(PPh3)4 (0.02 g) and Et3N (5 mL). The mixture was sealed in a vial and heated to 80° C. for 1 day. The solvent was evaporated in vacuum and the residue was purified by column to give 10 (44 mg, 0.09 mmol, yield: 60%). 1H NMR (400 MHz, CDCl3): 7.42 (s, 4H), 7.33 (d, J=7.87 Hz, 2H), 7.03 (d, J=16.18 Hz, 1H), 6.89 (d, J=16.24 Hz, 1H), 6.66 (d, J=8.08 Hz, 2H), 1.55 (m, 6H), 1.34 (m, 6H), 1.05 (t, 6H), 0.89 (t, 9H).
To a sealed vial were added 10 (50 μl, 50 μg in 50 μL of ethanol), [I-125] sodium iodide, and 1 N HCl (100 μL). Subsequently, 100 μL of H2O2 (3%, in water) was added via a syringe at room temperature. After 10 min, the iodination reaction was terminated by an addition of saturated NaHSO3, and the resulting solution was neutralized to pH 7-8 by adding a saturated NaHCO3 solution. The mixture was extracted with ethyl acetate (3×1 ml). The combined organic layers were dried over Na2SO4, and the solvent was removed by a stream of dry nitrogen gas. The residue was purified by high performance liquid chromatography (HPLC; C-18 column, acetonitrile: DMGA (5 mM, pH 7.4): 60/40, flow rate: 1 mL/min; retention time: 21 min) to get 18.5 MBq of final pure product with radiochemical purity over 98% and a specific activity near the theoretical limit (80 TBq/mmol). The chemical identity was verified by co-injection of the “cold standard” (nonradioactive compound).
Partition coefficients (PC) were measured by mixing the radioligands with 3 g (3.65 mL) 1-octanol and 3 g (3.0 mL) buffer (pH 7.40, 0.1 M phosphate) in a test tube. The test tube was vortexed for 3 min at room temperature and then centrifuged (3500 rpm, 5 min) 1 ml of samples from the 1-octanol and buffer layers were assayed for radioactivity content in a well γcounter. The partition coefficient was determined by calculating the ratio of cpm/g of 1-octanol to that of the buffer. Samples from the 1-octanol layer were repartitioned until consistent partitions of coefficient values were obtained. The measurement was repeated at least three times. PC was 2.5±0.1 at pH 7.40.
While under anesthesia, 0.1 mL of a saline solution (consisting of saline (2 mL, 9 mg/mL), propylene glycol (2 mL), ethanol (0.7 mL) and HCl (0.3 mL, 0.3 nM))25 containing 5 μCi of radioactive tracer, was injected into the tail veins of mice (Swiss-Webster, 2 month old, 2 mice per group). The mice were sacrificed by heart puncture at 2 min, 30 min, 60 min and 120 min post injection under anesthesia. Brains were rapidly removed and weighed, and the brain uptake was expressed as percentage of injection dose per gram organ (% ID/g), which was calculated by a ratio of per gram tissue counts to counts of 1% of the initial dose (100 times diluted aliquots of the injected radioligand) measured at the same time.
Mouse brain sections were incubated in [125I]9 (20% Ethanol, 4,380,000 cpm/16 ml) for 1 hr. The slides were quickly washed with PBS buffer (10 mM, pH 7.0) 3 times, saturated Li2CO3 in 40% ethanol (2×3 min), 40% ethanol (2 min) and H2O (30 sec). After drying by air, the slides were put in a cassette and exposed to film for 44 hrs to obtain images.
Normal control mice were deeply anesthetized and perfused transcardially with saline (10 mL) followed by fixation with 4% PFA in PBS (10 mL, 4° C., pH 7.6). Brain tissues were then removed, postfixed by immersion in 4% PFA overnight, dehydrated in 30% sucrose solution, embedded in freezing compound (OCT, Fisher Scientific, Suwanee, Ga.), cryostat sectioned at 10 μm on a microtome and mounted on superfrost slides (Fisher Scientific). The brain sections were incubated with compound 3, 6 and 7 (10 μM, 1% DMSO in PBS (10 mM, pH 7.0)) for 20 minutes at room temperature in dark. Excess compounds were washed by briefly rinsing the slides in PBS (10 mM, pH 7.0) and coversliped with fluoromount-G mounting media (Vector Laboratories, Burlingame, Calif.). Sections were then examined under a Leica DRMB microscope equipped for fluorescence.
Under anesthesia, wild-type mice were injected with compounds 6 and 7 (20˜80 mg/kg) via the tail vein, and the mice were then perfused transcardially with saline (10 ml) followed by 4% PFA in PBS (10 ml, 4° C., pH 7.6). Brain tissues were then removed, postfixed by immersion in 4% PFA overnight, dehydrated in 30% sucrose solution, cryostat sectioned at 16 μm on a microtome and mounted on superfrost slides (Fisher Scientific), and imaged directly under fluorescent microscopy without any further staining.
Sprague-Dawley (SD) rats were asphyxiated with CO2. When the rat had stopped breathing, the skin/fur over the neck was wetted with a spray of 70% ethanol. The brains were then taken out and put into 0.32 mol/L sucrose (1×Colman buffer) in the homogenizer, first with the loose pestle 5˜8 times and then with the tight pestle until the solution reached a uniform consistency. The solution was then transferred from the homogenizer to the corresponding tubes. The tubes were centrifuged at 1000 rpm (4° C.) for 10 minutes. The resulting supernatant was carefully removed and transferred into Beckmann tubes that were previously filled with 2.80 mol/L sucrose and mixed thoroughly. After carefully overlaying nearly to the top of the tube with 0.25 mol/L sucrose, the tube was spun in the Beckman ultracentrifuge for 2.5 hr at 35,000 rpm (4° C.). The 0.25 mol/L sucrose layer was drawn off and discarded. The myelin fraction was collected at 0.25 mol/L and 0.85 mol/L sucrose interface and the pellet was collected at 0.85 mol/L and/1.4 mol/L sucrose interface. Both myelin and pellet were washed with buffer (1×colman, 7˜8 mL) three times before suspended in buffer (1×colman, 5 mL) and kept in −80° C. freezer for future use. The concentration of myelin and pellet were determined by Bio-Rad Protein Assay.
In the spectrophotometry-based binding assays, a solution of 6 or 7 (800 μL, 12.5 μM) dissolved in 10% DMSO buffer solution containing 10 mM MgCl2 and 10 mM PBS (pH 7.4) was incubated with isolated myelin or pellets at different concentrations ranging from 0.06 to 14 μg/tube. Each tube contained 10 μM of 6 or 7, 10% DMSO buffer, and membrane fraction in a final volume of 1 mL. Following incubation at room temperature for 1 hr, the free and bound 6 or 7 was separated by centrifugation at 6000 rpm for 10 min The supernatant was then collected and the UV absorption of free 6 or 7 determined by UV spectrometer were at 350 nm or 363 nm. The concentration of free 6 or 7 was obtained by comparison to a standard curve. In parallel, non-specific binding was determined using pellets under the same condition. All assays were performed in triplicate.
The radioligand-based binding assays were carried out in 12×75 mm borosilicate glass tubes. For saturation studies, the reaction mixture contained 50 μL of myelin fraction (1˜2 μg, 1×PBS), 50 μL of [3H]BMB (diluted in 1×PBS, 0.25˜3.5 nM) in a final volume of 500 μL. Nonspecific binding was defined in the presence of cold BMB (1 μM, diluted in PBS (containing 1% DMSO) in the same assay tubes. For the competition binding, 10−5 to 10−10 M compounds and 1.87 nM [3H]BMB were used for the studies. The mixture was incubated at 37° C. for 2 hrs. The bound and free radioactivity were separated by rapid vacuum filtration through Whatman GF/B filters using a Brandel M-24R cell harvester followed by 3×2 mL washes of PBS at room temperature. Filters containing the bound radioligand were dissolved in 6 mL biodegradable counting cocktail overnight and the radioactivity was assayed next day in the scintillation counter (Beckman) with 42% counting efficiency. The results of saturation and inhibition experiments were subjected to nonlinear regression analysis using Graph Pad Prism 4 by which Kd and Ki values were calculated.
We screened a series of derivatives based on coumarin that possess the same pharmacophore as stilbene. Coumarin is a naturally occurring compound in plants with many important biological activities. We found that the Coumarin derivitive 3-(4-aminophenyl)-2H-chromen-2-one (CMC), is highly permeable across the BBB and selectively localizes in myelinated regions. Our studies in a hypermyelinated mouse model indicate that CMC is a sensitive probe that can be used for in situ staining of myelin.
3-(4-aminophenyl)-2H-chromen-2-one (CMC) was obtained from Matrix Scientific (Columbia, S.C.). The rest of compounds screened were obtained from Sigma-Aldrich (Milwaukee, Wis.), TCI American, (Portland, Oreg.) and used without further purification. The Coumarin derivatives are soluble in DMSO and other commonly used organic solvents. The coumarin derivatives are fluorescent compounds and the excitation and emission spectra of CMC was recorded using Fluorescence Spectrophotometers (Varian. Inc., Palo Alto, Calif.) as shown in
Swiss-Webster R/J mice were obtained from The Jackson Laboratory, Bar Harbor, Minn., and used as control. Transgenic mice expressing constitutively active Akt (HAAkt308D473D, Akt-DD; Ontario Cancer Institute, Toronto, Canada) driven by the Plp promoter (Wight P A, Duchala C S, Readhead C, Macklin W B (1993) A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice. J Cell Biol 123:443-454) were prepared and used as an animal model of hypermyelination. In this model, the Akt cDNA was inserted into the AscI/PacI sites of the modified Plp promoter cassette, and the Plp promoter/Akt-DD insert was injected to generate transgenics in SJL/SWR F1 mice to induce hypermyelination. Positive founders were identified by PCR amplification of tail DNA using IntronSV40F (5′-GCAGTGGACCACGGTCAT-3′) (SEQ ID NO:1) and Akt lower (5′-CTGGCAACTAGAAGGCACAG-3′) (SEQ ID NO:2) primer sequences. Analyses were done from littermatched mice in all developmental experiments, and where possible with older animals.
For immunohistochemistry, mice were deeply anesthetized and perfused with PBS followed by 4% paraformaldehyde in PBS via the ascending aorta. Brains were dissected out, incubated for 24 hrs in 4% paraformaldehyde at 4° C., cryoprotected and sectioned (30 μm) with a sliding microtome. Sections were immunostained overnight at 4° C. with rabbit anti-MBP antibody (Chemicon-Millipore, Bedford, Mass.) 1: 2000 dilution in 3% normal goat serum in PBS, followed by one hour incubation at room temperature with IRDye 800CW Goat Anti-Rabbit (LI-COR Biosciences, Lincoln, Nebr.) 1:5000 dilution. Images of the stained mouse brain sections were acquired on the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebr.).
Free floating sections were incubated in 1% H2O2/Triton-100 for 10 min, then incubated in a solution of test compounds (100 μM) in 1% DMSO/PBS for 30 min at room temperature. The sections were washed three times with PBS before cover-slipping with fluorescence mounting medium (Vectashield, Vector laboratories).
Following tissue staining with each test compound, images of mouse brain sections were acquired on a Leica DMI6000 inverted microscope (1.25× and 5× objectives) with a Hamamatsu Orca-ER digital camera, and operated with Improvision's Volocity software. Image J software was used to quantify pixel intensities values. The corpus callosum between the midline and below the apex of the cingulum was defined as region of interest (ROI). The density of myelin in the corpus callosum of wildtype mice was given the arbitrary value of 100, and the density of myelin in Plp-Akt-DD mice was determined as a percentage of wild-type mice. The data were analyzed using the GraphPad Prism, GraphPad Software, La Jolla, Calif., with a nonpaired Student's t test. For correlation, MBP immunoreactivity of the adjacent sections was also determined and quantified on the LI-COR Odyssey infrared imaging system, using 21 μm resolution, 1.2 mm offset with highest quality, and 3.0 channel sensitivity. The integrated densities of the midline corpus callosum were obtained using the associated Odyssey software. Statistical analysis was performed using a nonpaired Student's t-test (GraphPad Prism).
In this experiment, 25 mg/kg of CMC was administered through i.v. injection in the tail vein of 2-month old wild-type mice and Plp-Akt-DD mice Animals were sacrificed at 1 hr after injection through heart puncture. The brains were removed and fixed in 4% PFA. The brains were then sectioned and fluorescent images were directly acquired on a Zeiss Axiovert 200M inverted microscope (2.5× objective) with a AxioCam digital camera (Carl Zeiss Microlmagibg, Inc, Thomwood, N.Y.).
To date, we have screened several coumarin derivatives that potentially bind to myelin membranes. The structures of these coumarin derivatives are shown in
The fluorescent nature of these coumarin derivatives allows for staining of mouse brain tissue sections in a way similar to other conventional myelin stains. The tissue staining represents a direct approach to evaluate the binding specificity of the test compounds for myelin sheaths. The myelinated corpus callosum region was then examined by fluorescent microscopy. As shown in
The staining pattern was found consistent with immunohistochemical MBP staining and was proportional to the size of corpus callosum region as demonstrated in a hypermyelinated Plp-Akt-DD mouse model. Compared to the control sections, the hypermyelinated mouse brain showed that the corpus callosum region was significantly enlarged (
Following our in vitro studies, we investigated the ability of CMC to monitor myelin contents ex vivo in the mouse brain. A dose of 0.5 mg CMC (25 mg/kg) was injected via the tail vein into wild-type mice and Plp-Akt-DD mice. One hr post-injection, mice were perfused and brains were removed and sectioned as described above. CMC staining of myelin was then directly examined under fluorescent microscopy. As shown in
We evaluated a series of small molecule probes as PET agents for quantitative analysis of GL-3 deposition in the kidney. We found that some of the imaging agents can bind to GL-3 in vitro in the kidney of a transgenic mouse model of Fabry's disease. In this example, we determined the structural features of molecular probes that can be used for binding to GL-3 with high affinity and specificity. We then optimized the in vivo pharmacokinetic properties necessary for longitudinal monitoring GL-3 deposition in animal models of Fabry's disease. The optimized molecular probes can then be radiolabeled and used as radiotracers for PET imaging and quantification of GL-3 in vivo. This allows us to develop an imaging tool for both preclinical drug screening in animal models and clinical evaluations of therapeutic treatments in Fabry disease patients. In long term, it will enable us to apply the same imaging approach to study other lipid storage diseases.
To date, we have identified some prototypical structures that can be used for GL-3 imaging. Some of the GL-3 imaging agents we have designed are shown in
Given that some compounds are strongly fluorescent; we started evaluating its binding property by fluorescent tissue staining of kidney tissue sections of a transgenic mouse model of GL-3 deposition. We screened some compounds and preliminary results showed that a bis-stilbene derivative, termed CIC, exhibited high specificity for GL-3 as shown in
Following fluorescent tissue staining, we radiolabeled CIC with C-11 and conducted whole body microPET studies in wild-type rats. This study was designed to show if [11C]CIC can be used for PET imaging of the kidney. A series of coronal PET images are shown in
[11C]CIC-PET images showed a relative high background in the abdomen region. To reduce the background, we further evaluated a monostilbene derivative termed AIC. AIC is almost half of the size of CIC. Thus, it may clear faster than CIC from other organs. Because AIC is not strongly fluorescent, its binding properties could not be readily assessed by fluorescent tissue staining. To circumvent this problem, we radiolabeled AIC with carbon-11 and conducted autoradiography using both wild-type and GLA knockout mouse kidney tissue sections. As shown in
Encouraged by this result, we conducted in vivo [11C]AIC-PET imaging in wild-type rats to determine its biodistribution relative to kidney uptake. As shown in
The following Example illustrate the preparation and use of (E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl) vinyl)-N-methylaniline as myelin imaging probe. As shown below, (E)-4-(2-(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl) vinyl)-N-methylaniline was synthesized in accordance with the following reaction scheme:
Optical properties of Compound 9 were measured in a 50 uM solution (5% DMSO/95% H2O). As shown in
Compound 9 was used for chemical staining of frozen tissue sections of two-month-old Swiss-Webster R/J mouse brains. Axial sections of the whole mouse brain close to the bregma were used to examine the myelin-binding properties of compound 9 in both myelin-deficient gray and myelin-rich white matter regions. Axial sections were used to examine staining in the cerebellum.
As shown in
From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.
This application claims priority from U.S. Provisional Application No. 61/639,381, filed Apr. 27, 2012, and is a Continuation-in-Part of U.S. patent application Ser. No. 13/121,742, filed Mar. 30, 2011, which claims priority of U.S. Provisional Application No. 61/101,299, filed Sep. 30, 2008, and is also a Continuation-in-part of PCT/US2013/027667, filed Feb. 25, 2013, which claims priority from U.S. Provisional Application No. 61/602,988, filed Feb. 24, 2012, the subject matter of which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant No. R01 NS061837 awarded by The National Institutes of Health and National Multiple Sclerosis Society. The United States Government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61639381 | Apr 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13121742 | Mar 2011 | US |
| Child | 13872733 | US | |
| Parent | PCT/US2013/027667 | Feb 2013 | US |
| Child | 13121742 | US |
