EP0262212A1 - Psoralenes biotinyles - Google Patents

Psoralenes biotinyles

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Publication number
EP0262212A1
EP0262212A1 EP19870903009 EP87903009A EP0262212A1 EP 0262212 A1 EP0262212 A1 EP 0262212A1 EP 19870903009 EP19870903009 EP 19870903009 EP 87903009 A EP87903009 A EP 87903009A EP 0262212 A1 EP0262212 A1 EP 0262212A1
Authority
EP
European Patent Office
Prior art keywords
psoralen
nucleic acid
biotinylated
avidin
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19870903009
Other languages
German (de)
English (en)
Inventor
Wilma A. Saffran
Richard L. Edelson
Francis P. Gasparro
John T. Welsh
Charles R. Cantor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
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Columbia University in the City of New York
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Publication date
Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of EP0262212A1 publication Critical patent/EP0262212A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • Psoralen is a linear three ring heterocyclic compound having the structure
  • Psoralen's ability to react with DNA has given it clinical importance in the treatment of psoriasis and other skin disorders. Additionally, its ability to form interstrand crosslinks in double stranded DNA has made it a useful reagent in the study of nucleic acid structure and function.
  • Biotin a growth factor present in very minute amounts in every living cell and occurring mainly bound to proteins or polypeptides, has the structure
  • Avidin is a glycoprotein containing four essentially identical subunits, each of which is a single polypeptide chain of 128 amino acid residues with a carbohydrate moiety attached at position 17 (3).
  • Biotin combines with avidin and becomes inactive (4 , 5) .
  • antigens are recognized by specif ic antibodies, which are then bound to a biotinylated anti-immunoglobul in. This then forms a tight complex with avidin, either conj ugated directly to a signal of some kind, such as a fluorescent dye or enzyme, or in turn bound to a biotinylated label.
  • molecules that are themselves di rectly biotinylated can be recognized di rectly by avidin, omitting the antibody reactions, with their somewhat lower affinities.
  • Cimino et al. (6) described the synthesis of a psoralen derivative (aminomethyltrioxsalen-AMT) which contains a biotin moiety attached to the 4' position by various undisclosed linker chains. However, the purported methods for synthesizing these compounds were not disclosed. Furthermore, Cimino et al. reported only that preliminary studies of these undisclosed compunds indicated that they could be used to interchalate and crosslink double-stranded nucleic acid and that they can be detected colorimetrically or fluorescently by standard methods based on the avidinbiotin interaction.
  • biotinylated psoralens which retain the biological activity of psoralen and the binding specificity of biotin for avidin are not known. Furthermore, methods for synthesizing compounds which retain the biological activity of psoralen and the binding specificity of biotin are not known.
  • a quick, easy, efficient, and safe method for preparing a biotinylated psoralen would provide readily accessible amounts of biotinylated psoralens useful for psoralen modification of cellular components, the visualization of minute amounts of DNA, investigations of the uptake and distribution of psoralen within cells, the delivery of psoralen to specific cells, and the conversion of nucleic acid molecules to ligands for avidin.
  • the present invention provides a compound having the formula
  • Y is biotin or iminobiotin
  • X is CH 2
  • P is psoralen or a psoralen derivative
  • r is an integer equal to or greater than 2
  • s is an integer equal to or greater than 1.
  • the invention also provides a method for preparing a biotinylated psoralen derivative which comprises treating a compound having the structure
  • a positively charge multi-aminated linker having two amine groups separated by at least two carbon atoms under suitable conditions so as to allow the formation of a psoralen-linker complex.
  • the psoralen-linker complex is treated with the N-hydroxy succinimide ester of either biotin or iminobiotin under suitable conditions so as to allow the formation of a psoralen-linker-biotin or -iminobiotin complex.
  • This method comprises binding a suitable carrier molecule to biotin so as to form a biotinylated carrier.
  • the biotinylated carrier is bound to avidin so as to form an avidin-biotinylated carrier complex, which is then reversibly bound to an iminobiotinylated psoralen so as to form an iminobiotinylated psoralen-avidin-biotinylated carrier complex.
  • Cells are treated with a suitable amount of the iminobiotinylated psoralen-avidin-biotinylated carrier complex under suitable conditions so as to permit the iminobiotinylated psoralen-avidin-biotinylated carrier complex to become internalized.
  • the treated cells are incubated in the dark under suitable conditions so as to permit the iminobiotinylated psoralen moiety to dissociate from avidin and intercalate into a nucleic acid.
  • the incubated cells are then irradiated with near ultraviolet light under suitable conditions to allow the intercalated iminobiotinylated psoralen to covalently bind to the nucleic acid into which it has intercalated, thereby delivering to the cell an iminobiotinylated psoralen.
  • the invention further provides a method for treating leukemia in a subject.
  • This method comprises binding a suitable carrier molecule to biotin to form a biotinylated carrier.
  • the biotinylated carrier is bound to avidin so as to form an avidin-biotinylated carrier complex, which is then bound to an iminobiotinylated psoralen to form an iminobiotinylated psoralen-avidin-biotinylated carrier complex.
  • Separetely, plasma and red blood cells are separated from a sample of blood cells and a leukocyte enriched composition is recovered.
  • This leukocyte enriched composition is treated with a suitable amount of the iminobiotinylated psoralen avidin-biotinylated carrier complex, which is permitted to become internalized.
  • the treated leukocyte enriched composition is incubated in the dark under suitable conditions so as to permit the iminobiotinylated psoralen moiety to dissociate from avidin and intercalate into DNA within the cells.
  • the incubated, treated leukocyte enriched composition is then irradiated with near ultraviolet light so as to allow the intercalated iminobiotinylated psoralen moiety to covalently bind to the DNA into which is has intercalated.
  • a suitable amount of the irradiated leukocyte enriched composition is then administered to the subject, thereby treating the subject for leukemia by providing a toxic dose of psoralen to the leukemic DNA of the subject.
  • the invention additionally provides a method for detecting the presence of a nucleic acid in a sample.
  • This method comprises contacting the sample under suitable conditions in the dark with a biotinylated psoralen so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • the sample is then irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which is has intercalated.
  • the biotinylated psoralen which is covalently bound to the nucleic acid is contacted with a detectably marked avidin under suitable conditions so as to allow the avidin moiety to bind to the biotinylated psoralen which is covalently bound to the nucleic acid.
  • the presence of avidin bound to the biotinylated psoralen, which is also covalenty bound to the nucleic acid is detected and thereby the presence of the nucleic acid in the sample is detected.
  • the amount of a nucleic acid in a sample may be quantitatively determined by contacting the biotinylated psoralen which is covalently bound to the nucleic acid with a known amount of a detectably marked avidin. By determining the amount of avidin bound to the biotinylated psoralen which is also covalently bound to the nucleic acid, the amount of the nucleic acid in the sample is also determined.
  • the invention also provides a method for purifying or isolating nucleic acid from a sample.
  • This method comprises contacting the sample under suitable conditions in the dark with a biotinylated psoralen so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • the sample is irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which it has intercalated.
  • the biotinylated psoralen which is covalently bound to the nucleic acid, is then contacted with an immobilized avidin under suitable condi ti ons so as to allow the avidin moiety to bind to the biotinylated psoralen and form an avidin-biotinylated psoralen-nucleic acid complex.
  • the nucleic acid may then be recovered from the avidin-biotinylated psoralen-nucleic acid complex.
  • the invention provides a method for total nucleic acid pattern visualization of a sample.
  • This method comprises electrophoresing the nucleic acid of the sample and immobilizing it to a solid support.
  • the immobilized nucleic acid is contacted with a biotinylated psoralen and the solid support is incubated in the dark for an appropriate amount of time so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • Non-intercalated biotinylated psoralen is removed from the solid support and nucleic acid complexes bound to the solid support and intercalated with biotinylated psoralen are irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which it has intercalated.
  • the solid support is contacted with a detectably marked avidin under suitable conditions so as to allow the avidin moiety to bind the biotinylated psoralen, which is covalently bound to the nucleic acid.
  • the presence of avidin bound to the biotinylated psoralen is detected and thereby the total nucleic acid pattern of the sample is visualized.
  • the invention provides a method for visualizing a specific nucleic acid in a sample.
  • This method comprises electrophoresing the nucleic acid of the sample and immobilizing the electrophoresed nucleic acid to a solid support.
  • the immobilized nucleic acid is denatured so as to produced single stranded nucleic acids.
  • a single stranded nucleic acid clone of the specific nucleic acid to be visualized is contacted with a biotinylated psoralen.
  • the nucleic acid clone is incubated under suitable conditions in the dark so as to allow the formation of biotinylated psoralen-nucleic acid clone complexes, which are then irradiated with near ultraviolet light so as to permit the biotinylated psoralen to covalently bind the nucleic acid clone into which it has intercalated.
  • the immobilized single stranded nucleic acids are contacted with the nucleic acid clone which is covalently bound to the biotinylated psoralen so as to allow the nucleic acid clone and the immobilized single stranded nucleic acids to hybridize.
  • Fig. 1 Synthesis of biotinylated psoralen.
  • Fig. 2 DNA crossl inking by BPsor.
  • Linear pBR322 DNA (0.2 micrograms) was near UV irradiated in the presence BPsor.
  • Lane 1 no psoralen
  • lane 2 6 ng
  • lane 3 15 ng
  • lane 4 30 ng
  • lane 5 30 ng psoralen, but no irradiation.
  • Fig. 3 Detection of BPsor modification of DNA by ELISA. Modified DNA on microtiter dishes was incubated sequentially with streptavidin, biotinylated poly alkaline phosphatase, and phosphatase substrate.
  • Fig. 4 ELISA of BPsor modified DNA. Alkaline phosphatase activity in samples of DNA reacted with BPsor + near UV light ( ⁇ ) or BPsor alone, without irradiation ( ⁇ ).
  • the present invention pr ovi des a compound having th e f ormul a
  • Y is biotin or iminobiotin
  • X is CH 2
  • P is psoralen or a psoralen derivative
  • r is an integer equal to or greater than 2
  • s is an integer equal to or greater than 1.
  • X is bound to the 4' position of psoralen or a psoralen derivative.
  • the psoralen derivative may be 4, 5', 8 - trimethylpsoralen or 8 - methoxypsoralen.
  • r is the integer 2 and s is the integer 1.
  • the invention also provides a method for preparing a biotinylated psoralen or a biotinylated psoralen derivative which comprises treating a compound having the structure
  • L is chlorine, bromine, or iodine and P is psoralen or a psoralen derivative linked to L by CH 2 at the 4' position of P with a positively charge multi-ami nated linker having two amine groups separated by at least two carbon atoms under suitable conditions so as to allow the formation of a psoralen-linker complex.
  • the psoralen-linker complex is treated with the N-hydroxy succinimide ester of either biotin or iminobiotin under suitable conditions so as to allow the formation of a psoralen-linker-biotin or -iminobiotin complex.
  • This method comprises binding a suitable carrier molecule, i.e. a molecule which is capable of cellular internalization by receptor mediated endocytosis, to biotin so as to form a biotinylated carrier.
  • the biotinylated carrier is bound to avidin so as to form an avidin-biotinylated carrier complex, which is then reversibly bound to an iminobiotinylated psoralen so as to form an iminobiotinylated psoralen-avidin-biotinylated carrier complex.
  • Cells are treated with a suitable amount of the iminobiotinylated psoralen-avidin-biotinylated carrier complex under suitable conditions so as to permit the iminobiotinylated psoralen-avidin-biotinylated carrier complex to become internalized.
  • the treated cells are incubated in the dark under suitable conditions so as to permit the iminobiotinylated psoralen moiety to dissociate from avidin and intercalate into a nucleic acid.
  • suitable carrier molecule includes, but is not limited to, insulin and transferrin.
  • the invention further provides a method for treating leukemia in a subject.
  • This method comprises binding a suitable carrier molecule to biotin to form a biotinylated carrier.
  • the biotinylated carrier is bound to avidin so as to form an avidin-biotinylated carrier complex, which is then bound to an iminobiotinylated psoralen to form an iminobiotinylated psoralen-avidin-biotinylated carrier complex.
  • a leukocyte enriched composition is recovered.
  • This leukocyte enriched composition is treated with a suitable amount of the iminobiotinylated psoralen avidin-biotinylated carrier complex, which is permitted to become internalized.
  • the treated leukocyte enriched composition is incubated in the dark under suitable conditions so as to permit the iminobiotinylated psoralen moiety to dissociate from avidin and intercalate into DNA within the cells.
  • the invention additionally provides a method for detecting the presence of a nucleic acid in a sample.
  • This method comprises contacting the sample under suitable conditions in the dark with a biotinylated psoralen so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • the sample is then irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which is has intercalated.
  • the biotinylated psoralen which is covalently bound to the nucleic acid is contacted with a detectably marked avidin under suitable conditions so as to allow the avidin moiety to bind to the biotinylated psoralen which is covalently bound to the nucleic acid.
  • the presence of avidin bound to the biotinylated psoralen, which is also covalenty bound to the nucleic acid is detected and thereby the presence of the nucleic acid in the sample is detected.
  • the invention also provides a method for purifying or isolating nucleic acid from a sample.
  • This method comprises contacting the sample under suitable conditions in the dark with a biotinylated psoralen so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • the sample is irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which it has intercalated.
  • the biotinylated psoralen which is covalently bound to the nucleic acid, is then contacted with an immobilized avidin under suitable conditions so as to allow the avidin moiety to bind to the biotinylated psoralen and form an avidin-biotinylated psoralen-nucleic acid complex.
  • the nucleic acid may then be recovered from the avidin-biotinylated psoralen-nucleic acid complex.
  • the invention provides a method for total nucleic acid pattern visualization of a sample.
  • This method comprises electrophoresing the nucleic acid of the sample and immobilizing it to a solid support.
  • the immobilized nucleic acid is contacted with a biotinylated psoralen and the solid support is incubated in the dark for an appropriate amount of time so as to allow the biotinylated psoralen to intercalate into a nucleic acid.
  • Non-intercalated biotinylated psoralen is removed from the solid support and nucleic acid complexes bound to the solid support and intercalated with biotinylated psoralen are irradiated with near ultraviolet light to permit the intercalated biotinylated psoralen to covalently bind the nucleic acid into which it has intercalated.
  • the solid support is contacted with a detectably marked avidin under suitable conditions so as to allow the avidin moiety to bind the biotinylated psoralen, which is covalently bound to the nucleic acid.
  • the presence of avidin bound to the biotinylated psoralen is detected and thereby the total nucleic acid pattern of the sample is visualized.
  • the invention provides a method for visualizing a specific nucleic acid in a sample.
  • This method comprises electrophoresing the nucleic acid of the sample and immobilizing the electrophoresed nucleic acid to a solid support.
  • the immobilized nucleic acid is denatured so as to produced single stranded nucleic acids.
  • a single stranded nucleic acid clone of the specific nucleic acid to be visualized is contacted with a biotinylated psoralen.
  • the nucleic acid clone is incubated under suitable conditions in the dark so as to allow the formation of biotinylated psoralen-nucleic acid clone complexes, which are then irradiated with near ultraviolet light so as to permit the biotinylated psoralen to covalently bind the nucleic acid clone into which it has intercalated.
  • the immobilized single stranded nucleic acids are contacted with the nucleic acid clone which is covalently bound to the biotinylated psoralen so as to allow the nucleic acid clone and the immobilized single stranded nucleic acids to hybridize.
  • the invention also provides a cross-linked double stranded nucleic acid represented by the structure wherein A is a purine or a pyrimidine, B 2 and B 3 are purines, B 1 and B 4 are pyrimdines, W is a biotinylated psoralen cross-linked to B 1 and B 4 such that the 3, 4 and 4', 5' double bonds of W react with the 5, 6 double bonds of B 1 and B 4 to form cyclobutane products, Z is H or OH and m and n are integers from 0 to about 100,000.
  • A is a purine or a pyrimidine
  • B is a pyrimidine
  • W is a biotinylated psoralen linked to B such that either the 3, 4 or the 4', 5' double bond of W reacts with the 5, 6 double bond of B to form a cyclobutane product
  • Z is H or OH
  • m and n are integers from 0 to 100,000.
  • the invention further provides a method for detecting in a sample a biotinylated substance.
  • This method comprises contacting a cross-linked double stranded nucleic acid of the present invention with avidin under suitable conditions to allow the avidin to bind to the biotinylated psoralen moiety and form an avidin-biotinylated psoralen-nucleic acid complex.
  • the sample is contacted with the avidin-biotinylated psoralen-nucleic acid complex under suitable conditions to allow the formation of a biotinylated substance-avidin-biotinylated psoralen-nucleic acid complex, the nucleic acid moiety of which is detected, thereby detecting the presence of the biotinylated substances.
  • the biotinylated substance which is detected may be a biotinylated molecule, cell component, or intact cell.
  • nucleic acid moiety may be detected by a colorimetric, chemical, or radioactive technique.
  • a method for detecting in a sample an avidinylated substance is also provided by the present invention.
  • This method comprises contacting the sample with a cross-linked double stranded nucleic acid molecule of the present invention under suitable conditions to allow the formation of avidinylated substance-biotinylated psoralen-nucleic acid complexes. The presence of the nucleic acid moiety of the complexes is detected, thereby detecting the presence of the avidinylated substance.
  • the avidinylated substance may be an avidinylated molecule, cell component, or intact cell. Additionally, the nucleic acid moiety may be detected by a colorimeteric, chemical, or radioactive technique.
  • Diaminepsoralen was prepared from chloromethyl-trimethylpsoralen and sym-dimethylethylene diamine (Aldrich) as described by Welsh (7). Twelve mg of DAPS were dissolved in 0.4 ml dimethylformamide and 15 mg of NHS biotin (Pierce) was added as a solid. The reaction proceeded at room temperature. Reaction progress was followed by thin layer chromatography (TLC) on silica in CH 2 Cl 2 : NH 3 saturated methanol (15:1). The R f 's of DAPS, NHS biotin, and the reaction product were 0.25, 0 and 0.28 respectively. After one hour the reaction was complete and the solvent was rotoevaporated off, leaving a yellow oil. All steps were carried out under subdued light, and vessels were covered with aluminum foil when possible.
  • TLC thin layer chromatography
  • the product was purified by flash chromatography on a 30 x 2.5 cm column of silica gel in the TLC solvent system CH 2 Cl 2 : NH 3 saturated methanol (15:1) .
  • the reaction mixture did not dissolve directly in the running solvent, so the yellow oil was first taken up in 0.1 ml CH 3 OH, and then 1.5 ml CH 2 Cl 2 was added to the solution.
  • Fractions of about 10 ml were collected by hand and analyzed by TLC.
  • Fractions 15-22 contained the reaction product, running as a single spot with blue fluorescence at R f - 0.28.
  • Fractions 17-20 were pooled, dried down, taken up in running solvent as before, and rechromatographed.
  • [ 3 H] labelled biotinylated psoralen was prepared from diaminepsoralen and [ 3 H] NHS biotin (Amersham), and purified by preparative HPLC. The specific activity was 3.8 x 10 11 cpm/mmol.
  • Plasmid pBR322 DNA was linearized with Hindlll, and the DNA purified by phenol extraction, followed by ethanol precipitation and resuspension in 10mM Tris HCl, 1mM
  • BPsor biotinylated psoralen
  • the samples were then alkali denatured and run on a non-denaturing 1% agarose gel in the Tris acetate-EDTA, as described in (9) .
  • Calf thymus DNA at a concentration of 20 micrograms/ml (30 mM base pairs) was combined with 6 uM [ 3 H] BPsor in TE buffer. The sample was irradiated with near UV light, then phenol extracted and ethanol precipitated to remove unbound psoralen. The pellet was resuspended in phosphate buffered saline (PBS). The level of BPsor addition was 0.9%, or 1 psoralen/110 base pairs.
  • PBS phosphate buffered saline
  • the plates were washed three (3) times with IX PBS-0.5% Tween 20, and 200 microliters of 1% fetal calf serum in PBS-Tween was added to block the wells. After one hour at 37°C the solution was shaken off. 100 microliters of 5 microgram/ml streptavidin (Bethesda Research
  • Lymphocytes were isolated from 50 ml of whole blood by centrifugation on Ficoll Hypaque. They were washed twice in Roswell Park Memorial Institute (RPMI) medium, then resuspended in PBS and adjusted to 10 6 cells/ml in PBS.
  • RPMI Roswell Park Memorial Institute
  • BPsor in ethanol
  • BPsor in ethanol
  • the final ethanol concentration was 1%.
  • Negative controls contained no drug, while positive controls contained 10 ng/ml AMT, in ethanol.
  • 200 microliters of the cell suspension was added to wells in microtiter plates. Two dishes were prepared, each with 10 wells of each drug concentration. One was wrapped in aluminum foil and the second was irradiated with 3 J/cm 2 of near UV light. The microtiter plates were centrituged at 1200 rpm for seven minutes, then quickly inverted to discard the PBS.
  • lymphocytes in the wells were resuspended in 100 microliters RPMI, then half the wells received 100 microliters RPMI-20% fetal calf serum, while the other half received 100 microliters RPMI-20% fetal calf serum-2% PHA.
  • the dishes were incubated for three days at 37°C.
  • Psoralens intercalate into DNA in the dark and form covalent bonds at their 3, 4 and 4', 5' double bonds with pyrimidines upon near UV irradiation. If both ends of psoralen are reacted, the result is an interstrand DNA crosslink.
  • the ability of the BPsor to form DNA crosslinks was tested by reacting linear double strand plasmid DNA with this derivative and near UV light. The DNA was then alkali denatured and loaded onto a nondenaturing agarose gel. Crosslinked DNA immediately renatures in the gel buffer and runs as the double stranded form, while non-crosslinked DNA remains single stranded and runs with greater mobility. Increasing amounts of BPsor resulted in increased levels of crosslinking after UV irradiation, while even the greatest amounts of BPsor produced no crosslinking in the absence of light ( Figure 2).
  • the measured enzyme activity was proportional to the added BPsor and after a two hour incubation, alkaline phosphatase reaction at levels two times above background were seen in the wells with 2 fmol of bound BPsor, corresponding to 0.15ng ( Figure 4). After overnight incubation, 1 fmol could be detected above background. Both BPsor and UV irradiation were required for avidin binding. The control samples, which had been incubated with BPsor but not irradiated, stayed at background levels of alkaline phosphatase activity, as did DNA which had been exposed to light in the absence of BPsor.
  • the reagent binds covalently to DNA in the presence of UV light and subsequently binds tightly to avidin.
  • BPsor The biological effectiveness of BPsor was tested by assaying its ability to inhibit lymphocyte proliferation. Freshly prepared peripheral blood lymphocytes were incubated with BPsor and exposed to 3J/cm 2 of near UV light. PHA was then added to the treated cells to stimulate their proliferation, and after three days their growth was assayed by measuring [ 3 H] thymidine incorporation. Proliferation is expressed as the stimulation index, the ratio of [ 3 H] incorporation into cell s with and without PHA addition.
  • BPsor addition to lymphocytes at 1 microgram/ml decreased the stimulation index by more than 99% after irradiation, but had no effect in the dark. See Table I below. TABLE I
  • a biotin-containing psoralen derivative (BPsor) has been synthesized from commercially available reagents in a simple two-step reaction, producing a bifunctional nucleic acid- and avidin-binding reagent.
  • BPsor Like other psoralens, BPsor binds covalently to DNA in a near UV photoreaction, resulting in interstrand crosslinks, and like other biotinylated molecules it binds to avidin, even after it has been incorporated into DNA. The biotinylation does not interfere with its biological activity in lymphocytes; treatment with BPsor at 10 ng/ml plus near UV light inhibits PHA stimulation. BPsor shows a potency comparable to that of its immediate precursor, diaminepsoralen, and higher than that of the commonly used psoralen derivative 8-MOP.
  • BPsor is useful for the following:
  • BPsor Delivery of BPsor to cell s as an avidin-BPsor conj ugate with a readily internal ized mol ecul e, such as transferrin or insulin.
  • a r eversible iminobiotin-Psor shoul d be useful for this purpose , as the drug will bind to avidin wi th a K d of 5 x 10 -7 at extracellular pH, but only 10 -4 in the more acidic endosome, leading to dissociation of most of the psoralen.
  • This internalized compound may then be activated by ultraviolet A energy to kill or functionally impair target cells.
  • the DNA contains a clonable-selectable gene.
  • BPsor may be used to target psoralen to specific cells by attaching it to avidin and a biotinylated cell-specific carrier.
  • the biotinylation of psoralen does not interfere with its biological activity in lymphocytes; treatment with BPsor at 10 ng/ml plus near UV light inhibits cellular proliferation.
  • BPsor exhibits a potency comparable to that of its immediate precursor, diaminepsoralen, and greater than that of the clinically used derivative 8-MOP.

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Abstract

Composé de formule (I) dans laquelle Y représente biotine ou iminobiotine, X représente CH2, P représente psoralène ou un dérivé de psoralène, R est un nombre entier égal ou supérieur à 2 et s est un nombre entier égal ou supérieur à 1. Est également décrit un procédé de préparation de psoralène biotinylé ou d'un dérivé de psoralène biotinylé. Sont également décrits des procédés de détection, purification, isolation d'acides nucléiques, des procédés d'administration de psoralène iminobiotinylé à une cellule, ainsi que des procédés de traitement de la leucémie.
EP19870903009 1986-04-02 1987-03-30 Psoralenes biotinyles Withdrawn EP0262212A1 (fr)

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US847422 1986-04-02

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DK641487A (da) * 1987-12-07 1989-06-08 Gluetech Aps Fremgangsmaade til modificering af polymeroverflader

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US4206232A (en) * 1976-05-10 1980-06-03 E. R. Squibb & Sons, Inc. Relieving hypertension with carboxyalkylacylamino acids
US4656252A (en) * 1980-01-24 1987-04-07 Giese Roger W Amidobiotin compounds useful in a avidin-biotin multiple layering process

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See references of WO8705805A1 *

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