EP0641438A1 - Controle de medicaments assiste par fluorogene - Google Patents

Controle de medicaments assiste par fluorogene

Info

Publication number
EP0641438A1
EP0641438A1 EP92925368A EP92925368A EP0641438A1 EP 0641438 A1 EP0641438 A1 EP 0641438A1 EP 92925368 A EP92925368 A EP 92925368A EP 92925368 A EP92925368 A EP 92925368A EP 0641438 A1 EP0641438 A1 EP 0641438A1
Authority
EP
European Patent Office
Prior art keywords
pharmaceutical
fluorophore
modifiable
carbohydrate
pharmaceuticals
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Withdrawn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
EP92925368A
Other languages
German (de)
English (en)
Inventor
John C. Klock, Jr.
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.)
Glyko Inc
Original Assignee
Glyko Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Glyko Inc filed Critical Glyko Inc
Publication of EP0641438A1 publication Critical patent/EP0641438A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the subject invention is in the field of biological and chemical assays, in particular the measurement of levels therapeutic agents in the body.
  • the biologically available levels of a pharmaceutical may vary unpredictably in accordance with a number of factors including, clearance by the kidneys, binding to various serum components, rate of absorption into the body, breakdown rate, biological activity of the breakdown products, and the like.
  • Therapeutic drug monitoring TDM is currently performed for more than two dozen pharmaceutical treatments. Assays on a number of pharmaceutical chemicals are available; the total number of pharmaceutical chemical assays currently available is nearly 100.
  • Carbohydrates are increasingly recognized as important pharmaceutical agents. Convenient assays to measure the levels of carbohydrates and similar biologicals are not readily available. In most instances the analysis of such compounds is possible only with techniques employing advanced instrumentation, such as mass spectroscopy and NMR spectrometry. Furthermore, for some drugs, such as the anticoagulant carbohydrate drug "heparin", assays are difficult to develop because the drug is a mixture of biochemical components.
  • Heparin is a mixture of acidic polysaccharides derived from the proteolytic or autolytic digestion of animal connective tissue or mucosa (skin, gut, lung, or heart) ; for a review of heparin and its uses see Ofosu, et al. , Annals of the N.Y. Acad. Sci.. Vol. 556 (June 7, 1989) . Heparin is assayed only on the basis of its ability to inhibit blood coagulation protein reactions. Heparin assays are typically performed by either measuring whole blood clotting times or activated clotting assays (see for example, Williams, et al.. Hematolo ⁇ y. pgs.
  • anti-thrombin - III a specific clotting protease called anti-thrombin - III
  • FACE fluorophore-assisted carbohydrate electrophoresis
  • One aspect of the present invention is to apply the technique of FACE to quantitate various carbohydrate and non-carbohydrate pharmaceuticals in the body so as to provide for more successful therapy.
  • the present invention therefore provides for the quantitative detection of a variety of fluorophore- modifiable pharmaceutical molecules, pharmaceutical carbohydrates in particular, present in tissue samples of a patient exposed to a fluorophore- modifiable pharmaceutical in the course of treatment or that which may occur accidentally.
  • the present invention enables the dosage of the therapeutic carbohydrate administered to the patient may be adjusted so as maximize the desirable effects and minimize toxicity.
  • This quantitation of fluorophore-modifiable pharmaceuticals is accomplished by a process comprising derivatizing a functional side group of the pharmaceutical present in a tissue sample with a fluorescent label, separating the labeled carbohydrate (preferably by electrophoresis) , and identifying and quantitating the fluorophore-modified pharmaceutical, preferably by comparison with a derivatized pharmaceutical carbohydrate standard.
  • a preferred florescent label for modifying fluorophore-modifiable pharmaceuticals to be quantitated is 8-amino-naphthalene-l,3,6-trisulfonic acid (ANTS) .
  • the separation and quantification of the fluorophore-modified pharmaceuticals is preferably performed by polyacrylamide gel electrophoresis.
  • the separated fluorophore-modified pharmaceutical molecules may then be visualized using an imaging system based on photoelectric detection means, such means include the use of laser-scanner photomultiplier tubes and charge coupled device (CCD) cameras. Information from the CCD camera may subsequently be stored in digital form and analyzed by various computer programs for comparing banding patterns between samples and identification standards.
  • CCD charge coupled device
  • Gel-separated fluorophore-modified pharmaceuticals may optionally be transferred in situ to an immobilizing membrane; i.e., blotted, then probed with various specific-binding reagents whereby the identity and quantity of pharmaceutical molecules of interest may be determined.
  • fluorophore-modifiable pharmaceutical is defined as a pharmaceutical that is capable of being derivatized (through covalent bonds) by a fluorophore that can be employed in the technique of fluorophore-assisted carbohydrate electrophoresis.
  • the fluorophore-assisted carbohydrate electrophoresis technique is described in detail in U.S. patent 4,874,492 and in co-pending U.S. patent application
  • Fluorophores for modifying fluorophore-modifiable pharmaceuticals include 8- aminonapthalene-l,3,6-trisulphonic acid, 1-amino- 6,8-disulphonic acid, l-amino-4-naphthalene sulfonic acid, lucifer yellow, and 2-amino acridone. Fluorophore-modifiable pharmaceuticals may be derivatized using procedures described in the referenced patents and patent applications. Pharmaceuticals may be conveniently tested to determine if they are fluorophore-modifiable pharmaceuticals by attempting to fluorophore label the pharmaceuticals by the fluorophore labeling methods and labels useful for FACE.
  • Fluorophore- modifiable pharmaceuticals typically include carbohydrates having reducing sugars and other pharmaceuticals that have aldehyde functional groups. Fluorophore-modifiable pharmaceuticals include pharmaceutical carbohydrates and pharmaceuticals that are not carbohydrates. A fluorophore-modifiable pharmaceutical that has been labelled with a fluorophore is defined to be a "fluorophore-modified pharmaceutical.”
  • pharmaceutical carbohydrate is defined as carbohydrates that may be used as pharmaceuticals for human or non-human animals.
  • Pharmaceutical carbohydrates include the actual carbohydrate introduced into the body and any in vivo breakdown product or products of the carbohydrate introduced.
  • Pharmaceutical carbohydrates include heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, hyaluronic acid, dextran, dextran sulfate, lactulose, sucralose, sucralfate, sialic acids, glucose, galactose, mannose and the like.
  • Pharmaceutical carbohydrates may include carbohydrate antibiotics such as the aminoglycosides, beta-lactams and the like, and various phosphate and sulfate derivatives thereof.
  • Pharmaceutical carbohydrates include polyethylene glycol (and other similar water-soluble polymer) derivatives of non- carbohydrate pharmaceuticals.
  • Pharmaceutical carbohydrates may be covalently joined to polypeptides or lipids, i.e., glycoconjugates, or may be independent of other molecules.
  • Pharmaceutical carbohydrates may be monosaccharides, oligosaccharides, or polysaccharides.
  • Pharmaceutical carbohydrates may be branched or unbranched.
  • carbohydrate includes molecules that are completely carbohydrate, such as monosaccharides, oligosaccharides, and polysaccharides.
  • carbohydrate also includes glycoconjugates such as glycoproteins, glycolipids, proteoglycans, and the like.
  • biological level when used in reference to a pharmaceutical, is the concentration or quantity of that pharmaceutical found in a tissue sample.
  • drug when use in reference to pharmaceutical refers to both the absolute amount of pharmaceutical administered at a given point in time and the frequency of administration.
  • the subject invention provides for the convenient measurement of biological levels of fluorophore-modifiable pharmaceuticals used for the treatment of human and animal diseases.
  • Samples for analysis are removed from patients and subsequently treated so as to label any fluorophore-modifiable pharmaceutical present in the sample by a fluorophore suitable for use in the analytical technique known as fluorophore-assisted carbohydrate electrophoresis (FACE) .
  • FACE fluorophore-assisted carbohydrate electrophoresis
  • Pharmaceutical molecules modified by the fluorophore label are then quantitated, preferably based on fluorescence.
  • electrophoresis is used to separate and quantitate the fluorophore- modified pharmaceuticals in a patient sample.
  • the subject invention possesses a number of advantages over conventional techniques for measuring the biological levels of pharmaceuticals, particularly carbohydrate pharmaceuticals.
  • the present invention permits the simultaneous measurement of fluorophore-modifiable pharmaceutical quantities in multiple samples. Moreover, several pharmaceuticals in a single sample may be analyzed simultaneously. Additionally, the subject invention may be used to measure biological levels of pharmaceuticals that are chemically uncharacterized, since the method of the subject invention does not rely on highly specific reagents.
  • a further advantage of the subject invention is the high sensitivity of the detection system.
  • the method of the subject invention necessarily employs the step of fluorophore labeling fluorophore- modifiable pharmaceuticals.
  • the fluorophore labeling of fluorophore- modifiable pharmaceuticals enables the quantitation and separation, i.e., purification, of fluorophore-modifiable pharmaceuticals.
  • the fluorophores useful for labeling fluorophore- modifiable pharmaceuticals and the methods for performing the labeling are essentially the same as those fluorophores and methods using in the fluorophore labeling step of FACE.
  • One aspect of the subject invention is the measurement of the levels of fluorophore-modifiable pharmaceuticals present in a sample isolated from an individual being treated with the pharmaceutical of interest.
  • the quantity of fluorophore-modified pharmaceutical present in a sample may be measured by a variety of methods, these methods include the use of immunoassays specific for fluorophore-modifiable pharmaceuticals and fluorophore-modified pharmaceuticals, and the fluorescence measurement electrophoresis separation products.
  • the subject invention provides for several methods of quantitating fluorophore-modified pharmaceuticals, it is particularly preferred to apply the FACE technique to tissue samples from patients undergoing pharmaceutical treatment with various fluorophore-modifiable pharmaceutical.
  • the successful application of FACE to the detection of fluorophore-modifiable pharmaceuticals has a number of advantages over conventional measurement procedures for therapeutic carbohydrates, these advantages include the detection of drugs for which no specific assay is available, (possibly, for lack of antigenicity, lack of separability, lack of specifically reactive functional groups) , high sensitivity, the ability to simultaneously measure several drugs, and the ability to quantitate therapeutic carbohydrates from patient samples that contain molecules that bind to the drugs of interest.
  • fluorophore-assisted therapeutic drug monitoring of concentrations of fluorophore-modifiable pharmaceuticals in the body permits the controlled management of a variety of diseases conditions treatable by fluorophore- modifiable pharmaceuticals that require careful monitoring of biological levels in order to minimize adverse side-effects and maximize beneficial effects.
  • Fluorophore-modifiable pharmaceuticals may be separated by fluorophore-assisted carbohydrate electrophoresis in essentially the same way as the technique is used to separate carbohydrates.
  • a brief description of fluorophore-assisted carbohydrate electrophoresis is given below.
  • Fluorophore-assisted carbohydrate electrophoresis permits the electrophoretic separation of a complex mixture of carbohydrates into distinct bands on a gel or other electrophoresis separation matrix.
  • a carbohydrate mixture for analysis Prior to electrophoresis, a carbohydrate mixture for analysis is treated with a fluorophore label that combines with the reducing end of the carbohydrates for analysis. The fluorophore label permits the quantitative measurement of the labeled carbohydrates by fluorescence.
  • the fluorophore label either is charged or coupled with a charge imparting species when the fluorophore itself is uncharged.
  • the labelling process not only fluorescently tags the carbohydrates, but imparts an ionic charge, permitting hitherto uncharged carbohydrates to migrate in an electric field.
  • carbohydrates possessing a charge strong enough to permit migration in an electrophoresis system e.g., heparin
  • Suitable fluorescent labels include 8-aminonapthalene-l,3,6- trisulphonic acid (ANTS) , l-amino-4-naphthalene sulfonic acid (ANSA) , l-aminonapthalene-6,8- disulphonic acid (ANDA) , lucifer yellow, and 2- aminoacridone.
  • a description of fluorophores suitable for use in the subject invention can be found in ⁇ . S. patent application 07/483,043 file-; February 16, 1990 and UK patent: application GB/90/01448 filed September 20, 2990 and published as PCT application WO 91/05256, which are herein incorporated by reference.
  • the sample is subsequently subjected to polyacrylamide gel electrophoresis, or similar separation means, in order to separate and concentrate the labeled carbohydrates into bands.
  • the separated carbohydrates may be visualized directly (with staining) or by fluorescence under U.V. light and the banding patterns stored photographically.
  • the separated carbohydrates may be visualized by photoelectric means, including laser-scanner photomultiplier tube systems and cooled charge coupled devices (CCD) .
  • CCD's are semiconductor imaging devices that permit the sensitive detection of emitted light. CCD's and their uses are described in U.S. Patent 4,874,492 and U.S. Patent application 07/317,480, filed February 14, 1989, which are herein incorporated by reference.
  • the image produced by the CCD may be subsequently transferred to a computer so that the band information may be analyzed with respect to intensity, mobility, standards, and the like.
  • electrophoretic separation typically takes place to an extent sufficient to independently resolve bands of fluorophore-modifiable pharmaceuticals.
  • Electrophoresis may proceed past the point where some carbohydrates have been removed from the electrophoresis separation medium. Electrophoresis may be in one or in two dimensions. Two-dimensional separation of carbohydrates by fluorophore-assisted carbohydrate electrophoresis is described in U.S. patent 4,975,165, which is herein incorporated by reference. These two-dimensional electrophoresis techniques may also be applied to fluorophore-modified pharmaceuticals that are not carbohydrates.
  • fluorophore-modifiable pharmaceutical compositions administered to the body comprise a mixture of structurally related molecules, such as heparin, or glycoconjugates that differ from one another with respect to the extent of glycosylation
  • fluorophore-assisted carbohydrate electrophoresis of tissue samples containing the fluorophore-modifiable pharmaceutical may produce diffuse bands of labeled compounds rather than a narrow distinct band.
  • the amount of fluorophore-modified pharmaceutical present in diffuse bands such as the type produced by heparin, may be quantitated by determining the perimeter of the band and quantitating the florescence observed within the perimeter of the diffuse band.
  • Samples for analysis by fluorophore-assisted therapeutic drug monitoring may be prepared from many tissues removed from subjects.
  • tissue includes bodily fluids as well as solid cellular masses.
  • Tissues for analysis may contain at least one fluorophore-modifiable pharmaceutical (or the pharmaceutical breakdown product) .
  • Suitable tissues for use as samples include, blood, saliva, urine, skin, muscle, bone marrow, cerebrospinal fluid, synovial fluid, lymphatic fluid, amniotic fluid, and the like.
  • Preferred tissues for analysis are those tissues conveniently obtained from patients, particularly preferred tissues include urine and blood.
  • the selection of tissues for quantitation of fluorophore-modifiable pharmaceuticals by the subject invention vary in accordance with the disease and compound being analyzed.
  • Factors affecting the choice of tissues for analysis include: the quantity of therapeutic carbohydrates present in the tissue, the quantity of background carbohydrates in the tissue, and the presence of molecules in the tissue capable of interfering with electrophoretic separation of the fluorophore-modifiable pharmaceuticals.
  • Fluorophore-modifiable pharmaceuticals in a sample from a patient treated with a fluorophore- modifiable pharmaceutical may be present in a concentration that is higher than the concentration of fluorophore-modifiable pharmaceutical present in a sample from that individual prior to treatment.
  • the term "higher” includes the presence of pharmaceuticals that are present as opposed to completely absent. Differences in fluorophore- modifiable pharmaceutical concentrations between treated and untreated individuals do not necessarily exist in all body tissues.
  • Fluorophore-modifiable pharmaceuticals administered to a patient may vary in concentrations with respect to different tissues.
  • Preferred tissues for use as a source of samples for analysis by fluorophore-assisted therapeutic monitoring are those tissues that have the highest levels of the fluorophore-modifiable pharmaceuticals of interest; however, it may also be of interest to measure the presence of pharmaceutical carbohydrates in tissues that would be not be expected to contain appreciable amount of a specific fluorophore-modifiable pharmaceutical unless the patient has been administered too large, i.e., producing unacceptable side effects, a dose that fluorophore-modifiable pharmaceutical.
  • Samples for analysis may require processing prior to the fluorophore labeling step in the subject method.
  • the precise method of sample processing employed for a given assay may vary in accordance with a number of factors attributable to the choice of sample tissue and the identity of the specific fluorophore-modifiable pharmaceutical; these factors include: the concentration of the pharmaceutical, the concentration of background molecules, the presence of interfering molecules, i.e., molecules that adversely affect the mobility or fluorophore labelling of fluorophore-modifiable pharmaceutical molecules whether the pharmaceuticals are free or bound to other molecules, and similar problems.
  • Suitable methods for processing samples include: dialysis, to remove interfering molecules; filtration or centrifugation, to remove interfering particulates or concentrate cells; precipitation, to remove interfering molecules; and detergent solubilization, to release pharmaceutical carbohydrates from cells.
  • the pharmaceuticals of interest may then be transferred in situ to an immobilizing matrix, such as a nitrocellulose or nylon membrane.
  • an immobilizing matrix such as a nitrocellulose or nylon membrane.
  • the transfer may be by electroblotting or similar in situ transfer procedures.
  • Membranes containing the pharmaceuticals may subsequently be probed with antibodies or similar specific-binding reagents so as to indicate the presence and quantity of fluorophore-modified pharmaceuticals of interest.
  • the transfer of fluorophore-assisted carbohydrate electrophoresis separated carbohydrates onto immobilizing matrices is described in detail in U.S. patent application 07/481,367 filed February 16, 1990 and U.S. Patent 5,019,231 which is herein incorporated by reference. These membrane transfer techniques may be used with many fluorophore-modifiable pharmaceuticals, as well as carbohydrates in general.
  • Reasons for glycosidase treatment of samples include the liberation of carbohydrates from glycoconjugates, and the generation of pharmaceutical carbohydrates derivatives that have a more convenient gel migration rate, i.e., better separation from fluorophore-labeled compounds other than the pharmaceutical carbohydrates of interest. Similarly, it may be advantageous to use a glycosyl transferase, along with a donor sugar, to structurally modify a pharmaceutical carbohydrate so as to produce a compound with a more convenient gel migration rate.
  • antibodies specific for the polypeptide portion of a glycoprotein with many glycoforms, and present in a tissue with a high carbohydrate background may be used to immunoprecipitate the glycoprotein so that the precipitated glycoprotein may then be subjected to endoglycosylase treatment to release the carbohydrate moieties prior to performing fluorophore-assisted carbohydrate electrophoresis.
  • the purified glycoforms could thus be easily detected and quantitated in the presence of a reduced background.
  • fluorophore-modifiable pharmaceutical standards are included on the gels used to analyze pharmaceuticals in patient samples; however, the information embodied by the standards, e.g., band migration distance, band width, and intensity, may also be obtained by comparison with stored records made from fluorophore- modified pharmaceutical standards previously subjected to fluorophore-assisted carbohydrate electrophoresis under conditions similar to the conditions the samples for analysis are exposed.
  • Fluorophore-modifiable pharmaceutical standards may have a composition similar to that of samples for analysis in that they may contain both fluorophore- modified pharmaceuticals identical to the fluorophore-modified pharmaceuticals to be detected and background molecules with a composition similar to that found in actual samples.
  • fluorophore-modifiable pharmaceutical standards may contain one or more fluorophore-modified pharmaceuticals identical to the fluorophore- modified pharmaceuticals to be detected free of background carbohydrates.
  • Fluorophore-modifiable pharmaceutical standards for use in detecting and quantitating fluorophore-modifiable pharmaceutical that are the metabolic breakdown products of other fluorophore-modifiable pharmaceuticals may, among other methods, be produced by administering the pharmaceutical to an animal that produces the desired break down product or by treating the pharmaceutical with enzymes participating in the breakdown process.
  • Fluorophore-modifiable pharmaceutical standards are fluorophore labelled, i.e., fluorophore- modified, preferably labeled prior to the labelling of the samples for analysis; however, fluorophore- modifiable pharmaceutical standards are preferably labeled concomitantly with the labeling for the samples for analysis. Furthermore, fluorophore- modifiable pharmaceutical standards are preferably of known concentrations so as to provide for a quantitative determination of the amount of a given pharmaceutical carbohydrates in the samples of analysis. Preferably, several fluorophore-modifiable pharmaceutical standards of known concentration are present on a gel so as to provide a calibration curve for more accurate determination of the quantity of pharmaceutical carbohydrates in a samples for analysis.
  • kits for performing fluorophore-assisted therapeutic drug monitoring may, among other things, provide collections of reagents useful for performing fluorophore-assisted carbohydrate electrophoresis, or fluorophore-modifiable pharmaceutical standards.
  • Kits of the subject invention enable laboratories to conveniently and reproducibly perform fluorophore- assisted therapeutic drug monitoring.
  • Kits may include reagents for performing tests to identify one or more specific therapeutic carbohydrates.
  • Kits may include fluorophore-modifiable pharmaceutical standards, fluorescent label, blotting materials, (such as immobilizing membranes) carbohydrate- specific binding reagents, instructions, sample containers, polyacrylamide gel reagents, and the like.
  • More complete kits may also include equipment for performing fluorophore-assisted carbohydrate electrophoresis, such as polyacrylamide gel apparatus, CCDs, computers, software, and the like. Reagents included in fluorophore-assisted therapeutic drug monitoring kits are preferable provided in pre- measured quantities.
  • band data from the gels used to separate and quantitate fluorophore-modified pharmaceuticals are read by means of a CCD and stored in a computer usable form.
  • the image detected by the CCD, or other detection system may be analyzed by image analysis software such as Optimas* (Bioscan 1 ") or similar image analysis programs.
  • the data may be subjected to analysis by a variety of software programs.
  • Software programs of interest include those with the ability to quantitate band intensity, measure band mobility, determine the relative molecular weight of fluorophore-modifiable pharmaceuticals forming bands, compare the standards with the samples for analysis, prepare intensity/concentration calibration curves, remove unwanted background information, and perform various forms of statistical analysis.
  • quantitative data obtained from the fluorophore- assisted carbohydrate electrophoresis is manipulated and/or presented in electronic spreadsheet form, e.g., Lotus 1-2-3", Microsoft Excel".
  • An aspect of the subject invention is to provide for the improved management of the therapy of patients undergoing treatment with one or more fluorophore-modifiable pharmaceuticals. Improved management of patient therapy may be achieved by monitoring the concentration of the administered pharmaceutical and/or breakdown products of the administered pharmaceutical present in patient or tissues.
  • the amount of pharmaceutical administered may be altered by decreasing the dosage of fluorophore- modifiable pharmaceutical carbohydrate and/or the frequency of administration of the fluorophore- modifiable pharmaceutical.
  • the amount of fluorophore-modifiable pharmaceutical administered may be altered by increasing the dosage of pharmaceutical carbohydrate, and/or frequency of administration of the pharmaceutical carbohydrate.
  • Measuring the amount of a fluorophore- modifiable pharmaceutical (and/or its metabolic breakdown product) by fluorophore-assisted electrophoresis may also be used as a method of diagnosing the dysfunction of an organ that is involved in the metabolic processing of fluorophore- modifiable pharmaceuticals, especially the liver or kidneys.
  • the concentration of a particular fluorophore-modifiable pharmaceutical (and/or its metabolic breakdown product) found in a tissue is known to be a function of how well a particular organ(s) is functioning, e.g., if the organ metabolizes or clears the pharmaceutical, then determining the difference between the amount of a pharmaceutical carbohydrate administered and the amount found in the sample tissue, considering the amount of time that has elapsed since administration, serves to indicate how well that organ(s) is functioning.
  • a cirrhotic liver (possibly damaged as a result of a pharmaceutical carbohydrate) might not be able to degrade a particular pharmaceutical carbohydrate as fast as a healthy liver, thus blood levels of that pharmaceutical carbohydrate are found to be virtually the same at both 1 hour and 12 hours after administration, liver damage would be indicated.
  • Human serum was partially purified using a Millipore Ultra Free centrifugal concentrator, specifically to remove the high molecular weight compounds.
  • the sample was then placed in a microcentrifuge tube and dried using a centrifugal vacuum evaporator centrifuge vacuum evaporator.
  • To each dried sample was added 5 ⁇ l of 0.2M aminonapthalene-l,3,6-trisulphonic acid (ANTS) solution in acetic acid/water (3:17 v/v) and 5 ⁇ l of l.OM NaCNBH 3 solution in dimethyl sulfoxide (DMSO) .
  • the solution was vortex mixed, centrifuged at 10,000 g to ensure all the reactants are in the tips of the tubes, and incubated at 37°C for 15 hours.
  • the reaction mixture was dried under a vacuum for 4 hours in a centrifuge vacuum evaporator at approximately 45°C.
  • the reaction mixture was then dissolved in a suitable concentration of electrophoresis sample buffer.
  • Dextran sulfate has been used as an experimental treatment for HIV infections. Blood samples from several patients undergoing treatment with dextran sulfate are removed. Serum is isolated from the blood samples. Known quantities of dextran sulfate are added to serum so as to produce multiple quantitation standards. The serum samples and standards are partially purified using a centrifugal concentrator. The samples and standards are then placed in microcentrifuge tubes and dried. To each sample is added 5 ⁇ l of a 0.2M 2-amino acridone solution and 5 ⁇ l of l.OM NaCNBH 3 in DMSO. The solutions are mixed by vortexing, centrifuged, and incubated overnight at 37°C. The reaction mixtures are dried.
  • the reaction mix is then resuspended in electrophoresis loading buffering.
  • the samples and standards are then loaded on to a vertical discontinuous 8% polyacrylamide Laemli gel (lacking SDS) .
  • the gel is run at constant voltage with cooling.
  • the individual labeled dextran sulfate bands are imaged based on their fluorescence by means of a CCD camera and software designed to recognize and quantitate bands.
  • the quantity of fluorescence observed from the standards is plotted against the known concentration of dextran sulfate in the standards.
  • the quantity of fluorescence observed from the labeled dextran sulfate in the patient samples is then compared with the plots obtained from the standards in order to determine the quantity of dextran sulfate present in the patient samples.

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Abstract

L'invention décrit des procédés servant à quantifier les niveaux biologiques de différents produits pharmaceutiques, particulièrement des produits pharmaceutiques à base de glucides. Lesdits procédés comprennent l'étape de marquage fluorescent du produit pharmaceutique présent dans un échantillon d'analyse. On peut ensuite quantifier le produit pharmaceutique marqué au fluorogène en se basant sur des mesures de fluorescence. De plus, on peut séparer lesdits produits pharmaceutiques d'autres composés de l'échantillon par électrophorèse. On utilise la quantification des niveaux biologiques de produits pharmaceutiques, afin de déterminer le dosage optimum desdits produits.
EP92925368A 1991-11-21 1992-11-20 Controle de medicaments assiste par fluorogene Withdrawn EP0641438A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US79572691A 1991-11-21 1991-11-21
US795726 1991-11-21
PCT/US1992/010061 WO1993010450A1 (fr) 1991-11-21 1992-11-20 Controle de medicaments assiste par fluorogene

Publications (1)

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EP0641438A1 true EP0641438A1 (fr) 1995-03-08

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EP92925368A Withdrawn EP0641438A1 (fr) 1991-11-21 1992-11-20 Controle de medicaments assiste par fluorogene

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EP (1) EP0641438A1 (fr)
AU (1) AU3144993A (fr)
WO (1) WO1993010450A1 (fr)

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