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WO1999061910A1 - Criblage de composes par ultrafiltration et spectrometrie de masse - Google Patents

Criblage de composes par ultrafiltration et spectrometrie de masse Download PDF

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Publication number
WO1999061910A1
WO1999061910A1 PCT/US1999/011493 US9911493W WO9961910A1 WO 1999061910 A1 WO1999061910 A1 WO 1999061910A1 US 9911493 W US9911493 W US 9911493W WO 9961910 A1 WO9961910 A1 WO 9961910A1
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Prior art keywords
ultrafiltration
sample
solution
biological material
metabolites
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PCT/US1999/011493
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English (en)
Inventor
Richard B. Van Breemen
Judy L. Bolton
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Board Of Trustees Of The University Of Illinois
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Application filed by Board Of Trustees Of The University Of Illinois filed Critical Board Of Trustees Of The University Of Illinois
Publication of WO1999061910A1 publication Critical patent/WO1999061910A1/fr
Priority to US09/471,523 priority Critical patent/US6995022B1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • This invention relates to a high throughput, on-line, pulsed ultrafiltration method useful for drug development and screening for metabolic parameters, bioactivation and potential toxicity of compounds and molecules. Enzyme assays and bioavailability studies are aspects of the invention.
  • vi tro assays have been developed to study drug metabolism using hepatic microsomes, reconstituted purified isozymes, primary culture hepatocytes, tissue slices, and cytochrome P450 overexpressed in whole cells (Parkinson, 1996; aurel, 1996).
  • these in vi tro assays lack sufficient throughput to keep pace with the large number of lead compounds being identified through combinatorial chemistry drug discovery programs.
  • the fastest current method to assess drug metabolism utilizes hepatic microsomes, which are incubated in a test tube with a drug and the cofactor NADPH. After at least 10 minutes, the solution is extracted (5-30 min) , the extract may be concentrated (0-60 min) , then the extract is analyzed using HPLC, GC, LC-MS or GC-MS (10-60 min) . Therefore, the fastest current method requires from 35-170 min, depending upon the sample handling and analysis procedures. There is a need to streamline (reduce the number of separate steps, reduce the sample handling requirements, and integrate the process into a faster, simpler and automated assay) and increase the throughput of existing in vi tro metabolism assays.
  • the present invention provides a method of determining whether a compound has predetermined characteristics that make it a candidate for drug 5 development or a substrate for a particular enzyme.
  • the method is a novel, high-throughput , on-line analysis of compounds added to a continuous flow system through biological materials in solution-that interact with the compounds. "High-throughput" is defined herein to be of
  • Results of the biological interactions are separated for analysis by an ultrafiltration membrane. This method is useful for characterization of, e . g.
  • 1M Xenobiotic is defined herein as compositions foreign to a living organism.
  • second solution and analyzing the second solution to determine whether the compound in the sample had the predetermined characteristics.
  • the biological sample includes a protein, a peptide, an oligonucleotide, an oligosaccharide, a microsome, a cell, a tissue, an enzyme, a receptor, DNA and RNA.
  • the compound is generally a candidate for drug development produced by combinatorial chemistry or a natural product.
  • the supportive solution includes a buffer, a nutrient medium, or a combination thereof.
  • the supportive solution is capable of maintaining the biological material in a state wherein the biological material can interact with a compound in the sample.
  • the continuous flow facilitates the interaction of the compound (s) with the biological material and then facilitates the removal of the compounds and metabolites thereof by washing them through the ultrafiltration membrane and into the second solution for analysis.
  • the predetermined characteristics of a compound to be analyzed include functioning as substrate for an enzyme in the biological material, showing desirable rates of enzyme catalysis, showing desirable rates of cell membrane permeability or transport, or showing activation to reactive or toxic metabolites.
  • the sample is added to the continuous flow by means of injection or infusion.
  • the suitable conditions for interaction of the biological material in the first solution with the compound in the sample include mixing the sample with the biological material to achieve a homogeneous distribution, controlling temperature to maintain function of the biological material, providing adequate sample concentration and a sufficient amount of biological material to facilitate analysis, allowing sufficient time for interaction, and controlling atmospheric gases to maintain function of biological material .
  • the ultrafiltration membrane pore sizes allow the sample to pass through but not the biological material.
  • the analyzing of the second solution is generally by mass spectrometry, UV spectrophotometer, electrochemistry, IR, radioactivity, fluorescence spectrophotometry or NMR.
  • a kit for analyzing compounds in a sample includes, in separate containers, an ultrafiltration membrane, a first solution containing a biological material, a buffer, a test solution, a set of standard solutions with predetermined characteristics and a receptacle for the second solution that results from interactions of the compounds with the biological material .
  • the high throughput, on-line, ultrafiltration- mass spectrometric method is useful to generate, identify, and quantify metabolites of compounds formed by drug metabolizing enzymes such as cytochrome P450, UDP- glucuronyltransferases, and glutathione transferases.
  • This method designated pulsed ultrafiltration-mass spectrometry, may be used for rapid screening of drugs or other compounds to determine the extent of their metabolism and to characterize their primary metabolites. If reactive and potentially toxic metabolites are formed during reactions, e . g. cytochrome P450 oxidation, the metabolites can be reacted with glutathione and then detected on-line using mass spectrometry in a rapid assay to assess the potential for toxicity.
  • the method is useful for the determination of the inherent bioavailability of xenobiotic compounds.
  • FIG. 1A is a diagrammatic representation of pulsed ultrafiltration-mass spectrometric on-line screening for metabolism and toxicity or bioavailability of a compound.
  • FIG. IB is a diagrammatic representation of a stirred pulsed ultrafiltration chamber.
  • FIG. 2 is a diagrammatic representation of a high throughput pulsed ultrafiltration-mass spectrometry system to screen xenobiotic compounds for products of drug metabolism.
  • FIG. 3 shows results of an on-line pulsed ultrafiltration positive ion electrospray mass spectrometric analysis of chlorpromazine oxidation by rat liver microsomal cytochromes P450.
  • Chlorpromazine (3 ⁇ g, molecular structure shown) was injected into an ultrafiltration chamber containing uninduced rat liver microsomes. NADPH was added to the continuous flow.
  • FIG. 4 shows results of pulsed ultrafiltration mass spectrometric analysis of the O-dealkylation of pentoxyresorufin (molecular structure shown) by uninduced rat liver microsomes .
  • the deprotonated molecules of pentoxyresorufin and the metabolite resorufin (modular structure shown) were monitored on-line using negative ion electrospray.
  • the control experiment was identical except that no NADPH was present .
  • FIG. 5 shows results of high throughput pulsed ultrafiltration mass spectrometric screening obtained using the format described in FIG. 2.
  • FIG. 6 shows computer-reconstructed mass chromatograms of the positive ion electrospray reversed phase LC-MS (liquid chromatography-mass spectrometry) analyses of the major imipramine metabolites, which were formed by 1) incubation of imipramine with rat liver cytochromes P450 during pulsed ultrafiltration, 2) collection of the effluent containing the metabolites, and 3) reinjection onto the LC-MS. Tandem mass spectra of each of the isomeric peaks of m/z 297 are summarized in Table 1.
  • FIG. 7 shows results of screening for formation of glutathione adducts as indicators of toxic
  • metabolites using pulsed ultrafiltration- mass spectrometry.
  • the chamber was loaded with rat liver microsomes (1 mg/mL protein) containing cytochromes P450 and icrosomal glutathione S- transferase.
  • the substrate, butyldimethyl phenol was injected along With NADPH and glutathione as cofactors .
  • the formation of metabolites was monitored by using negative ion electrospray mass spectrometry.
  • FIG. 8 illustrates constant neutral loss LC-MS- MS analysis of the glutathione adducts of 3-methylindole formed during pulsed ultrafiltration toxicity screening.
  • A isomeric glutathione ducts detected at m/z 437
  • B conjugation with glutathione
  • FIG. 9 illustrates constant neutral loss tandem mass spectrum obtained in 1 min using on-line pulsed ultrafiltration-MS-MS showing the detection of a butyldimethyl phenol-glutathione adduct; in this high throughput analysis, glutathione adducts are selectively detected by the elimination of the gamma-glutamyl group weighing 129 Daltons; background ions corresponding to unreacted butyldimethyl phenol, NADPH, buffer and contaminants are eliminated by the quadrupole mass filters during this MS-MS analysis because they do not fragment to eliminate the characteristic glutathione group weighing 129 Daltons.
  • FIG. 10 shows elution profiles for propranolol and aspirin during a pulsed ultrafiltration assay to determine their relative permeability to intestinal epithelial Caco-2 cells.
  • the permeability of intestinal epithelial cells is a fundamental factor that determines the absorption of compounds from the gut and affects their bioavailability; data in the shaded area of the elution curve were used to calculate the relative elution rates shown in FIG. 11.
  • FIG. 11 shows elution rates of propranolol and aspirin from the ultrafiltration chamber containing Caco- 2 cells; the data used to generate this plot were obtained from the shaded area of the elution curves shown in FIG. 10.
  • a high throughput, on-line, pulsed ultrafiltration-mass spectrometric method has been developed to determine whether a compound has predetermined characteristics that would make it suitable for a specific purpose, e . g. drug development.
  • the method is used to generate, identify, and quantify metabolites of compounds formed by drug metabolizing enzymes such as cytochrome P450, UDP- glucuronyltransferases, and glutathione transferases.
  • the method is useful for rapid screening of drugs or other compounds to determine the extent of their metabolism and to characterize their primary metabolites. If reactive and potentially toxic metabolites are formed during, e.g.
  • cytochrome P450 oxidation the metabolites can be reacted with glutathione and then detected on-line using mass spectrometry in a rapid assay to assess the potential for toxicity.
  • rates of cellular uptake, absorption and permeability may be measured using pulsed ultrafiltration as a measure of bioavailability.
  • Biomaterial such as enzymes responsible for metabolism of drugs and other xenobiotic compounds is in a first solution in a first chamber (an
  • the biological material includes enzymes which may consist of single, purified enzymes or mixtures of enzymes. Alternatively, the enzymes may be contained in microsomal preparations such as liver microsomes, tissue homogenates or intact, living cells such as hepatocytes.
  • the enzymes may include soluble enzymes such as proteases or glutathione transferases, microsomal enzymes such as cytochromes P450 or UDP-glucuronyl transferases, or enzymes contained in intact cells.
  • the first chamber may contain intact cells or membrane preparations for the study of drug uptake, transport, absorption and bioavailability.
  • the pore size of the membrane can, however, be closer to retain molecules of whatever molecular weight that is suitable for a particular application.
  • a continuous flow of supportive solution such as a buffer and/or nutrient media passes through the first chamber.
  • a sample with a compound (or molecule (s) or a mixture of compounds) to be tested is joined with the continuous stream of a supportive solution, as a pulse.
  • the sample to be tested is injected into the stream to interact with the biological material in the first solution.
  • the results of the interaction subsequently flow through an ultrafiltration membrane to the analyzer.
  • the pore sizes of the membrane are selected based on the size of the predetermined resulting compounds or molecules and biological material. Pore sizes may range from those allowing only very low molecular weight products to pass through, e . g. acetone or benzene (50- 100D) to larger molecules in the 12,000-20,0000 range e . g. enzymes, myoglobin, to very large molecules (100, 000-million D) .
  • the pore size must be small enough to prevent the biological material from passing through, e.gr. a 10,000 molecular weight cut-off pore size prevents adenosine deaminase from passing through and a 100,000 molecular weight cut-off membrane prevents microsomes from passing through.
  • a preferred analyzer is a mass spectrometer.
  • Other suitable analyzers are UV- spectrophotometers, fluorescence spectrophotometers, electrochemical detectors, NMR spectrometers, radioactivity monitors, and IR spectrophotometers.
  • a continuous-flow of a buffer (usually a volatile buffer such as ammonium acetate at physiological pH) is pumped through the ultrafiltration chamber and into an electrospray mass spectrometer detector (or other suitable LC-MS or LC-MS-MS instrument) .
  • the metabolism of xenobiotic compounds such as drugs is investigated by flow-injecting an aliquot of the compound (or mixture of xenobiotic compounds) through the first ultrafiltration chamber containing the trapped enzymes and then detecting the metabolites as they elute from the chamber using on-line electrospray mass spectrometry.
  • the solutions in the first chamber are stirred.
  • Any necessary cofactors, such as NADPH (for cytochrome P-450 oxidation) , UDPGA for glucuronyl transferase, or glutathione for glutathione transferase, are either included in the continuous phase or co-injected with the xenobiotic substrate.
  • Electrospray mass spectrometric detection facilitates 1) the determination of whether metabolites of the xenobiotic compound (or compound mixture) are formed by a particular enzyme preparation, 2) the determination of the molecular weight of each metabolite, and 3) quantitation of each metabolite and unchanged substrate. Measurement of the rate of disappearance of the substrate provides a measure of how extensively one compound is metabolized compared to another, which is an important consideration during drug development.
  • Tandem mass spectrometry may be used to confirm structures of metabolites, and if isomers of metabolites are formed (such as mixtures of monooxygenated metabolites with identical molecular weights) , then an aliquot of the effluent from the ultrafiltration chamber may be analyzed using LC-MS-MS to separate each isomer and then obtain its tandem mass spectrum for identification.
  • reactive metabolites may be trapped by reaction with nucleophiles such as N-acetyl cysteine or as phase II (reaction with conjugating enzymes such as glutathione-S-transferase, UDP-glucuronyl transferases, or sulfo transferases) conjugates such as glutathione adducts.
  • nucleophiles such as N-acetyl cysteine or as phase II (reaction with conjugating enzymes such as glutathione-S-transferase, UDP-glucuronyl transferases, or sulfo transferases) conjugates such as glutathione adducts.
  • conjugating enzymes such as glutathione-S-transferase, UDP-glucuronyl transferases, or sulfo transferases
  • conjugates such as glutathione adducts.
  • High throughput bioavailability measurements are carried out using, for example, live epithelial cells as the biological material in the ultrafiltration chamber.
  • live epithelial cells For example, absorption from the human intestine may be predicted using human intestinal epithelial (Caco-2) cells.
  • Xenobiotic compounds are injected through the ultrafiltration chamber and their elution profiles are recorded using mass spectrometric detection. Compounds that are excluded from entering the cells elute from the chamber first, and compounds that easily diffuse into, or are actively transported into, the cells have a larger volume of distribution and elute later. Bioavailability correlates inversely with elution time, and the most bioavailable compounds are those that elute last .
  • Ultrafiltration mass spectrometry may be used for the analysis of one compound or one mixture of compounds at a time per ultrafiltration chamber.
  • higher throughput ultrafiltration mass spectrometric metabolic and toxicity screening may be carried out using multiple ultrafiltration chambers ⁇ e . g. up to 60 at a time) arranged in parallel with a single mass spectrometer.
  • FIG. 2 By connecting each ultrafiltration chamber on-line to the mass spectrometer for only 1 minute, efficient use of the mass spectrometric detector may be obtained with a throughput of up to 60 ultrafiltration experiments per hour.
  • the compounds that may be screened include drugs, drug candidates, combinatorial libraries, natural products, pesticides, herbicides, other xenobiotic compounds and endogenous biological compounds.
  • On-line ultrafiltration electrospray mass spectrometry offers a streamlined, high-throughput method for in vi tro formation and mass spectrometric characterization of drug metabolites.
  • a scheme of pulsed ultrafiltration-mass spectrometric screening for metabolism and toxicity or bioavailability is shown in diagrammatic form in FIG. 1A.
  • a compound (or mixture of compounds) 1 is flow-injected 2 into an ultrafiltration chamber 3 containing, e . g. an enzyme, enzyme mixture or cells 7.
  • Any necessary cofactors, such as NADPH, UDPGA, glutathione, etc. are injected along with the compound (s) 1 or added to the continuously flowing buffer solution.
  • Cofactors and cellular nutrients that are essential for cell viability must be continuously introduced to the ultrafiltration chamber.
  • cofactors such as NADPH or UDPGA that are only needed for enzymatic activity may be co-injected with the-compound (s) 1 under study.
  • Co-injection with the compound (s) 1 is the most economical approach.
  • the enzymes which are too large to pass through the ultrafiltration membrane may be in pure form or contained in microsomes, tissue homogenate, or living cells.
  • the living cells used for bioavailability measurements cannot pass through the ultrafiltration membrane. Metabolites formed in the ultrafiltration chamber are washed out by the buffer solution being pumped through the ultrafiltration membrane and are detected by the on-line electrospray mass spectrometer 4. Unchanged compound (s) 1 may be washed out into the detector and measured during bioavailability studies or quantitative metabolism studies.
  • part of the effluent from the chamber may be collected for LC-MS or LC-MS-MS analysis 5.
  • bioavailability measurements may be carried out by using living cells such as Caco-2 intestinal epithelial cells in the ultrafiltration chamber.
  • FIG. IB shows details of a stirred pulse ultrafiltration chamber.
  • FIG. 2 A diagrammatic representation for a high throughput pulsed ultrafiltration-mass spectrometry system to screen xenobiotic compounds for drug metabolism is shown in FIG. 2.
  • Multiple ultrafiltration chambers 13 are connected in parallel 12 to a single mass spectrometer detector 15.
  • a different xenobiotic compound or mixture is injected into each chamber at intervals of e . g. 1 min. (for 60 screens per hour using 60 chambers) or 3 min. (for 20 screens per hour using 20 chambers) .
  • Constant flow of incubation buffer is maintained through all chambers by HPLC pumps 10, but only one chamber at a time is connected 16 to the mass spectrometer 15.
  • Metabolite profiles are obtained by recording mass spectra over a period of up to, e . g. 1 min. (if screening 60 samples per hour, or up to three minutes (20 samples per hour) when the maximum concentration of metabolites is eluting (approximately 20-30 min after the xenobiotic compound is injected) . Waste 14 is discarded.
  • Results of on-line pulsed ultrafiltration positive ion electrospray mass spectrometric analysis of chlorpromazine oxidation by rat liver microsomal cytochromes P450 is shown in FIG. 3.
  • Chlorpromazine (3 ⁇ g, molecular structure shown) was injected into an ultrafiltration chamber containing uninduced rat liver microsomes. NADPH was added to the mobile phase.
  • This experiment demonstrates that drug metabolism by microsomal cytochromes P450 may be investigated directly using on-line ultrafiltration mass spectrometry without the need for sample extraction or chromatography. Quantitative analysis of the extent of drug metabolism may be carried out by measuring consumption of NADPH or sample, chlorpromazine.
  • results of pulsed ultrafiltration mass spectrometric analysis of the O-dealkylation of pentoxyresorufin by uninduced rat liver microsomes is shown in FIG. 4.
  • the cofactor NADPH was coinjected along with the sample pentoxyresorufin.
  • the deprotonated molecules of pentoxyresorufin and the metabolite, resorufin (molecular structure shown) were monitored on-line using negative ion electrospray.
  • the control experiment was identical except that no NADPH was present. This experiment demonstrates that cofactors such as NADPH may be co-injected with the sample instead of being added continuously in the mobile phase.
  • High throughput pulsed ultrafiltration mass spectrometric screening as shown in FIG. 5 was obtained using the 20 analyses per hour format disclosed for FIG. 2.
  • the ultrafiltration chamber contained rat liver microsomes. Imipramine and NADPH were coinjected through the chamber, and on-line mass spectrometric detection was used for only 2 minutes. Metabolism of imipramine by rat liver microsomes was demonstrated by the appearance of monoxygenated imipramine at m/z 297. Mass spectrometric analysis for 2 minutes of effluent from the parallel control incubation (containing NADPH and inactive microsomes) showed no metabolites of imipramine.
  • Results of screening for formation of glutathione adducts as indicators of toxic (electrophilic) metabolites using pulsed ultrafiltration- mass spectrometry are shown in FIG. 7.
  • the chamber was loaded with rat liver microsomes (1 mg/mL protein) containing cytochromes P450 and microsomal glutathione S-transferase .
  • the substrate, butyldimethyl phenol was injected along with NADPH and glutathione as cofactors.
  • the formation of metabolites was monitored by using negative ion electrospray mass spectrometry.
  • Oxidation of butyldimethyl phenol by cytochromes P450 produced a reactive quinone methide intermediate which either reacted with water to form the oxidation product detected at m/z 193, or reacted with glutathione to form the adduct detected at m/z 482.
  • glutathione adducts were observed only in embodiments containing both glutathione and NADPH.
  • FIGS. 1A and B A schematic diagram of the pulsed ultrafiltration-mass spectrometry system is shown in FIGS. 1A and B.
  • the ultrafiltration chamber is built out of a solvent resistant polymer with low protein and drug binding properties for example, the polymer polyetheretherketone (PEEK) and contains a Teflon-coated magnetic stirring bar and a Teflon or Viton 0-ring, which forms a seal around the ultrafiltration membrane.
  • PEEK polymer polyetheretherketone
  • the surface area of the ultrafiltration membrane should be as large as possible to reduce back pressure that might rupture the ultrafiltration membrane and to allow for high flow rates through the chamber (up to 100 ⁇ L/min) . Flow rates may be a low as desired for enzyme reaction or cell permeation (i . e .
  • the ultrafiltration chamber should have a small volume compared to the large surface area of the ultrafiltration membrane. Therefore, the ultrafiltration chamber is shallow but wide, and the ultrafiltration membrane covers the exit side of the wide face of the chamber. (FIG. IB) . Volumes of, e . g. ( ⁇ 1 mL) minimize the amount of enzymes and substrates required per experiment, thereby reducing costs, which is particularly useful when enzymes and substrates are difficult to obtain in larger quantities.
  • a prototype chamber (FIG.
  • the ultrafiltration chamber is preferably stirred to rapidly mix substrates and enzymes and to prevent build up of enzymes (or microsomes or cells 9) on the underside of the ultrafiltration membrane.
  • a commercially available methylcellulose ultrafiltration membrane is suitable (i.e., Amicon; Beverly, MA) , and the molecular weight cut-off of the membrane must be small enough to prevent the metabolizing enzymes from leaving the first chamber, but large enough to allow the xenobiotic compounds under study and their metabolites to pass through to form a second solution.
  • the enzyme adenosine deaminase may be trapped in the first chamber using an ultrafiltration membrane with a molecular weight cut-off of 10,000
  • liver microsomes may be trapped in the first chamber using an ultrafiltration membrane with a molecular weight cut-off of 100,000
  • cells may be trapped in the first chamber using an ultrafiltration membrane with a MWCO of 10,000 or 1 million.
  • Standard chromatography tubing and fittings made from polyetheretherketone (PEEK) are suitable to connect the HPLC pump, injector, ultrafiltration chamber, and mass spectrometer.
  • biological material is loaded into the chamber using an HPLC injector.
  • Suitable material includes cells, enzymes, microsomes or other material.
  • the biological material may consist of enzymes, cells, microsomes, tissue homogenate, tissue slices, RNA, DNA or other macromolecules.
  • a constant flow of buffer i.e., 50-100 ⁇ L/min
  • the buffer should be volatile (i . e . , ammonium acetate buffer) in order to minimize contamination of the mass spectrometer ion source.
  • non-volatile buffers may be used if a desalting column is used between the ultrafiltration chamber and the mass spectrometer, or if a salt-resistant LC-MS interface is used such as the Z-sprayTM interface (Micromass, Manchester, UK) .
  • a xenobiotic compound (or mixture of compounds) is flow- injected into an ultrafiltration chamber using the HPLC injector.
  • Cofactors such as NADPH for cytochrome P450 oxidation, UDPGA for glucuronidation by UDP- glucuronyltransferases, or glutathione for glutathione-S- transferases, are injected along with the compound (s) under study or added to the continuously flowing buffer solution.
  • phase I oxidation by cytochromes P450 is often followed immediately in vivo by conjugation by phase II enzymes such as glucuronyltransferases or glutathione-S-transferases .
  • phase II enzymes such as glucuronyltransferases or glutathione-S-transferases
  • phase I and phase II enzyme systems may be included in an on-line pulsed ultrafiltration-mass spectrometry assay.
  • microsomes containing complex mixtures of enzymes such as liver microsomes may be used in the chamber. Living cells may be used in the chamber as well for studies of metabolism or bioavailability.
  • Metabolites As metabolites are formed in the ultrafiltration chamber, they are separated from the enzymes and washed out of the chamber by the continuously flowing buffer solution. Metabolites may be detected, identified and quantified on-line using a mass spectrometer equipped with an appropriate LC-MS interface such as electrospray or atmospheric pressure chemical ionization. Identification and quantification of metabolites using LC-MS is routine in the art of mass spectrometry. For additional characterization of metabolite mixtures, part of the effluent from the chamber may be collected for LC-MS or LC-MS-MS analysis. The addition of a chromatography step is necessary for the analysis of isomeric metabolites (i.e., metabolites with identical molecular weights) .
  • An aliquot of the effluent may be directly injected onto an HPLC column for LC-MS-MS analysis, or a short HPLC column may be used to concentrate and desalt a larger volume of effluent prior to LC-MS or LC-MS-MS analysis. Since the compound (s) 1 and their metabolites might be in low concentration in the second solution (i.e., the ultrafiltrate) and since the second solution will contain buffer salts, a trapping/desalting column may be used to concentrate the compound (s) 1 and their metabolites and at the same time remove the buffer. The desalted and concentrated extract may then be analyzed directly by mass spectrometry, LC- MS, or LC-MS-MS. (See Materials and Methods for more details.) Alternatively, the metabolites may be extracted from the effluent prior to analysis using LC-MS or LC-MS-MS.
  • Living cells may be used in the ultrafiltration chamber either as a source of enzymes for metabolism and other enzymatic assays, or for on-line bioavailability measurements. Because of the large size of cells compared to enzymes, much larger pore sizes may be used for the ultrafiltration membrane and molecular weight cutoffs of 500,000 or 1,000,000 daltons work well with low back pressure.
  • the ultrafiltration chamber is preferably stirred in order to maintain the cells in suspension, otherwise the cells will adhere to the ultrafiltration membrane. For some types of assays, it may be desirable to have a layer of cells on the membrane. In order to maintain cell viability for several hours, the buffer must be isotonic with the cells, but it does not need to be isotonic with respect to sodium and potassium nor must the buffer contain cell nutrients.
  • the buffer must function as a complete cell growth medium and should contain appropriate amounts of sodium and potassium to support cell survival.
  • the effluent from the chamber may be directed through an IAPLC column to extract and concentrate the desired compounds and remove the buffer salts and cell nutrients. Then, the desired compounds may be eluted directly into analyzer such as a mass spectrometer for identification and quantification.
  • Bioavailability assays are generally carried out in one of two ways.
  • the chamber is loaded with cells (i.e., Caco-2 human intestinal epithelial cells) such that the volume of the cells is significant compared to the volume of the chamber ( e . g. , 1 million cells/mL) , and a size exclusion experiment is carried out. (Hidalgo, et al . 1989).
  • An aliquot of a drug mixture is injected through the chamber and the elution profile is recorded by the mass spectrometer detector.
  • the mixture may contain a single drug or a mixture of drugs under investigation and two internal controls:
  • the negative control compound such as mannitol
  • the positive control compound such as certain steroids
  • Compounds that are excluded from the cells are washed out of the ultrafiltration chamber faster and are detected first, while compounds that enter the cells are distributed into a larger volume within the chamber and require longer to elute. In this manner, quantitative bioavailability or absorption data may be determined.
  • a monolayer of cells i.e., Caco-2 cells
  • Caco-2 cells a monolayer of cells (i.e., Caco-2 cells) is grown on an ultrafiltration membrane, which is then mounted in the ultrafiltration chamber with the cells facing the interior of the chamber. Then, compounds are injected through the chamber at a very low flow rate that does not disturb the integrity of the cell membranes. Compounds that readily enter and pass through the cells are detected first.
  • pulsed ultrafiltration-mass spectrometry facilitates 1) the determination of whether enzymatic products or metabolites of a xenobiotic compound (or compound mixture) are formed by a particular enzyme preparation, 2) the identification of the metabolites, and 3) quantitation of each product or metabolite and the amount of unchanged substrate. Measurement of the rate of disappearance of the substrate provides a measure of how extensively one compound is metabolized compared to another. Additional mass spectrometric analysis using pulsed ultrafiltration-tandern mass spectrometry or LC- MS-MS may be used for confirmation of the structures of each enzymatic product or metabolite.
  • pulsed ultrafiltration-mass spectrometry may be used as a method for screening for the formation of toxic drug metabolites .
  • mixtures of xenobiotic compounds may be injected simultaneously into the continuous stream flowing through the ultrafiltration chamber, and the metabolism or bioavailability of multiple compounds at a time may be investigated.
  • the throughput may be increased 10-fold by injecting mixtures containing 10 compounds at a time.
  • the concentration of each compound must be kept low enough so that the enzyme (s) do not become saturated, or the cell receptors or other sites of entry into the cells do not become saturated.
  • On- line ultrafiltration electrospray mass spectrometry offers a streamlined, high-throughput method for in vi tro formation and mass spectrometric characterization of metabolites of xenobiotic compounds.
  • the system is easily automated for high throughput screening to rapidly determine 1) if a compound is a substrate for a drug metabolizing enzyme or enzyme system, 2) how rapidly and extensively a compound is metabolized relative to other substrates, 3) if reactive (electrophilic) and potentially toxic metabolites are formed, 4) what are the structures of each metabolite, and finally, 5) what is the relative bioavilability of each compound.
  • Chlorpromazine is an antidepressant-drug that undergoes extensive hepatic metabolism. (De Vane, 1995) .
  • the continuous mobile phase consisted of the volatile buffer, 50 mM ammonium acetate, pH 7.4, and cofactor, NADPH (0.1 mM) at a flow rate of 70 ⁇ L/min.
  • Protonated molecules of chlorpromazine, the cofactor NADPH, and the microsomal metabolic products were recorded continuously using positive ion electrospray mass spectrometry on a Hewlett-Packard (Palo Alto, CA)
  • Electrospray mass spectrometry was used to identify all major metabolites, and the computer-reconstructed mass chromatograms of selected ions are shown in FIG. 3 to illustrate the appearance of the oxidized chlorpromazine metabolites, monitor the consumption of NADPH, and monitor the consumption of unreacted chlorpromazine.
  • the area under the curve for unreacted chlorpromazine is proportional to the amount of chlorpromazine consumed during the on-line metabolism experiment.
  • the appearance of abundant metabolites, such as the oxidized chlorpromazine shown in FIG. 3 is evidence that chlorpromazine is extensively metabolized by hepatic cytochromes P450 and that this method may be used to identify metabolites or types of metabolites (such as oxidation products) and determine the extent of metabolism.
  • Pentoxyresorufin is a standard substrate used in the assay of cytochrome P450 2B-catalyzed 0- dealkylation activity (Burke et al . , 1985), and enzymatic O-dealkylation results in the loss of the pentoxy group to form resorufin.
  • Pentoxyresorufin (1 ⁇ g) was injected into the ultrafiltration chamber containing hepatic microsomes as described herein. Negative ion electrospray mass spectrometry was used to monitor the appearance of resorufin, the O-dealkylated product, at m/z 212.
  • the mass chromatograms for the elution of the metabolite resorufin and unreacted pentoxyresorufin are shown in FIG. 4.
  • NADPH was injected simultaneously with substrate instead of being added to the mobile phase to produce a maximum concentration in the ultrafiltration chamber of 0.46 mM NADPH.
  • This approach consumes less NADPH cofactor than the example shown in Example 1. If NADPH was not injected with the drug, no metabolism occurred as shown in the control profile of resorufin in FIG. 4.
  • This example shows that other types of drug metabolites, such as dealkylation products, may be identified using this method.
  • liver microsomes were used from a rat that had not been treated with any compounds to induce cytochrome P450 activity.
  • the on-line observation of metabolites formed using uninduced rat cytochromes P450 illustrates the great sensitivity of this method and indicates that uninduced human microsomes are a practical alternative.
  • the chamber is rapidly flushed out to waste and then reloaded with microsomes for another assay.
  • Metabolism of imipramine, chlorpromazine, quinidine and naringenin was evaluated using the 20 analyses per hour format described in FIG. 2, and control assays were carried out using heat inactivated microsomes.
  • imipramine and cofactor NADPH were injected simultaneously into a pulsed ultrafiltration chamber containing rat liver microsomes as described herein to give a maximum chamber concentration of 1.53 ⁇ g/mL (5.46 ⁇ M) imipramine and 0.46 mM NADPH. Similar injections were carried out for other compounds, but the effluent from each ultrafiltration chamber was connected on-line to the electrospray mass spectrometer for only 3 min. each, so that 20 analyses per hour could be carried out.
  • FIG. 5 shows the mass spectra for the assay of imipramine obtained during these high throughput metabolic screening experiments. Formation of metabolites was determined by comparing a control incubation to an incubation with active microsomes. As expected, background ions, but no ions of metabolites were observed in the control experiments (See FIG. 5A) . Metabolites of imipramine and chlorpromazine were observed (for example, see the ions of m/z 297 corresponding to monooxygenated imipramine in FIG. 5B) , but no metabolites of naringenin or quinidine were detected.
  • the high throughput analyses of the present invention may also be used quantitatively to assess the extent of metabolism by measuring the disappearance of drug during incubation compared to control incubations that use inactive microsomes or no NADPH cofactor.
  • imipramine metabolites were generated using rat liver microsomes and pulsed ultrafiltration as described in Example 2, except that the effluent from the ultrafiltration chamber was reinjected onto a reversed phase HPLC column for LC-MS and LC-MS-MS analysis.
  • the major metabolites of cytochrome P450 oxidation of imipramine were N- desmethylimipramine and the isomeric monooxygenated species 10-hydroxyimipramine, 2 -hydroxyimipramine and imipramine N-oxide as shown in the LC-MS analysis of the ultrafiltrate in FIG. 6.
  • Each metabolite shown in FIG. 6 was identified based on its tandem mass spectrum (obtained during LC-MS-MS) as shown in Table 1.
  • the pulsed ultrafiltration mass spectrometry system described in Examples 1 and 2 was used with rat liver microsomes and butyldimethyl phenol as the substrate. Coinjection with NADPH resulted in formation of a reactive metabolite, a quinone methide oxidation product of butyldimethyl phenol which may be detected after its reaction with water (FIG. 7) .
  • An advantage of this on-line metabolism-mass spectrometric detection system is the ability to detect and identify metabolites just seconds after they are formed. Because so little time elapses and no sample manipulation is necessary, some reactive (and potentially toxic) metabolites that might not be identified using any other method might be observed directly before they decompose.
  • reactive metabolites may be trapped as phase II conjugates such as glutathione adducts.
  • Phase II conjugates such as glutathione adducts.
  • Coinjection of butyldimethyl phenol with NADPH and glutathione resulted in the formation of a quinone methide metabolite (a phase 1 oxidation reaction) followed by formation of a glutathione adduct (a phase II conjugation reaction) of the electrophilic metabolite with the nucleophilic glutathione.
  • the glutathione product was detected online using pulsed ultrafiltration electrospray mass spectrometry (FIG. 7) .
  • the observation of glutathione adducts during pulsed ultrafiltration mass spectrometry indicates that reactive metabolites are formed by cytochrome P450 metabolism, and therefore, this assay system is appropriate for toxicity screening of drugs and other compounds .
  • Glutathione adducts formed from reactive drugs or their activated metabolites in the ultrafiltrate may be trapped and concentrated on a trapping column and then analyzed using LC-MS or LC-MS-MS for identification of the metabolites and for the differentiation of isomeric products.
  • This application is similar to Example 4, which describes the use of LC-MS and LC-MS-MS for the identification of isomeric metabolites of imipramine.
  • the metabolism of 3-methylindole was investigated using pulsed ultrafiltration mass spectrometry as an example of formation and detection of adducts of a toxic, electrophilic imine methide intermediate (see Skiles et al .1996 for additional information about the metabolism of 3-methylindole) .
  • the eluate from the ultrafiltration chamber was trapped on a short HPLC column, concentrated and then eluted and analyzed using LC-MS-MS.
  • Constant neutral loss MS-MS was used to selectively detect glutathione adducts by scanning for ions that fragment to eliminate a group weighing 129 D, which is characteristic of glutathione adducts (Ramanathan et al .
  • Pulsed ultrafiltration mass spectrometric screening may be used on-line for high throughput detection of glutathione adducts for rapid toxicity screening of compounds or compound mixtures.
  • the effluent from the ultrafiltration chamber needs to be monitored only long enough to obtain a tandem mass spectrum that would indicate the presence of glutathione adducts, if any were formed.
  • constant neutral loss mass spectrometry on a triple quadrupole mass spectrometer was used for 1 min. to detect the presence of a glutathione conjugate of butyldimethyl phenol.
  • the metabolic activation of butyldimethyl phenol and reaction with glutathione are described in Example 5. If 60 ultrafiltration chambers were arranged in parallel as shown in FIG.
  • Microsomes may be prepared from any types of cells or tissues, which are obtained from any species including humans .
  • male or female Sprague-Dawley rats (180- 200 g) were obtained from Sasco Inc. (Omaha, NE) .
  • Microsomes were prepared from rat liver, and protein and cytochrome P450 concentrations were determined using standard procedures (1) .
  • the microsomes were diluted (typically three-fold) with 50 mM ammonium acetate buffer at pH 7.4 immediately before use to make the protein concentration approximately 10 mg/mL.
  • microsomes were washed for 30 min at 70 ⁇ L/min with 50 mM ammonium acetate buffer at pH 7.4 to remove low molecular weight contaminants . This washing step may be accelerated by using a higher flow rate, but care should be taken to avoid excessive pressure and rupture of the ultrafiltration membrane.
  • the ultrafiltration chamber (FIG. IB) was built in-house out of polyetheretherketone (PEEK) and contained a Teflon ® -coated magnetic stirring bar and a Viton ® 0- ring, which formed a seal around the ultrafiltration membrane.
  • the methylcellulose ultrafiltration membrane was purchased from Amicon (Beverly, MA) and had a molecular weight cut-off of 100,000. Ultrafiltration membranes with smaller pore sizes (i . e . , 10,000 molecular weight cut-off) were investigated but could not be used without clogging. Chromatography tubing and fittings were made from polyetheretherketone (PEEK, Upchurch Scientific, Oak Harbor, WA) .
  • NADPH and drug substrates were purchased from Sigma Chemical Company (St. Louis, MO). All solvents were HPLC grade.
  • chlorpromazine 3 ⁇ g
  • 50 ⁇ L buffer was injected into the ultrafiltration chamber.
  • Protonated molecules of chlorpromazine, cofactor NADPH, and the microsomal metabolic products were recorded continuously using positive ion electrospray mass spectrometry.
  • 1 ⁇ g of pentoxyresorufin was injected into the ultrafiltration chamber containing hepatic microsomes as described above.
  • Negative ion electrospray mass spectrometry was used to monitor the appearance of resorufin, the O-dealkylated product, at m/z 212.
  • NADPH was injected simultaneously with substrate instead of being added to the mobile phase.
  • imipramine, and cofactor NADPH were injected simultaneously to give a maximum chamber concentration of 1.53 ⁇ g/mL (5.46 ⁇ M) imipramine and 0.46 mM NADPH.
  • a wide mass range was scanned by the mass spectrometer in order to identify the major metabolites (if unknown) .
  • selected ion monitoring mass spectrometry is used to follow the appearance of metabolites, monitor the consumption of NADPH, and/or monitor the profile of unreacted compound (s) .
  • Control experiments are typically carried without cofactor or with heat inactivated microsomes.
  • Mass spectrometry An Applied Biosystems (Foster City, CA) 140A dual syringe pump with a Rheodyne 8125 injector was used for all analyses. Mass spectra were acquired using either a Micromass (Manchester, UK) Quattro II triple quadrupole mass spectrometer equipped with an electrospray ionization source, or a Hewlett- Packard (Palo Alto, CA) 5989B quadrupole mass spectrometer with an Analytica (Branford, CT) electrospray ion source. Both instruments were tuned to a peak width of 0.6 u over the entire mass range.
  • LC-MS Liquid chromatography-mass spectrometry
  • LC- MS-MS liquid chromatography-tandem mass spectrometry
  • the solvent system consisted of a gradient from 75% water (containing 0.5% acetic acid and adjusted to pH 3.5) to 70% methanol in 50 min and then to 90% methanol over an additional 10 min at a flow rate of 180 ⁇ L/min.
  • Table 1 shows a summary of tandem mass spectra of imipramine metabolites that were generated by cytochrome P450 metabolism during pulsed ultrafiltration.
  • tandem mass spectrometric analysis of the various imipramine metabolites facilitated the determination of metabolite structures such as the localization of hydroxyl groups or site of demethylation. For example, only the 10-hydroxyimipramine metabolite eliminated water during collision induced dissociation, so that this metabolite was easily distinguished from 2- hydroxyimipramine or imipramine N-oxide.
  • Parkinson, A. An overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Toxicol . Pathol . 24:45-57 (1996) . Ramanathan, R. , Cao, K, Cavalieri, E. and Gross, M. : Mass spectrometric methods for distinguishing structural isomers of glutahione conjugates of estrone and estradiol . J. Am . Soc. Mass Spectrom . 9 : 612 - 619. Sequeira, D. J., Strobel, H. W.: High-performance liquid chromatographic method for the analysis of imipramine metabolism in vitro by liver and brain microsomes. J " . Chromatogr. B. 673:251-258 (1995).

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Abstract

L'invention concerne une technique de spectrométrie de masse/ultrafiltration pulsée, en ligne et à haut rendement, qui permet de déterminer si un composé possède une caractéristique prédéterminée susceptible de le rendre intéressant pour un objectif spécifique, tel que le développement de médicaments. Cette technique est utile pour générer, identifier et quantifier les métabolites des composés formés par des enzymes métabolisant les médicaments, telles que le cytochrome P450, les UDP-glucuronyltransférases et les glutathion transférases. Elle est également utile pour cribler rapidement des médicaments ou d'autres composés afin de déterminer l'étendue de leur métabolisme et de caractériser leurs métabolites primaires. Si des métabolites réactifs et potentiellement toxiques sont formés durant, par exemple, l'oxydation par le cytochrome P450, on peut les faire réagir avec le glutathion et les détecter en ligne par spectrométrie de masse, en une analyse rapide, afin d'étudier leur éventuelle toxicité. Cette technique est également utile pour déterminer la biodisponibilité, l'absorption et la perméabilité cellulaire des composés.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6979545B2 (en) 2000-10-05 2005-12-27 Pharmacia & Upjohn Company Method for determining chemical reactivity
RU2232387C1 (ru) * 2002-11-04 2004-07-10 Научно-исследовательский институт по изучению лепры Способ оценки общетоксического действия лекарственных средств на организм

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