| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 11.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 23.11.99 |
| PATENT TITLE |
ADME analysis of mixtures |
| PATENT ABSTRACT |
A method for analyzing the ADME/PK properties of a mixture of compounds is (1) perfusing an animal or organ with a perfluorocarbon emulsion blood substitute, (2) administering the mixture of test compounds, (3) withdrawing an aliquot of the perfusate, (4) disrupting the emulsion, and (5) analyzing the aqueous phase of the perfusate for the concentration of test compounds. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 28.08.97 |
| PATENT REFERENCES CITED |
Pellegrin et al., European Journal of Drug Metabolism and Pharmacokinetics., 1985., vol. 10., No. 2., pp. 113-120. Takahashi et al., "The Use of a Perfluorochemical Emulsion as a Vascular Perfusate in Drug Absorption" J. Pharm. Pharmacol. 40:252-257, 1988. Geyer, "`Bloodless` Rats Through the Use of Artificial Blood Substitutes" Federation Proceedings 34(6):1499-1505, May, 1975. Baba et al., "Ex-Vivo Perfusion of Surgically Removed Organs" Biomat. Art. Cells Art. Org. 16(1-3): 623-624, 1988. Faithfull, "Fluorocarbons Current Status and Future Applications" Anaesthesia 42:234-242, 1987. Dietz et al., "ADME Characterization of Complex Chemical Mixtures in vivo: Development of a Method to Analyze Mixtures of 24 Molecules in an Oxygen-Carrying Blood Substitute" Pharmaceutical Research 13(9 Suppl), 1996, Abstract No. PPDM 8426. Spence, "Perfluorocarbons in the Twenty-First Century: Clinical Applications as Transfusion Alternatives" Art. Cells, Blood Subs., and Immob. Biotech. 23(3):367-380, 1995. Spence, "Perfluorocarbons as Blood Substitutes: the Early Years Experience with Fluosol DA-20% in the 1980s" Art. Cells, Blood Subs., and Immob. Biotech. 22(4):955-963, 1994. Noble, "Artificial Blood" Analytical Chemistry 67(1):31A-33A, Jan., 1995. Flaim, "Pharmacokinetics and Side Effects of Perfluorocarbon-Based Blood Substitutes" Art. Cells, Blood Subs., and Immob. Biotech. 22(4):1043-1054, 1994. Marchbank, "Fluorocarbon Emulsions" Perfusion 10:67-88, 1995. Luo et al., Poster Presented at the 14th Symposium on Liquid Chromatography/Mass Spectrometry (L.C./MS, CF/MS, MS/MS) at Cornell University, Ithaca, New York, Jul. 23-25, 1997, poster entitled "Development of a Quantitative Method for Measuring Chemical Mixtures in a Perfluorocarbon Based Oxygen-Carrying Blood Substitute Using LC/MS". |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed: 1. A method for determining pharmacokinetics of a test compound in an isolated live organ, comprising: (a) perfusing said isolated organ with a blood substitute, wherein said blood substitute is a fluorocarbon emulsion; (b) administering said test compound to said isolated organ; (c) removing a portion of said blood substitute from said isolated organ; (d) adding a water-miscible organic solvent to said portion to disrupt said emulsion; and (e) analyzing said portion for the presence of said test compound or its metabolites. 2. The method of claim 1, wherein said fluorocarbon emulsion is selected from the group of perfluorocarbons consisting of perfluorotributylamine, perfluorodecalin, perfluorooctyl bromide, perfluorotripropylamine, bis(perfluorobutyl)ethylene, perfluoro-N,N-dimethylcyclohexylmethylamine, perfluorotrimethylbicyclo(3.3.1)-nonane, perfluorobicyclo(5.3.0)decane, and perfluorobutyltetrahydrofuran. 3. The method of claim 2, wherein said fluorocarbon emulsion is perfluorotributylamine. 4. The method of claim 1, wherein said water-miscible organic solvent is selected from the group consisting of an alcohol, an amine, tetrahydrofuran, and pyridine. 5. The method of claim 1, wherein said water-miscible organic solvent is selected from the group consisting of isopropanol and acetonitrile. 6. The method of claim 1, wherein said test compound is administered as a mixture of test compounds. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to the fields of pharmacokinetics and pharmacological research. More specifically, the invention describes the concept of using perfluorocarbon emulsions for examining the ADME/PK (absorption, distribution, metabolism, excretion and pharmacokinetics) properties of chemical mixtures in animals and a method for preparing the emulsions for direct analysis by techniques such as high performance liquid chromatography (HPLC), mass spectrometry and capillary electrophoresis. BACKGROUND OF THE INVENTION Drug development begins with the identification of a lead compound, based on the ability of the compound to exhibit a desired biological effect, such as the ability to inhibit bacterial growth, inhibit the activity of a target enzyme, increase or modulate the uptake of neurotransmitters, and the like. Biological activity is typically determined on the basis of in vitro experimentation or assays designed to rapidly identify candidate drugs. Typically, only a small percentage of the compounds tested will demonstrate sufficient activity and selectivity to merit further investigation. Once a candidate or lead compound has been identified and selected for further development, its ADME/PK characteristics are determined. ADME/PK concerns the absorption, distribution, metabolism, excretion and pharmacokinetics of drugs in the body. The ADME/PK properties of a drug are critical, and often serve to distinguish pharmaceutical products from mere lead compounds. For example, a drug that is poorly absorbed orally may require intravenous (or other parenteral) administration to be effective, which may be unacceptable for the condition to be treated. A compound effective as an antibiotic may be ineffective to treat bacterial meningitis if its distribution does not carry it to the central nervous system. A compound that is rapidly metabolized and/or excreted may not reside in the body long enough to serve its intended purpose. These properties are all independent of the drug candidate's in vitro activity, and are difficult or impossible to predict based on current information. The complex factors that influence ADME/PK make it hard to model accurately, and necessitate the use of living tissues and research animals before a compound may proceed with clinical trials. To enhance the speed of drug discovery and reduce the number of animals required, it is desirable to characterize the ADME/PK of mixtures of lead compounds (rather than single compounds) in procedures that involve either living animals (i.e., in vivo), or isolated organs or organ systems from animals. In in vivo analyses of ADME/PK, plasma is generally the biophase used as the analytical endpoint. Measurement of individual drug candidates in plasma typically involves a unique extraction method based on the physicochemical properties of each molecule, in order to separate and quantify the compound from the numerous plasma components. Optimization of one plasma extraction method for all components of a chemical mixture poses a major problem for rapid screening. SUMMARY OF THE INVENTION We have now invented a method for improving the ADME/PK analysis of candidate compounds, by replacing the blood of a test animal or tissue with an emulsified blood substitute, administering a test compound, and analyzing the resulting blood substitute. Preferably, the test compound is administered as a mixture of test compounds. Another aspect of the invention is the method for designing libraries of pharmaceutical candidates based on ADME/PK properties. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the uv RP-HPLC trace obtained from a mixture of compounds added to a perfluorocarbon emulsion before ("test sample") or after ("reference sample") the emulsion is disrupted. FIG. 2 shows the uv RP-HPLC trace obtained from a mixture of compounds added to a perfluorocarbon emulsion, comparing disruption of the emulsion using isopropanol or acetonitrile. FIG. 3 shows the RP-HPLC trace obtained from a mixture of compounds added to a perfluorocarbon emulsion, obtained with and without added proteins. FIG. 4 shows the RP-HPLC trace obtained from perfusion of a perfluorocarbon emulsion through a portion of isolated rat gastrointestinal tract at 0, 5, and 45 minutes after beginning perfusion. DETAILED DESCRIPTION Definitions The term "ADME/PK" refers to the absorption, distribution, metabolism, and excretion pharmacokinetics of a pharmaceutical compound or potential pharmaceutical compound, upon administration to an animal, isolated organ, or a tissue. "Animal" includes all animals useful in pharmaceutical research, preferably warm-blooded animals, more preferably mammals, up to and including humans. The term "blood substitute" refers to a fluid which may be used to replace the blood of a test animal, or may be used to perfuse an isolated organ in the absence of blood, and is sufficient to sustain the life of the animal, organ or tissue for the duration of the experiment. Preferred blood substitutes for the purposes of this invention are those which contain few or no components that are chemically similar to the pharmaceutical candidates under evaluation. Presently preferred blood substitutes are fluorocarbon emulsions, such as those described by Geyer, Fed Proc (1975) 34:1499-1505; Geyer, "Advances in Blood Substitute Research" (Alan R. Liss, Inc., NY, N.Y., 1983), pp. 157-68; Spence, Art. Cells Blood Subs., and Immob. Biotech. (1994) 22(4):955-963; Spence, Art. Cells, Blood Subs., and Immob. Biotech. (1995) 23(3): 367-380; and Dracker, Immunol Invest (1995) 24:403-10, all of which are incorporated herein by reference. The term "perfluorocarbon" as used herein refers to an organic molecule in which all hydrogens bound to carbon atoms have been replaced with fluorine atoms. Thus, for example, perfluoroethane has the formula F.sub.3 C--CF.sub.3. As a group, they are characterized by insolubility in aqueous solutions, low viscosity and a specific gravity approximately twice that of water. Liquid perfluorocarbons dissolve considerable quantities of oxygen and carbon dioxide (and other non-polar gasses) in simple solution, the dissolved concentration in direct proportion to the partial pressure according to Henry's Law. Perfluorocarbon compounds are generally heat-stable and are biochemically inert due to the high carbon-fluorine bond energy. The hydrophobic nature of the fluoride renders it impervious to enzymatic attack. Perfluorocarbons are used in vivo by emulsifying the compounds into small particles (typically 0.1-0.3 microns in diameter) by sonication in the presence of a detergent or surfactant. Perfluorocarbons are suitable for the practice of the instant invention if they make acceptable blood substitutes. Exemplary perfluorocarbons include, without limitation, perfluorotributylamine, perfluorodecalin, perfluorooctyl bromide, perfluorotripropyl-amine, bis(perfluorobutyl)ethylene, perfluoro-N,N-dimethylcyclohexylmethylamine, perfluorotrimethylbicyclo(3.3.1)nonane, perfluorobicyclo(5.3.0)decane, and perfluorobutyltetrahydrofuran. The term "surfactant" refers to a "surface-active" agent, in the present case, one useful for emulsifying perfluorocarbon compounds in an aqueous suspension. Preferred surfactants will be generally non-toxic (for the duration of the experiments contemplated). Suitable surfactants include, without limitation, lecithin-based surfactants, egg yolk phospholipids, and synthetic surfactants such as poloxamers (available commercially as Pluronic.RTM. polyols). Poloxamers are polyoxyethylene-polyoxypropylene block polymers, which are available in a wide range of molecular weights and HLB (hydrophilic/lipophilic balance) values. The currently preferred poloxamer is poloxamer-188 (also known as Pluronic F68). See, for example, Hammerschmidt et al., "Blood Substitutes" (T. M. S. Chang, ed., Marcel Dekker, Inc., NY 1988) pp. 431-38; Breuninger et al., J. Pediatr Surg (1993) 28:144-50; Mattrey et al., Crit Care Med (1989) 17:652-56, all of which are incorporated herein by reference. The term "water-miscible organic solvent" refers to an organic solvent which may be mixed with aqueous solutions, and which disrupt (or break) perfluorocarbon emulsions. Suitable water-miscible organic solvents include alcohols such as propanol, isopropanol, butanol, cyclohexanol, and the like; amines such as triethylamine; and other solvents such as acetonitrile, tetrahydrofuran, pyridine, and the like. Presently preferred water-miscible organic solvents are isopropanol and acetonitrile. The term "isolated organ" refers to a living organ or organ system, such as a kidney, lung, gastrointestinal tract, heart, liver, brain, and the like, with the accompanying vasculature. In general, isolated organs will be selected for their utility in demonstrating or suggesting ADME/PK properties for pharmaceutical compounds (or potential pharmaceutical compounds). General Methods and Detailed Description By replacing blood in rats with a perfluorocarbon-containing blood substitute, the quantitation of diverse chemical mixtures can be accomplished by an assortment of analytical methods without extraction steps specific to each component of a mixture. In ADME/PK procedures involving isolated organs and organ systems, the organs are typically oxygenated by perfusion with either blood or an aqueous buffer. Viability of the organs is generally good with blood as the perfusate, but, as noted above, this medium is not readily amenable to facile quantitation of the components of chemical mixtures. In contrast, aqueous buffers have the potential for simple analysis of mixture components, but may result in loss of tissue viability due to the poor capacity of such media to transport oxygen. Thus, the use of perfluorocarbon-containing blood substitutes in isolated organs or organ systems holds two key benefits: 1) facile measurement of multiple analytes in the perfusate; and 2) enhanced tissue viability relative to that achieved with aqueous buffers. The invention provides for characterization of ADME/PK of chemical mixtures in experiments with either living animals or isolated organs or organ systems from animals. Two processes are included, one involving the use of perfluorocarbon emulsions to examine chemical mixtures in animals, and one involving the preparation of perfluorocarbon emulsions for assay by standard analytical methods. The process of the invention involving animals entails the replacement of blood in the blood vasculature with a perfluorocarbon emulsion, followed by administration of a chemical mixture to the test animal or isolated organ or organ system. The ADME/PK fate of the mixture components is then followed over time in the perfluorocarbon emulsion using a new analytical procedure. In the analytical procedure, there is an initial chemical disruption of the emulsion by addition of a water-miscible organic solvent (such as isopropanol), followed by centrifugation to separate the medium into discrete phases. The supernatant phase is then analyzed directly by any of a number of methods, including HPLC, mass spectrometry and capillary electrophoresis. A variety of blood substitutes are taught in the literature and/or are commercially available. An appropriate blood substitute for the present invention is selected to (1) provide sufficient oxygen to the animal or tissues under investigation for at least the duration of the experiment, and (2) contribute the least number of interfering substances, i.e., compounds which might interact with the compounds being tested, or which might be confused with the compounds being tested under the experimental conditions. Presently preferred blood substitutes are perfluorocarbon emulsions. Perfluorocarbon emulsions employed in the practice of the present invention typically contain at least about 5% (w/v) of a perfluorocarbon, more typically at least about 10% (w/v), even more typically at least about 12% (w/v), yet even more typically at least about 15% (w/v), and optionally, they contain at least about 20% (w/v) of a perfluorocarbon. Suitable perfluorocarbon emulsions typically contain less than about 90% (w/v) of a perfluorocarbon, more typically less than about 85% (w/v), even more typically less than about 80% (w/v), yet even more typically less than about 75% (w/v), more typically less than about 70% (w/v), and optionally, they contain less than about 65% (w/v) of a perfluorocarbon. Preferably, perfluorocarbon emulsions of the present invention contain at least about 20% (w/v) of a perfluorocarbon. The experimental design will, of course, depend upon the compounds being tested, the intended route of administration, and the particular ADME/PK characteristics to be determined. For example, one may study the absorption of test compounds from the gut by perfusing an animal or an isolated portion of gastrointestinal tract, administering the compound or mixture of compounds, and monitoring the rate of appearance of the compound(s) in the perfusate. Similarly, one might study the rate of removal from the system by adding the compound(s) directly to the perfusion fluid, and measuring the rate at which the compounds disappear from the perfusate. First-pass metabolism may be studied through perfusion experiments employing the liver. This information can then be used to screen compounds (or whole libraries of compounds) for potential bioavailability, permitting one to research compounds that have the best chance of being useful as biopharmaceutical agents. For example, one might find that all triazine compounds in a diverse library were metabolized rapidly by the liver. This information may lead one to forego the examination of triazine libraries in future applications in which first pass metabolism is possible. Alternatively, one might find that compounds having a particular moiety were rapidly absorbed, which may lead one to design libraries of compounds which all employ that moiety. Conversely, one might find that all compounds from a library that included a large hydrophobic group were not orally absorbed, leading one to design compound libraries that eschew that feature when designing compounds for oral administration. Thus, one may employ the data generated to select particular libraries for particular applications, and may in fact design libraries to incorporate chemical features that provide the desired ADME/PK characteristics. In this method, a target property is identified (for example, transmission across the blood-brain barrier), and the ADME/PK characteristics of a mixture of compounds having diverse structural and chemical properties is determined. The data resulting from the ADME/PK determination are analyzed for structural and chemical features that maximize transmission across the blood-brain barrier, and that information is then used to design a library of compounds which incorporate the appropriate features. Mixtures of compounds may be prepared by any of a variety of methods, for example by the techniques taught in WO91/09735 and WO94/0645 1, both incorporated herein by reference, or by simply mixing compounds together. The techniques of the invention are useful for analysis of both compound mixtures and single compounds, as the invention provides a biophase essentially free of contaminating proteins (apart from 1-2 well-defined peaks), and a universal extraction procedure. In the method of the invention, the perfusate is preferably analyzed by disrupting the emulsion, separating the aqueous and non-aqueous phases, and analyzing the aqueous phase (e.g., by reverse-phase HPLC, thin layer chromatography, and the like). Perfluorocarbon emulsions may typically be disrupted by adding a water-miscible organic solvent such as isopropanol or acetonitrile, optionally in the presence of a saline solution. Once disrupted, the two phases of the emulsion may be allowed to separate on standing, but are preferably separated by centrifugation. Other solvents and methods for disrupting emulsions may be employed, if desired. In general, the analysis method will be advantageous if it (1) partitions the compounds under investigation into the aqueous phase, and (2) preferably causes precipitation of any plasma proteins in the non-aqueous phase. One may also deliberately incorporate a plasma protein to study the interaction between the test compounds and the protein, for example to examine binding to serum albumin and its effect on the plasma half-life of the test compounds. The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way. |
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| PATENT PHOTOCOPY | Available on request |
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