WO2001016598A2 - Evaluation a fort rendement du profil d'un produit chimique - Google Patents

Evaluation a fort rendement du profil d'un produit chimique Download PDF

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Publication number
WO2001016598A2
WO2001016598A2 PCT/US2000/024093 US0024093W WO0116598A2 WO 2001016598 A2 WO2001016598 A2 WO 2001016598A2 US 0024093 W US0024093 W US 0024093W WO 0116598 A2 WO0116598 A2 WO 0116598A2
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agent
effectors
binding
pharmaceutical composition
biological
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PCT/US2000/024093
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WO2001016598A3 (fr
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Dennis Brown
Lev Leytes
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Medalys Corporation
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Publication of WO2001016598A3 publication Critical patent/WO2001016598A3/fr

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/466—Flow patterns using more than one column with separation columns in parallel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography

Definitions

  • the present invention is concerned with massively parallel testing of chemicals to develop a profile of potential lead compounds as a way of identifying and characterizing bioactive leads for the development of pharmaceuticals, agrochemicals, and the like.
  • Drug development processes are lengthy: drug discovery, preclinical testing, human clinical trials and FDA approval constitute the majority of time expended in the process of new drug development.
  • a conventional process involves testing and screening thousands of individual compounds for a desired therapeutic activity. Historically, less than 1 in 10,000 potential compounds have successfully made it to the drug market, at costs of greater than $200 million per drug; it typically requires greater than 10 years from discovery to approval.
  • the present invention is directed to a method for high throughput profiling of biological, physical and chemical characteristics of a test agent to produce a database that can be used to predict or model for such critical development criteria as bioavailability, carcinogenicity, potential toxicities and pharmaceutical properties such as diluent/excipient interactions, general molecular interactions and general drug stabilities.
  • the method includes the steps of contacting multiple replicates of the agent under dynamic conditions with a parallel array of effectors and for a time sufficient for a migration pattern to develop for each agent as a result of interaction with each of the effectors as compared to one or more controls.
  • Several parallel arrays of effectors can be used and the information combined to construct a multidimensional space of characteristics for the agent.
  • Also included is a method for identifying agents which bind to a target molecule of interest which includes the step of contacting a parallel array of the agents with multiple replicates of the target molecule of interest.
  • the invention finds use in providing a preview of the pharmaceutical potential of an agent, as well as, insight into the pharmacokinetic and/or pharmacodynamic properties of the agent, and for identifying agents which have particular characteristics of interest.
  • the method can be used to screen many compounds in vitro with assays that help predict whether oral bioavailability is possible for a potential pharmaceutical.
  • Figure 1 shows a photograph of a TLC plate viewed under chartwave UV light for test described in Tables 4 and 6.
  • the migration pattern shows that Epinephrine (Epi) as a test drug was affected by KMnO 4 and to a lesser extent by DNA in terms of the development of a degradant.
  • Figure 2 shows a photograph of a TLC plate viewed under shortwave UV light for test described in Tables 4 and 6.
  • the migration pattern shows that Doxycycline (Doxy) was affected by an interaction with KMnO 4 and an with phosphatidyl choline (PC).
  • FIG. 3 shows a photograph of a TLC plate viewed under shortwave UV light for test described in Tables 5 and 7.
  • the migration pattern shows that Ethidium bromide (EtBr) bound to DNA, and that EtBr was sequestered by G-C, and A-T nucleic acid polymers.
  • EtBr Ethidium bromide
  • Methods are described for high throughput profiling of characteristics of an agent to provide information about the agent including its physical stability, chemical stability and biological availability and for identifying agents which bind to target molecules of interest.
  • To obtain profiles of the characteristics of an agent multiple replicates of small amounts of an agent are contacted simultaneously on a solid support generally under dynamic conditions with a parallel array of effectors, and any interactions evaluated, using for example TLC methods. electrical charge, capillary action or a combination of these methods.
  • a parallel array of effectors is intended a multiplicity of agents in a single assay panel.
  • agent a compound or a composition which is a molecule of interest, for example a candidate drug, a pesticide, an environmental contaminant, a member of a specific binding pair such as a receptor ligand, an antigen or an antibody.
  • effector any molecule or condition which has the potential to alter a test agent, for example by promoting degradation of the agent, by complexing with the agent, and the like.
  • the effectors include physical effectors, such as increased or decreased temperature, and lighting changes; chemical effectors, such as agents which alter pH, oxidizing agents, reducing agents, and metal ions; effectors which alter bioavailability, particularly biomolecules such as DNA, RNA, and proteins such as serum albumin, membrane lipids and components of biological fluids and degradative enzymes.
  • the complex can be as a result of covalent binding, specific binding or non-specific binding.
  • specific binding is intended binding such as that between members of a specific binding pair that has a high affinity, usually, a kD of about 1 x 10 " > to 1 x 10 " ' 2 M, generally greater than about 1 x 10 "9 M.
  • non-specific binding is intended non-covalent binding that is generally of low affinity, usually a kD of about 1 x lO ' ⁇ M to 1 x 10 "6 M.
  • Nonspecific binding may result from a hydrophilic or a hydrophobic interaction between molecules.
  • fingerprint is intended information as to an array of individual characteristics of a particular agent which can be visualized as a multidimensional space of characteristics.
  • agents which bind to target molecules of interest such as DNA, receptor sites, substrates for particular enzymes, and the like, a parallel array of agents is contacted with multiple replicates of the target molecule of interest.
  • the subject invention offers several advantages over existing technology.
  • the method provides a fundamental characterization of information required for an early understanding of a test compound ' s pharmaceutical or other potential. Early elimination of false positive leads is achieved by screening to identify and rank order possible candidates and thereby eliminate false positives in a dramatically shortened time which provides an early go or no go decision on more extensive in vivo testing.
  • enhanced quantitative structure activity relationship (QSAR) analyses typically used by medicinal and synthetic chemists to identify trends of critical parameters which correlate with biological activity can now be developed with many rather than a limited number of test parameters (C. Hansch and A. Leo "Exploring QSAR", 1995, ACS Prof. Ref. Book, American Chemical Society).
  • the technology also has the advantage that massively parallel in vitro assays can be run using automated analysis systems to provide multiparameter profiles for drug candidates which can be coupled with other chemical and bioinformatics and data management systems. Furthermore, the information can provide chemists with better direction for additional chemical syntheses for lead drug optimization.
  • the system is high throughput.
  • the multiparameter profiles that are developed yield increased confidence in lead drug discovery and optimization because drug candidate selection is made with substantial data support.
  • the high failure rate and extensive time and investment of developing new drugs thus can be averted by obtaining more valuable information about a test compound's attributes and vulnerabilities during the initial drug-screening phase.
  • This aspect is particularly important in view of the rapidly growing number of leads being developed using combinatorial chemistry; by clearing the path for "true/winning" positive leads to be evaluated, new drug development is accelerated.
  • the methodology has the advantage that it identifies alternate potential biological targets or sources of toxicity for a given test compound and provides guidance as to the potential of more value- added routes of administration, for example, oral rather than intravenous delivery.
  • a compound under preclinical development be orally bioavailable because oral bioavailability affords a number of important benefits.
  • Testing for oral availability can be performed rapidly using the subject methodology with a small quantity of test drug, typically less than about 10 mg. This is in contrast to traditional testing where the determination of whether a lead compound is orally bioavailable typically requires initial testing in rodent experimental models. This determination can require that a radioactive label to be attached to the test compound so that the fate of the molecule can be followed after oral dosing. This testing is costly, time consuming, often requires additional synthesis, and requires that the researchers make the decision that the compound chosen was a ''best bet".
  • the amount of biological chemical and physical information about the compound is limited and in most cases there would be other compound candidates that may be also worthy of additional testing, particularly, in vivo.
  • Additional advantages of the subject methodology include that the data generated from test agents can be compared to data generated from established drugs with known oral bioavailability, thus providing/identifying those effectors/conditions that are critical for predicting bioavailability.
  • the information whether favorable or not, provides insights to medicinal chemists as to the direction of future synthesis for lead optimization in a more timely, cost effective manner than is currently practiced.
  • an assay device such as that described in copending application attorney docket No. MECO.002.00USP filed on even date herewith, can be used.
  • the device used has at least three primary regions, a central region containing a means for separate massively parallel analysis of an interaction between a test compound and select effectors and, in fluid connection at either end, a starting reservoir and a receiving reservoir.
  • the means preferably is multiple racetracks, such as used in the above-identified assay device.
  • the starting reservoir contains a test agent in a suitable solvent and the racetracks are each filled with different effectors, the effectors generally are provided as parallel arrays of conditions and/or compounds for assessing particular characteristics of interest relating to a test agent.
  • the starting reservoir contains an effector in a suitable solvent and the racetracks are each filled with different test agents.
  • the starting reservoir contains a suitable solvent and the racetracks each contain one or more layers of absorbent material which have been impregnated with a test agent and an effector. Either the test agent or the effector can be present in multiple replicates, depending upon the experimental design and purpose of the experiment.
  • at least one racetrack contains test agent alone (no effector) or effector alone (no test agent), whichever is present in multiple replicates.
  • Table 1 A listing of some examples of effectors is presented in Table 1 , below.
  • the agent can be any compound for which it is desired to obtain a profile of characteristics, including drug leads and other compounds intended as antineoplastic agents. pesticides, fungicides, antibiotics, antiviral agents and the like. Agents can include known drugs as an aid in discovering new uses or new routes of administration, and potential drug candidates, such as drug candidates which are structurally related to known drugs, particularly those which have undesirable side effects, which lack a desired specificity, and / or which are marginally efficacious.
  • the test agent is dissolved in a suitable solvent for a pharmaceutical, such as physiological saline, ethanol, ethylacetate, or dimethylsulfoxide; for agricultural products, diluents used for sprays, and the like can be used.
  • the amount of test agent used per assay generally is in a concentration of about 0.001 mg/ml to 10 mg/ml, preferably about 0.01 mg/ml to 1 mg/ml, more preferably about 0.1 to 0.5 mg/ml.
  • Reaction mixture volumes can range from about 1 to 2000 ⁇ l, more usually about 2 tolOOO ⁇ l.
  • temperatures are normally employed for carrying out the method.
  • the temperature employed during the method is usually about 4°C to 50°C, more usually about 15°C to 40°C. For most biological applications, a temperature of about 20°C to 37°C is preferred.
  • the ionic strength and viscosity of the medium are optimized for a particular application.
  • the ionic strength of the medium is usually about 0.005 to 0.6 mM, more typically, about 0.02 to 0.10 mM. Isotonic conditions are preferable for most biological assay applications.
  • the time period for carrying out the method is usually from about 0.2 seconds to 24 hours, more usually, from about 2 minutes to about 4 hours, generally about 5 minutes to 1 hour. The time period depends on the temperature and the anticipated rate of interaction of the test agent with the effector.
  • Structural Proteins a. albumin b. fibrogen / tofibin / thrombin c. collagens d. tubulin e. elastin
  • nucleic acids and polymers a. individual nucleotides A T C G U b. homopolymers single strand poly A, T, G, L c. homopolymers double strand G-C, A-T
  • pancreatic juice 11. feces
  • Effectors may be prepared in a variety of ways. If stable, stock materials generally are made in a defined concentration and preapplied to the components of the solid support used in the testing device. Other effectors, if unstable long term in solution, are prepared and lyophilized, then rehydrated and applied to the testing device prior to use and exposure with the compounds of interest to profile. Each effector can be made in a reproducible, stable fashion that allows for inter experiment analysis. The test agent and effector can be mixed prior to application to the absorbent material, or can be spotted onto the absorbent material individually at the same or different locations within individual racetracks.
  • test agent and the effector are on separate layers of absorbent material, the layers are overlaid so that the test agent and the effector are in contact either prior to or during induction of solvent flow from the starting reservoir through the racetracks in the direction of the receiving reservoir.
  • agent and the effectors potentially are each members of a specific binding pair
  • the test agent is present in an amount sufficient to saturate the binding site for the test agent on the effector.
  • Preferred ratios range from 1 : 1 to 5-6 log excesses of the test agent or effector.
  • the range of molar ratios of test drug to effectors may encompass 10 " '° to 1 moles.
  • An array of effectors for evaluating chemical effects can include compounds to alter the pH to which the agent is exposed, including a pH range of about 2-10; in the acidic range, effectors can include acids such as hydrochloric acid, acetic acid and sulphuric acid, and in the alkaline range, effectors can include bases such as sodium hydroxide and sodium carbonate; compounds which are oxidizers, such as sodium persulfate, potassium permanganate; sodium hypochlorite, sodium perchlorate, sodium perborate, ferric ion, iodine, osmium tetroxide, and transition metals such as vanadium, chromium, manganese and the like; compounds which are reducers, for example, sodium borohydride and mercaptoethanol, sodium hydride, Raney nickel, amalgams of mercury and sodium or potassium; metal ions involved in chelations and/or catalytic redox reactions, such as ferrous and ferric ions, magnesium and aluminum ions, lead
  • An array of effectors for evaluating potential efficacy of a test agent can include biological targets of the test agent, including macromolecules such as DNA and RNA, particularly from a potential target organism, i.e. mammalian, bacterial, etc.
  • biological targets of the test agent including macromolecules such as DNA and RNA, particularly from a potential target organism, i.e. mammalian, bacterial, etc.
  • the biological functions of some classes of small molecules can be correlated with their interactions with nucleic acids including DNA and RNA, double stranded or single stranded in varying sizes and polymers including single stranded homopolymers (poly A,T,G, C) and double stranded homopolymers (G-C, A-T), nucleotide and individual nucleotides. Defined sequences and specific genes also can be evaluated.
  • binding to DNA can be either a positive or a negative trait.
  • an ordered, double-stranded polymer structure is a prerequisite for binding, e.g., acridines (Blake and Peacocke (1968) Biopolymers 61 : 1225-1253). actinomycin (Muller and Crothers (1968) J. Molec. Biol. 35:251-290) and ethidium bromide (LePecq and Paoletti (1967) J. Molec. Biol. 27:87-106).
  • acridines Brown and Peacocke (1968) Biopolymers 61 : 1225-1253
  • actinomycin Meler and Crothers (1968) J. Molec. Biol. 35:251-290
  • ethidium bromide LePecq and Paoletti (1967) J. Molec. Biol. 27:87-106.
  • Such binding indicates that the test compound is intercalating between nearest neighbor bases and may be a good antineoplastic
  • actinomycin has a preference for guanine (Kersten (1961) Biochem. Biophys. Ada 47:610-61 1 ; Goldberg et al. (1961) Proc. Nat ' l. Acad. Sci. USA 48:2094-2101 ). Actinomycin binding to DNA additionally appears to depend not only on guanine but also on the base sequence (Wells (1969) Science 165:75-76). However, in general, unique base sequences are not required for the binding of small ligands.
  • the biomolecule to be used as an effector can be a native biomolecule or the biomolecule can be denatured so as to observe whether there is an interaction with the single-stranded molecule, particularly if there is no interaction with the double-stranded molecule.
  • steroid binding to DNA requires the denatured form (Cohen and kidson (1969) Proc. Nat 'I. Acad. Sci. USA 63:458-464).
  • the polymers can be denatured, for example, by heating to about 100°C for 10 minutes, followed by rapid cooling in an ice-salt bath. Binding also can be evaluated in both high (about 0.51M sodium ion) and low (about 0.01M sodium ion) salt.
  • Binding to deoxyribopolynucleotides such as poly dAT or poly dA:dC, both double- stranded and single-stranded, also can be evaluated.
  • the four standard homodeoxyribonucleotides also can be tested. Binding to denatured DNA or to disordered deoxyribopolymers can indicate a requirement for a less-ordered structure of the polymer or for a more hydrophobic environment: both may be true. It also can indicate a requirement for binding sites involving reactive groups on the bases that are not available in double-stranded structures.
  • rRNA viral RNA
  • TMV tobacco mosaic virus
  • sRNA yeast soluble RNA
  • Binding generally is carried out prior to introduction to the assay device in an aqueous solution at moderate salt concentrations. If binding to DNA is observed only with single- stranded DNA, synthetic ribocopolymers can be used, particularly poly UC and poly AC, because any base pairing in these copolymers would be non-ideal and hence minimal and would enhance the ability to detect any binding to the ribocopolymers.
  • the role of hydrophobic forces in the association of a test compound with a polynucleotide can be evaluated by varying the solvent used for binding. See, for example, Seiger (1992) Advances Protein Chem.
  • macromolecules are also of interest as effectors, including proteins such as receptor sites, substrates for particular enzymes, structural proteins such as fibrinogen, fibrin, thrombin. albumin, collagen, tubulin, elastin, etc; antigens; antibodies; viral core and coat proteins; extracellular matrix components such as hyaluronic acid, heparin, and synovial fluids; cellular membranes and lipids, such as cholesterol, phosphatidyl choline, brain lipids, nucleic acids; and carbohydrates, such as heparin and the like.
  • proteins such as receptor sites, substrates for particular enzymes, structural proteins such as fibrinogen, fibrin, thrombin. albumin, collagen, tubulin, elastin, etc; antigens; antibodies; viral core and coat proteins; extracellular matrix components such as hyaluronic acid, heparin, and synovial fluids; cellular membranes and lipids, such as cholesterol, phosphatidyl choline, brain lipid
  • the amount of the effector generally is less than the amount of test agents by at least one order of magnitude, usually two orders of magnitude, and in the range of about 10 "9 M to 10 "6 M.
  • Subcellular affinity can be evaluated by analyzing differential binding to subcellular organelles, such as cell membranes or other cellular components, cells and tissues or tissue homogenates.
  • the effector used can be prepared by standard methods for preparation of tissue homogenates and/or subcellular fractions from biological sources. A defined concentration on a weight per volume or total protein/volume basis is used.
  • Selective tissue affinities also can be evaluated, for example normal vs tumor tissue and affinity for particular blood components; cellular affinities/affinity to various cell types and/or tissues.
  • An array of effectors for evaluating the potential bioavailability of a test agent include compositions that mimic one or more biological system which affects the bioavailability of a test agent, for example by degradation or by sequestering of the test agent. Sequestering of the test agent includes binding to the test agent, to form a complex which is not available biologically and/or is eliminated from the body, or to transport the complex into a tissue other than the intended biological target, e.g. adipose tissue instead of brain tissue.
  • Route of administration modeling can be performed by using components integral to a particular route as the effectors. Examples of such bioavailability effectors include those present at the site of intended introduction into a biological arena of a test agent.
  • the sites of introduction can include lungs, oral, intravenous, intrathecal, rectal, vaginal, intramuscular, subcutaneous, and ocular.
  • the bioavailability following introduction at these sites can be evaluated by exposure of the agent to effectors that are biological fluids such as saliva, using samples and/or extracts of whole blood, serum, plasma, lymph, gastric fluids, synovial fluids, tears, bile, pancreatic juice, feces, and various tissues and extracts such as organ homogenates and/or supematants from brain, glandular tissue, visceral organs (such as liver), skin, muscle, and gastrointestinal tract (e.g.
  • stomach and intestines and adipose tissue or proteins, particularly proteolytic enzymes, or proteins known to bind to drugs.
  • drugs such as serum albumin
  • surrogate systems are available commercially.
  • Surrogate systems that can be employed include vinegar; olive oil; hydrogen peroxide (3%); soluble fiber; starch; and egg/milk protein. These materials represent absorbents in the gastrointestinal tract or agents that my degrade a test compound if it is administered orally.
  • effectors can be used to specifically evaluate whether a drug candidate would be efficacious when formulated for administration by a particular route.
  • differential interaction with and/or affinity for components of saliva, gastric enzymes, acid conditions, gastrointestinal tract, and proteolytic enzymes can be evaluated.
  • ingested food stuffs can alter absorption and ultimately bioavailability following oral drug administration and can be evaluated by using as effectors foodstuffs such as carbohydrates, fiber, fats, oils and protein.
  • effectors foodstuffs such as carbohydrates, fiber, fats, oils and protein.
  • affinity for blood components and interaction with liver derived fractions such as a liver homogenate or liver enzymes, preferably purified enzymes, is evaluated using these substances as effectors.
  • Drugs administered intravenously are often dramatically affected by binding to serum albumin which can be evaluated as an effector.
  • Intrarterial administration of drugs is affected by liver enzymes and subcutaneous drug administration is affected by adipose tissue which can sequester the drug; liver and adipose components can be used as appropriate as effectors.
  • affinity for muscle tissue and components thereof can be evaluated using appropriate samples and/or extracts as effectors.
  • bioavailability can be evaluated, depending upon the site of application, by exposure to soil organisms for application to the rhizosphere, or to organisms which colonize a plant part such as leaves, fruit or flowers, for application to a plant part.
  • this evaluation can be combined with an evaluation of the effects of temperature and/or light vs. time with or without stressors such as metal ions or altered salinity on bioavailability to determine persistence, potential breakdown products, and the like.
  • stressors such as metal ions or altered salinity
  • Toxicological modelling studies also can be performed by evaluating the differential binding of a test agent as an example to DNA, reproductive tissue, brain, and liver.
  • the effect of binding to any of these tissues by an agent also can be used to evaluate e.g. metabolic conversion by the liver of an agent to a metabolite(s), particularly an active metabolite(s).
  • interaction with pharmaceutical excipients can be profiled with this system
  • An array of effectors for evaluating physical stability as a means of assessing, for example, shelf life of an agent under various conditions of temperature and/or light vs. time can include temperatures from about 4 ° C to 50 ° C and are tested by positioning the test apparatus over a controlled heating or cooling means, such as contact heating or cooling, for example electrical; convection heating; forced hot air circulation; immersion of at least a portion of the apparatus in a liquid, for example water of the appropriate temperature, or by incorporating a heating or cooling means into the test apparatus.
  • the effect of light can be assessed by exposing the apparatus to light of a wavelength in the infrared through ultraviolet, generally in the visible range, by mounting a light source of the appropriate wavelength over the test apparatus.
  • the light can be constant or can be provided as light cycles. The effect of both temperature and light together also can be assessed.
  • the method used can involve any of a number of separation techniques, including capillary flow electrophoresis, solvent partitioning and/or conventional chromatography, including liquid column chromatography. thin layer chromatography. high performance liquid chromatography, affinity chromatography, or ion exchange chromatography. These systems can be exploited in miniature and/or automated.
  • the solvent is an aqueous solvent, although other solvents such as acetonitrile can be used depending upon the nature of test agent and the effectors and/or the nature of the interaction under evaluation, such as specific binding, hydrophobic interactions and the like.
  • the solvent used should be one which is able to move the test agent.
  • tissue homogenates, or viable cells a culture medium with or without serum or a balanced salt solution can be used so as to maintain viability of the cells.
  • the pH of an aqueous solvent is usually about 4 to 10, preferably, about 5 to 9, more preferably, 6 to 8.
  • the pH is chosen to maintain a significant amount of binding between a test agent and an effector and/or chemical interaction between a test agent and an effector as well as optimal signal generation by a signal producing system used to detect any flow of the test agent through the racetracks.
  • Various buffers can be used to achieve the desired pH and maintain the pH during the method.
  • Illustrative buffers include borate, acetate, phosphate, carbonate, tris, barbital and the like.
  • the particular buffer employed is not critical, but in individual analyses, one buffer may be preferred over another. Choice of solvents, pH and buffers is within the level of skill of one skilled in the art.
  • test agent If there is binding of some or all of the test agent to the effector, flow of the test agent is impeded. If the effector alters the chemical characteristics of the test agent, there is loss of the test agent and formation of at least one new compound, including a conjugate between the test agent and the effector, or a degradation product of the test agent.
  • Methods of detecting any interactron(s) between an effector and a test agent include visual inspection, densitometry, two-dimensional imaging and video and other methods known to those of skill in the art.
  • the methods of detection can be performed using fluorescence, UV light, visible range or another wavelength.
  • a signal producing system can be used.
  • the test agent is labeled and the signal produced from the labeled ligand bound to the effector or the signal produced by the labeled ligand that is chemically or physically sequestered is detected.
  • the label can be any detectable label, including a radionuclide, a fluorescer, or any other detectable label known to those of skill in the art.
  • test drug/effector interactions can be scored, for example, as unaffected, sequestered, bound or degraded or whatever other interaction is appropriate for the effector and / or test agent under evaluation.
  • percent of test control compound that is bound or degraded, etc. can be determined either from a single test concentration or in a dose-response type of testing system. Binding constants can be derived from such tests and analyses.
  • metabolites or degradation products derived during the testing can be separated or extracted and chemically analyzed by techniques such as mass spectroscopy and NMR, or other methods known to those of skill in the art.
  • % Match Total Number of Parameters X 100
  • a particular minimum percent match can be specified as a cut-off and in addition some parameters that are "must have " can be specified so that any candidate chemical which lacks these "must have” parameters is omitted from the list, regardless of percent match.
  • the desirable set of profile parameters are nonbinding (B), sequestering (S) or degradating (D) with effectors phosphatidyl choline (P). bovine serum albumin (B), DNA (D), and potassium permanganate (K)
  • this information is entered as a search profile and a search conducted for all compounds with a 50% or better match. All compounds in the database are searched, and those that have a percent match of 50% or greater are printed out as shown in Table 2. below.
  • 5-Fluorouracil (Sigma F-6627 (lg) Lot 55H05201)
  • Epinephrine bitartrate (Epi) (Sigma) ( ⁇ l-5mg/ml in H 2 0) Actinomycin D (ActD) (Sigma)
  • Oxidizers Potassium permangenate (KMnO4 )( ⁇ l-5mg/ml in H20); 3% hydrogen peroxide Blood Protein: Bovine Serum Albumin (BSA) (Fraction V) Sigma A-2153 (5g) Lot
  • DNA Deoxyribonucleic acid
  • RNA Ribonucleic acid
  • G-C Guanine-Cytosine Nucleic acid polymer
  • A-T Adenine-Thymine (A-T) Nucleic acid polymer (Sigma)
  • Tissue Homogenates mouse stomach; liver; intestine; gastrointestinal fluids/substances; saliva, urine, feces, acidic solution (vinegar).
  • Food Stuffs Soluble fiber; milk/egg protein; starch; olive oil; mayonnaise Solvents:
  • TLC plates silica gel + fluorescent marker Fisher 5x20 cm Testing Techniques Used In The Examples Technique A
  • a 2-5 ⁇ l spot of a test drug was placed on 5 x 20 cm TLC plate at origin ( ⁇ 5 mm from plate edge).
  • a ⁇ 5-10 ⁇ l spot of an effector was placed 1-2 cm from the test drug spot in the direction of solvent flow.
  • the plate was developed in a solvent system such that the test drug would come in contact with the effector spot.
  • the plate was developed to 5-10 cm from the origin. The plate was inspected visually by visible or short or longwave UV light.
  • Technique B A 1 : 1 mixture of test compound with an effector was made in a 1.5 ml Eppendorf centrifuge tube. As a function of time, a 2-5 ⁇ l sample of the combined mixture was spotted on a TLC plate at the origin. The spot was air dried and then developed in a TLC tank with a solvent system. The solvent front rose from 5-15 cm. After development, the excess solvent was dried in air from the TLC plate. The plate was inspected visually by visible light or short or longwave UV light.
  • test drugs were Doxy, 5-FU, EtBr, and Epi.
  • test solutions of test drugs with biomolecule or KMnO 4 were mixed 1 : 1 ( ⁇ 100 ⁇ l of each). After mixing, 2-5 ⁇ l of the mixture were spotted onto 5 x 20 silica cm TLC plates and then separated using Acetonitrile: H 2 O (3:1) as described in Example 1 as a solvent system. The mixture was incubated for approximately 5-10 min. prior to spotting. The results are shown in Table 4, and Figures 1 and 2.
  • the purpose of this study was to determine whether binding between test drug substances, nucleic acids or nucleotide copolymers can be detected using small reaction mixtures and different time exposures for their interaction by TLC.
  • the test drugs used were actinomycin D, mitomycin C, and EtBr.
  • Test mixtures were made 1 : 1 into Eppendorf tubes, then mixed and the samples incubated at room temperature for -20-30 minutes or -24 hours. After incubation, 5 ⁇ l samples were spotted onto silica TLC plates, the spots were air dried and developed in a solvent system (Acetonitrile:H 2 0 (3:1) in a TLC developing tank -2-5 minutes). The plates were air dried (origin and solvent fronts were first marked). The plates were examined under UV light, either short wave (SW) or long wave (LW). The results are shown in Table 5 below.
  • the purpose of this study was to determine whether binding between test drug substances and substances/effectors that may affect oral drug delivery could be detected using small reaction mixtures and separation by TLC.
  • the effectors used included biological fluids (BF) such as saliva, feces, urine, foodstuffs (F) such as carbohydrates, proteins, oils, fats, etc. or tissues, homogenates/enzymes (TH) found in the Gl tract or metabolic organs such as liver.
  • the test drugs used were Vancomycin (Vanco), Doxycycline (Doxy) and Epinephrine (epi).
  • Tissue homogenates were generated from mouse tissues diluted -1 :1 with phosphate buffered saline and frozen prior to use in the experiment. Test mixtures were made 1 :1 into Eppendorf tubes then mixed and the samples incubated at room temperature for -20-30 minutes or -24 hours. After incubation,
  • vancomycin which is a drug given orally but which is not absorbed into the blood stream, is degraded by food stuffs (soluble fiber) and stomach tissue homogenates and would be eliminated in favor of other drugs with a better profile.
  • doxycycline which is 100% bioavailable after oral administration, is not degraded or bound by any of the test substances studied although it is sequestered by some food stuffs and substances in the stomach homogenate. This drug then would be retained as a possible drug candidate for oral administration for further development.
  • Doxycycline Acetonitrile/ H 2 0 3 1 binding 7 mayonnaise (Pecnutoulter) Doxycycline Acetonitrile/ H,0 3 1 binding 8 soluble fiber Doxycycline Acetonitrile/ H,0 3 1 sequestration 9 starch Doxycycline Acetonitrile/ H,0 3 1 sequestration 10 egg/milk protein Doxycycline Acetonitrile/ H,0 3 1 binding stomach homogenate Doxycycline Acetonitrile/ H 2 0 3 1 degradation 4 intestine homogenate Doxycyclme Acetonitrile/ H 2 0 3: 11 sequestration liver homogenate Doxycycline Acetonitrile/ H 2 0 3.1 sequestration degradation liver supernatant Doxycycline Acetonitrile/ H,0 3:1 seqiestration degradation drug control Doxycycline Acetonitrile/ H 2 0 3.1
  • Tables 9-12 summarize the types of interactions identified by the rapid chemical profiling technique in the experiments outlined in Examples 1 -4. Using a chemical stress effector, potassium permanganate, degradation of doxycycline, ethidium bromide and epinephrine was demonstrated. (Table 9). This is consistent with the known properties of these compounds relating to sensitivity to oxidation.
  • Example 6 Evaluation of Profiles to Identify Chemicals with Desired Characteristics
  • each of four drugs tested in the examples above was scored for each effector tested (see Table 1 1). The following scoring system was applied to describe the test results in the examples provided below:
  • the desired chemical profile is that of an antimetabolite, such as an anticancer agent
  • the desired set of characteristics are nonbinding and/or non-sequestering by any effector except DNA

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Abstract

L'invention porte sur des procédés rapides d'évaluation des caractéristiques de composés chimiques fournissant des indications utiles: a) sur l'efficacité potentielle de composants chimiques individuels destinés à un usage particulier, b) et/ou sur leurs voies d'administration, et c) sur leur stabilité chimique ou physique potentielle. Lesdits procédés requièrent de petites quantités (<10 mg) de composés chimiques pour obtenir un profil complet de leurs caractéristiques. On peut compulser une base de données constituée à partir de ces profils pour identifier des composés chimiques présentant un ensemble de caractéristiques désirées.
PCT/US2000/024093 1999-09-01 2000-09-01 Evaluation a fort rendement du profil d'un produit chimique WO2001016598A2 (fr)

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WO2004011939A1 (fr) * 2002-07-30 2004-02-05 Amersham Biosiciences Uk Limited Reconnaissance de la reponse d'un systeme biologique a une perturbation
CN103411893A (zh) * 2013-07-29 2013-11-27 陕西步长制药有限公司 一种脑心通胶囊近红外光谱的检测方法

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US4671870A (en) * 1985-04-04 1987-06-09 Tompa Ildiko F Apparatus for overpressured thin-layer chromatographic technique
WO1989003430A1 (fr) * 1987-10-13 1989-04-20 Terrapin Diagnostics, Ltd. Procede de production de reactifs pour diagnostics d'immunite
GB2281122A (en) * 1993-08-18 1995-02-22 Zeneca Ltd Complementary binding of receptor and ligand or test compound involving capillary action
WO1997007245A1 (fr) * 1995-08-14 1997-02-27 Ely Michael Rabani Procedes et dispositifs de sequençage multiplex parallele de polynucleotides
WO1998059360A1 (fr) * 1997-06-20 1998-12-30 Ciphergen Biosystems, Inc. Chromatographie par retentat et ensembles de detection de proteines ayant des applications en biologie et en medecine
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WO2004011939A1 (fr) * 2002-07-30 2004-02-05 Amersham Biosiciences Uk Limited Reconnaissance de la reponse d'un systeme biologique a une perturbation
GB2406195A (en) * 2002-07-30 2005-03-23 Amersham Biosciences Uk Ltd Perturbation effect recognition in a biological system
CN103411893A (zh) * 2013-07-29 2013-11-27 陕西步长制药有限公司 一种脑心通胶囊近红外光谱的检测方法

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