US20070009883A1 - Low fluorescence assay platforms and related methods for drug discovery - Google Patents

Low fluorescence assay platforms and related methods for drug discovery Download PDF

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US20070009883A1
US20070009883A1 US11520869 US52086906A US2007009883A1 US 20070009883 A1 US20070009883 A1 US 20070009883A1 US 11520869 US11520869 US 11520869 US 52086906 A US52086906 A US 52086906A US 2007009883 A1 US2007009883 A1 US 2007009883A1
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well
multi
composition
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wells
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Peter Coassin
Alec Harootunian
Andrew Pham
Harry Stylli
Roger Tsien
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Aurora Discovery Inc
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Aurora Discovery Inc
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    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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    • C12M23/00Constructional details, e.g. recesses, hinges
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    • C12M23/12Well or multiwell plates
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    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
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    • G01N33/536Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00306Reactor vessels in a multiple arrangement
    • B01J2219/00313Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315Microtiter plates
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00277Apparatus
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00306Reactor vessels in a multiple arrangement
    • B01J2219/00313Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315Microtiter plates
    • B01J2219/00317Microwell devices, i.e. having large numbers of wells
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
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    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0829Multi-well plates; Microtitration plates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL CHEMISTRY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
    • C40B60/00Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N2021/0346Capillary cells; Microcells
    • GPHYSICS
    • G01MEASURING; TESTING
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    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions

Abstract

One aspect of the present invention is a multi-well platform for fluorescence measurements, comprising a plurality of wells within a frame, wherein the multi-well platform has low fluorescence background. Another aspect of the present invention is a system for spectroscopic measurements, comprising reagents for an assay and a multi-well platform for fluorescence measurements. A further aspect of the present invention is a method for detecting the presence of an analyte in a sample contained in a multi-well platform by detecting light emitted from the sample. Another aspect of the present invention is a method from identifying a modulator of a biological process or target in a sample contained in a multi-well platform by detecting light emitted from the sample. Another aspect of the present invention is a composition identified by this method. A further aspect of the present invention is a method to identify a therapeutic. A further aspect of the present invention is a method of testing a therapeutic for therapeutic activity and toxicology by identifying a therapeutic using a method of the present invention and monitoring the toxicology and efficacy of the therapeutic in an in vivo model.

Description

  • This section describes materials, selection criteria, and rapid tests to facilitate choosing a material for the multi-well plates described herein.
  • Materials
  • The present inventors conducted extensive research on different polymers in search of polymers that offer the appropriate properties for detecting spectroscopic signals, particularly fluorescence signals. Although any suitable material can be used, such as polymers or other materials such as glass or quartz, some of the materials used in the present invention have not been used in the commercially available multi-well platforms listed in Table 1. Surprisingly, these materials offer exceptional properties, including low intrinsic fluorescence, which was demonstrated herein for the first time.
  • The methods described herein to identify cycloolefin copolymers as low fluorescent materials can be used to screen other materials, such as other polymers and other materials such as glasses and quartz, in a variety of configurations, such as in plates, sheets, or films, to determine if they possess desirable optical or fluorescent properties. Thus, these teachings should not be construed to be limited to cycloolefins.
  • Polymers that are compatible with cycloolefin can be used in regions of the multi-well platform in physical contact with cycloolefin. In some embodiments, the frame can be manufactured with a material other than a cycloolefin polymer and the cycloolefin bonded, welded or otherwise fused to the second material. Polymers with glass transition temperatures suitable for heat induced fusion with cycloolefin can be selected for manufacturing the wells and other portions of the plate.
  • Typically, cycloolefins can be used as films, plates, or resins to make various embodiments of present invention. Resins and films based on cycloolefin polymers can be used in various manufacturing processes known in the relevant art and described herein. Selection criteria for cycloolefin films or resins are described more fully below.
  • Suitable cycloolefins for many embodiments of the present invention include those described in U.S. Pat. No. 5,278,238 (Lee B. L. et al.); U.S. Pat. No. 4,874,808 (Minami et al.; U.S. Pat. No. 4,918,133 (Moriya et al.); U.S. Pat. No. 4,935,475 (Kishimura et al.); U.S. Pat. No. 4,948,856 (Minchak et al.); U.S. Pat. No. 5,115,052 (Wamura et al.); U.S. Pat. No. 5,206,306 (Shen); U.S. Pat. No. 5,270,393 (Sagane et al.); U.S. Pat. No. 5,272,235 (Wakatsuru et al.); U.S. Pat. No. 5,278,214 (Monya et al.); U.S. Pat. No. 5,534,606 (Bennett et al.); U.S. Pat. No. 5,532,030 (Hirose et al.); U.S. Pat. No. 4,689,380 (Nahm et al.); and U.S. Pat. No. 4,899,005 (Lane et al.). Cycloolefins available from Hoechst (Summerville, N.J.) are preferred, especially cycloolefin (e.g., cyclopentane, cyclohexane, and cycloheptene) and their polyethylene copolymers, as well as the thermoplastic olefin polymers of amorphous structure (TOPAS line).
  • Multilayer laminates are preferred when multiple functional requirements are difficult to obtain from a single laminate (e.g., layer or film). The properties of transmittance, rigidity, heat sealability, fluorescence, moisture penetration can be blended by the use of films of differing resins. Blended resins known in the art and developed in the future can be used when multilaminate films or blended resins have properties consistent with those of the present invention. For example, U.S. Pat. No. 5,532,030 (Hirose et al.) describes the manufacture of certain cycloolefin films, both single and multilaminate, that can be adapted for use in the devices described herein. The present invention includes multilaminates of any suitable material, such as polymers and other materials, such as glass or quartz.
  • Selection Criteria and Testing
  • Desirable properties for materials used in the present invention will vary depending on the type of multi-well plate desired. Generally, the materials are selected to yield a final product with low fluorescence, high transmittance, sufficient rigidity to resist deformity, and to allow for substantially single plane (especially for spectroscopic embodiments), good chemical inertness to, for example, DMSO, relatively low cytotoxicity, low water absorption, heat resistance/deflection up to about 150° C., and resistance to acids and bases. Starting materials with good molding properties are particularly desirable.
  • Fluorescence of the materials or final product can be readily measured. Such measurements proceed rapidly and a number of plates or films (e.g., 20 to 80 films), or prototype products, can be rapidly tested within a matter of hours or days, usually less than a one person week. Consequently, films or resins used to make final products can rapidly be selected for the desired properties that are important in a particular application. The fluorescence measurements can be used as described herein or those known in the art, so long as the measurements are comparable (or better) in sensitivity to the measurements described herein. A standard reference point for relative fluorescence, such as the standard described herein, is particularly useful for comparing different cycloolefins and for determining their applicability to certain applications. Relative fluorescence properties described herein are particularly desirable. Similarly, transmittance of films, plates, or final products can be measured using techniques known in the relevant art.
  • In the final product, layer thicknesses of generally, about 20 to 500 micrometers, are most likely to impart the properties desirable for use in the devices described herein, especially low fluorescence and high transmittance. Although thinner or thicker films, such as about 10 to 1,500 micrometers, can be used in applications where the demands for extremely low fluorescence and high transmittance films are less stringent, or when e desired properties as a function of film thickness. Preferably, film thickness is between about 30 to 200 micrometers for multi-well platform applications, and more preferably between about 50 to 150 micrometers, and most preferably between about 80 to 100 micrometers. Preferably, film thickness is between about 30 to 600 micrometers for scaffolding applications where the film typically contributes to a structural function in the device that usually demands more strength or rigidity, and more preferably between about 100 to 500 micrometers, and most preferably between about 120 to 200 micrometers. Preferably, film thickness is between about 75 to 600 micrometers for the thinner regions of injection molded applications where the film typically contributes to a structural function, more preferably between about 100 to 500 micrometers and most preferably between about 120 to 200 micrometers. Film thickness refers to the thickness of the film used (or material thickness). Layer thickness is generally about 100 to 200 percent of film thickness, preferably about 100 to 150 percent of film thickness and more preferably about 100 to 125 percent of film thickness.
  • In the final product, breaking stresses (Kg/cm2 at 22° C.) of generally, about 400 to 3,000 Kg/cm2 are most likely to impart the properties desirable for use in the devices described herein, especially rigid devices of low fluorescence and high transmittance. Although weaker or stronger films, such as about 200 to 3,500 Kg/cm2, can be used in different applications based on the demands for breaking strength of the device. For example, the breaking strength of the film, generally need not be as great for the bottoms of multi-well platforms as compared to applications where the film is part of the frame in a multi-well platform. Preferably, breaking stress is between about 500 to 2,000 Kg/cm2 for multi-well plate applications, and more preferably between about 800 to 1,600 Kg/cm2 and most preferably between about 900 to 1,400 Kg/cm2. Preferably, breaking stress for platform/scaffolding applications is about 15 to 60 percent higher than for multi-well platform applications. Breaking stresses can be measured by standard techniques as known in the art. In addition to cycloolefins, materials such as other polymers such as polystyrene, polycarbonate, polypropylene, poly-methyl pentene, copolymers of and of the above-mentioned polymers, or any other polymer appropriate for an intended use of a multi-well platform of the present invention, or other materials, such as glass or quartz, can be used to make the frame or bottom of a multi-well platform of the present invention.
  • Manufacturing Methods
  • The present invention includes a process for making cycloolefin based multi-well platforms. Other methods appropriate for other materials, such as other polymers or other materials such as glasses or quartz, are readily apparent to those skilled in the art based on the properties of the material or materials selected.
  • A variety of processes can be used, including heat welding, insert molding, injection molding and other processes described herein and known in the art. One process comprises heat welding a frame to a bottom exhibiting low fluorescence and high transmittance, such as a cycloolefin copolymer. Processes typically use a cycloolefin copolymer selected from the group of cyclopentane polyethylene copolymer, cyclohexane polyethylene copolymer, and cycloheptene polyethylene copolymer. The process can alternatively, or optionally, comprise the step of exposing the layer and the polymer to a sufficient amount of radio frequency energy to promote internal heating of the layer and the polymer, or ultrasonic welding. Alternatively the process can entail heating the layer and the polymer that forms the wells to about 320° C. for a sufficient amount of time to allow fusion of the polymers. Pressure can be applied to enhance the welding process (e.g., about 100 and 1,000 psi of pressure to the layer and the polymer for low pressure processes using low viscosity monomer solutions and about 10,000 to 25,000 psi for high pressure processes such as insert molding).
  • In another embodiment, the invention provides for a process for making multi-well plates by injection molding or insert molding. Injection molding techniques known in the art or developed in the future can be applied. The process comprises insert molding at least a well to a bottom of the well of the multi-well plate, wherein the bottom is a cycloolefin copolymer. Using this method, cycloolefin films can be heat fused to the supporting structure (e.g., the frame) to make a multi-well platform. The entire frame or platform can also be made of a cycloolefin. Inserting molding can be performed between about 195 and 350° C. degrees, preferably resins are heated to 260° to 320° C. Pressures used are typically between 10,000 and 25,000 psi and preferably about 15,000 to 22,000 psi.
  • Methods for preparing of cycloolefins and their polymers have been described. Other methods and cycloolefins were described in U.S. Pat. Nos. 4,002,815; 4,069,376; 4,110,528; 4,262,103 and 4,380,617 (by Robert J. Minchak and co-workers). A number of catalysts can be used in the manufacture of cycloolefins as known in the art or developed in the future and can be used to manufacture materials for various embodiments of the present invention. Such catalysts include those described in U.S. Pat. No. 5,278,238 (Lee et al.) and U.S. Pat. No. 5,278,214 (Moriya et al.). Regardless of the exact type of catalyst system utilized, cycloolefin monomers can be polymerized in the presence of a catalyst and the ethylene based functional copolymers to make embodiments of the invention suitable for injection molding. Polymerization can carried out preferably in bulk. Bulk polymerization such as reaction injection molding (RIM), liquid injection molding (LIM), reinforced reaction injection molding (RRIM), and resin transfer molding (RTM), and combinations thereof are known to the art well as those techniques developed in the future. Bulk polymerization is polymerization conducted in the absence of a solvent or a diluent. Reaction injection molding is a type of bulk polymerization wherein a monomer in a liquid state is transferred or is injected into a mold where polymerization of the monomer takes place in the presence of a catalyst system. RIM is not conventional injection molding for melt polymers and is readily distinguishable therefrom.
  • RIM is a low pressure, one-step or one-shot, mix and injection of two or more liquid components into a closed mold where rapid polymerization occurs resulting in a molded plastic product. RIM differs from conventional injection molding in a number of important aspects. Conventional injection molding is conducted at pressures of about 10,000 to 20,000 psi in the mold cavity by melting a solid resin and conveying it into a mold maintained at a temperature less than the melt temperature of the resin. At an injection temperature of about 150° to 350° C., viscosity of the molten resin in conventional injection molding process is generally in the range of 50,000 to 1,000,000 and typically about 200,000 cps. In the injection molding process, solidification of the resin occurs in about 10 to 90 seconds, depending on the size of the molded product, following which, the molded product is removed from the mold. There is no chemical reaction occurring in a conventional injection molding process when the resin is introduced into a mold.
  • In a RIM process, the viscosity of the materials fed to a mix chamber is about 1 to 10,000 cps, preferably 1 to about 1500 cps, at injection temperatures varying from room temperature to about 100° C. for different cycloolefin monomer systems. Mold temperatures in a RIM process are in the range of about 50° C. to 150° C. and pressures in the mold are generally in the range of about 50 to 150 psi. At least one component in the RIM formulation is a monomer that is polymerized to a polymer in the mold. The main distinction between conventional injection molding and RIM resides in the fact that in RIM, a chemical reaction is initiated on mixing, with optional heating, and is completed in the mold to transform monomers to a polymeric state. For practical purposes, the chemical reaction must take place rapidly in less than about 2 minutes.
  • Conventional injection molding can also be used to make various embodiments of the invention. The term injection molding refers to both conventional injection molding and the other types of injection molding described herein and known or developed in the art.
  • A LIM process is similar to a RIM system except that generally an impingement head is not utilized. Instead, a simple mixer is utilized such as a static mixer, an agitating mixer, and the like. Moreover, in a LIM system, the injection molding cycle is carried out over a longer period of time and thus the chemical reaction can take place in a period of up to about 5 or 10 minutes.
  • Various reinforcing particles can also be utilized, that is injected with the solution when utilizing either the RIM or the LIM process. As a practical manner, the RIM process is not always suitable and hence reinforced particles are generally utilized only in a LIM process, that is a reinforced liquid injection molding process. Another alternative is to utilize a mat that already exists in a mold, for example a fiberglass mat, or the like. Accordingly, such systems are called RMRIM, RMLIM, or RTM. Due to the reaction cure times as well as injection molding times, the RMLIM system is generally preferred for some operations, RMRIM or RTM for others.
  • Hence, the blends or alloys of cycloolefins and suitable copolymers can he utilized in any of the above noted bulk polymerization systems as well as variations thereof. In as much as the above systems are generally conventional or known to the art as well as to the literature, they have not been discussed in detail, but rather briefly discussed herein for purposes or brevity.
  • U.S. Pat. No. 4,426,502 to Minchak describes bulk (e.g., RIM) polymerization of cycloolefins using a modified co-catalyst with a catalyst whereby polymerization of the cycloolefin monomers can be conducted in absence of a solvent or a diluent. The alkylaluminum halide co-catalyst is modified by pre-reacting it with an alcohol or an active hydroxy-containing compound to form an alkyoxyalkylaluminum halide or an aryloxyalk-ylaluminum halide that is then used in the polymerization reaction. The pre-reaction can be accomplished by using oxygen, an alcohol, or a phenol. Such modification of the co-catalyst results in lowering of its reducing potential of the catalyst.
  • Regardless of whether the halide metathesis or the halogen-free metathesis catalyst system is utilized, the reaction rate is generally slowed down by utilization of the above-described alcohols. Thus, depending if little or no alcohol is utilized, the halide metathesis catalyst system can cure the various cycloolefins in a matter of minutes and even seconds. If high amounts of alcohol are utilized, the cure can be a matter of hours and even days.
  • It is important to lower the reducing power of the co-catalyst of either metathesis system in order to make such bulk polymerization reactions practical. When a monomer diluted with unmodified alkylaluminum co-catalyst is mixed with a monomer-diluted catalyst to polymerize a cycloolefin, the reaction is very rapid. In such systems, the polymerization is usually unacceptable because polymer formed at the interfaces or the two streams during intermingling prevents thorough mixing and results in poor conversions. Modifying the co-catalyst by pre-reaction with hydroxy-containing materials reduces the activity of the co-catalyst to the point where adequate mixing of the liquid components can occur and acceptable polymer products can be produced. Sometimes, a cycloolefinic monomer will contain various impurities that naturally reduce the activity of the co-catalyst. In such cases, it is not necessary to add active hydroxy-containing materials to reduce the activity of the co-catalyst. With the modified co-catalyst, mixing or the cycloolefins, and other components, can be carried out at lower temperatures, such as room temperature, without immediately initiating polymerization. The co-catalyst can be formulated to allow a reasonable pot life at room temperature and thermal activation in the mold of the mixed liquid components. The co-catalyst can also be formulated to give mixing initiated RIM systems.
  • When utilizing a bulk polymerization method, the blend of the cycloolefin monomers and the ethylene-based functional copolymers as well as the catalyst and any optional additives therein can be added to a bulk polymerizing mold having a temperature well below the Tg of the polymerized cycloolefin polymers. This is especially desirable since the reaction is usually exothermic and can result in a temperature increase of the mold up to about 120° C. The final mold temperature is thus from about 50° C. to about 200° C., generally from about 50° C. to about 150° C., and preferably from about 50° C. to about 90° C. Of course, such temperatures will vary depending upon the specific type of catalyst system utilized, the specific type of cycloolefin monomers, and the like. When utilizing the catalyst systems described herein above, the cycloolefin monomer and ethylene-based functional co-polymer mixture has a good shelf life that is up to about 24 hours. Should longer times be desirable, the catalyst system is not added to the mixture but kept separate. Thus, upon the point in time of carrying out the polymerization of the cycloolefin monomers, the catalyst system is added to the mixture and polymerized in bulk. A preferred method of polymerization includes the above noted RIM method.
  • A System for Spectroscopic Measurements
  • The present invention is a system for spectroscopic measurement, comprising: a reagent for an assay, and a device comprising at least one multi-well platform of the present invention, and a second platform to hold said multi-well platform for detecting a signal from a sample. The system can further comprise a detector. In this context, a reagent for an assay includes any reagent useful to perform biochemical or biological in vitro or in vivo testing procedures, such as, for example, buffers, proteins, carbohydrates, lipids, nucleic acids, active fragments thereof, organic solvents such as DMSO, chemicals, analytes, therapeutics, compositions, cells, antibodies, ligands, and the like. In this context, an active fragment is a portion of a reagent that has substantially the activity of the parent reagent. The choice of a reagent depends on the type of assay to be performed. For example, an immunoassay would include an immuno-reagent, such as an antibody, or an active fragment thereof.
  • The present invention is directed to systems and methods that utilize automated and integratable workstations for detecting the presence of an analyte and identifying modulators or chemicals having useful activity. The present invention is also directed to chemical entities and information (e.g., modulators or chemical or biological activities of chemicals) generated or discovered by operation of workstations of the present invention.
  • The present invention includes automated workstations that are programmably controlled to minimize processing times at each workstation and that can be integrated to minimize the processing time of the liquid samples from the start to finish of the process. Typically, a system of the present invention would include: A) a storage and retrieval module comprising storage locations for storing a plurality of chemicals in solution in addressable chemical wells, a chemical well retriever and having programmable selection and retrieval of the addressable chemical wells and having a storage capacity for at least 100,000 the addressable wells, B) a sample distribution module comprising a liquid handler to aspirate or dispense solutions from selected the addressable chemical wells, the chemical distribution module having programmable selection of, and aspiration from, the selected addressable chemical wells and programmable dispensation into selected addressable sample wells (including dispensation into arrays of addressable wells with different densities of addressable wells per centimeter squared), C) a sample transporter to transport the selected addressable chemical wells to the sample distribution module and optionally having programmable control of transport of the selected addressable chemical wells (including adaptive routing and parallel processing), D) a reaction module comprising either a reagent dispenser to dispense reagents into the selected addressable sample wells or a fluorescent detector to detect chemical reactions in the selected addressable sample wells, and a data processing and integration module.
  • The present invention can be used with systems and methods that utilize automated and integratable workstations for identifying modulators, pathways, chemicals having useful activity and other methods described herein. Such systems are described generally in the art (see, U.S. Pat. No. 4,000,976 to Kramer et al. (issued Jan. 4, 1977), U.S. Pat. No. 5,104,621 to Pfost et al. (issued Apr. 14, 1992), U.S. Pat. No. 5,125,748 to Bjornson et al. (issued Jun. 30, 1992), U.S. Pat. No. 5,139,744 to Kowalski (issued Aug. 18, 1992), U.S. Pat. No. 5,206,568 Bjornson et al. (issued Apr. 27, 1993), U.S. Pat. No. 5,350,564 to Mazza et al. (Sep. 27, 1994), U.S. Pat. No. 5,589,351 to Harootunian (issued Dec. 31, 1996), and PCT Application Nos: WO 93/20612 to Baxter Deutschland GMBH (published Oct. 14, 1993), WO 96/05488 to McNeil et al. (published Feb. 22, 1996) and WO 93/13423 to Agong et al. (published Jul. 8, 1993).
  • The storage and retrieval module, the sample distribution module, and the reaction module are integrated and programmably controlled by the data processing and integration module. The storage and retrieval module, the sample distribution module, the sample transporter, the reaction module and the data processing and integration module are operably linked to facilitate rapid processing of the addressable sample wells. Typically, devices of the invention can process at least 100,000 addressable wells in 24 hours. This type of system is described in U.S. Ser. No. 08/858,016 by Stylli et al., filed May 16, 1997, entitled “Systems and method for rapidly identifying useful chemicals in liquid samples,” which has attorney docket no. 08366/008001, which is incorporated herein by reference.
  • If desired, each separate module is integrated and programmably controlled to facilitate the rapid processing of liquid samples, as well as being operably linked to facilitate the rapid processing of liquid samples.
  • In one embodiment the invention provides for a reaction module that is a fluorescence detector to monitor fluorescence. The fluorescence detector is integrated to other workstations with the data processing and integration module and operably linked with the sample transporter. Preferably, the fluorescence detector is of the type described herein and can be used for epi-fluorescence. Other fluorescence detectors that are compatible with the data processing and integration module and the sample transporter, if operable linkage to the sample transporter is desired, can be used as known in the art or developed in the future. For some embodiments of the invention, particularly for plates with 96, 192, 384 and 864 wells per plate, detectors are available for integration into the system. Such detectors are described in U.S. Pat. No. 5,589,351 (Harootunian), U.S. Pat. No. 5,355,215 (Schroeder), and PCT patent application WO 93/13423 (Akong). Each well of a multi-well platform can be “read” sequentially. Alternatively, a portion of, or the entire plate, can be “read” simultaneously using an imager, such as a Molecular Dynamics Fluor-Imager 595 (Sunnyvale, Calif.).
  • Fluorescence Measurements
  • It is recognized that different types of fluorescent monitoring systems can be used to practice the invention with fluorescent probes, such as fluorescent dyes or substrates. Preferably, systems dedicated to high throughput screening, e.g., 96-well or greater microtiter plates, are used. Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B. Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361 and the Molecular Probes Catalog (1997), OR, USA.
  • Fluorescence in a sample can be measured using a detector described herein or known in the art for multi-well platforms. In general, excitation radiation from an excitation source having a first wavelength, passes through excitation optics. The excitation optics causes the excitation radiation to excite the sample. In response, fluorescent probes in the sample emit radiation that has a wavelength that is different from the excitation wavelength. Collection optics then collect the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned. According to one embodiment, a multi-axis translation stage (e.g., a dedicated X,Y positioner) moves a multi-well platform holding a plurality of samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer also can transform the data collected during the assay into another format for presentation.
  • Preferably, FRET (fluorescence resonance energy transfer) is used as a way of monitoring probes in a sample (cellular or biochemical). The degree of FRET can be determined by any spectral or fluorescence lifetime characteristic of the excited construct, for example, by determining the intensity of the fluorescent signal from the donor, the intensity of fluorescent signal from the acceptor, the ratio of the fluorescence amplitudes near the acceptor's emission maxima to the fluorescence amplitudes near the donor's emission maximum, or the excited state lifetime of the donor. For example, cleavage of the linker increases the intensity of fluorescence from the donor, decreases the intensity of fluorescence from the acceptor, decreases the ratio of fluorescence amplitudes from the acceptor to that from the donor, and increases the excited state lifetime of the donor.
  • Preferably, changes in signal are determined as the ratio of fluorescence at two different emission wavelengths, a process referred to as “ratioing.” Differences in the absolute amount of probe (or substrate), cells, excitation intensity, and turbidity or other background absorbances between addressable wells can affect the fluorescence signal. Therefore, the ratio of the two emission intensities is a more robust and preferred measure of activity than emission intensity alone.
  • A ratiometric fluorescent probe system can be used with the invention. For instance the reporter system described in PCT publication WO 96/30540 (Tsien and Zlokarnik) has significant advantages over existing reporters for gene integration analysis, as it allows sensitive detection and isolation of both expressing and non-expressing single living cells. This assay system uses a non-toxic, non-polar fluorescent substrate that is easily loaded and then trapped intracellularly. Cleavage of the fluorescent substrate by β-lactamase yields a fluorescent emission shift as substrate is converted to product. Because the β-lactamase reporter readout is ratiometric, it is unique among reporter gene assays in that it controls variables such as the amount of substrate loaded into individual cells. The stable, easily detected, intracellular readout simplifies assay procedures by eliminating the need for washing steps, which facilitates screening with cells using the invention.
  • Methods for Detecting the Presence an Analyte in a Sample
  • A method of the present invention uses targets for detecting the presence of an analyte, such as chemicals that are useful in modulating the activity of a target, in a sample. Typically, as discussed below targets can be proteins such as cell surface proteins or enzymes. A biological process or a target can be assayed in either biochemical assays (targets free of cells), or cell based assays (targets associated with a cell). This method can also be used to identify a modulator of a biological process or target in a sample. This method detects the presence of an analyte in a sample contained in a multi-well platform of the present invention by detecting light emitted from the sample. The method comprises the steps of: exciting at least one sample with radiation of a first wavelength, wherein at least one sample suspected of containing an analyte is placed into at least one well of a multi-well platform of the present invention, which can contain a biological process or target. The sample and biological process or target can be contacted within the well, or outside of the well and later placed within the well. The emission of radiation of a second wavelength emitted from the sample is measured, wherein the amount of radiation of a second wavelength measured indicates the presence or absence of the analyte in the sample.
  • Targets can be cells, which may be loaded with ion or voltage sensitive dyes to report receptor or ion channel activity, such as calcium channels or N-methyl-D-aspartate (NMDA) receptors, GABA receptors, kainate/AMPA receptors, nicotinic acetylcholine receptors, sodium channels, calcium channels, potassium channels excitatory amino acid (EAA) receptors, nicotinic acetylcholine receptors. Assays for determining activity of such receptors can also use agonists and antagonists to use as negative or positive controls to assess activity of tested chemicals. In preferred embodiments of automated assays for identifying chemicals that have the capacity to modulate the function of receptors or ion channels (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion-sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed, are those disclosed in the Molecular Probes 1997 Catalog, herein incorporated by reference.
  • Other methods of the present invention concern determining the activity of receptors. Receptor activation can sometimes initiate subsequent intracellular events that release intracellular stores of calcium ions for use as a second messenger. Activation of some G-protein-coupled receptors stimulates the formation of inositol triphosphate (IP3 a G-protein coupled receptor second messenger) through phospholipase C-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine (1984), Nature 312: 315-21. IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels caused by release of calcium ions from intracellular stores can be used to reliably determine G-protein-coupled receptor function. Among G-protein-coupled receptors are muscarinic acetylcholine receptors (mAChR), adrenergic receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors and the like. Cells expressing such G-protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable, although not necessary, to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such 3s EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
  • Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., CAMP, cGMP. For example, activation of some dopamine, serotonin, metabotropic glutamate receptors and muscarinic acetylcholine receptors results in a decrease in the CAMP or cGMP levels of the cytoplasm. Furthermore, there are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels (see, Altenhofen. W. et al. (1991) Proc. Natl. Acad. Sci. USA 88:9868-9872 and Dhallan et al. (1990) Nature 347:184-187) that are permeable to cations upon activation by binding of cAMP or cGMP. In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel and DNA encoding a receptor (e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, cause a change in cyclic nucleotide levels in the cytoplasm.
  • Any cell expressing a protein target in sufficient quantity for measurement in a cellular assay can be used with the invention. Cells endogenously expressing a protein can work as well as cells expressing a protein from heterologous nucleic acids. For example, cells may be transfected with a suitable vector encoding one or more such targets that are known to those of skill in the art or may be identified by those of skill in the art. Although essentially any cell which expresses endogenous ion channel or receptor activity may be used, when using receptors or channels as targets it is preferred to use cells transformed or transfected with heterologous DNAs encoding such ion channels and/or receptors so as to express predominantly a single type of ion channel or receptor. Many cells that can be genetically engineered to express a heterologous cell surface protein are known. Such cells include, but are not limited to, baby hamster kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44 cells (see, Chasin (1986) Cell. Molec. Genet. 12: 555) human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamster ovary (CHO) cells (ATCC Nos. CRL96 18, CCL61, CRL9096), PC 12 cells (ATCC No. CRL 17.21) and COS-7 cells (ATCC No. CRL 1651). Preferred cells for heterologous cell surface protein expression are those that can be readily and efficiently transfected. Preferred cells include Jurkat cells and HEK 293 cells, such as those described in U.S. Pat. No. 5,024,939 and by Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060.
  • Exemplary membrane proteins include, but are not limited to, surface receptors and ion channels. Surface receptors include, but are not limited to, muscarinic receptors, e.g., human M2 (GenBank accession #M16404); rat M3 (GenBank accession #M16407); human M4 (GenBank accession #M16405); human M5 (Bonner, et al., (1988) Neuron 1, pp. 403-410); and the like. Neuronal nicotinic acetylcholine receptors include, but are not limited to, e.g., the human α2, α3, and β2, subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990, which is hereby expressly incorporated by reference herein in its entirety); the human α5 subtype (Chini, et al. (1992) Proc. Natl. Acad Sci. USA 89:1572-1 576), the rat α2 subunit (Wada, et al. (1988) Science 240, pp. 330-334); the rat α3 subunit (Boulter, et al. (1986) Nature 319, pp. 368-374); the rat α4 subunit (Goldman, et al. (1987) Cell 48, pp. 965-973); the rat α5 subunit (Boulter, et al. (1990) J. Biol. Chem. 265, pp. 4472-4482); the chicken α7 subunit (Couturier et al. (1990) Neuron 5:847-856); the rat β2 subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat β3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp. 6268-6272); the rat β4 subunit (Duvoisin, et al. (1989) Neuron 3, pp. 487-496); combinations of the rat α subunits, β subunits and a and p subunits; GABA receptors, e.g., the bovine n, and p, subunits (Schofield, et al. (1987) Nature 328, pp. 221-227); the bovine n, and a, subunits (Levitan, et al. (1988) Nature 335, pp-76-79); the γ-subunit (Pritchett, et al. (1989) Nature 338, pp-582-585); the p, and p, subunits (Ymer, et al. (1989) EMBO J. 8, pp. 1665-1670); the 6 subunit (Shivers, B. D. (1989) Neuron 3, pp. 327-337); and the like. Glutamate receptors include, but are not limited to, e.g., rat GluR1 receptor (Hollman, et al. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3 receptors (Boulter et al. (1990) Science 249:1033-1037; rat GluR4 receptor (Keinanen et al. (1990) Science 249:556-560 ); rat GluRS receptor (Bettler et al. (1990) Neuron 5:583-595) g rat GluR6 receptor (Egebjerg et al. (1991) Nature 351:745-748); rat GluR7 receptor (Bettler et al. (1992) Neuron 8:257-265); rat NMDARI receptor (Moriyoshi et al. (1991) Nature 354:31-37 and Sugihara et al. (1992) Biochem. Biophys. Res. Comm. 185:826-832); mouse NMDA el receptor (Meguro et al. (1992) Nature 357:70-74): rat NMDAR2A. NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science 256:1217-1221); rat metabotropic mGluR1 receptor (Houamed et al. (1991) Science 252:1318-1321); rat metabotropic mGluR2, mGluR3 and mGluR4 receptors (Tanabe et al. (1992) Neuron 8:169-179); rat metabotropic mGluR5 receptor (Abe et al. (1992) J. Biol. Chem. 267:13361-13368); and the like. Adrenergic receptors include, but are not limited to, e.g., human pl (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924); human α2 (Kobilka, et al. (1987) Science 238, pp. 650-656); hamster β2 (Dixon, et al. (1986) Nature 321, pp. 75-79); and the like. Dopamine receptors include, but are not limited to, e.g., human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6); mammalian dopamine D2 receptor (U.S. Pat. No. 5,128,254); rat (Bunzow, et al. (1988) Nature 336, pp. 783-787); and the like. NGF receptors include, but are not limited to, e.g., human NGF receptors (Johnson, et al. (1986) Cell 47, pp. 545-554); and the like. Serotonin receptors include, but are not limited to, e.g., human 5HT1a (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Pat. No. 4,985,352); human 5HT1D (U.S. Pat. No. 5,155,218); rat 5HT2 (Julius, et al. (1990) PNAS 87, pp. 928-932); rat 5HT1c (Julius, et al. (1988) Science 241, pp. 558-564); and the like.
  • Ion channels include, but are not limited to, calcium channels comprised of the human calcium channel α2 β and/or γ-subunits disclosed in commonly owned U.S. application Ser. Nos. 07/745,206 and 07/868,354, filed Aug. 15, 1991 and Apr. 10, 1992, respectively, the contents of which are hereby incorporated by reference; (see also, W089/09834; human neuronal α2 subunit); rabbit skeletal muscle a1 subunit (Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal muscle α2 subunit (Ellis, et al. (1988) Science 241, pp. 1661 -1664); rabbit skeletal muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118); rabbit skeletal muscle γ subunit (Jay, et al. (1990) Science 248, pp. 490-492); and the like. Potassium ion channels include, but are not limited to, e.g., rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp, 9230-8236); mouse brain (BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839); and the like. Sodium ion channels include, but are not limited to, e.g., rat brain I and II (Noda, et al. (1986) Nature 320, pp. 188-192); rat brain III (Kayano, et al. (1988) FEBS Lett. 228, pp. 187-194); human II (ATCC No. 59742, 59743 and Genomics 5:204-208 (1989); chloride ion channels (Thiemann, et al. (1992), Nature 356, pp. 57-60 and Paulmichl, et al. (1992) Nature 356, pp. 238-241), and others known or developed in the art.
  • Intracellular receptors may also be used as targets, such as estrogen receptors, glucocorticoid receptors, androgen receptors, progesterone receptors, and mineralocorticoid receptors, in the invention. Transcription factors and kinases can also be used as targets, as well as plant targets.
  • Various methods of identifying activity of chemical with respect to a target can be applied, including: ion channels (PCT publication WO 93/13423) and intracellular receptors (PCT publication WO 96/41013, U.S. Pat. No. 5,548,063, U.S. Pat. No. 5,171,671, U.S. Pat. No. 5,274,077, U.S. Pat. No. 4,981,784, EP 0 540 065 A1, U.S. Pat. No. 5,071,773, and U.S. Par. No. 5,298,429). All of the foregoing references are herein incorporated by reference in their entirety.
  • If the analyte is present in the sample, then the target will exhibit increased or decreased fluorescence. Such fluorescence can be detected using the methods of the present invention by exciting the sample with radiation of a first wavelength, which excites a fluorescent reporter in the sample, which emits radiation of a second wavelength, which can be detected. The amount of the emission is measured, and compared to proper control or background values. The amount of emitted radiation that differs from the background and control levels: either increased or decreased, correlates with the amount or potency of the analyte in the sample. Standard curves can be determined to make the assay more quantitative.
  • Testing a Therapeutic for Therapeutic Activity and Toxicology
  • The present invention also provides a method for testing a therapeutic for therapeutic activity and toxicology. A therapeutic is identified by contacting a test chemical suspected of having a modulating activity of a biological process or target with a biological process or target in a multi-well platform of the present invention. If the sample contains a modulator, then the amount of a fluorescent reporter product in the sample, such as inside or outside of the cell, will either increase or decrease relative to background or control levels. The amount of the fluorescent reporter product is measured by exciting the fluorescent reporter product with an appropriate radiation of a first wavelength and measuring the emission of radiation of a second wavelength emitted from said sample. The amount of emission is compared to background or control levels of emission If the sample having the test chemical exhibits increased or decreased emission relative to that of the control or background levels, then a candidate modulator has been identified. The amount of emission is related to the amount or potency of the therapeutic in the sample. Such methods are described in, for example. Tsien (PCT/US90/04059) The candidate modulator can be further characterized and monitored for structure, potency, toxicology, and pharmacology using well known methods.
  • The structure of a candidate modulator identified by the invention can be determined or confirmed by methods known in the art, such as mass spectroscopy. For putative modulators stored for extended periods of time, the structure, activity, and potency of the putative modulator can be confirmed.
  • Depending on the system used to identify a candidate modulator, the candidate modulator will have putative pharmacological activity. For example, if the candidate modulator is found to inhibit T-cell proliferation (activation) in vitro, then the candidate modulator would have presumptive pharmacological properties as an immunosuppressant or anti-inflammatory (see, Suthanthiran et al., Am. J. Kidney Disease 28:159-172 (1996)). Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, as well as toxicology, can be selected. The methods described herein can also be used to assess pharmacological selectivity and specificity, and toxicity.
  • Toxicology of Candidate Modulators
  • Once identified, candidate modulators can be evaluated for toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Pat. Nos. 5,196,313 to Culbreth (issued Mar. 23, 1993) and U.S. Pat. No. 5,567,952 to Benet (issued Oct. 22, 1996). For example, toxicology of a candidate modulator can be established by determining in vitro toxicity towards a cell line, such as a mammalian i.e. human cell line. Candidate modulators can be treated with, for example, tissue extracts such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the chemical after being metabolized by a whole organism. The results of these types of studies are often predictive of toxicological properties of chemicals in animals, such as mammals, including humans.
  • Alternatively, or in addition to these in vitro studies, the toxicological properties of a candidate modulator in an animal model, such as mice, rats, rabbits, or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)). Depending on the toxicity, target organ, tissue, locus, and presumptive mechanism of the candidate modulator, the skilled artisan would not be burdened to determine appropriate doses, LD50 values, routes of administration, and regimes that would be appropriate to determine the toxicological properties of the candidate modulator. In addition to animal models, human clinical trials can be performed following established procedures, such as those set forth by the United States Food and Drug Administration (USFDA) or equivalents of other governments. These toxicity studies provide the basis for determining the efficacy of a candidate modulator in vivo.
  • Efficacy of Candidate Modulators
  • Efficacy of a candidate modulator can be established using several art recognized methods, such as in vitro methods, animal models, or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a chemical to extend the life-span of HIV-infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIV infection or AIDS (see, Daluge et al., Antimicro. Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of cyclosporin A (CsA) to prevent proliferation of T-cells in vitro has been established as an acceptable model to identify chemicals expected to be efficacious as immunosuppressants (see, Suthanthiran et al., supra, (1996)). For nearly every class of therapeutic, disease, or condition, an acceptable in vitro or animal model is available. Such models exist, for example, for gastro-intestinal disorders, cancers, cardiology, neurobiology, and immunology. In addition, these in v i m methods can use tissue extracts, such as preparations of liver, such as microsomal preparations, to provide a reliable indication of the effects of metabolism on the candidate modulator. Similarly, acceptable animal models may be used to establish efficacy of I treat various diseases or conditions. For example, the rabbit knee is an accepted model for testing chemicals for efficacy in treating arthritis (see, Shaw and Lacy, J. Bone Joint Surg. (Br) 55:197-205 (1973)). Hydrocortisone, which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an appropriate model to determine efficacy of a candidate modulator, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, regime, and endpoint and as such would not be unduly burdened.
  • In addition to animal models, human clinical trials can be used to determine the candidate modulator in humans. The USFDA, or equivalent governmental we established procedures for such studies.
  • Selectivity of Candidate Modulators
  • The in vitro and in vivo methods described above also establish the selectivity of a candidate modulator. It is recognized that chemicals can modulate a wide variety of biological processes or be selective. Panels of cells based on the present invention can be used to determine the specificity of the candidate modulator. Selectivity is evident, for example, in the field of chemotherapy, where the selectivity of a chemical to be toxic towards cancerous cells, but not towards non-cancerous cells, is obviously desirable. Selective modulators are preferable because they have fewer side effects in the clinical setting. The selectivity of a candidate modulator can be established in vitro by testing the toxicity and effect of a candidate modulator on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities. The data obtained from these in vitro toxicity studies can be extended animal model studies, including human clinical trials, to determine toxicity, efficacy, and selectivity of the candidate modulator.
  • Identified Compositions
  • The invention includes compositions such as novel chemicals, and therapeutics identified as having activity by the operation of methods, systems or components described herein. Novel chemicals, as used herein, do not include chemicals already publicly known in the art as of the filing date of this application. Typically, a chemical would be identified as having activity from using the invention and then its structure revealed from a proprietary database of chemical structures or determined using analytical techniques such as mass spectroscopy.
  • One embodiment of the invention is a chemical with useful activity, comprising a chemical identified by the method described above. Such compositions include small organic molecules, nucleic acids, peptides and other molecules readily synthesized by techniques available in the art and developed in the future. For example, the following combinatorial compounds are suitable for screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication No. WO 93/20242, 14 Oct. 1993), random bioooligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidyl peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc. 114: 9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, C. Y. et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon, E. M. et al., J. Med. Chem. 37: 1385 (1994). The contents of all of the aforementioned publications are incorporated herein by reference.
  • The present invention also encompasses the identified compositions in a pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration, which have a pharmaceutically effective amount of he products disclosed above in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.
  • The compositions of the present invention may be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; and the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g., liposomes), may be utilized.
  • The pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • In practicing the methods of the invention, the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, the products or compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. Such methods may also be applied to testing chemical activity in vivo.
  • As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
  • In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage for the products of the present invention can range broadly depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 kg/kg and 100 mg/kg body weight, preferably between about 100 μg/kg and 10 mg/kg body weight. Administration is preferably oral on a daily basis.
  • The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., in The Pharmacological Basis of Therapeutics, 1975.) It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the seventy of the condition to be treated and to the route of administration. The seventy of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parented delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragées, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragée-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical formulations for parented administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragée cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragée cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragée coatings for identification or to characterize different combinations of active compound doses.
  • EXAMPLES Example 1 Fluorescence Properties of Cycloolefins Compared to Glass and Other Polymeric Materials
  • To investigate the fluorescence properties of various selected materials, different polymeric films were tested for fluorescence emission at predetermined excitation wavelengths and compared to two types of glass sheets (standard). These experiments were conducted using a SPEX Fluorolog 111 Fluorimeter with excitation wavelengths between 315 and 425 nm. The films and glass materials were disposed on a holder. The sample was positioned with the excitation beam perpendicular to the sample face. The fluorescence emission from the sample was collected off angle at about 12.5 degrees. The material's fluorescence emission was reflected off of a mirror and onto a monochrometer. The emission radiation was selected by the monochromatic grating and was detected by the photomultiplier tube of the instrument. The SPEX Fluorolog 111 Fluorometer utilizes Raman radiation lines of water to calibrate and background correct the instrument measurements from day to day. This background correction was performed each day before instrument use for calibration. The calibration file is stored with the measurements made that day and then subsequent measurements with the SPEX instrument can be compared directly and corrected for instrument fluctuation.
  • The materials tested were 1) glass sheets (Coming Glass Works cover-slip No 1 (catalog number 2935/58333 1) (average thickness between about 130 and 170 micrometers), 2) polystyrene films (ps1, ps2 (Gom Plastic Suppliers) and ps3 (from Dow Chemical Company), 3) polycarbonate films (pc1 (from General Electric Corporation) and pc2 (from Plastic Suppliers); 4) non-aromatic, alkyl polymers (nap; obtained from Mobil Oil Company), 5) cycloolefin copolymer film (coc; obtained from Hoechst, Topas (2 mil, or 50 micrometers thick)) and 6) Aclar (a fluorocarbon material from Allied Signal).
  • Table 2 shows the fluorescence normalized emission data over 400 to 650 nm at three different excitation wavelengths. The data is normalized to glass and to correct for instrumentation fluctuation. Polystyrene, which is often used as a component of multi-well plates (see Table 1), generated high background fluorescence levels, consistent with its aromatic structure. Surprisingly, polycarbonate, which is often a biocompatible polymer, was generally better than polystyrene, especially at longer wavelengths. Surprisingly, the non-aromatic, alkyl polymer was generally the second best polymer across the range of wavelengths tested. Also surprisingly, the cycloolefin copolymer produced the best results and nearly approached the extremely low fluorescence levels of glass.
    TABLE 2
    Material
    Ex = 315 Em = 400 Em = 425 Em = 450 Em = 475 Em = 500 Em = 550 Em = 600 Em = 650
    Glass 0.22513 0.25824 0.26817 0.30459 0.33107 0.38735 0.51316
    Pc1 - 5 mil 3.31071 2.10230 2.01953 1.78778 1.41036 0.66876 0.60586
    Pc2 - 5 mil 11.04128 7.04943 6.11517 5.18091 3.79367 1.70432 1.05317
    Ps1 - 2 mil 2.45986 1.96447 1.93714 1.78340 1.52374 1.02494 1.18893
    Ps2 - 2 mil 2.20826 1.72697 1.69866 1.64204 1.48633 1.07582 1.18906
    Ps3 - 2 mil 4.55807 3.29823 3.00096 2.72352 2.34132 1.57409 1.98743
    Nap - 1.5 mil 1.01919 0.75307 0.62850 0.52942 0.50110 0.56622 1.12111
    Nap - 1.5 mil 0.52658 0.48978 0.42466 0.37654 0.38220 0.50960 1.00787
    Coc - 2 mil 0.40485 0.40485 0.34256 0.31142 0.31142 0.41617 0.83234
    Aclar - .75 mil 0.08473 0.08875 0.07864 0.07368 0.07503 0.09701 0.22497
    Aclar - 3 mil 0.27245 0.28586 0.26367 0.26522 0.29309 0.44479 1.03199
    Material
    Ex = 350 Em = 400 Em = 425 Em = 450 Em = 475 Em = 500 Em = 550 Em = 600 Em = 650
    Glass 0.30790 0.20526 0.23837 0.17547 0.16222 0.17878 0.25492
    Pc1 - 5 mil 0.77802 0.62572 0.60586 0.50323 0.42708 0.31452 0.33769
    Pc2 - 5 mil 3.96354 2.74616 2.20826 1.61373 1.24568 0.75024 0.62284
    Ps1 - 2 mil 1.28801 1.44858 2.22754 2.06013 1.78340 1.06594 0.84387
    Ps2 - 2 mil 1.01919 1.34477 1.85437 1.84021 1.64204 1.08997 0.89180
    Ps3 - 2 mil 2.13182 2.68388 3.47092 3.14252 2.68388 1.57692 1.29381
    Nap - 1.5 mil 0.95408 0.80120 0.81536 0.59170 0.53508 0.58321 0.79554
    Nap - 1.5 mil 0.53791 0.48695 0.55206 0.39918 0.39918 0.48129 0.69079
    Coc - 2 mil 0.42466 0.38220 0.43033 0.31142 0.31142 0.38503 0.56056
    Aclar - .75 mil 0.08689 0.08710 0.08669 0.07327 0.07224 0.08050 0.10733
    Aclar -3 mil 0.24045 0.23323 0.24974 0.21981 0.23375 0.31373 0.43756
    Material
    Ex = 400 Em = 400 Em = 425 Em = 450 Em = 475 Em = 500 Em = 550 Em = 600 Em = 650
    Glass 0.29134 0.21520 0.25492 0.18540 0.26817 0.43039
    Pc1 - 5 mil 0.38073 0.30459 0.32114 0.22844 0.31783 0.48667
    Pc2 - 5 mil 0.65115 0.59736 0.62284 0.43033 0.53791 0.77855
    Ps1 - 2 mil 0.55347 0.55347 0.67646 0.43731 0.61155 0.91561
    Ps2 - 2 mil 0.49544 0.50960 0.60869 0.46996 0.65115 1.00221
    Ps3 - 2 mil 0.75873 0.80120 0.97107 0.63417 0.86065 1.24568
    Nap - 1.5 mil 0.57754 0.59170 0.67663 0.50110 0.72476 1.08431
    Nap - 1.5 mil 0.41900 0.39635 0.50394 0.42466 0.66248 1.05883
    Coc - 2 mil 0.32558 0.33407 0.41900 0.37087 0.55489 0.87198
    Aclar - .75 mil 0.06295 0.06295 0.07121 0.06966 0.10010 0.15686
    Aclar - 3 mil 0.14138 0.14654 0.17750 0.20433 0.32405 0.47988
  • Example 2 Fluorescence Properties of Cycloolefins Compared to Glass and Other Polymeric Materials
  • To further investigate fluorescence properties of various selected materials, different polymeric films were tested for fluorescence emission at predetermined excitation wavelengths and compared to two types off used silica glass sheets (standard). These experiments were conducted to simulate biochemical or cell-based assays that involve aqueous media. Therefore, films were mounted on a horizontal plastic holder to permit addition of a drop of aqueous media. Three milliliters of water were dispensed onto the film and fluorescence recorded using a Zeiss inverted fluorescence microscope. Background in the absence of a film was recorded and subtracted from signals in the presence of a film.
  • The materials tested were 1) glass sheets (Fisher cover-slip Number 1 (Fisher Catalog number 12-542B (1996)), 2) polystyrene films (ps1, ps2 (from Plastic Suppliers) and ps3 (from Dow Chemical Company), 3) polycarbonate films (pc1 (from General Electric Corporation) and pc2 (from Plastic Suppliers); 4) non-aromatic, alkyl polymers (obtained from Mobil), 5) cycloolefin copolymer film (coc; obtained from Hoechst, Topas), and 6) Aclar (a fluorocarbon material from Allied Signal) and 7) Syran Wrap.
  • Table 3 shows the fluorescence normalized emission data at 460 nm at 350 and 405 nm (excitation wavelengths). The data is normalized to glass. Polystyrene, which is often used as a component of multi-well plates (see Table 1), generated high background fluorescence levels, consistent with its aromatic structure as in Example 1. In contrast to Example 1, polycarbonate, which is often a biocompatible polymer, was worse than polystyrene, especially at longer wavelengths. Generally consistent with Example 1, the non-aromatic, alkyl polymer was generally better than polystyrene across the range of wavelengths tested. Generally consistent with Example 1, the cycloolefin copolymer produced the best results and surprisingly out performed the extremely low fluorescence levels of glass. Aclar film also surprisingly produced either low or extremely low fluorescence values relative to glass. However, Aclar films were later found to have undesirable manufacturing characteristics, such as bonding to other materials and suitability for use in injection molding.
    TABLE 3
    350ex/ 405ex/
    Material 460em Rank Material 460em Rank
    Fisher #1 1.02 1 Fisher #1 1.03 1
    coverslip coverslip
    Polycarbonate 6.91 6 Polycarbonate 19.79 6
    5 mil 5 mil
    Polystyrene 3.57 5 Polystyrene 3.36 4
    2 mil 2 mil
    NAP 1.5 ml 2.06 3 NAP 1.5 ml 5.76 3
    NAP 1.5 ml 1.33 3 NAP 1.5 ml 3.51 3
    coc#2 2 mil 1.58 2 coc#2 2 mil 2.60 2
    coc#1 2 mil 1.22 2 coc#1 2 mil 1.59 2
    Aclar sample 2.62 4 Aclar sample 9.08 5
    (>2 yrs old) (>2 yrs old)
    Fisher #1 1.00 5 Fisher #1 1.00 1
    coverslip coverslip
    Polycarbonate 5.15 9 Polycarbonate 17.75 8
    5 mil 5 mil
    Polystyrene 2.01 7 Polystyrene 2.53 7
    2 mil 2mil
    coc#2 A 2 mil 1.09 6 coc#2 A 2 mil 1.71 4
    coc#2 B 2 mil 0.89 4 coc#2 B 2 mil 1.65 3
    coc#1 2 mil 0.86 3 coc#1 2 mil 1.47 2
    Aclar 3 mil 0.71 1 Aclar 3 mil 2.34 6
    (<1 yr old) (<1 yr old)
    Aclar 0.75 mil 0.64 1 Aclar 0.75 mil 2.14 5
    (<1 yr old) (<1 yr old)
    Syran wrap 4.18 8 Syran wrap 22.12 9
  • Example 3 Cycloolefins are Not CytoToxic to Cultured Cells
  • The cytotoxicity of cycloolefin was evaluated by incubating cells in cycloolefin multi-well plates for 60 hours at 37° C. 1.8 μL volumes of media containing about 90 Chinese hamster ovary (CHO) were placed in cycloolefin multi-well plates using a tapered pipette. A glass cover was placed over the wells to prevent evaporation. Cells were incubated for 60 hours in a 5% CO2, 37° C., 90% RH incubator. Cells were then tested for viability by loading with the vital dye calcein. The CHO cells were loaded by incubation in a solution containing 4 μM calcein/AM for 30 minutes at room temperature. Cells were inspected using both phase contrast microscopy to determine the total number of cells and fluorescence microscopy to determine the number of live cells. Approximately, greater than 95% of cells were alive as indicated by loading with calcein dye (approximately 200 cells/well).
  • Example 4 Cycloolefins Are Not CytoToxic to Cultured Cells and Can be Used for Drug Screening Assays
  • To investigate the cytotoxic properties of cyclolefins, cycloolefin film were tested using an assay for cell viability. CCF2 a vital dye, as described in PCT publication WO 96/30540 (Tsien), diffuses into cells and is trapped by living cells having esterase activity that cleaves ester groups on the molecules which results in a negatively charged molecule that is trapped inside the cell. Trapped dye appears green inside of living cells and turns blue in the presence of beta-lactarnase. CCF2 was incubated with Jurkat cells for at least hour in a 1 microliter well having black walls and a cycloolefin bottom, and fluorescence was appropriately monitored. These Jurkat cells were constitutively expressing β-lactamase. Cells were cultured for 60 hours in the conditions of Example 3. After 60 hours, β-lactamase activity was measured using CCF2. Cells appeared blue indicating that β-lactamase was indeed active in these cells, which normally do not contain β-lactamase. These results demonstrate that cycloolefins can be used with sensitive fluorescent assays because the films yield low fluorescent backgrounds. This is particularly beneficial because it permits smaller assay volumes (e.g., 2 microliters or less) and the measurement of smaller signals (e.g., from fewer cells or fewer number of isolated biochemical targets).
  • Example 5 High Density Multi-Well Platforms
  • FIG. 2 shows a preferred multi-well platform of the present invention. A 240 well (5×48 wells) injection molded multi-well platform and a 45 well (three sets of 3×5 wells) multi-well platform, each having a well-center-to-well-center distance of 1.5 mm, were made.
  • Injection Molded Multi-Well Platform
  • This multi-well platform comprised a frame, wherein the wall of a well was disposed in the frame. The frame was made of cycloolefin copolymer, which was made optically opaque with about 2% black pigment (OmniColor® IM0055 Reed Spectrum, Holden, Mass.). The frame was about 3.25 mm thick and was made by injection molding. The bottom of the frame was substantially flat.
  • Each well had a bottom, which had a high transmittance portion. The bottom had a thickness of about 50 micrometers and was made of clear, flat, cycloolefin copolymer film. The frame and bottom were joined by heat-sealing to from the wells. The wall of each well was chamfered at about 2.87 degrees and the well-center-to-well-center distance was about 1.5 millimeters. The diameter of the wells at the bottom of the frame was about 0.95 mm.
  • The multi-well platform further comprised a groove 21 that surrounded three of the four sides of the well matrix 22. These multi-well platforms were used in fluorescent based assays as described in the following examples.
  • Machined Multi-Well Platform
  • Alternatively, the frame was machined from an acrylic plate (black Acrylic butyl styrene (black ABS)) made optically opaque with about 2% to 4% of back pigment, and a bottom. The bottom was a glass plate 0.01 mm thick (borosilicate glass, Precision Glass) attached to the bottom of the frame by adhesive silicone. The frame and the bottom were combined for form a multi-well platform 13 mm thick. In this multi-well platform the wells were not chamfered and the well-center-to-well center distance was about 1.5 millimeters. The diameter of each well was about 0.95 mm.
  • Example 6 Detection of Protease Activity in a Machined Multi-Well Platform
  • In this example, trypsin activity in the machined multi-well platform described in Example 5 was detected using a green fluorescent protein tandem construct comprising two green fluorescent protein molecules coupled by a linker as reported by Tsien et al. (WO 97/28261). The two green-fluorescent protein molecules can exhibit fluorescence resonance energy transfer between themselves, and the linker comprises a trypsin substrate. When a sample comprises this intact construct, fluorescence resonance energy transfer between the green fluorescent protein molecules causes the sample to fluoresce at 535 nm when excited with light of about 400 nm. When the linker is cleaved with a protease such as trypsin, the green fluorescent protein molecules no longer exhibit fluorescence resonance energy transfer, and the sample will fluoresce at 460 nm when exited with light of about 400 nm. The increase in the ratio of the emission of 460 nm and 535 nm correlates with the protease activity in the sample.
  • To individual wells, 2.0 μL of the same 1 μM solution of the tandem construct with or without 0.015 nM trypsin were added. The bottom of each well was excited with light of 400 nm, and the emission at 460 nm and 535 nm measured through the bottom of each well. The samples were incubated at room temperature for thirty minutes. The bottom of each well was exited again with light of 400 nm, and the emission at 460 nm arid 535 nm measured through the bottom of each well. As shown in Table 4, addition of trypsin to the wells consistently elicited a greater than four fold increase in the emission ratio.
    TABLE 4
    460/535 Emission Ratio
    Well Number No Trypsin Trypsin Added
    1 0.20 1.00
    2 0.20 0.94
    3 0.20 0.94
    4 0.20 0.98
    5 0.20 0.93
  • Example 7 Detection of an Activated Reporter Gene in a Cell
  • In this example, a concentration response of carbachol in a Jurkat cell line stably transfected with a plasmid encoding the M1 muscarinic receptor and a NF-AT-β-lactamase reporter gene. In this transfected cell line, carbachol acts to stimulate the M1 muscarinic receptor so that the NFAT-β-lactamase reporter gene is expressed. When expressed, this gene produces β-lactamase, which can then be detected using a fluorescent probe, such as CCF2/AM, that exhibits different emissions when intact and cleaved by β-lactamase as reported by, for example, Tsien et al. (WO 96/30540).
  • Jurkat cells used in this example were made using the following procedures. Wild-type Jurkat cells were transfected with plasmid 3XNFAT-blax by electroporation (regarding the plasmid 3XNFAT-blax, see generally Fiering, Genes and Development, 4:1823-1834 (1990)). This plasmid is driven by the 1L-2 minimum promoter. A portion of this population of transfected cells was seeded into 96-well plates with limited dilution and selected by Zeocin® (250 μg/ml). The clones in each well were screened for CCF2-AM staining in the presence and the absence of 10 nm PMA/2 μM ionomycin (Calbiochem). FACS sorting was used to isolate individual clones, which were further transfected with pcDNA3-M1, which comprises pcDNA3 (Invitrogen) configured such that nucleic acids encoding M1 can be expressed. These transfected cells were selected using G418 (1 mg/ml) for about 3 weeks. The clones in each well were screened for CCF2-AM staining in the presence and absence of 30 μM Carbachol (Calbiochem). FACS sorting was used to isolate individual clones.
  • Transfected Jurkat cells in 1.8 μL of RPMT buffer were dispensed at approximately 500 cells per well into individual wells of injection molded multi-well platform described in Example 5. These wells contained 0.3 to 31 nL of stock carbachol solution. Cells were incubated for three hours at 37° C. The solution in each well was made one μM CCF2/AM. The bottom of each well was excited with light of 400 nm and the emission of light at 460 nm and 535 nm was detected and measured through the bottom of the well. The ratio of the emission at these wavelengths is correlated with β-lactamase activity in the cell, which is correlated with the stimulation of the cell. As shown in Table 5, the stimulation of the cells, as measured by the ratio of emission at 460 nm and 535 nm, was dependent upon the concentration of carbachol provided in the well.
    TABLE 5
    Carbachol Concentration (μM) Emission Ratio (460/535)
    0.09 1.28
    0.17 2.44
    0.43 3.38
    0.87 5.90
    1.70 7.70
    4.30 8.90
  • Example 8 Detection of an Activated Reporter
  • In this example, a Jurkat cell line stably transfected with a CMV-β-lactamase reporter gene. When expressed, this gene produces β-lactamase, which can then be detected using a fluorescent probe, CCF2/AM, that exhibits different fluorescent emissions when intact or cleaved by β-lactamase as reported by Tsien et al. (WO 96/30540). The same Jurkat cell line without the CMV-β-lactamase reporter gene was used as a control.
  • The Jurkat cells used in this example were obtained in a similar way as described in the example above. Briefly, pcDNA3-bla, which encodes β-lactamase operatively linked to the CMV promoter, was transfected into wild type Jurkat cells. The G418 (1 mg/ml) selected population were stained with CCF2-AM and FACS sorted.
  • Control and transfected Jurkat cells in RPMI buffer were dispensed at approximately 800 cells per well into individual wells of the machined multi-well platform described in Example 5. Cells were incubated for ninety minutes at 23° C. in an RPMI buffer containing 10 μM CCF2/AM as described above. The bottom of each well was excited with light of 400 nm and the emission of light at 460 and 535 nm was detected and measured through the bottom of the well. The emission at that wavelength is correlated with β-lactamase activity in the cell, which is correlated with the expression of β-lactamase in the cell. As shown in Table 6, about 800 cells expressing β-lactamase showed at least a twelve-fold increase in the ration of emission at 460 and 535 compared to control cells.
    TABLE 6
    Well Number Cell Type Emission Ratio 460/535
    1 Wild Type 1
    2 Wild Type 1
    3 Wild Type 1
    4 Wild Type 1
    5 Wild Type 1
    6 CMV-β-lactamase 13
    7 CMV-β-lactamase 13
    8 CMV-β-lactamase 13
    9 CMV-β-lactamase 13
    10 CMV-β-lactamase 13

    Publications
  • All publications, including patent documents and scientific articles, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference.
  • All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims (20)

  1. 1. A composition identified by the method comprising the steps of:
    a) contacting a test chemical suspected of having modulating activity of a biological process or target with a biological process or target in a multi-well platform comprising:
    i) a plurality of wells, each well comprising:
    a) a wall having less fluorescence than a polystyrene-wall of at least about 90 percent of said wall's thickness and
    b) a bottom;
    b) exciting said sample with radiation of a first wavelength, and
    c) measuring the emission of radiation of a second wavelength emitted from said sample, wherein said test chemical has a modulating activity with respect to said process or target.
  2. 2. The composition of claim 1, wherein said multi-well platform further comprises a frame, wherein said wells are disposed in said frame.
  3. 3. The composition of claim 1, wherein said multi-well platform further comprises a recessed groove surrounding said plurality of wells, said recessed groove being filled with a fluid.
  4. 4. The composition of claim 1, wherein said multi-well platform further comprises an identification structure.
  5. 5. The composition of claim 4, wherein said identification structure includes a barcode, numbering or lettering.
  6. 6. The composition of claim 1, further comprising comparing said measured emission of radiation to a background or control level of emission.
  7. 7. The composition of claim 19, further comprising a pharmaceutically acceptable carrier.
  8. 8. A composition identified by the method comprising the steps of:
    a) contacting a test chemical suspected of having modulating activity of a biological process or target with a biological process or target in a multi-well platform comprising:
    i) a plurality of wells, each well comprising:
    a) a wall; and
    b) a bottom formed of a cycloolefin material;
    b) exciting said sample with radiation of a first wavelength; and
    c) measuring the emission of radiation of a second wavelength emitted from said sample; wherein said test chemical has a modulating activity with respect to said process or target.
  9. 9. The composition of claim 8, wherein said well wall has less fluorescence than a polystyrene-wall of at least about 90 percent of said wall's thickness
  10. 10. The composition of claim 8, wherein said multi-well platform further comprises a recessed groove surrounding said plurality of wells, said recessed groove being filled with a fluid.
  11. 11. The composition of claim 8, wherein said multi-well platform further comprises an identification structure.
  12. 12. The composition of claim 8, wherein said identification structure may include a barcode, numbering or lettering.
  13. 13. The composition of claim 8, further comprising comparing said measured emission of radiation to a background or control level of emission.
  14. 14. A therapeutic identified by the method comprising the steps of:
    a) identifying a therapeutic using the method comprising the steps of:
    i) contacting a test chemical suspected of having modulating activity of a biological process or target with a biological process or target in a multi-well platform comprising:
    a) a plurality of wells, each well comprising:
    (1) a wall having less fluorescence than a polystyrene-wall of at least about 90 percent of said wall's thickness;
    (2) a bottom, and
    b) a frame, wherein said wells are disposed in said frame,
    ii) exciting said sample with radiation of a first wavelength, and
    iii) measuring the emission of radiation of a second wavelength emitted from said sample, wherein said test chemical has a modulating activity with respect to said process or target;
    b) monitoring the toxicology of said therapeutic in an in vitro or in vivo model; and
    c) monitoring the efficacy of said therapeutic in an in vitro or in vivo model.
  15. 15. The composition of claim 14, wherein said well bottom formed of a cycloolefin material
  16. 16. The composition of claim 14, wherein said multi-well platform further comprises a recessed groove surrounding said plurality of wells, said recessed groove being filled with a fluid.
  17. 17. The composition of claim 14, wherein said multi-well platform further comprises an identification structure.
  18. 18. The composition of claim 17, wherein said identification structure may include a barcode, numbering or lettering.
  19. 19. The composition of claim 14, further comprising comparing said measured emission of radiation to a background or control level of emission.
  20. 20. The therapeutic of claim 14, further comprising a pharmaceutically acceptable carrier.
US11520869 1997-06-02 2006-09-13 Low fluorescence assay platforms and related methods for drug discovery Abandoned US20070009883A1 (en)

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US10120644 US6730520B2 (en) 1998-02-24 2002-04-09 Low fluorescence assay platforms and related methods for drug discovery
US10830253 US20040202582A1 (en) 1998-02-24 2004-04-21 Low fluorescence assay platforms and related methods for drug discovery
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