U.S. patent application number 11/520869 was filed with the patent office on 2007-01-11 for low fluorescence assay platforms and related methods for drug discovery.
This patent application is currently assigned to AURORA DISCOVERY, INC.. Invention is credited to Peter J. Coassin, Alec Tate Harootunian, Andrew A. Pham, Harry Stylli, Roger Y. Tsien.
Application Number | 20070009883 11/520869 |
Document ID | / |
Family ID | 26706197 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009883 |
Kind Code |
A1 |
Coassin; Peter J. ; et
al. |
January 11, 2007 |
Low fluorescence assay platforms and related methods for drug
discovery
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.
Inventors: |
Coassin; Peter J.;
(Encinitas, CA) ; Harootunian; Alec Tate; (Del
Mar, CA) ; Pham; Andrew A.; (Del Mar, CA) ;
Stylli; Harry; (San Diego, CA) ; Tsien; Roger Y.;
(La Jolla, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.;DLA PIPER US LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
AURORA DISCOVERY, INC.
|
Family ID: |
26706197 |
Appl. No.: |
11/520869 |
Filed: |
September 13, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10830253 |
Apr 21, 2004 |
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11520869 |
Sep 13, 2006 |
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10120644 |
Apr 9, 2002 |
6730520 |
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10830253 |
Apr 21, 2004 |
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09476959 |
Jan 3, 2000 |
6517781 |
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10120644 |
Apr 9, 2002 |
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09030578 |
Feb 24, 1998 |
6171780 |
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09476959 |
Jan 3, 2000 |
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Current U.S.
Class: |
435/4 ;
435/287.1 |
Current CPC
Class: |
B01J 2219/00315
20130101; B01L 2300/0829 20130101; B01J 2219/00317 20130101; B01L
3/5085 20130101; B01J 2219/00707 20130101; G01N 21/03 20130101;
C12M 23/12 20130101; G01N 21/6428 20130101; B01L 2300/021 20130101;
G01N 33/542 20130101; B01L 9/523 20130101; G01N 2021/0346 20130101;
B01J 2219/00659 20130101; G01N 21/6452 20130101; G01N 1/30
20130101; G01N 33/5308 20130101; B01L 2300/12 20130101; C40B 60/14
20130101; C12Q 1/00 20130101; B01J 2219/0072 20130101 |
Class at
Publication: |
435/004 ;
435/287.1 |
International
Class: |
C12M 1/34 20060101
C12M001/34; C12Q 1/00 20060101 C12Q001/00 |
Claims
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. The composition of claim 1, wherein said multi-well platform
further comprises a frame, wherein said wells are disposed in said
frame.
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. The composition of claim 1, wherein said multi-well platform
further comprises an identification structure.
5. The composition of claim 4, wherein said identification
structure includes a barcode, numbering or lettering.
6. The composition of claim 1, further comprising comparing said
measured emission of radiation to a background or control level of
emission.
7. The composition of claim 19, further comprising a
pharmaceutically acceptable carrier.
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. 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. 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. The composition of claim 8, wherein said multi-well platform
further comprises an identification structure.
12. The composition of claim 8, wherein said identification
structure may include a barcode, numbering or lettering.
13. The composition of claim 8, further comprising comparing said
measured emission of radiation to a background or control level of
emission.
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. The composition of claim 14, wherein said well bottom formed of
a cycloolefin material
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. The composition of claim 14, wherein said multi-well platform
further comprises an identification structure.
18. The composition of claim 17, wherein said identification
structure may include a barcode, numbering or lettering.
19. The composition of claim 14, further comprising comparing said
measured emission of radiation to a background or control level of
emission.
20. The therapeutic of claim 14, further comprising a
pharmaceutically acceptable carrier.
Description
[0001] This section describes materials, selection criteria, and
rapid tests to facilitate choosing a material for the multi-well
plates described herein.
Materials
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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).
[0007] 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
[0008] 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.degree. C., and resistance to acids and bases. Starting
materials with good molding properties are particularly
desirable.
[0009] 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.
[0010] 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.
[0011] In the final product, breaking stresses (Kg/cm.sup.2 at
22.degree. C.) of generally, about 400 to 3,000 Kg/cm.sup.2 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/cm.sup.2, 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/cm.sup.2 for multi-well plate
applications, and more preferably between about 800 to 1,600
Kg/cm.sup.2 and most preferably between about 900 to 1,400
Kg/cm.sup.2. 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
[0012] 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.
[0013] 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.degree. 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).
[0014] 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.degree. C. degrees,
preferably resins are heated to 260.degree. to 320.degree. C.
Pressures used are typically between 10,000 and 25,000 psi and
preferably about 15,000 to 22,000 psi.
[0015] 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.
[0016] 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.degree. to 350.degree. 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.
[0017] 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.degree. C. for different cycloolefin monomer systems.
Mold temperatures in a RIM process are in the range of about
50.degree. C. to 150.degree. 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.degree. C. The final mold temperature is thus
from about 50.degree. C. to about 200.degree. C., generally from
about 50.degree. C. to about 150.degree. C., and preferably from
about 50.degree. C. to about 90.degree. 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
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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).
[0030] 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.
[0031] 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.
[0032] 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
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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 .beta.-lactamase yields a fluorescent emission shift
as substrate is converted to product. Because the .beta.-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
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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 .alpha..sub.2, .alpha..sub.3, and .beta..sub.2, 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 .alpha..sub.5 subtype (Chini, et al. (1992) Proc. Natl.
Acad Sci. USA 89:1572-1 576), the rat .alpha..sub.2 subunit (Wada,
et al. (1988) Science 240, pp. 330-334); the rat .alpha..sub.3
subunit (Boulter, et al. (1986) Nature 319, pp. 368-374); the rat
.alpha..sub.4 subunit (Goldman, et al. (1987) Cell 48, pp.
965-973); the rat .alpha..sub.5 subunit (Boulter, et al. (1990) J.
Biol. Chem. 265, pp. 4472-4482); the chicken .alpha..sub.7 subunit
(Couturier et al. (1990) Neuron 5:847-856); the rat .beta..sub.2
subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat
.beta.3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp.
6268-6272); the rat .beta..sub.4 subunit (Duvoisin, et al. (1989)
Neuron 3, pp. 487-496); combinations of the rat .alpha. subunits,
.beta. 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 .gamma.-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 .alpha..sub.2 (Kobilka,
et al. (1987) Science 238, pp. 650-656); hamster .beta..sub.2
(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.
[0044] Ion channels include, but are not limited to, calcium
channels comprised of the human calcium channel .alpha..sub.2
.beta. and/or .gamma.-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 .alpha..sub.2 subunit); rabbit skeletal muscle a1 subunit
(Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal
muscle .alpha..sub.2 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 .gamma. 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.
[0045] 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.
[0046] 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.
[0047] 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
[0048] 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.
[0049] 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.
[0050] 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
[0051] 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.
[0052] 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, LD.sub.50 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
[0053] 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.
[0054] 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
[0055] 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
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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
.mu.g/kg and 10 mg/kg body weight. Administration is preferably
oral on a daily basis.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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, dragees, 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, dragee-making, levitating, emulsifying,
encapsulating, entrapping, or lyophilizing processes.
[0069] 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.
[0070] 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 dragee 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. Dragee 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 dragee 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
[0071] 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.
[0072] 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).
[0073] 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-US-00001 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
[0074] 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.
[0075] 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.
[0076] 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-US-00002 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
[0077] The cytotoxicity of cycloolefin was evaluated by incubating
cells in cycloolefin multi-well plates for 60 hours at 37.degree.
C. 1.8 .mu.L 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% CO.sub.2,
37.degree. 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 .mu.M 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
[0078] 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
.beta.-lactamase. Cells were cultured for 60 hours in the
conditions of Example 3. After 60 hours, .beta.-lactamase activity
was measured using CCF2. Cells appeared blue indicating that
.beta.-lactamase was indeed active in these cells, which normally
do not contain .beta.-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
[0079] FIG. 2 shows a preferred multi-well platform of the present
invention. A 240 well (5.times.48 wells) injection molded
multi-well platform and a 45 well (three sets of 3.times.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
[0080] 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.RTM. 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.
[0081] 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.
[0082] 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
[0083] 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
[0084] 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.
[0085] To individual wells, 2.0 .mu.L of the same 1 .mu.M 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-US-00003 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
[0086] 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-.beta.-lactamase reporter gene. In
this transfected cell line, carbachol acts to stimulate the M1
muscarinic receptor so that the NFAT-.beta.-lactamase reporter gene
is expressed. When expressed, this gene produces .beta.-lactamase,
which can then be detected using a fluorescent probe, such as
CCF2/AM, that exhibits different emissions when intact and cleaved
by .beta.-lactamase as reported by, for example, Tsien et al. (WO
96/30540).
[0087] 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.RTM. (250 .mu.g/ml). The clones in each well were screened
for CCF2-AM staining in the presence and the absence of 10 nm PMA/2
.mu.M 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 .mu.M Carbachol (Calbiochem). FACS sorting was used to
isolate individual clones.
[0088] Transfected Jurkat cells in 1.8 .mu.L 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.degree. C. The solution
in each well was made one .mu.M 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 .beta.-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-US-00004 TABLE 5 Carbachol
Concentration (.mu.M) 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
[0089] In this example, a Jurkat cell line stably transfected with
a CMV-.beta.-lactamase reporter gene. When expressed, this gene
produces .beta.-lactamase, which can then be detected using a
fluorescent probe, CCF2/AM, that exhibits different fluorescent
emissions when intact or cleaved by .beta.-lactamase as reported by
Tsien et al. (WO 96/30540). The same Jurkat cell line without the
CMV-.beta.-lactamase reporter gene was used as a control.
[0090] The Jurkat cells used in this example were obtained in a
similar way as described in the example above. Briefly, pcDNA3-bla,
which encodes .beta.-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.
[0091] 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.degree. C. in an RPMI
buffer containing 10 .mu.M 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 .beta.-lactamase activity in the cell, which is correlated
with the expression of .beta.-lactamase in the cell. As shown in
Table 6, about 800 cells expressing .beta.-lactamase showed at
least a twelve-fold increase in the ration of emission at 460 and
535 compared to control cells. TABLE-US-00005 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-.beta.-lactamase 13 7
CMV-.beta.-lactamase 13 8 CMV-.beta.-lactamase 13 9
CMV-.beta.-lactamase 13 10 CMV-.beta.-lactamase 13
Publications
[0092] 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.
[0093] 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.
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