U.S. patent application number 10/703143 was filed with the patent office on 2004-11-04 for systems and methods for characterizing a biological conditions or agent using selected gene expression profiles.
Invention is credited to Bankaitis-Davis, Danute M., Bevilacqua, Michael P., Cheronis, John C., Tryon, Victor.
Application Number | 20040219568 10/703143 |
Document ID | / |
Family ID | 46279930 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219568 |
Kind Code |
A1 |
Bevilacqua, Michael P. ; et
al. |
November 4, 2004 |
Systems and methods for characterizing a biological conditions or
agent using selected gene expression profiles
Abstract
Methods are provided for evaluating a biological condition of a
subject using a calibrated profile data set derived from a data set
having a plurality of members, each member being a quantitative
measure of the amount of a subject's RNA or protein as distinct
constituents in a panel of constituents. The biological condition
may be a naturally occurring physiological state or may be
responsive to treatment of the subject with one or more agents.
Calibrated profile data sets may be used as a descriptive record
for an agent.
Inventors: |
Bevilacqua, Michael P.;
(Boulder, CO) ; Cheronis, John C.; (Conifer,
CO) ; Tryon, Victor; (Loveland, CO) ;
Bankaitis-Davis, Danute M.; (Longmont, CO) |
Correspondence
Address: |
Barbara J. Carter
Bromberg & Sunstein LLP
125 Summer Street
Boston
MA
02110-1618
US
|
Family ID: |
46279930 |
Appl. No.: |
10/703143 |
Filed: |
November 3, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10703143 |
Nov 3, 2003 |
|
|
|
09821850 |
Mar 29, 2001 |
|
|
|
6692916 |
|
|
|
|
09821850 |
Mar 29, 2001 |
|
|
|
09605581 |
Jun 28, 2000 |
|
|
|
60141542 |
Jun 28, 1999 |
|
|
|
60195522 |
Apr 7, 2000 |
|
|
|
Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
G01N 33/6803 20130101;
G01N 33/5088 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. A method, for evaluating a biological condition of a subject,
based on a sample from the subject, the sample providing a source
of RNAs, the method comprising: deriving from the sample a first
profile data set, the first profile dataset including a plurality
of members, each member being a quantitative measure of the amount
of a distinct RNA constituent in a panel of constituents selected
so that measurement of the constituents enables evaluation of the
biological condition wherein such measure for each constituent is
obtained under measurement conditions that are substantially
reproducible; and producing a calibrated profile data set for the
panel, wherein each member of the calibrated profile data set is a
function of a corresponding member of the first profile data set
and a corresponding member of a baseline profile data set for the
panel, and wherein the baseline profile data set is related to the
biological condition to be evaluated, the calibrated profile data
set being a comparison between the first profile data set and the
baseline profile data set, thereby providing an evaluation of the
biological condition of the subject.
2. A method according to claim 1, wherein the measurement
conditions are such that the coefficient of variation, on
repeatedly obtaining such measure, is less than approximately 3
percent.
3. A method according to claim 1, wherein the measurement
conditions are such that the coefficient of variation, on
repeatedly obtaining such measure, is less than approximately 2
percent.
4. A method according to claim 1, wherein the baseline profile data
set is derived from one or more other samples from the same subject
taken under circumstances different from those of the sample.
5. A method according to claim 4, wherein the circumstances are
selected from the group consisting of (i) the time at which a given
sample is taken, (ii) the site from which a given sample is taken,
(iii) the biological condition of the subject when a given sample
is taken.
6. A method according to claim 4, wherein the one or more other
samples are taken over an interval of time between an initial
sample and the sample.
7. A method according to claim 4, wherein the one or more other
samples are taken over an interval of time that is at least twelve
months between an initial sample and the sample.
8. A method according to claim 4, wherein the one or more other
samples are taken over an interval of time that is at least one
month between an initial sample and the sample.
9. A method according to claim 1, wherein the sample is derived
from blood and the baseline profile data set is derived from tissue
or body fluid of the subject other than blood.
10. A method according to claim 4, wherein the baseline profile
data set is derived from one or more other samples from the same
subject, taken when the subject is in a biological condition
different from that in which the subject was at the time the sample
was taken, with respect to at least one of age, diet, medication,
and environmental exposure.
11. A method according to claim 1, wherein the baseline profile
data set is derived from one or more other samples from one or more
different subjects.
12. A method according to claim 11, wherein the one or more
different subjects have in common with the subject at least one of
age group, gender, ethnicity, geographic location, diet, medical
disorder, clinical indicator, medication, physical activity, body
mass, and environmental exposure.
13. A method according to claim 12 wherein a clinical indicator has
been used to assess biological condition of the one or more
different subjects, further comprising: interpreting the calibrated
profile data set in the context of at least one other clinical
indicator
14. A method according to claim 13, wherein the at least one other
clinical indicator is selected from the group consisting of blood
chemistry, urinalysis, X-ray, other chemical assays, and physical
findings.
15. A method according to claim 1, wherein the biological condition
is a complex disease process, involving multiple genes, the disease
being of a type involving at least one of inflammation, auto-immune
disease, degenerative disease, allergy, vascular disease, ischemia,
cancer, developmental disease, hormonal condition, aging and
infectious diseases.
16. A method according to claim 15, wherein the biological
condition is one of arthritis, asthma, multiple sclerosis, and
perimenopausal change.
17. A method according to claim 1, wherein the subject is a living
organism.
18. A method according to claim 17, wherein the subject is a
mammal.
19. A method according to claim 1, wherein the sample is derived
from one or more of body fluid and tissue.
20. A method according to claim 1, wherein the sample is derived
from blood.
21. A method according to claim 1, wherein the sample is derived
from one of a biopsy, a needle aspirate, a lavage specimen, a
scraping, and a surgical specimen.
22. A method according to claim 1, wherein the sample is derived
from tissue or fluid of a type distinct from that with respect to
which the condition is clinically manifested.
23. A method according to claim 1, wherein the condition is a
disease and the sample is derived from tissue or fluid of a type
distinct from that which is a primary target of the disease.
24. A method according to claim 1, wherein the function is other
than a simple difference.
25. A method according to claim 24, wherein the function is a
second function of the ratio of the corresponding member of first
profile data set to the corresponding member of the baseline
profile data set.
26. A method according to claim 25, wherein the function is a
logarithmic function.
27. A method according to claim 25, wherein each member of the
calibrated profile data set has biological significance if it has a
value differing by more than an amount D, where D=F(1.1)-F(0.9),
and F is the second function.
28. A method according to claim 1, wherein the biological condition
concerns an organ and the panel of constituents enables measurement
of the condition in relation to the organ.
29. A method according to claim 1, wherein the biological condition
concerns a system of the subject, the system selected from the
group consisting of respiratory, vascular, nervous, metabolic,
urinary, reproductive, structural, and immunological systems, and
the panel of constituents enables measurement of the condition of
the subject in relation to the system.
30. A method according to claim 1, wherein the number of
constituents in the panel is at least three but less than 100.
31. A method according to claim 1, wherein the number of
constituents in the panel is at least four but less than 100.
32. A method according to claim 1, wherein the number of
constituents in the panel is at least at least five but less than
100.
33. A method according to claim 1, wherein the number of
constituents in the panel is at least is at least six.
34. A method according to claim 1, wherein the number of
constituents in the panel is at least 4.
35. A method according to claim 1, wherein the number of
constituents in the panel is at least 3.
36. A method according to claim 1, wherein the number of
constituents in the panel is at least 2.
37. A method according to claim 1, wherein deriving the first
profile data set from the sample includes hybridizing the sample
with a set of nucleic acid probes.
38. A method according to claim 37, wherein the probes are attached
to an insoluble matrix and the sample is applied to the matrix.
39. A method according to claim 1, wherein obtaining the sample and
quantifying the first profile data set are performed at a first
location, and producing the calibrated profile data set includes
using a network to access a database stored on a digital storage
medium in a second location.
40. A method according to claim 39, further comprising updating the
database to reflect the first profile data set quantified from the
sample.
41. A method according to claim 39, wherein using a network
includes accessing a global computer network.
42. A method according to claim 1, wherein the quantitative measure
is determined by amplification, and the measurement conditions are
such that efficiencies of amplification for all constituents differ
by less than approximately ten percent.
43. A method according to claim 1, wherein the quantitative measure
is determined by amplification, and the measurement conditions are
such that efficiencies of amplification for all constituents differ
by less than approximately 2 percent.
44. A method according to claim 1, wherein the quantitative measure
is determined by amplification, and the measurement conditions are
such that efficiencies of amplification for all constituents differ
by less than approximately 1 percent.
45. A method according to claim 1, further comprising: accessing a
data in a condition database, the condition database having a
plurality of records relating to a population of subjects, each
record corresponding to a distinct instance of the calibrated
profile data set; and evaluating the first instance of the
calibrated profile data set in relation to data in the condition
database.
46. A method according to claim 44, wherein accessing the condition
database includes accessing the condition database over a
network.
47. A method according to claim 46, wherein the network is a global
computer network.
48. A method according to claim 46, further comprising
supplementing the condition database based on data associated with
the first instance of the calibrated profile data set.
49. A method according to claim 46, wherein the biological
condition concerns a system of the subject, the system selected
from the group consisting of respiratory, vascular, nervous,
metabolic, urinary, reproductive, structural, and immunological
systems and the panel of constituents enables measurement of the
condition of the subject in relation to the system.
50. A method according to claim 46, wherein each record also
references a characteristic of the population relative to the
record, the characteristic being at least one of age group, gender,
ethnicity, geographic location, diet, medical disorder, medication,
physical activity, body mass, and environmental exposure.
51. A method according to claim 46, wherein the characteristic
includes a clinical indicator.
52. A method according to claim 51, wherein the function is a
logarithmic function.
53. A method according to claim 51, wherein the graphical format is
a bar graph for each member of the calibrated profile data set.
54. A method according to claim 1 wherein the biological condition
to be evaluated relates to inflammation and the panel includes at
least half of the constituents of the Inflammation Precision Panel
of Table 1.
55. A method according to claim 54, wherein the panel includes at
least eighty percent of the constituents of the Inflammation
Precision Panel of Table 1.
56. A method according to claim 1 wherein the biological condition
to be evaluated relates to cell growth and differentiation and the
panel includes at least half of the constituents of the Cell Growth
and Differentiation Precision Panel of Table 2.
57. A method according to claim 56, wherein the panel includes at
least eighty percent of the constituents of the Cell Growth and
Differentiation Precision Panel of Table 2.
58. A method according to claim 1 wherein the biological condition
to be evaluated relates to metabolism and toxicity and the panel
includes at least half of the constituents of the Liver Metabolism
and Toxicity Precision Panel of Table 3.
59. A method according to claim 58, wherein the panel includes at
least eighty percent of the constituents of the Metabolism and
Toxicity Precision Panel of Table 3.
60. A method according to claim 1 wherein the biological condition
to be evaluated relates to skin response and the panel includes at
least half of the constituents of the Skin Response Precision
Panel.
61. A method according to claim 60, wherein the panel includes at
least eighty percent of the constituents of the Skin Response
Precision Panel of Table 4.
62. A method according to claim 1 wherein the biological condition
to be evaluated relates to the vascular system and the panel
includes at least half of the constituents of the Vascular
Precision Panel of Table 6.
63. A method according to claim 62, wherein the panel includes at
least eighty percent of the constituents of the Vascular Precision
Panel of Table 6.
64. A method according to claim 1 wherein the biological condition
to be evaluated relates to the prostate health and disease and the
panel includes at least half of the constituents of the Prostate
Precision Panel of Table 5.
65. A method according to claim 64, wherein the panel includes at
least eighty percent of the constituents of the Prostate Precision
Panel of Table 5.
66. A method according to claim 1 wherein the biological condition
to be evaluated relates to the prostate health and disease and the
panel includes at least half of the constituents of the Prostate
Precision Panel of Table 7.
67. A method according to claim 66, wherein the panel includes at
least eighty percent of the constituents of the Prostate Precision
Panel of Table 7.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of United states
application Ser. No. 09/821,850, filed Mar. 29, 2001, which is a
continuation in part of U.S. application Ser. No. 09/605,581, filed
Jun. 28, 2000, by the same inventors herein, which application
claims priority from provisional application Ser. No. 60/141,542,
filed Jun. 28, 1999 and provisional application Ser. No. 60/195,522
filed Apr. 7, 2000. These related applications are hereby
incorporated herein by reference.
TECHNICAL FIELD
[0002] Embodiments of the recent invention provide systems and
methods utilizing gene expression analysis for characterizing a
biological condition or agent.
BACKGROUND ART
[0003] There has been substantial discussion including
congressional hearings concerning medical errors. One source of
medical errors include errors with medications. Upwards of 98,000
hospitalized patients annually have been documented to be victims
of medication errors (Statement of the American Pharmaceutical
Association to the Senate Appropriations Committee Labor, health
and Human Services Education Subcommittee Hearing on Medical Errors
Dec. 13, 1999). These errors include problems arising from drug
interactions for a particular patient taking more than one drug,
problems concerning the response of an individual to a particular
drug and incorrect medication for a particular condition. Medical
errors further arise as a result of misdiagnosis. This may occur as
a result of insensitive diagnostic techniques or a wide range of
interpersonal variability in the manner in which a clinical state
is manifest. At present, there are few tools available for
optimizing prognosis, diagnosis and treatment of a medical
condition taking into account the particular phenotype and genotype
of an individual.
[0004] There has been increasing interest in herbal drugs or
nutraceuticals. These compounds are grown and collected from around
the world, and consequently the compounds are subject to regional
and temporal differences in collection and preparation that are
difficult to control. It is frequently the case that one batch of a
nutraceutical may be effective, there is no assurance that a second
batch will be effective. Moreover, analysis of nutraceuticals is
problematic because these drugs are complex mixtures in which
little is known with respect to the active agent.
[0005] All new therapeutic agents require some form of clinical
trials. It is known that a drug for treating tumor that is tested
in a clinical trial using standard recruiting techniques for
patients, may in fact show only limited efficacy. If the beneficial
effect observed in a clinical population is too small, the drug
will not receive approval by the Food and Drug Administration for
use in the population at large. However, the small beneficial
effect observed may in fact be an artifact of the clinical trial
design or the clinical endpoint in the population of patients. It
would be desirable to have criteria for screening patients as they
enter a clinical trial to ensure that the beneficial effect of a
drug if it exists may be detected and quantified.
SUMMARY OF THE INVENTION
[0006] In a first embodiment of the invention there is provided a
method, for evaluating a biological condition of a subject, that
includes: obtaining from the subject a sample having at least one
of RNAs and proteins; deriving from the sample a first profile data
set, the first profile data set including a plurality of members,
each member being a quantitative measure of the amount of a
distinct RNA or protein constituent in a panel of constituents
selected so that measurement of the constituents enables
measurement of the biological condition; and producing a calibrated
profile data set for the panel, wherein each member of the
calibrated profile data set is a function of a corresponding member
of the first profile data set and a corresponding member of a
baseline profile data set for the panel, the calibrated profile
data set providing a measure of the biological condition of the
subject.
[0007] In another embodiment, a method is provided for evaluating a
biological condition of a subject, that includes obtaining from the
subject a first sample having at least one of fluid, cells and
active agents; applying the first sample or a portion thereof to a
defined population of indicator cells; obtaining from the indicator
cells a second sample containing at least one of RNAs or proteins;
deriving from the second sample a first profile data set, the first
profile data set including a plurality of members, each member
being a quantitative measure of the amount of a distinct RNA or
protein constituent in a panel of constituents selected so that
measurement of the constituents enables measurement of the
biological condition; and producing a calibrated profile data set
for the panel, wherein each member of the calibrated profile data
set is a function of a corresponding member of the first profile
data set and a corresponding member of a baseline profile data set
for the panel, the calibrated profile data set providing a measure
of the biological condition of the subject.
[0008] In a another embodiment, a method is provided for evaluating
a biological condition affected by an agent, the method including:
obtaining, from a target population of cells to which the agent has
been administered, a sample having at least one of RNAs and
proteins; deriving from the sample a first profile data set, the
first profile data set including a plurality of members, each
member being a quantitative measure of the amount of a distinct RNA
or protein constituent in a panel of constituents selected so that
measurement of the constituents enables measurement of the
biological condition; and producing a calibrated profile data set
for the panel, wherein each member of the calibrated profile data
set is a function of a corresponding member of the first profile
data set and a corresponding member of a baseline profile data set
for the panel, the calibrated profile data set providing a measure
of the biological condition as affected by the agent.
[0009] In a another embodiment, a method is provided for evaluating
the effect on a biological condition by a first agent in relation
to the effect by a second agent, including: obtaining, from first
and second target populations of cells to which the first and
second agents have been respectively administered, first and second
samples respectively, each sample having at least one of RNAs and
proteins; deriving from the first sample a first profile data set
and from the second sample a second profile data set, the profile
data sets each including a plurality of members, each member being
a quantitative measure of the amount of a distinct RNA or protein
constituent in a panel of constituents selected so that measurement
of the constituents enables measurement of the biological
condition; and producing for the panel a first calibrated profile
data set and a second profile data set, wherein (i) each member of
the first calibrated profile data set is a function of a
corresponding member of the first profile data set and a
corresponding member of a first baseline profile data set for the
panel, and (ii) each member of the second calibrated profile data
set is a function of a corresponding member of the second profile
data set and a corresponding member of a second baseline profile
data set for the panel, the calibrated profile data sets providing
a measure of the effect by the first agent on the biological
condition in relation to the effect by the second agent.
[0010] In a further embodiment, a method of conducting a clinical
trial of an agent, is provided, including: causing the blind
administration of a selected one of a placebo and the agent to each
candidate of a pool of subjects; and using quantitative gene
expression to monitor an effect of such administration.
[0011] In another embodiment, a digital storage medium is provided
on which is stored a computer readable calibrated profile data set,
wherein: the calibrated profile data set relates to a sample having
at least one of RNAs and proteins derived from a target cell
population to which an agent has been administered; the calibrated
profile data set includes a first plurality of members, each member
being a quantitative measure of a change in an amount of a distinct
RNA or protein constituent in a panel of constituents selected so
that measurement of the constituents enables measurement of a
biological condition as affected by administration of the
agent.
[0012] In another embodiment, a digital storage medium is provided
on which is stored a plurality of records R.sub.i relating to a
population of subjects, each record R.sub.i corresponding to a
distinct instance P.sub.i of a computer readable profile data set P
wherein: each instance P.sub.i of the profile data set P relates to
a distinct sample derived from a subject, the sample having at
least one of RNAs and proteins; the profile data P set includes a
plurality of members M.sub.j, each member M.sub.j being a
quantitative measure of the amount of a distinct RNA or protein
constituent in a panel of constituents selected so that measurement
of the constituents enables measurement of a biological condition;
each record R.sub.i includes, for each member M.sub.ij of a
corresponding distinct instance P.sub.i of the profile data set P,
a value corresponding to the value of the member M.sub.ij; and each
record R.sub.i also includes a reference to a characteristic of the
subject relative to the record, the characteristic being at least
one of age group, gender, ethnicity, geographic location, diet,
medical disorder, clinical indicator, medication, physical
activity, body mass, and environmental exposure.
[0013] In a further embodiment, a digital storage medium is
provided on which is stored a large number of computer readable
profile data sets, wherein each profile data set relates to a
sample derived from a target cell population to which has been
administered an agent, the sample having at least one of RNAs and
proteins; each profile data set includes a plurality of members,
each member being a quantitative measure of the amount of a
distinct RNA or protein constituent in a panel of constituents
selected so that measurement of the constituents enables
measurement of a biological condition; and the panel is the same
for all profile data sets.
[0014] In a another embodiment of the invention, a method is
provided for evaluating a biological condition of a subject, based
on a sample from the subject, the sample having at least one of
RNAs and proteins, the method including: deriving from the sample a
first instance of a profile data set, the profile data set
including a plurality of members, each member being a quantitative
measure of the amount of a distinct RNA or protein constituent in a
panel of constituents selected so that measurement of the
constituents enables measurement of the biological condition; and
producing a first instance of a calibrated profile data set for the
panel, wherein each member of an instance of the calibrated profile
data set is a function of a corresponding member of an instance of
the profile data set and a corresponding member of an instance of a
baseline profile data set for the panel, the calibrated profile
data set providing a measure of the biological condition of the
subject; accessing data in a condition database, the condition
database having a plurality of records relating to a population of
subjects, each record corresponding to a distinct instance of the
calibrated profile data set; and evaluating the first instance of
the calibrated profile data set in relation to data in the
condition database.
[0015] In another embodiment of the invention, a method is provided
of displaying quantitative gene expression analysis data associated
with measurement of a biological condition, the method including:
identifying a first profile data set pertinent to the gene
expression analysis data, the first profile data set including a
plurality of members, each member being a quantitative measure of
the amount of a distinct RNA or protein constituent in a panel of
constituents selected so that measurement of the constituents
enables measurement of the biological condition; producing a
calibrated profile data set for the panel, wherein each member of
the calibrated profile data set is a function of a corresponding
member of the first profile data set and a corresponding member of
a baseline profile data set for the panel, the calibrated profile
data set providing a measure of the biological condition of the
subject; and displaying the calibrated profile data set in a
graphical format.
[0016] Another embodiment is directed to a descriptive record of a
change in a biological condition, that includes: a first set of
numerical gene expression values for a panel of gene loci, each
value in the set corresponding to a single gene locus in a panel of
gene loci, the set of values forming a profile data set for a
population of cells subjected to a first biological condition; a
second set of numerical gene expression values for the panel of
gene loci, each value in the set corresponding to a single gene
locus, the set of values forming a baseline profile data set for a
second population of cells subjected to a second biological
condition, the second set of values optionally being an average for
multiple gene expression values from multiple populations of cells
for each locus in the panel; and a third set of numbers
corresponding to the ratio of the first set of values and the
second set of values with respect to each gene locus in the panel,
the third set being a calibrated profile data set; the profile data
set and the calibrated profile data set being descriptive of the
first biological condition with respect to the second biological
condition.
[0017] In another embodiment, a method for diagnosing a biological
condition of a subject is provided that includes: obtaining a
sample from a subject; subjecting a population of cells to the
sample and determining the presence of a first biological condition
with respect to a second biological condition according to any of
the above claims.
[0018] In another embodiment, a method is provided for diagnosing a
susceptibility for a biological condition in a subject, that
includes obtaining a sample from the subject; creating a
descriptive record, according to the above, wherein the baseline
set of values is an average of second values contained in a library
of descriptive records for the second biological condition; the
library containing a plurality of descriptive records grouped
according to a predetermined biological condition; comparing the
calibrated profile data set of the subject with the library of
calibrated profile data sets and diagnosing the susceptibility of
the subject.
[0019] In another embodiment, a method is provided for monitoring
the progress of a biological condition, including: creating a
plurality of descriptive records, according to the above; wherein
each set of first values is determined at preselected time
intervals with respect to the first record; comparing each
calibrated profile data set with a library of calibrated profile
data sets, the plurality of calibrated profile data sets being
grouped according to a predetermined biological condition; and
determining the progress of the biological condition with respect
to gene expression.
[0020] In another embodiment, a method is provided for establishing
the biological activity of a composition, including: selecting a
population of cells; subjecting the cells to the composition; and
determining the record according to the above description using a
standardized baseline profile data set for the biological
condition.
[0021] In another embodiment, a method is provided for determining
which therapeutic agent from a choice of a plurality of therapeutic
agents to administer to a subject so as to change a biological
condition in a subject from a first biological condition to a
second biological condition; including: subjecting a sample from
the subject to each of a plurality of therapeutic agents;
determining a descriptive record for each of the samples according
to any of the above described methods, comparing each of the
calibrated profile data sets to a library of calibrated profile
data sets, the library of calibrated data sets being grouped
according to a predetermined biological condition; and determining
which of the therapeutic agents is capable of changing the first
biological condition in the subject to the second biological
condition in the subject.
[0022] In another embodiment, a method is provided for
characterizing the biological effectiveness of a single batch of a
composition produced by a manufacturing process, comprising:
providing a fingerprint or signature profile according to any of
the above methods; and labeling the batch of the composition by
placing the fingerprint (signature profile) on each container in
the batch.
[0023] In another embodiment, a method is provided for accessing
biological information on a digital storage medium as described
above, including: making the information available to a user.
[0024] In another embodiment, a method is provided for consumer
evaluation of a product, wherein the consumer evaluation is
dependent on a signature profile, including: identifying the
product using the signature profile.
[0025] In another embodiment, a computer program product is
provided for evaluating a biological condition of a subject or for
evaluating a biological condition resulting from the use of an
agent, including a computer usable medium having computer readable
program code thereon, the computer program code; including: a
program code for classifying a sample from the subject or the agent
for an identifiable record; a program code for deriving a first
data set, the first profile data set including a plurality of
members, each member being a quantitative measure of the amount of
a distinct RNA or protein constituent in a panel of constituents
selected so that measurement of the constituents enables
measurement of the biological condition; the profile data set being
stored in the record; and a program code for optionally producing a
calibrated profile data set for the panel, for storage in the
record, each member of the calibrated profile data set being a
function of a corresponding member of the first profile data set
and a corresponding member of a baseline profile data set for the
panel, the calibrated profile data set providing a measure of the
biological condition of the subject.
[0026] In another embodiment of the invention, a computer system
for evaluating a biological condition of a subject or for
evaluating a biological condition resulting from the use of an
agent is provided, the computer system, including: a classification
module for classifying a sample from the subject or the agent in an
identifiable record; a derivative module for deriving a first data
set, the first profile data set including a plurality of members,
each member being a quantitative measure of the amount of a
distinct RNA or protein constituent in a panel of constituents
selected so that measurement of the constituents enables
measurement of the biological condition; and a production module
for producing a calibrated profile data set for the panel, wherein
each member of the calibrated profile data set is a function of a
corresponding member of the first profile data set and a
corresponding member of a baseline profile data set for the panel,
the calibrated profile data set providing a measure of the
biological condition of the subject.
[0027] In another embodiment, a method is provided for analyzing a
patient for a biological condition at a remote site, including:
providing a kit for measuring a profile data base for evaluating a
biological condition, the kit including reagents for quantitative
analysis of RNA or protein for a panel of gene loci; accessing a
centralized database containing baseline profile data sets
corresponding to the panel; determining the calibrated profile data
set for the patient; and analyzing the biological condition of the
patient.
[0028] Further embodiments of the invention include the use of
calibrated profile data bases for determining the biological
condition at one site in a subject from a sample taken from a
second remote site. The biological condition may include disease,
therapeutic interventions, aging, health conditioning and exercise,
exposure to toxins, status of infection and health status. For
example, calibrated precision profiles may be used to measure a
biological condition(s) in one site (for example, the liver) by
sampling cells from the same subject, but at a different site not
generally considered a target for the biological condition, for
example, peripheral blood cells in the case of liver disease.
[0029] Further embodiments of the invention include the use of
calibrated profile data bases for determining the biological
condition of the subject that includes placing a cell or fluid
sample on indicator cells to assess the biological condition, the
biological condition including disease, therapeutic interventions,
aging, health conditioning and exercise, exposure to toxins, status
of infection and health status.
[0030] Further embodiments of the invention include the use of
calibrated profile data bases and profiles to assess, compare and
contrast the bioactivities of therapeutic agents and therapeutic
agent candidates including comparison of two agents having unknown
properties; comparison of agents that are complex mixtures against
those that are simple mixtures and comparisons of a single agent
against a class of agents.
[0031] Further embodiments of the invention include the use of
calibrated profile databases derived from in vitro dosing of an
agent in indicator cells, or fluids or cells ex vivo to predict in
vivo activities, activities including efficacy and toxicity and
further permitting data on short term in vivo dosing of agent to
predict long-term activities as described herein.
[0032] Another embodiment of the invention is at least one
databases and its uses, the databases containing at least one of
calibrated profile data sets and baseline profile data sets for
discrete populations identified according to factors including
diseases, geography, ethnicity, age and state of health.
[0033] A further embodiment of the invention is a database
corresponding to an individual over time, the uses including
managing a personalized health care program.
[0034] Additional embodiments include methods of running a clinical
trial using calibrated profile data and databases containing
calibrated profile data from in vitro and in vivo studies of the
effect of the agent on populations of cells and methods of building
a clinical research network that uses calibrated profile data and
traditional medical data.
[0035] Another embodiment of the invention provides a method, for
evaluating a biological condition of a subject. This method
includes:
[0036] a. obtaining from the subject a sample having at least one
of RNAs and proteins;
[0037] b. deriving from the sample a first profile data set, the
first profile dataset including a plurality of members, each member
being a quantitative measure of the amount of a distinct RNA or
protein constituent in a panel of constituents selected so that
measurement of the constituents enables measurement of the
biological condition; and
[0038] c. producing a calibrated profile data set for the panel,
wherein each member of the calibrated profile data set is a
function of a corresponding member of the first profile data set
and a corresponding member of a baseline profile data set for the
panel, the calibrated profile data set providing a measure of the
biological condition of the subject.
[0039] In this embodiment, the biological condition relates to
inflammation and the panel includes at least half, and, optionally,
at least eighty percent of the constituents of the Inflammation
Selected Panel of Table 1. In a related embodiment, the biological
condition relates to cell growth and differentiation and the panel
includes at least half, and optionally at least eighty percent, of
the constituents of the Cell Growth and Differentiation Selected
Panel of Table 2. In other related embodiments, the biological
condition relates to metabolism and toxicity and the panel includes
at least half, and optionally at least eighty percent, of the
constituents of the Liver Metabolism and Toxicity Selected Panel of
Tables 3 or 7. In another related embodiment, the biological
condition relates to skin response and the panel includes at least
half, and optionally at least eighty percent, of the constituents
of the Skin Response Selected Panel of Table 4. In another related
embodiment, the biological condition relates to the vascular system
and the panel includes at least half, and optionally, at least
eighty percent, of the constituents of the Vascular Selected Panel
of Table 6. In a further related embodiment, the biological
condition relates to the prostate health and disease and the panel
includes at least half, and optionally at least eighty percent of
the constituents of the Prostate Selected Panel of Table 5.
[0040] Another embodiment of the invention provides a method, for
evaluating a biological condition of a subject, that includes:
obtaining from the subject a sample having at least one of RNAs and
proteins; deriving from the sample a profile data set, the profile
data set including a plurality of members, each member being a
quantitative measure of the amount of a distinct RNA or protein
constituent in a panel of constituents selected so that measurement
of the constituents enables measurement of the biological
condition; wherein such measurement is performed for each
constituent under conditions wherein efficiencies of amplification
for all constituents are substantially similar, the profile data
set providing a measure of the biological condition of the
subject.
[0041] Another embodiment of the invention provides a method, for
evaluating a biological condition of a subject, that includes:
obtaining from the subject a first sample having at least one of
fluid, cells and active agents; applying the first sample or a
portion thereof to a defined population of indicator cells;
obtaining from the indicator cells a second sample containing at
least one of RNAs or proteins; deriving from the second sample a
profile data set, the profile data set including a plurality of
members, each member being a quantitative measure of the amount of
a distinct RNA or protein constituent in a panel of constituents
selected so that measurement of the constituents enables
measurement of the biological condition; wherein such measure is
performed for each constituent under conditions wherein
efficiencies of amplification for all constituents are
substantially similar, the profile data set providing a measure of
the biological condition of the subject.
[0042] Another embodiment of the invention provides method for
evaluating a biological condition affected by an agent, the method
that includes obtaining, from a target population of cells to which
the agent has been administered, a sample having at least one of
RNAs and proteins; deriving from the sample a profile data set, the
profile data set including a plurality of members, each member
being a quantitative measure of the amount of a distinct RNA or
protein constituent in a panel of constituents selected so that
measurement of the constituents enables measurement of the
biological condition; wherein such measure is performed for each
constituent under conditions wherein efficiencies of amplification
for all constituents are substantially similar, the profile data
set providing a measure of the biological condition as affected by
the agent.
[0043] Efficiencies of amplification of all constituents may differ
by less than approximately 2%. The efficiencies of amplification
may differ by less than approximately 1%. Moreover, in any of the
embodiments of the invention described above which refers to a
panel, the panel may include at least four constituents selected
from any one of Tables 1 through 7. For example, at least four
constituents may be selected from the group consisting of
expression products of TNF-.alpha., IL-1-.alpha., IL-.beta.,
IFN-.gamma., IL-8, and IL-10.
[0044] In another embodiment of the invention, a kit is provided
having primer-probe combinations for measuring expression products
of at least four constituents selected from any one of Tables 1
through 7. The kit may further include a primer probe combination
constructed so as to hybridize only to at least one of cDNA and
mRNA at a biologically relevant locus. Moreover, in each
combination, a reverse primer may be selected which is
complementary to a coding DNA strand located across an intron-exon
junction, with not more than three bases of a three-prime end of
the reverse primer being complementary to a proximal exon.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The foregoing features of the invention will be more readily
understood by reference to the following detailed description,
taken with reference to the accompanying drawings, in which:
[0046] FIG. 1 is a diagram showing the flow of information from
data acquired in molecular pharmacology and toxicology, clinical
testing, and use of the data for the application to individualized
medicine.
[0047] FIG. 2 is a diagram showing the drug discovery pathway of
new compounds from early leads to likely drug candidates. Although
calibrated profile data sets are indicated at the pre-clinical
step, gene expression data can be acquired and is useful at any of
the stages shown. IND refers to investigative new drug and refers
to an early stage in regulatory review.
[0048] FIG. 3 is a diagram presenting a comparison of in vivo and
in vitro protocols for forming calibrated profile data sets for
rapidly assessing product candidate toxicity and efficacy in
accordance with several embodiments of the present invention.
[0049] FIG. 4 is a diagram showing the application of gene
expression profiling as a guide to pre-clinical and clinical
studies in accordance with an embodiment of the present
invention.
[0050] FIG. 5 is a diagram showing a method in accordance with an
embodiment of the present invention for obtaining profile data in
the absence of a stimulus and in the presence of a stimulus.
[0051] FIG. 6 is a diagram showing the creation of a library of
profile data associated with a plurality of subjects in accordance
with an embodiment of the present invention.
[0052] FIG. 7 is a diagram illustrating the structure of a profile
data record in accordance with an embodiment of the present
invention.
[0053] FIG. 8 is a diagram illustrating a data entry screen for a
data record of the type shown in FIG. 7 and typical contexts in
which data records may be compiled in accordance with embodiments
of the present invention.
[0054] FIG. 9 shows an embodiment of the present invention in which
profile data, in either the raw or calibrated form, is evaluated
using data from a database that is remotely accessed over a
network.
[0055] FIG. 10 shows a schematic of a phase two clinical trial that
utilizes gene expression profiling (a). The right hand panel (b)
indicates that the same information may be used in Phase IV or post
marketing studies to compare the efficacy of already approved and
marketed drugs or to guide the marketing of such therapies; to
guide the choice of therapy for an individual subject or population
from within a class of appropriate compounds.
[0056] FIG. 11 is a bar graph that shows a graphical representation
in the form of a histogram representing calibrated profile data
sets based on quantitative expression of RNA in cells of a whole
blood sample using a panel of 12 constituents where each
constituent corresponds to a unique gene locus. (a) The blood
sample is stimulated ex vivo with heat killed staphylococci are
further exposed H7-TPCK, H9-UT-77, or H16-Dex as indicated. The
baseline profile data set is a blood sample stimulated ex vivo (in
vitro) with heat killed staphylococci (b) The blood sample is
stimulated ex vivo with lipopolysaccharide (LPS) and is then
further exposed to compounds H7-TPCK, H9-UT-77, or H16-Dex as
indicated.
[0057] FIG. 12 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for whole
blood stimulated ex vivo with lipopolysaccharide (LPS), using a
panel of 9 constituents, each constituent corresponding to a gene
locus encoding the gene products indicated, the blood being further
exposed to anti-inflammatory agents: methotrexate, meclofenamate
and methylprednisolone. The baseline profile data set is derived
from LPS stimulated (but otherwise untreated) cells.
[0058] FIG. 13 are bar graphs with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for two
different samples of whole blood (a) 991116 and (b) 991028
reflecting the biological condition of the cells using a panel of
24 members, each member corresponding to a gene locus, the baseline
profile data set being derived from untreated cells. The calibrated
data sets for cells exposed for six hours to three inflammation
inducing agents (lipopolysaccharide, heat killed staphylococci, and
phytohemagglutinin) are compared for each sample. (c) shows a
direct comparison of LPS stimulated 991116 with respect to 991028
as the baseline profile data set (d) shows a direct comparison
between unstimulated 991116 and 991028.
[0059] FIG. 14 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets using a
panel of 22 constituents, each constituent corresponding to a gene
locus, the baseline profile data set being derived from untreated
cells. Whole blood is exposed for six hours ex vivo to three
inflammation inducing agents (lipopolysaccharide, heat killed
staphylococci, and phytohemagglutinin) which are then treated with
a single anti-inflammatory agent (methyl prednisolone) to reveal
similarities and differences in the effect of a single agent on
cell populations differing in their biological condition.
[0060] FIG. 15 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for whole
blood where one calibrated data set refers to a subject (subject 2)
who has been treated in vivo with a corticosteroid (dexamethasone),
a second data set refers to the treatment of a blood sample from
the same subject prior to in vivo treatment where that sample has
been treated ex vivo (in vitro) and the third data set refers to a
second subject treated in vivo with dexamethasone (subject 1). The
data sets demonstrate the reproducibility and predictability of an
ex vivo (in vitro) treatment of blood compared to in vivo treatment
with the same agent. The figure also shows minor variation between
samples from different subjects reflecting interpersonal
variability. A panel of 14 constituents is provided. The baseline
profile data set is derived from untreated whole blood from the
cognate subject.
[0061] FIG. 16 is a bar graph with a logarithmic y axis that shows
a graphical representation of calibrated profile data sets for
whole blood where one calibrated data set refers to (a) 2 subjects
who have been treated in vivo with an inactive placebo for 3 days
and (b) active prednisolone for 3 days at 100 mg/day. The data set
shows some variation between samples from different subjects
treated with the same drug. The data sets demonstrate similarity of
responses across the same gene loci, as well as, quantitative
variation at other loci suggesting quantifiable interpersonal
variation. A panel of eight members is provided. The baseline
profile data set is derived from untreated whole blood.
[0062] FIG. 17 is a bar graph with logarithmic y axis that shows a
graphical representation of calibrated selected profile data sets
for two samples taken from a single subject within a 19 day period
using a panel (e.g., inflammation panel) of 24 members where each
member corresponds to a unique gene locus. The baseline profile
data set relates to peripheral blood taken from the subject prior
to treatment.
[0063] FIGS. 18(a) through 18(e) are bar graphs with a logarithmic
axis that show a graphical representation of calibrated profile
data sets for each of 5 subjects from which a blood sample has been
taken. Each of the blood samples was exposed to the inflammatory
agent phytohemagglutinin (PHA) or to a therapeutic agent
(anti-inflammatory agent) at different concentrations: 0.1 .mu.M,
0.3 .mu.M, 1 .mu.M, 3 .mu.M and 5 .mu.M, for a 4 hour period ex
vivo (in vitro) so as to determine the optimum dose for treating
the subject. A panel of 6 constituents was used corresponding to 6
gene loci. The baseline profile data set was an untreated sample
obtained from the cognate donor.
[0064] FIG. 19 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for three
different subjects having different biological conditions using a
panel with 24 constituents. The profile data sets show variability
according to these conditions providing the basis for a diagnostic
signature panel: (a) shows a calibrated profile data set for a
smoker against a baseline for a non-smoker. (b) shows a calibrated
profile data set for a subject with chronic obstructive pulmonary
disease against a baseline for a subject lacking this disease. The
baseline profile data set is derived from a subject that is
"normal" with respect to these conditions.
[0065] FIG. 20 illustrates that an individual responses can be
distinguished from a similarly treated population. A comparison of
the response of a single animal compared to its experimental cohort
(n=5 animals) with respect to a single locus (GST-P) is provided.
The baseline data set is the cohort average. The figures shows that
this animal varied significantly from the daily, population average
in the first two days of the study, but became more similar to the
cohort average with time after treatment with acetaminophen.
[0066] FIG. 21 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for
samples of blood treated ex vivo with LPS or LPS and one of three
anti-inflammatory herbals (Echinacea, Arnica or Siberian Ginseng)
at a concentration of 200 ug/ml. A panel of 24 constituents is
used. The baseline profile data set is derived from LPS stimulated
cells absent a herbal treatment. The figure illustrates the
effectiveness of the use of the calibrated selected profile to
investigate the overall effects of complex compounds such as
nutraceuticals whose biological effect is a summation of more than
one activity. In this case, each of the herbals is consumed as an
immunostimulant, however the calibrated selected profiles reveal a
unique pattern shows a mixture of both immunostimulatory and
anti-inflammatory effects.
[0067] FIG. 22 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for
samples of blood treated ex vivo with LPS or LPS and
methylprednisolone or LPS and Arnica. The baseline profile data set
is LPS treated blood sample.
[0068] FIG. 23 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for
samples of THP-1 cells treated with LPS or LPS and Arnica at three
different concentrations using a panel of 22 constituents. The
baseline profile data set is untreated THP-1 cells. The figure
illustrates a concentration response with respect to the gene
expression across the calibrated profile.
[0069] FIG. 24 is a bar graph with a logarithmic axis that shows a
graphical representation of calibrated profile data sets for
samples of THP-1 cells treated ex vivo with four different
commercial brands of Echinacea using a panel of 8 constituents. The
baseline profile data set is untreated THP-1 cells.
[0070] FIG. 25 illustrates the use of the calibrated profile to
compare relative efficacy across brands, or different formulations.
Calibrated profile data sets for herbal preparations from different
manufacturing sources with respect to an indicator monocytic cell
line (THP-1) are shown graphically, the baseline profile data set
being THP-1 cells absent the herbal. (a) Three commercial herbal
Echinacea preparations at 250 (.mu.g/ml); (b) three herbal
preparations at different concentrations (250 .mu.g/ml, 50 .mu.g/ml
and 3-10 .mu.g/ml) (c) four commercial Echinacea brands at 250
.mu.g/ml).
[0071] FIGS. 26(a) through 26(d) illustrate calibrated profile data
sets, using a subset of the Inflammation Selected Panel, that show
the effect of administration of a steroid.
[0072] FIGS. 27(a) through 27(d) illustrate calibrated profile data
sets, using a subset of the Inflammation Selected Panel, providing
a comparison of the effects of administration of methylprednisolone
and Ibuprofen.
[0073] FIGS. 28(a) through 28(d) illustrate calibrated profile data
sets, using a subset of the Inflammation Selected Panel, in
identifying chronic obstructive pulmonary disease (COPD)
patients.
[0074] FIGS. 29(a) and 29(b) provide illustrations in which
evaluations of the effects of drug exposure performed in vitro
correspond closely with evaluations performed in vivo, employing in
each case calibrated profile data sets, using a subset of the
Inflammation Selected Panel.
[0075] FIG. 30 illustrates the effect of different agents evaluated
using a subset of the Selected Prostate Panel, and shows broad
functions of constituents of the panel.
[0076] FIG. 31 illustrates the effect of the pharmaceutical agent,
clofibrate, as measured on a rat liver metabolism selected panel.
The profiles for six rats are provided as indicated on the z axis.
The control (baseline) is a set of rats treated only with the
carrier control.
[0077] FIG. 32 illustrates the ability of the rat metabolism
selected panel to differentiate drug responses (clofibrate versus
benzo[a]pyrene) in Spraque-Dawley rats. Clofibrate (right hand
bars) and Benzopyrene (left hand bars). The control (baseline) is a
set of rats treated only with the carrier control. FIG. 33
illustrates the effect of administration of a stimulant
(TNF-.alpha.) as measured by a combination of constitutents
selected from the inflammation, skin/epithelial, and vascular
selected panels. The target is human keratinocytes in culture. The
baseline is non stimulated cells. The baseline is a set of rats
that were non-stimulated. FIG. 34 illustrates the effect of
administration of benzo[a]pyrene on cryo-preserved human
hepatocytes over time as measured by the human liver selected
panel. The control (baseline) are cells treated similarly but
without the addition of benzo[a]pyrene. FIG. 35 demonstrates the
effect of treating human umbilical vein endothelial cells in
culture with TNF.alpha.. for 24 hours. The control or baseline is
established from cells handled similarly but without the addition
of the stimulant.
[0078] FIG. 36 illustrates the protective effect of the antioxidant
n-acetylcysteine (NAC) on human keratinocytes in culture after
exposure the UVB energy. The dark bars indicate the effect of UVB
exposure only. Cells that were treated with NAC followed by
exposure to the same UVB energy show a decreased induction of
expression at most of the gene loci covered by the skin selected
panel. The baseline corresponds to cells exposed to assay media
only.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0079] As used in this description and the accompanying claims, the
following terms shall have the meanings indicated, unless the
context otherwise requires:
[0080] A "collection of cells" is a set of cells, wherein the set
has at least one constituent.
[0081] A "population of cells" includes one or more cells. A
population of cells may refer to cells in vivo or to in vitro
cultures. In vitro cultures may include organ cultures or cell
cultures where cell cultures may be primary or continuous cell
cultures of eukaryotic or prokaryotic cells. Cell lines can be
primary cultures or cell samples, e.g. from a tumor, from blood or
a blood fraction, or biopsy explants from an organ, or can be
established cell lines or microbial strains.
[0082] A "region of the subject" from which proteins are obtained
may (but is not required to be) the same part of the subject from
which has been obtained a collection of cells or a population of
cells. The cells and the proteins may both be obtained from blood
of the subject, for example. Alternatively, for example, the cells
may be obtained from blood and the proteins may be obtained from a
scraping of tissue or vice versa. Similarly, the proteins may be
obtained from urine of the subject, for example, whereas the cells
may obtained elsewhere, as, for example, from blood.
[0083] A "panel" of genes is a set of genes including at least two
constituents.
[0084] A "normative" condition of a subject to whom a composition
is to be administered means the condition of a subject before
administration, even if the subject happens to be suffering from a
disease.
[0085] An "expression" of a gene includes the gene product whether
RNA or protein resulting from translation of the messenger RNA.
[0086] A "large number" of data sets based on a common panel of
genes is a number of data sets sufficiently large to permit a
statistically significant conclusion to be drawn with respect to an
instance of a data set based on the same panel.
[0087] A "biological condition" of a subject is the condition of
the subject in a pertinent realm that is under observation, and
such realm may include any aspect of the subject capable of being
monitored for change in condition, such as health, disease
including cancer; trauma; aging; infection; tissue degeneration;
developmental steps; physical fitness; obesity, or mood. As can be
seen, the conditions may be chronic or acute or simply transient.
Moreover, a targeted biological condition may be manifest
throughout the organism or population of cells or may be restricted
to a specific organ (such as skin, heart, eye or blood). The term
"biological condition" includes a "physiological condition".
[0088] The "blind administration" of a selected one of a
composition or placebo to a subject in a clinical trial involves
administering the composition or placebo to the subject in
accordance with a protocol pursuant to which the subject lacks
knowledge whether the substance administered is the composition or
a placebo.
[0089] An "organism" is any living cell including microorganisms,
animals and plants. An animal is commonly in this context a mammal,
but may be a vertebrate non-mammal, as e.g., a zebra fish, or an
invertebrate, as, e.g. Caenorhabditis elegans.
[0090] An "agent" is a composition or a stimulus. A "stimulus" may
include, for example ultraviolet A or B, or light therapy for
seasonal affective disorder, or treatment of psoriasis with
psoralen or treatment of melanoma with embedded radioactive seeds,
other radiation exposure, etc. A "composition" includes a chemical
compound, a nutraceutical, a combination of compounds, or a complex
mixture.
[0091] A "clinical indicator" is any physiological datum used alone
or in conjunction with other data in evaluating the physiological
condition of a collection of cells or of an organism. This term
includes pre-clinical indicators.
[0092] A "selected panel" is an experimentally verified set of
constituents, each constituent being a distinct expressed product
of a gene, whether RNA or protein, wherein constituents of the set
are selected so that their measurement provides a measurement of a
targeted biological condition A "selected profile" is a set of
values associated with constituents of a selected panel resulting
from evaluation of a biological sample (or population of
samples).
[0093] A "signature profile" is an experimentally verified subset
of a selected profile selected to discriminate a biological
condition, agent or physiological mechanism of action. A "signature
panel" is a subset of a selected panel, the constituents of which
are selected to permit discrimination of a biological condition,
agent or physiological mechanism of action.
[0094] "Distinct RNA or protein constituent" in a panel of
constituents is a distinct expressed product of a gene, whether RNA
or protein.
[0095] An embodiment of the invention includes the formation of
calibrated data sets that describe a biological condition or an
effect of an agent on a biological condition. A calibrated data set
represents a set of values that correspond to variations in gene
expression where the variations are informative. This approach does
not require comprehensive analysis of all gene expression in target
cells associated with a particular condition. Nor is any one single
gene locus necessarily of particular significance. Rather a pattern
of variation (a profile) is sought that correlates, in a
reproducible manner, with a particular condition. There may be no a
priori knowledge of a correlation but rather a correlation may be
established by evaluating a panel of constituents of reasonable
size (for example up to 100 constituents) and iteratively testing
the gene expression profiles for different subjects or for the same
subject from which the most informative loci for a particular
condition may be selected. An informative subgroup of constituents
in a panel may be selected that consistently vary for a particular
condition and this subgroup may then become the signature panel,
the signature panel giving rise to a signature profile.
[0096] In further embodiments of the invention, any calibrated data
set for an individual that has more members than reflective of a
single signature panel may be mined for calibrated profiles that
correspond to additional signature panels, thereby potentially
providing new insights into mechanisms of action of a biological
condition on sets of genes. Measurement of changes in transcribed
RNA in a cell as a result of an environmental change or aging is an
exquisitely sensitive measure of the response of a cell. Techniques
available today to quantify transcribed RNA in a cell add to the
sensitivity of the approach. Embodiments of the invention that are
directed to patterns of change in amounts of transcribed RNA
provide a means to focus and interpret this rich information.
[0097] In contrast to the above approach, much attention in the
prior art has been directed to the sequencing of the human genome
and the identification of all the genes encoded therein.
Accompanying the growing amount of sequence data, microarrays
provide a means to survey many hundreds to thousands of gene
sequences. Microarrays are being used to provide DNA profiles that
identify mutations in an individual and those mutations will be
associated with predictions concerning development of disease in
those individuals.
[0098] Transcriptomics and proteomics are now the focus of
increasing attention. These studies are directed to analyzing the
entire body of RNA and protein produced by living cells.
Microarrays provide a method for analyzing many thousands of
different human RNAs as to whether they are expressed and by which
cells. For example, a project undertaken by the National Cancer
Institute and others to examine mRNAs produced by various types of
cancer cells, have revealed 50,000 genes that are active in one or
more cancers. The goal of these studies is to identify novel cancer
drugs that are directed to knocking out or enhancing the production
of certain proteins. (Kathryn Brown, The Human Genome Business
Today, Scientific American, July 2000, p.50; Julia Karow, The
"Other" Genomes, Scientific American, July 2000, p.53; Ken Howard,
"The Bioinformatics Gold Rush, Scientific American, July 2000,
p.58; Carol Ezzell, Beyond the Human Genome, Scientific American,
July 2000, p.64; all incorporated by reference.) Major efforts in
correlating genetic variation of individuals and the functional
interrelationships of genes in health and disease are being
conducted in a variety of consortia including the single nucleotide
polymorphism consortium and the Human Epigenome Consortium (Beck et
al. Nature BioTechnology 17 (1999) p 1144). The Epigenome
Consortium plans to analyze sets of genome fragments from both
healthy and diseased individuals in the 500 different human tissues
(Bioworld International: Dec. 22, 1999). This approach seeks to
correlate absolute expression of genes associated with a particular
condition with the presence of that condition. Examples of prior
art that seek to measure gene expression in absolute amounts
including by subtractive methods or by determining amounts with
respect to housekeeping genes or by targeting a single gene
expression system are U.S. Pat. No. 5,643,765; U.S. Pat. No.
5,811,231; U.S. Pat. No. 5,846,720; U.S. Pat. No. 5,866,330; U.S.
Pat. No. 5,968,784; U.S. Pat. No. 5,994,076; WO 97/41261; WO
98/24935; WO 99/11822; WO 99/44063; WO 99/46403; WO 99/57130;
WO00/22172 and WO00/11208.
[0099] We have taken a different and novel approach to the above by
identifying reproducible patterns of gene expression that are
informative by virtue of the degree of variation between a sample
and a baseline, for example, in a subject with the condition and a
subject without the condition. The variations may be correlated
with other non-genetic indications such as clinical indicators (for
humans) of a traditional nature but are not required per se to be
causative. Accordingly, the amount of gene expression product (for
example RNA transcript) produced by a gene locus in a cell under
certain circumstances is measured and then stored as a value in a
first profile data set. This value is calibrated with respect to a
second value (a baseline profile data set) to provide a member of a
calibrated profile data set. The values recorded for the profile
data set, relying on a particular baseline data set to produce a
calibrated data set, become part of the descriptive record; any or
all of these results can be stored in a database which may be
accessed through a global network. In this way any new data in the
form of a profile data set or a calibrated profile data set
measured at any global location can be directly compared to an
archive of descriptive records including calibrated profile data
sets and baseline data sets so as to extend the stored library of
profiles and provide predictive, diagnostic, or evaluative data
about a particular biological condition or agent.
[0100] We have exemplified the use of selected panels of
constituents corresponding to gene loci from which quantitative
gene expression is determined by, for example, quantitatively
measuring the transcribed RNA in a sample of a subject, for
applications that include: (a) measurement of therapeutic efficacy
of natural or synthetic compositions or stimuli that may be
formulated individually or in combinations or mixtures for a range
of targeted physiological conditions; (b) predictions of
toxicological effects and dose effectiveness of a composition or
mixture of compositions for an individual or in a population; (c)
determining how two or more different agents administered in a
single treatment might interact so as to detect any of synergistic,
additive, negative, neutral or toxic activity (d) performing
pre-clinical and clinical trials by providing new criteria for
pre-selecting subjects according to informative profile data sets
for revealing disease status and conducting preliminary dosage
studies for these patients prior to conducting phase 1 or 2 trials.
Gene expression profiling may be used to reduce the cost of phase 3
clinical trials and may be used beyond phase 3 trials; (e) labeling
for approved drugs; (f) selection of suitable medication in a class
of medications for a particular patient that is directed to their
unique physiology; (g) diagnosing or determining a prognosis of a
medical condition or an infection which may precede onset of
symptoms or alternatively diagnosing adverse side effects
associated with administration of a therapeutic agent; (h) managing
the health care of a patient; and (i) quality control for different
batches of an agent or a mixture of agents.
[0101] The Subject
[0102] The methods herein can be applied to a subject that includes
any living organism where a living organism includes a prokaryote
such as a bacterium or a eukaryote including single celled
eukaryotic organisms at one end of the spectrum and humans at the
other and everything in between including plants. The figures
relate to calibrated profile data sets obtained from humans and
mammals. Nonetheless, the methods disclosed here may be applied to
cells of other organism without the need for undue experimentation
by one of ordinary skill in the art because all cells transcribe
RNA and it is known in the art how to extract RNA from all types of
cells.
[0103] A tissue sample may include a single cell or multiple cells
or fragments of cells. Body fluid includes blood, urine, spinal
fluid, lymph, mucosal secretions, hemolymph or any other body fluid
known in the art for a subject. For an animal subject, a tissue or
fluid sample may be obtained by means of a biopsy needle aspirate,
a lavage sample, scrapings and surgical incisions or other means
known in the art.
[0104] Selected Panels
[0105] Steps in selecting constituents in a selected panel may
include searching publicly available medical literature for RNA or
proteins or sets of RNAs or proteins that directly or indirectly
vary with a particular biological condition. A selected panel
containing up to 100 constituents may be selected. According to the
condition being examined, just a small subset of the selected panel
constituents may be informative. In determining membership of the
selected panel of genes, it is not necessary for the panel to be an
exhaustive selection. Rather it is desired to obtain from the
selected panel an expression profile that discriminates
consistently with respect to the targeted physiological or
biological condition. Moreover, a selected panel is not necessarily
selected according to an expected profile of gene expression in
cells that directly respond to a biological effect. For example,
gene expression associated with liver metabolism may be analyzed in
a blood sample. FIGS. 20 and 22 provide calibrated profiles of
whole blood treated with herbal agents using markers for liver
metabolism.
[0106] The number of constituents in a selected panel can vary.
According to the examples provided below, selected panels of up to
24-96 genes are selected for evaluating expression levels. Although
a selected panel may be as large as 100 constituents, it is
desirable for a particular selected panel to have no more than 24
constituents, more particularly, less than 12 constituents. For
example, subsets of no more than 8 genes have been used that may be
derived from a larger panel but which are sufficiently informative
to effectuate discrimination. The number of constituents in a
selected panel for which expression is monitored may vary widely
depending on the context. For example, FIG. 1 describes data
acquisition from in vitro cell culture and from animal toxicology
studies, which includes expression of about 25 to 100 or more
genes. In contrast, selection of markers or surrogate markers
include, for example, three to 100 genes, preferably five to 50 or
five to 25 genes to be analyzed from samples obtained in clinical
studies. In this manner markers or surrogate markers having
predictive value for a medical condition, such as a genetic
predisposition, a response to therapeutic agent, an inflammatory
condition, or an infection, etc. can be identified and cumulatively
larger populations can be obtained to refine the correlations. A
health profile can then be generated for an individual subject
using a low volume blood sample. The blood sample can be analyzed
for expression profile data of about 100-500 genes, comprising
markers or surrogate markers of a number of medical conditions
(FIG. 1: right panel). Selected panels of varying sizes may be
utilized as necessary and subsequent refinements in methodology may
lead to selection of subsets having selected panels as large as 15
genes or 12 genes or as small as 6, 5, 4, 3 or 2 genes.
[0107] We have found that we can measure concentrations of
constituents in selected panels in a manner that is both highly
precise and reproducible in samples taken from the same individual
under the same conditions. We have similarly found that such
concentration measurements are reproducible in samples that are
repeatedly tested.
[0108] We commonly run a sample through a panel in quadruplicate;
that is, a sample is divided into aliquots and for each aliquot we
measure concentrations of each constituent in a selected panel.
Over a total of 900 constituent assays, with each assay conducted
in quadruplicate, we found an average coefficient of variation,
(standard deviation/average)*100, of less than 1 percent among
results for each assay. This figure is a measure of what we call
"intra-assay variability". We have also conducted assays on
different occasions using the same sample material. With 72 assays,
resulting from concentration measurements of constituents in a
panel of 24 members, and such concentration measurements determined
on three different occasions over time, we found an average
coefficient of variation of less than 2 percent. We regard this as
a measure of what we call "inter-assay variability".
[0109] We have found it valuable in using the quadruplicate test
results to identify and eliminate data points that are statistical
"outliers"; such data points are those that differ by a percentage
greater, for example, than 3% of the average of all four values and
that do not result from any systematic skew that is greater, for
example, than 1%. Moreover, if more than one data point in a set of
four is excluded by this procedure, then all data for the relevant
constituent is discarded.
[0110] As discussed in further detail below under "Gene
Expression", we have found it valuable to optimize the efficiency
of amplification for all constituents of a panel in a manner to
achieve comparable amplification efficiencies (that is,
amplification efficiencies that are substantially similar as
described below under "Gene Expression") for all constituents, so
that precise quantification of gene expression of all panel
constituents may be determined consistently on successive
occasions. In this manner, there may result data that is useful
because it is precise and reliable.
[0111] What this approach means, among other things, is that by
utilizing a relatively small panel, and by controlling
amplification efficiency and other parameters, we create a panel
that is uniquely informative. This approach differs from prior art
endeavors where specificity is optimized only on a per-constituent
basis and reaction conditions are not optimized for the panel as a
whole.
[0112] It is envisaged that any single biological condition may be
described by a signature panel having a small number of highly
informative constituents providing a signature calibrated profile
(also referred to as a fingerprint). The presence of highly
informative loci is demonstrated in several of the accompanying
figures. For example, FIG. 11(a) Il-2, Il-4 and Il-5 are highly
informative. Highly informative constituents in FIG. 21 include the
pro-inflammatory-interleukins. The signature panel may provide a
signature profile or fingerprint which is sufficiently robust to
serve as a standard in describing a particular biological condition
or an effect of a particular agent on a biological condition
[0113] For purposes of illustrating a signature panel, constituents
of a selected panel for measuring inflammation have been provided
that are informative with respect to a particular biological
condition. For example, we have used a selected panel for
inflammation that has 6 constituents-Il-1.alpha., Il-6, Il-8,
Il-18, GMCSF and IFN-.gamma. in FIG. 18(a)-(e) to determine the
response of 5 subjects to varying concentrations of drugs. This
group of constituents is a subset of a larger selected panel of
inflammation related gene loci such as shown in FIG. 19a and FIG.
19b where the Inflammation Selected Panel includes Il-.alpha.,
Il-.beta., Il-2, Il-3, Il-4, Il-6, Il-7, Il-8, Il-10, Il-12p40,
Il-15, Il-15, Il-18, GM-CSF, Ifn-gamma, TGF-.beta., cox-2, ICE,
MMP-9, ICAM, TNF-.alpha. and TNF-.beta.. The subset of constituents
were selected on the basis of the information sought concerning the
biological condition.
[0114] Embodiments of the invention provide examples of numerous
different selected panels which may be used separately or together.
These selected panels include an Inflammation Selected Panel (Table
1) a Cell Growth and Differentiation Selected Panel (Table 2), a
Liver Metabolism and Toxicity Selected Panel (Table 3). We have
developed additional selected panels including Skin Response
Selected Panel (Table 4), Prostate Selected Panel (Table 5)(for
measuring prostate health and disease), Vascular Selected Panel
(Table 6)(for measuring condition of the vascular system and
endothelial cells). It is a significant property of each of these
selected panels that measurement of the selected panel's
constituents provides a measurement of the physiological condition
to which the selected panel is targeted. Selected panels may also
provide useful information concerning gene response outside the
target condition. In these tables the left-hand column identifies
the particular gene loci, and the right-hand column describes
proteins expressed by these loci. However, as described in detail
below, embodiments of the present invention may utilize, for
example, mRNA or protein expression products as constituents. While
below we provide examples based primarily on the Inflammation
Selected Panel and subsets of it, the approaches set forth herein
are equally applicable to the other selected panels described
above. Although provided as examples, the above selected panels are
not intended to be limiting.
[0115] Gene Expression
[0116] For measuring the amount of a particular RNA in a sample, we
have used methods known to one of ordinary skill in the art to
extract and quantify transcribed RNA from a sample with respect to
a constituent of a selected panel (See detailed protocols below.)
Briefly, RNA is extracted from a sample such as a tissue, body
fluid (see Example 11 below), or culture medium in which a
population of a subject might be growing. For example, cells may be
lysed and RNA eluted in a suitable solution in which to conduct a
DNAse reaction. First strand synthesis (see Example 10 below) may
then be performed using a reverse transcriptase. Gene
amplification, more specifically quantitative PCR assays, can then
be conducted and the gene of interest size calibrated against a
marker such as 18S rRNA (Hirayama et al., Blood 92, 1998: 46-52).
Samples are measured in multiple duplicates, for example, 4
replicates. Relative quantitation of the mRNA is determined by the
difference in threshhold cycles between the internal control and
the gene of interest (see Example 12 below). In an embodiment of
the invention, quantitative PCR is performed using amplification,
reporting agents and instruments such as those supplied
commercially by Applied Biosystems (Foster City, Calif.). Given a
defined efficiency of amplification of target transcripts, the
point (e.g., cycle number) that signal from amplified target
template is detectable may be directly related to the amount of
specific message transcript in the measured sample. Similarly,
other quantifiable signals such as fluorescence, enzyme activity,
disintegrations per minute, absorbance, etc., when correlated to a
known concentration of target templates (e.g., a reference standard
curve) or normalized to a standard with limited variability can be
used to quantify the number of target templates in an unknown
sample.
[0117] Although not limited to amplification methods, quantitative
gene expression techniques may utilize amplification of the target
transcript. Alternatively or in combination with amplification of
the target transcript, amplification of the reporter signal may
also be used. Amplification of the target template may be
accomplished by isothermic gene amplification strategies, or by
gene amplification by thermal cycling such as PCR.
[0118] It is desirable to obtain a definable and reproducible
correlation between the amplified target or reporter and the
concentration of starting templates. We have discovered that this
objective can be achieved by careful attention to, for example,
consistent primer-template ratios and a strict adherence to a
narrow permissible level of amplification efficiencies (for example
99.8 to 100% relative efficiency). For example, in determining gene
expression levels with regard to a single selected profile, it is
necessary that all constituents of the panels maintain a similar
and limited range of primer template ratios (for example, within a
10-fold range) and amplification efficiencies (within, for example,
less than 1%) to permit accurate and precise relative measurements
for each constituent. We regard amplification efficiencies as being
"substantially similar", for the pursposes of this description and
the following claims, if they differ by no more than approximately
10%. Preferably they should differ by less than approximately 2%
and more preferably by less than approximately 1%. These
constraints should be observed over the entire range of
concentration levels to be measured associated with the relevant
biological condition. In practice, we run tests to assure that
these conditions are satisfied. For example, we typically design
and manufacture a number primer-probe sets, and determine
experimentally which set gives the best performance. Even though
primer-probe design and manufacture can be enhanced using computer
techniques known in the art, and notwithstanding common practice,
we still find that experimental validation is useful. Moreover, in
the course of experimental validation, we associate with the
selected primer-probe combination a set of features:
[0119] (i) The reverse primer should be complementary to the coding
DNA strand; located across an intron-exon junction, with not more
than three bases of the three-prime end of the reverse primer
complementary to the proximal exon. (If more than three bases are
complementary, then it would tend to competitively amplify genomic
DNA.)
[0120] (ii) The primer probe should amplify cDNA of less than 110
bases in length.
[0121] (iii) The primer probe should not amplify genomic DNA or
transcripts or cDNA from related but biologically irrelevant
loci.
[0122] A suitable target of the selected primer probe is first
strand cDNA, which may be prepared, in one embodiment, according to
Example 1 below. In Example 11 below, we illustrate use of the
primer probe with the first strand cDNA of Example 1 to permit
measurement of constituents of a selected panel.
[0123] It is envisaged that techniques in the art using
microfluidics for example and highly sensitive markers will enable
quantitation of RNA to occur directly from a single cell or lysed
cell. This may rely on amplification of a marker but may not
require amplification of the transcripts themselves. The amount of
transcript measured for any particular locus is a data point or
member of the first profile data set for a particular selected
panel.
[0124] According to embodiments of the invention, a first profile
data set is derived from the sample, the first profile data set
including a plurality of members, each member being a quantitative
measure of the amount of a RNA transcribed from a gene locus, the
gene locus being a constituent in a panel of constituents. A first
profile data set may be obtained from a quantitative measure of the
amount of a distinct RNA or protein corresponding to a gene locus.
The figures provided here are directed to RNA. However, methods
herein may also be applied using proteins where sensitive
quantitative techniques, such as an Enzyme Linked ImmunoSorbent
Assay (ELISA), are available and well-known in the art for
measuring the amount of a protein constituent.
[0125] Baseline Profile Data Sets
[0126] The analyses of samples from single individuals and from
large groups of individuals provide a library of profile data sets
relating to a particular panel or series of panels. These profile
data sets may be stored as records in a library for use as baseline
profile data sets. As the term "baseline" suggests, the stored
baseline profile data sets serve as comparators for providing a
calibrated profile data set that is informative about a biological
condition or agent. Baseline profile data sets may be stored in
libraries and classified in a number of cross-referential ways. One
form of classification may rely on the characteristics of the
panels from which the data sets are derived. Another form of
classification may be by particular biological condition. The
concept of biological condition encompasses any state in which a
cell or population of cells may be found at any one time. This
state may reflect geography of samples, sex of subjects or any
other discriminator. Some of the discriminators may overlap. The
libraries may also be accessed for records associated with a single
subject or particular clinical trial. The classification of
baseline profile data sets may further be annotated with medical
information about a particular subject, a medical condition, a
particular agent etc.
[0127] The choice of a baseline profile data set for creating a
calibrated profile data set is related to the biological condition
to be evaluated, monitored, or predicted, as well as, the intended
use of the calibrated panel, e.g., as to monitor drug development,
quality control or other uses. It may be desirable to access
baseline profile data sets from the same subject for whom a first
profile data set is obtained or from different subject at varying
times, exposures to stimuli, drugs or complex compounds; or may be
derived from like or dissimilar populations.
[0128] The profile data set may arise from the same subject for
which the first data set is obtained, where the sample is taken at
a separate or similar time, a different or similar site or in a
different or similar physiological condition. For example, FIG. 5
provides a protocol in which the sample is taken before stimulation
or after stimulation. The profile data set obtained from the
unstimulated sample may serve as a baseline profile data set for
the sample taken after stimulation. The baseline data set may also
be derived from a library containing profile data sets of a
population of subjects having some defining characteristic or
biological condition. The baseline profile data set may also
correspond to some ex vivo or in vitro properties associated with
an in vitro cell culture. The resultant calibrated profile data
sets may then be stored as a record in a database or library (FIG.
6) along with or separate from the baseline profile data base and
optionally the first profile data set although the first profile
data set would normally become incorporated into a baseline profile
data set under suitable classification criteria.
[0129] Selected baseline profile data sets may be also be used as a
standard by which to judge manufacturing lots in terms of efficacy,
toxicity, etc. Where the effect of a therapeutic agent is being
measured, the baseline data set may correspond to gene expression
profiles taken before administration of the agent. Where quality
control for a newly manufactured product is being determined, the
baseline data set may correspond with a gold standard for that
product. However, any suitable normalization techniques may be
employed. For example, an average baseline profile data set is
obtained from authentic material of a naturally grown herbal
nutraceutical and compared over time and over different lots in
order to demonstrate consistency, or lack of consistency, in lots
of compounds prepared for release.
[0130] Calibrated Data
[0131] A calibrated profile data set may be described as a function
of a member of a first profile data set and a corresponding member
of a baseline profile data set for a given gene locus in a panel.
For example, calibrated profile data sets may be derived by
calculating a ratio of the amount of RNA transcribed for a panel
constituent in a cell sample in an environmental including
intervention such as a therapeutic treatment or at a particular
time (first profile data set) with respect to the amount of RNA
transcribed for the same panel constituent in a cell that differs
in some manner from the sample (baseline profile data set) (FIGS. 5
and 6). Given the precision we have achieved in measurement of gene
expression, described above in connection with "selected panels"
and "gene amplification", we conclude that where differences occur
in measurement under such conditions, the differences are
attributable to differences in biological condition. Thus we have
found that calibrated profile data sets are highly reproducible in
samples taken from the same individual under the same conditions.
We have similarly found that calibrated profile data sets are
reproducible in samples that are repeatedly tested. We have also
found repeated instances wherein calibrated profile data sets
obtained when samples from a subject are exposed ex vivo to a
compound are comparable to calibrated profile data from a sample
that has been exposed to a sample in vivo (FIG. 14, FIG. 16(a),
(b), and FIGS. 29(a) and 29(b)). We have also found, importantly,
that an indicator cell line treated with an agent can in many cases
provide calibrated profile data sets comparable to those obtained
from in vivo or ex vivo populations of cells (FIG. 15). Moreover,
we have found that administering a sample from a subject onto
indicator cells can provide informative calibrated profile data
sets with respect to the biological condition of the subject
including the health, disease states, therapeutic interventions,
aging or exposure to environmental stimuli or toxins of the subject
(FIG. 25).
[0132] A use of a calibrated profile data set is to evaluate a
biological condition of a subject. This may be for purposes of
diagnosis or prognosis of a clinical disorder. It is desirable to
obtain a calibrated data set that describes a state of health or
alternatively a state of age or body mass or any condition or state
that an individual subject might find themselves to be in. For
example, the biological condition may relate to physical activity,
conditioning or exercise, mental state, environmental factor such
as medication, diet, or geography or exposure to radiation or
environmental contamination or infectious agent, biological or
environmental toxin. If health or conversely a clinical disorder is
being evaluated, calibrated profiles data sets may be used for
monitoring change in health status by periodic or regular
comparison of profiles; the disorder may be a complex disease
process possibly involving multiple gene including inflammation,
autoimmune disease, degenerative disease, allergy, vascular
disease, ischemia, developmental disease, hormonal conditions and
infectious diseases. The clinical disorder may further include
arthritis, asthma, multiple sclerosis and perimenopausal changes.
The biological condition may affect a system of a subject including
a respiratory, vascular, nervous, metabolic, urinary, reproductive,
structural and immunological system or other metabolic state. The
above examples of a biological condition are given by way of
illustration and are not intended to be limiting.
[0133] Similarly, calibrated profile data sets may be used to
measure, monitor or predict the host response to an infectious
agent for purposes of identifying the infectious agent, assessing
the duration of infection, the extent of exposure or making
therapeutic decisions.
[0134] The evaluation of activity of an agent may require a series
of calibrated profiles. It is here shown that calibrated profile
data sets may be used to describe the biological activity of an
agent that may be a single compound or a complex compound such as a
nutraceutical or herbal. The agent may be assayed using indicator
cells, ex vivo cell populations or by in vivo administration. These
assays may rely on a series of signature panels or enlarged panels
for different biological conditions. The resultant calibrated
profiles may then be used to infer likely in vivo activity from the
in vitro study. Insights into toxicity and mechanisms of action can
also be inferred from calibration profile data sets. For example,
the herbal Echinacea is believed to have both immunostimulatory and
anti-inflammatory properties although neither has been measured
systematically. We have provided a systematic approach to
investigate the biological activities of these and other herbs. We
investigated the alleged immunostimulatory properties of the herbs
by comparing the effect of treating the indicator cell line THP-1
or peripheral blood cells with the agent to untreated cells.
Untreated cells include LPS stimulated untreated cells. Untreated
cells were used as a baseline profile data set to measure the
difference in gene expression between a baseline profile data set
and the experimental treatment with the compound. Baseline profile
data sets included a single sample or an average value from a
series of experiments. The resultant calibrated profile data sets
could then be compared with a library of calibrated profile data
sets for a particular herb or/and libraries associated with
different agents or conditions.
[0135] From the information obtained about a previously undescribed
agent, a signature panel may be derived optionally together with a
signature profile to serve as a gold standard for testing other
batches of the same agent.
[0136] Calculation of Calibrated Profile Data Sets and
Computational Aids
[0137] The function relating the baseline and profile data sets is,
in an embodiment of the invention, a ratio expressed as a
logarithm. The calibrated profile data set may be expressed in a
spreadsheet or represented graphically for example, in a bar chart
or tabular form but may also be expressed in a three dimensional
representation. The constituent may be itemized on the x-axis and
the logarithmic scale may be on the y-axis. Members of a calibrated
data set may be expressed as a positive value representing a
relative enhancement of gene expression or as a negative value
representing a relative reduction in gene expression with respect
to the baseline.
[0138] Each member of the calibrated profile data set should be
reproducible within a range with respect to similar samples taken
from the subject under similar conditions. For example, the
calibrated profile data sets may be reproducible within one order
of magnitude with respect to similar samples taken from the subject
under similar conditions. More particularly, the members may be
reproducible within 50%, more particularly reproducible within 20%,
and sometimes even 10%. In accordance with embodiments of the
invention, a pattern of increasing, decreasing and no change in
relative gene expression from each of a plurality of gene loci
examined in the precision panel may be used to prepare a calibrated
profile set that is informative with regards to a biological
condition, biological efficacy of an agent treatment conditions or
for comparison to populations. Patterns of this nature may be used
to identify likely candidates for a drug trial, used in combination
with other clinical indicators to be diagnostic or prognostic with
respect to a biological condition or may be used to guide the
development of a pharmaceutical or nutraceutical through
manufacture, testing and marketing.
[0139] The numerical data obtained from quantitative gene
expression and numerical data from calibrated gene expression
relative to a baseline profile data set may be stored in databases
or digital storage mediums and may retrieved for purposes including
managing patient health care or for conducting clinical trials or
for characterizing a drug. The data may be transferred in networks
via the World Wide Web, email, or internet access site for example
or by hard copy so as to be collected and pooled from distant
geographic sites (FIG. 8).
[0140] In an embodiment of the present invention, a descriptive
record is stored in a single database or multiple databases where
the stored data includes the raw gene expression data (first
profile data set) prior to transformation by use of a baseline
profile data set, as well as a record of the baseline profile data
set used to generate the calibrated profile data set including for
example, annotations regarding whether the baseline profile data
set is derived from a particular signature panel and any other
annotation that facilitates interpretation and use of the data.
[0141] Because the data is in a universal format, data handling may
readily be done with a computer. The data is organized so as to
provide an output optionally corresponding to a graphical
representation of a calibrated data set.
[0142] For example, a distinct sample derived from a subject being
at least one of RNA or protein may be denoted as P.sub.1. The first
profile data set consists of M.sub.j where Mj is a quantitative
measure of a distinct RNA or protein constituent. The record Ri is
a ratio of M and P and may be annotated with additional data on the
subject relating to for example, age, diet, ethnicity, gender,
geographic location, medical disorder, mental disorder, medication,
physical activity, body mass and environmental exposure. Moreover,
data handling may further include accessing data from a second
condition database which may contain additional medical data not
presently held with the calibrated profile data sets. In this
context, data access may be via a computer network.
[0143] The above described data storage on a computer may provide
the information in a form that can be accessed by a user.
Accordingly, the user may load the information onto a second access
site including downloading the information. However, access may be
restricted to users having a password or other security device so
as to protect the medical records contained within. A feature of
this embodiment of the invention is the ability of a user to add
new or annotated records to the data set so the records become part
of the biological information.
[0144] The graphical representation of calibrated profile data sets
pertaining to a product such as a drug provides an opportunity for
standardizing a product by means of the calibrated profile, more
particularly a signature profile. The profile may be used as a
feature with which to demonstrate relative efficacy, differences in
mechanisms of actions, etc. compared to other drugs approved for
similar or different uses.
[0145] The various embodiments of the invention may be also
implemented as a computer program product for use with a computer
system. The product may include program code for deriving a first
profile data set and for producing calibrated profiles. Such
implementation may include a series of computer instructions fixed
either on a tangible medium, such as a computer readable medium
(for example, a diskette, CD-ROM, ROM, or fixed disk), or
transmittable to a computer system via a modem or other interface
device, such as a communications adapter coupled to a network. The
network coupling may be for example, over optical or wired
communications lines or via wireless techniques (for example,
microwave, infrared or other transmission techniques) or some
combination of these. The series of computer instructions
preferably embodies all or part of the functionality previously
described herein with respect to the system. Those skilled in the
art should appreciate that such computer instructions can be
written in a number of programming languages for use with many
computer architectures or operating systems. Furthermore, such
instructions may be stored in any memory device, such as
semiconductor, magnetic, optical or other memory devices, and may
be transmitted using any communications technology, such as
optical, infrared, microwave, or other transmission technologies.
It is expected that such a computer program product may be
distributed as a removable medium with accompanying printed or
electronic documentation (for example, shrink wrapped software),
preloaded with a computer system (for example, on system ROM or
fixed disk), or distributed from a server or electronic bulletin
board over a network (for example, the Internet or World Wide Web).
In addition, a computer system is further provided including
derivative modules for deriving a first data set and a calibration
profile data set.
[0146] Clinical Trials
[0147] The use of calibrated profile data sets for performing
clinical trials is illustrated in FIG. 10 using the above-described
methods and procedures for running a clinical trial or managing
patient care. Moreover, standardization between laboratories may be
achieved by using a particular indicator cell line such as THP-1
which is stimulated by a known stimulator such as
lipopolysaccharide so that resultant profile acts as a measure that
the laboratory is performing the protocol correctly. Of course this
is one single example, and other cells lines, tissues, or
biological samples or combinations of the foregoing may be used as
standards.
[0148] A further embodiment of the invention provides a method for
patient selection for augmenting clinical trials. Clinical trials
in which candidate subjects are included or excluded according to a
predetermined optimum calibrated profile for a given biological
condition can result in more precise monitoring than would be
otherwise possible. It can also result in a greater efficiency in
clinical trial design because unsuitable patients that have for
example complicating factors or conditions can be screened out. The
calibrated profile data will also enhance the "signal to noise" by
removing non-responders from clinical studies. The basic structure
of a clinical trial design using gene expression profiling may
follow any of several formats. These include testing body fluid
from a candidate patient in the trial ex vivo against a new
therapeutic agent and analyzing the calibrated profiles with
respect to an agent-treated and placebo-treated samples using a
predetermined selected panel and evaluating whether the candidate
patient would be likely to respond without adverse effects to the
composition being tested. In selected indications, profile data
obtained from in vitro cell cultures or organ cultures may be
desired where the cell originates from a target subject or from
another subject or from an established cell line, or from a cell
samples removed from the target subject where the cell samples may
be obtained from any body fluid including a blood, urine, semen,
amniotic, or a cerebrospinal fluid sample, or from a scraping from
mucosal membranes such as from the buccal cavity, the eye, nose,
vagina or by means of a biopsy including epithelial, liver, sternum
marrow, testicular, or from tumor tissue removed surgically from a
tumor at any location. The above-described sources of samples are
applicable to any medical use in which calibrated profile data sets
are desired.
[0149] In vitro dosage and toxicity studies using calibrated
profile data sets obtained from indicator cell lines or samples of
the patient tested ex vivo may provide useful information prior to
initiation of the clinical trial and may significantly reduce the
cost and time of a clinical trial while increasing the likelihood
of identifying the presence of beneficial effect(s). In particular,
the dose may be optimized on an individualized basis to maximize
the impact on therapeutic outcome. For example, FIG. 12 shows how
ex vivo blood cells respond to the stimulatory effect of LPS and
the subsequent treatment with an anti-inflammatory drug
(methotrexate, meclofenamate or methylprednisolone). The data show
how the effect of methotrexate and meclofenamate generates similar
calibrated profile data sets where the baseline is LPS treated
blood. In contrast, methylprednisolone has a substantially
different effect from the other two compounds. A similar type of
analysis can be performed with complex mixtures, as illustrated in
FIG. 21, in which the calibrated profiles obtained when Echinacea,
Arnica and Siberian Ginseng applied to LPS stimulated blood ex vivo
are compared. In this example, all three agents appear to act
differently from each other with respect to a sample from a single
subject. Similar analyses can be used to compare compounds with
unknown targets or activities or metabolic patterns to compounds,
complex or simple, with known or pre-determined profiles.
[0150] The above methods and procedures may be utilized in the
design and running of clinical trials or as a supplemental tool.
Moreover, the above methods and procedures may be used to monitor
the patients' health as well as the patient's responsiveness to an
agent before during and after the clinical trial. This includes
monitoring whether multiple agents interfere with each other, act
synergistically or additively or are toxic or neural with respect
to each other. This type of information is very important as
individuals take an increasing number of medications.
[0151] Similarly, the methods and procedures described above may be
used to manage patient care for an individual or a population. Such
methods and procedures may also be used to develop a regional or
global research network that uses calibrated profile data sets and
the resulting databases to conduct research or trials.
[0152] Both the calibration profile data sets in graphical form and
the associated databases together with information extracted from
both are commodities that can be sold together or separately for a
variety of purposes. For example, graphic representations of
calibration profile data sets may provide a description of a
product with respect to its activity that may be used to promote
the product. Alternatively, the graphical form of the calibrated
profile data sets and access to baseline profile databases provide
a means for manufacturers to test discrete batches of product
against a gold standard.
[0153] The data may be used strategically for design of clinical
trials. It may also be useful for physicians practicing at remote
sites to offer personalized healthcare to a patient. Accordingly,
the physician may set up personalized databases for calibrated
profile data sets prior to and after treatment of a particular
condition. New data on the subject could be added to the
personalized database at each visit to the doctor. The data may be
generated at remote sites by the use of kits that permit a
physician to obtain a first profile data set on a sample from a
patient. For remote users to access the site, it is envisaged that
secured access to the global network containing libraries of
baseline profile data sets and calibrated profile data sets,
classified by particular criteria and representing data from larger
populations than a single individual, would be necessary. The
access to the global database may be password protected thereby
protecting the database from corrupted records and safeguarding
personal medical data. The graphical form provided by the
calibrated data sets may be used to create catalogs of compounds in
a pharmacopiae complete with toxic effects that might arise for
particular individuals as well as other types of drug
interactions.
[0154] Access to the global database may include the option to load
selected data onto a second access site. This process may include
downloading the information to whatever site is desired by the user
and could include securing hard copies of information. It is
desirable to control how and what data is offloaded or copied to
maintain the integrity of the database. It is envisaged that while
a global network of clinical data would be an informational
resource, it would have utility in conducting research that may
include epidemiological studies and studies concerning the
mechanism of action of an agent, as well as studies concerning the
nature of interpersonal variability as determined by calibrated
profile data sets.
[0155] Examples of Medical Uses
[0156] (a) Early detection of infectious diseases: Markers or
surrogate markers from mice may be obtained for measuring gene
expression in humans that indicate early or immediate response to
infection, for example, to a virus such as hepatitis virus, or to a
bacterium such as Mycobacterium tuberculosis (the etiologic agent
of tuberculosis) (see FIG. 4). Candidate genes are identified and
changes in expression of those genes in the presence of a challenge
provide a set of markers. The set of markers can combine markers
encoded by the genome of the subject and one more distinctive
markers encoded by the genome of the infectious agent. For example,
changes in expression of an immediate early gene of a virus, e.g. a
gene encoding an enzyme of viral replication, and a host gene such
as the gene for any or all of IL-2, IL-4 and IL-5, may comprise
markers or surrogate markers for a medical condition capable of
detecting that condition prior to the onset of medical symptoms.
This method may afford earlier detection of an infection than is
possible using current diagnostic techniques.
[0157] (b) Toxicity profiles and mechanistic profiles obtained from
an in vitro assay and in vivo assays. Toxicity and mechanistic
information arising from the administration of a compound to a
population of cells may be monitored using calibrated profile data
sets. The following is an example of an experimental protocol for
obtaining this information. Firstly; an experimental group is
established: (1) control cells maintained without therapeutic agent
and without stimulus; (2) cells treated with therapeutic agent but
without stimulus; (3) cells without therapeutic agent but with
stimulus, (4) sample with therapeutic agent and with stimulus. The
population of cells can be selected from primary cell cultures
prepared in culture plates using methods well established in the
art; or mature differentiated cell preparation from whole blood or
isolated monocytes from the target organism.
[0158] The cells are stimulated so as to present a targeted
physiological condition by pretreatment with LPS purified from a
Gram-negative bacterium (a variety of LPS preparations from
pathogenic bacteria, for example, from Salmonella typhimurium and
from Escherichia coli O1157:H7, are available from Sigma, St.
Louis, Mo.). The therapeutic agent administered to the cell samples
in this example is an inhibitor of an enzyme known to be key in
disease etiology, namely an inhibitor of a protease or a nucleic
acid polymerase. Following treatment by addition of the therapeutic
agent and further incubation for four to six hours, samples of the
cells are harvested and analyzed for gene expression. Nucleic acid,
specifically mRNA, can be prepared from the sample by methods known
to one or ordinary skill in the art (see, for example, the
Lyse-N-Go.TM. reagent, Pierce Chem. Co., Rockford, Ill.). Samples
are analyzed by QPCR according to a quantitative replicative
procedure, (for example, quantitative polymerase chain reaction
procedure (QPCR)) (see, for example, Gibson, U. 1996 Genome Res.
6:995-1001, and references cited therein). Total RNA was assessed
using universal primers. Toxicity of the agent for cells can be
measured in untreated cells by vital stain uptake, rate of DNA
synthesis (autoradiography of labeled nucleic compared to cells
stained), stain by DNA-specific eyes (Hoechst), etc. Mechanistic
profiles can be determined by analysis of the identifies of de novo
up- or down-regulated genes. Further, in the presence of a
therapeutic agent, some genes are not expressed or differentially
expressed, indicating potential efficacy of the therapeutic agent
in suppressing the effects of stimulation by the LPS. For example,
in FIG. 21, levels of ICE that are somewhat stimulated in the
presence of LPS+Echinacea are substantially depressed by LPS+Arnica
relative to LPS stimulated cells absent agent. Levels of HSP 70
which are depressed in the presence of LPS+Echinacea are
substantially stimulated in the presence of LPS+Arnica, and
LPS+Siberian Ginseng relative to LPS stimulated cells absent the
addition of an agent. Levels of IL-12p40 which are slightly
increased in the presence of LPS+Echinacea are substantially
depressed in the presence of LPS+Arnica and LPS+Siberian Ginseng
relative to LPS stimulation. Similarly, FIG. 16 shows a much
enhanced reduction of gene expression in whole blood for
IL-1.alpha., Il-1.beta., Il-7, Il-10, IL-IL-15, IFN-.gamma.,
TGF-.beta., TNF-.beta. cox-2, and ICAM in the presence of
prednisolone +LPS when compared to arnica +LPS or nothing +LPS.
[0159] (c) Quantitation of gene expression in a blood cell to
predict toxicity in another tissue or organ.
[0160] Leukocytes, for example, may be obtained from a blood sample
of a subject, for the purpose of assessing the appearance of a
pathological condition in another organ, for example, the liver. A
profile data set is obtained of genes expressed in the leukocytes,
for example, genes encoding a set of lymphokines and cytokines. The
data set is compared to that of the database, to examine
correlations, for example to other subjects, and to the subject
prior to administration of a therapeutic agent.
[0161] By this method, a correlation can be drawn between, for
example, administration of acetaminophen (Tylenol) and sensitivity
to this therapeutic agent and manifested by liver damage. An early
prediction of therapeutic agent sensitivity, detected prior to the
onset of actual damage to the liver, may be clinically available so
that the subject receives no further administration of
acetaminophen. The database may be used to detect a correlation or
correlations prior to the onset of traditional medical assessments,
such as increase in bilirubin level or other indication of liver
pathology.
[0162] (d) Calibrated profiles from blood cells for prognosis of
severity and prediction of adverse reactions in treatment of an
autoimmune disease.
[0163] The probability and timing of onset of symptoms of an
autoimmune disease, for example, rheumatoid arthritis, may be
monitored by appearance of expression of markers or surrogate
markers as determined by the methods of gene expression profiling
of markers or surrogate markers and comparison to a profile
database as described above. Thus an indication of onset may be
obtained, and advance management by utilization of preventive
measures to forestall onset, can be taken. Further, the user may
choose a set of potential therapeutic agents, and assess for a
given agent, the probability that a subject will present an adverse
reaction if given a full course of treatment, prior to that full
course. For example, using embodiments of the invention, a single
dose or a few doses of the agent methotrexate may be administered
to a subject having arthritis and in need of a therapeutic agent.
If the gene expression profile data set of the subject in response
to the short course of methotrexate correlates with data sets from
subjects having adverse reactions to this agent, then
administration of a full course of methotrexate is
counterindicated. Conversely, if the gene expression profile data
set correlates with those of subjects who have responded positively
to administration of a course of methotrexate treatment, then this
therapeutic agent can be administered to the subject with much
lower probability of adverse reaction.
[0164] Discussion of Figures
[0165] FIGS. 1-4 illustrate some of the applications of calibrated
profile data sets. In FIG. 1, three possible scenarios are
provided. Firstly, a candidate therapeutic agent may be tested to
determine its molecular pharmacology and toxicology profiles. The
test might include obtaining calibrated profile data sets for a
series of selected panels selected on the basis of what activity is
predicted for the drug. The population of cells exposed to the
agent may be the result of in vivo administration as depicted by
the mouse or direct exposure in vitro where the cells may be an
indicator cell line or an ex vivo sample from the subject. The
result of the screen is the identification of more effective drug
candidates for testing in human subjects.
[0166] The second scenario in FIG. 1 is the use of calibrated
profile data sets to identify a suitable clinical population for
screening a potential therapeutic agent. Both demonstration of lack
of toxicity and demonstration of clinical efficacy require certain
assumptions about the clinical population. The calibrated profile
data sets provide a means for establishing those assumptions with
respect to the biological condition of the individuals selected for
the clinical trials.
[0167] The third scenario in FIG. 1 involves the practice of
individualized medicine, which may include creating an archive of
calibrate profile data sets on the individual in a state of health
such that changes can be identified using signature panels so as to
permit evaluation, prognosis, or diagnosis of a particular
condition. Moreover, stored information about the patient in the
form of calibrated profile data sets permits selecting one of a
group of possible therapeutic agents most likely to be effective
for the patient, optimizing dosage of drug, and detecting adverse
effects that might arise through drug-drug interactions before
symptoms arise. Use of calibrated profile data sets may provide
more efficient and cost-effective health care management.
[0168] The novel approach described above for evaluating a
biological condition of a subject may be applied to an ex vivo or
in vitro assay for measuring the effect of an agent on a biological
condition as illustrated in FIGS. 2-4. A sample from the patient
may measured directly ex vivo or tested ex vivo against an agent to
predict an effect in the patient. This provides a quick and
effective way to determine which drug, chosen from within a single
class of drugs that all may be used to treat a particular
condition, may be most effective for a given subject.
Alternatively, an agent may be tested on an indicator cell line
that can provide a quantitative measure of therapeutic performance
in a class of individuals.
[0169] FIG. 2 illustrates how calibrated profile data sets may
assist in screening a library of candidate compounds to discover
candidate drugs. Starting with for example, 500 candidate drugs,
these can be tested in indicator cells or ex vivo body fluid or
tissues against signature panels for in vitro toxicology or
metabolic indicators. The figure illustrates the large number of
compounds that entered in late stages in the development process
only to ultimately be rejected due to adverse biological
interactions. Use of calibrated profile data sets may in many
instances more readily identify likely successful candidates and
thereby reduce the expense and untoward effects of animal and human
experimentation for compounds that could have been predicted to
fail.
[0170] FIG. 3 illustrates how a compound may be administered to an
experimental animal such as a mouse or to an indicator cell line.
The in vivo or ex vivo or indicator cell sample may further be
treated with a stimulus. The result of both the compound and the
stimulus may then be detected, for example, using signature
profiles for toxicity or for mechanism to compare the effect of no
drug +/-stimulus or +/-drug and no stimulus. Both in vitro (left
panel of FIG. 3) and in vivo (right panel of FIG. 3) studies can be
used to evaluate the effect of a compound (drug, nutraceutical,
environmental stimuli, etc.). The right hand panel also illustrates
the specific embodiment of an "in vitro clinical trial", that is,
treatment of cells obtained from a subject and treated with a
compound (with or without a stimulus) in vitro (or ex vivo) in
order to predict the outcome of similar treatment of the subject in
vivo (see FIG. 15 for a specific example). The output from both
panels is described as toxicity and mechanistic profiles. Either
experimental course may be used to both evaluate potential
toxicity, e.g., using the toxicity, or liver metabolism selected
panels, and to determine or confirm likely mechanism of action by a
critical selection of a gene panel(s) that illustrates and
differentiates molecular mechanisms of action (see FIG. 12 for a
specific example). These are merely examples, and other selected
panels may be employed to evaluate or characterize other biological
effects or conditions. FIG. 4 illustrates a bioassay in which cells
are removed from the subject and tested ex vivo with the addition
of a compound and also a challenge or stimulus. The ex vivo effect
of stimulus and then drug on whole blood taken from a human subject
is shown in FIG. 12 in which the stimulus is lipopolysaccharide (an
inflammatory agent) while the drug is any of methotrexate,
meclofenamate or methylprednisolone using a signature panel for
inflammation. Methylprednisolone, a drug commonly used in the
treatment of acute exacerbations of COPD as well as in the chronic
management of this disease, is considered to be a potent by
non-specific anti-inflammatory agent. However, as demonstrated in
FIG. 22, its effects on gene expression are dependent on the
stimulus. While there are general qualitative similarities between
the effects on gene expression across these three stimuli, there
are both quantitative and qualitative differences that may be
important in understanding when glucocorticoid intervention is
warranted.
[0171] According to embodiments of the invention, an indicator cell
population is used to measure quantitative gene expression the
effect of an agent or a biological sample may influence the choice
of which indicator cell line will be most informative. For example,
a cloned cell line such as THP-1 or a primary cell population
(peripheral mononuclear cells) may provide information that is
comparable to that obtained from a body sample directly (see FIG.
15). The normal state of gene expression may range from zero or few
transcripts to 10.sup.5 or more transcripts.
[0172] Similarly, an agent may be evaluated for its effect on any
population of cells, either in vivo, ex vivo or in vitro, by
administering the agent and then determining a calibrate profile
data set for those cells under the selected conditions. Examples of
this approach are provided in FIGS. 10-16 and 18. FIG. 18 further
provides calibrated profile data sets for different concentrations
of a single agent showing that the transcription of selected
constituents vary with dose and therefore the anticipated
effectiveness with respect to the biological condition.
[0173] The above description of determining a biological condition
is exemplified as follows: the action of a pharmaceutical or
nutraceutical is measured with respect to its anti-inflammatory
properties. The measurement of the effect may be established using
a selected panel of constituent gene loci for example, an
inflammation selected panel, including, Interleukin 1 alpha
(IL-1.alpha.) or Tumor Necrosis Factor alpha (TNF-.alpha.). The
anti-inflammatory effect may first be established by treating
indicator cells or sample cells ex vivo with a known inflammation
inducers (for example, lipopolysaccharide or other mitogens)
followed by treatment with the experimental agent or condition
expected to affect the expression from the appropriate gene loci.
Accordingly, a baseline profile data set may be established in this
case as the gene expression for a particular panel of constituents
resulting in the presence of the inflammation inducer. The addition
of a potential anti-inflammatory agent results in a change relative
to the baseline. This approach is illustrated for example in FIG.
12. Methylprednisolone has a substantial down regulation effect on
IL-2 in blood cells stimulated ex vivo with LPS where the baseline
data set is LPS stimulated cells. In this case the effect is shown
as negative. In contrast, as shown in FIG. 16b, IL-2 appears to be
upregulated in whole blood not previously exposed to LPS, where the
baseline data set is unstimulated cells. These results are
consistent with the observation that methylprednisolone stimulates
IL-2 production.
[0174] The determination of the biological condition of a subject
may include measuring and storing additional data about the
subject. For example, if the subject is a human or mammalian
patient, additional clinical indicators may be determined from
blood chemistry, urinalysis, X-ray, other chemical assays and
physical or sociological findings.
[0175] FIG. 7 illustrates how the accumulation of calibrated
profile data sets may improve the predictive power of the database
and thereby increase its value in generating information about a
biological condition or agent. The figure indicates the use of the
database in terms of its power, for example, to predict the course
of a therapeutic intervention or follow the course of an individual
subject compared to a population. Information from the database may
be used to predict a likely mechanism of metabolism or molecular
mechanism of action, and to compare a single profile to a
collection of signature, calibrated selected profiles.
[0176] Use of a database in accordance with an embodiment of the
present invention is illustrated in FIG. 8. FIG. 8 illustrates a
data profile set from the database. Entries for input include a
name, an Experimental Type, and whether the entry is a New
Reference; Cell/Tissue/Species and whether these are new;
Therapeutic agent (compound), Dose, and additional parameters and
whether the therapeutic agent is new. Observations are recorded
according to the identity of a Gene (New Gene) and a Protein (New
Protein). The Stimulus or other Treatment, if any, and the Dose are
entered. Gene (and/or Protein) Expression, Expression Value,
Expression Units if appropriate and Expression Time are shown. The
figure specifically illustrates the range of applicable fields of
investigation from complex natural products to clinical trails in
humans, linkage to traditional forms of measurement and evaluation
such as literature citations, clinical indicators and traditional
pharmacokinetic measurements. Expert analysis of the selected
profile data contained in the database may then be used to guide
product development and marketing, or used to improve the clinical
decision making concerning the health of a single individual or
population of individuals.
[0177] One form of record may provide information about a subject
or agent with respect to identity, medical history including
traditional pharmaceutical/medical data, clinical indications as
determined from literature data, reference to additional types of
analysis in the database, etc.
[0178] FIG. 9 shows an embodiment of the present invention in which
profile data is evaluated using data from a database that is
remotely accessed over a network. Using the approach of this figure
data may be derived at one or more locations (such as location 1
shown here), compared using information retrieved over
communication path 1109 from a central database at location 2, and
the result of the comparison may be used to affect, for example,
the course of treatment of an individual or population. The
communication path 1109 between location 1 and location 2 is
two-way, so that information resulting from determinations made at
location 1 may delivered over the path 1109 to update the database
1108. The consequence is an iterative process whereby the
information from database is used in a determination that may
affect the course of treatment, evaluation, or development, and the
results of the determination become part of the database. In a
first location, as in FIG. 5, from a tissue sample procured in
process 1101, there are derived multiple RNA species pursuant to
process 1102, and then in process 1103, profile data are quantified
to produce a profile data set that is pertinent to the tissue
sample obtained in process 1101. In order to evaluate the profile
data set, in process 1104 information is retrieved from database
1108, which is located in a second location. In fact the database
may be in communication with a large number of locations, each of
which is generating profile data that must be evaluated. The
retrieval of information from the database is accomplished over a
communication path 1109, which may include a network such as the
Internet, in a manner known in the art. Once information has been
obtained from the database 1108, the information is used in
evaluating the quantified profile data in process 1105, with the
result in process 1106 that the medical condition of the subject
may be assessed. In process 1107, the database 1108 is updated over
the communication path 1109 to reflect the profile data that have
been quantified in process 1103. In this manner the database 1108
may be updated to reflect the profile data obtained over all
locations, and each location has the benefit of the data obtained
from all of the locations. While, for simplicity, all of the
processes in FIG. 9 are shown as taking place at location 1, some
or even all of the processes may be implemented elsewhere, for
example location 2, or in multiple locations. At location 2,
associated with the database, for example, may be a server that is
used for hosting these processes, including evaluation of the
quantified profile data.
EXAMPLES
Example 1
[0179] (a) Use of whole blood for ex vivo assessment of a
biological condition affected by an agent.
[0180] Human blood is obtained by venipuncture and prepared for
assay by aliquoting samples for baseline, no stimulus, and stimulus
with sufficient volume for at least three time points. Typical
stimuli include lipopolysaccharide (LPS), phytohemagglutinin (PHA)
and heat-killed staphylococci (HKS) or carrageean and may be used
individually (typically) or in combination. The aliquots of
heparinized, whole blood are mixed without stimulus and held at
37.degree. C. in an atmosphere of 5% CO.sub.2 for 30 minutes.
Stimulus is added at varying concentrations, mixed and held loosely
capped at 37.degree. C. for 30 min. Additional test compounds may
be added at this point and held for varying times depending on the
expected pharmacokinetics of the test compound. At defined times,
cells are collected by centrifugation, the plasma removed and RNA
extracted by various standard means.
[0181] Nucleic acids, RNA and or DNA are purified from cells,
tissues or fluids of the test population or indicator cell lines.
RNA is preferentially obtained from the nucleic acid mix using a
variety of standard procedures (or RNA Isolation Strategies, pp.
55-104, in RNA Methodologies, A laboratory guide for isolation and
characterization, 2nd edition, 1998, Robert E. Farrell, Jr., Ed.,
Academic Press); in the present use using a filter-based RNA
isolation system from Ambion (RNAqueous.TM., Phenol-free Total RNA
Isolation Kit, Catalog #1912, version 9908; Austin, Tex.).
[0182] In accordance with one procedure, the whole blood assay for
selected profiles determination was carried out as follows: Human
whole blood was drawn into 10 mL Vacutainer tubes with Sodium
Heparin. Blood samples were mixed by gently inverting tubes 4-5
times. The blood was used within 10-15 minutes of draw. In the
experiments, blood was diluted 2-fold, i.e. per sample per time
point, 0.6 mL whole blood+0.6 mL stimulus. The assay medium was
prepared and the stimulus added as appropriate.
[0183] A quantity (0.6 mL) of whole blood was then added into each
12.times.75 mm polypropylene tube. 0.6 mL of 2.times. LPS (from E.
coli serotye 0127:B8, Sigma#L3880 or serotype 055, Sigma #L4005, 10
ng/ml, subject to change in different lots) into LPS tubes was
added. Next, 0.6 mL assay medium was added to the "control" tubes
with duplicate tubes for each condition. The caps were closed
tightly. The tubes were inverted 2-3 times to mix samples. Caps
were loosened to first stop and the tubes incubated @ 37.degree.
C., 5% CO.sub.2 for 6 hours. At 6 hours, samples were gently mixed
to resuspend blood cells, and 1 mL was removed from each tube
(using a micropipettor with barrier tip), and transfered to a 2 mL
"dolphin" microfuge tube (Costar #3213).
[0184] The samples were then centrifuged for 5 min at 500.times.g,
ambient temperature (IEC centrifuge or equivalent, in microfuge
tube adapters in swinging bucket), and as much serum from each tube
was removed as possible and discarded. Cell pellets were placed on
ice; and RNA extracted as soon as possible using an Ambion
RNAqueous kit
[0185] (b) Amplification Strategies.
[0186] Specific RNAs are amplified using message specific primers
or random primers. The specific primers are synthesized from data
obtained from public databases (e.g., Unigene, National Center for
Biotechnology Information, National Library of Medicine, Bethesda,
Md.), including information from genomic and cDNA libraries
obtained from humans and other animals. Primers are chosen to
preferentially amplify from specific RNAs obtained from the test or
indicator samples, see, for example, RT PCR, Chapter 15 in RNA
Methodologies, A laboratory guide for isolation and
characterization, 2nd edition, 1998, Robert E. Farrell, Jr., Ed.,
Academic Press; or Chapter 22 pp.143-151, RNA isolation and
characterization protocols, Methods in molecular biology, Volume
86, 1998, R. Rapley and D. L. Manning Eds., Human Press, or 14 in
Statistical refinement of primer design parameters, Chapter 5,
pp.55-72, PCR applications: protocols for functional genomics, M.
A. Innis, D. H. Gelfand and J. J. Sninsky, Eds., 1999, Academic
Press). Amplifications are carried out in either isothermic
conditions or using a thermal cycler (for example, a ABI 9600 or
9700 or 7700 obtained from Applied Biosystems, Foster City, Calif.;
see Nucleic acid detection methods, pp. 1-24, in Molecular methods
for virus detection, D. L. Wiedbrauk and D. H., Farkas, Eds., 1995,
Academic Press). Amplified nucleic acids are detected using
fluorescent-tagged detection primers (see, for example, Taqman.TM.
PCR Reagent Kit, Protocol, part number 402823 revision A, 1996,
Applied Biosystems, Foster City Calif.) that are identified and
synthesized from publicly known databases as described for the
amplification primers. In the present case, amplified DNA is
detected and quantified using the ABI Prism 7700 Sequence Detection
System obtained from Applied Biosystems (Foster City, Calif.).
Amounts of specific RNAs contained in the test sample or obtained
from the indicator cell lines can be related to the relative
quantity of fluorescence observed (see for example, Advances in
quantitative PCR technology: 5' nuclease assays, Y. S. Lie and C.
J. Petropolus, Current Opinion in Biotechnology, 1998, 9:43-48, or
Rapid thermal cycling and PCR kinetics, pp. 211-229, chapter 14 in
PCR applications: protocols for functional genomics, M. A. Innis,
D. H. Gelfand and J. J. Sninsky, Eds., 1999, Academic Press.
[0187] As a particular implementation of the approach described
here, we describe in detail a procedure for synthesis of first
strand cDNA for use in PCR. This procedure can be used for both
whole blood RNA and RNA extracted from cultured cells (i.e. THP-1
cells).
[0188] Materials
[0189] (1) Applied Biosystems TAQMAN Reverse Transcription Reagents
Kit (P/N 808
[0190] 0234). Kit Components: 10.times.TaqMan RT Buffer, 25 mM
Magnesium chloride, deoxyNTPs mixture, Random Hexamers, RNase
Inhibitor, MultiScribe Reverse Transcriptase (50 U/mL) (2)
RNase/DNase free water (DEPC Treated Water from Ambion (P/N 9915G),
or equivalent)
[0191] Methods
[0192] 1 Place RNase Inhibitor and MultiScribe Reverse
Transcriptase on ice immediately. All other reagents can be thawed
at room temperature and then placed on ice.
[0193] 2 Remove RNA samples from -80.degree. C. freezer and thaw at
room temperature and then place immediately on ice.
[0194] 3 Prepare the following cocktail of Reverse Transcriptase
Reagents for each 100 .mu.L RT reaction (for multiple samples,
prepare extra cocktail to allow for pipetting error):
1 1 reaction(.mu.L) 11X, e.g. 10 samples(.mu.L) 10X RT Buffer 10.0
110.0 25 mM MgCl.sub.2 22.0 242.0 dNTPs 20.0 220.0 Random Hexamers
5.0 55.0 RNAse Inhibitor 2.0 22.0 Reverse Transcriptase 2.5 27.5
Water 18.5 203.5 Total: 80.0 880.0 (80 .mu.L per sample)
[0195] 4 Bring each RNA sample to a total volume of 20 .mu.L in a
1.5 mL microcentrifuge tube (for example, for THP-1 RNA, remove 10
.mu.L RNA and dilute to 20 .mu.L with RNase/DNase free water . . .
for whole blood RNA use 20 .mu.L total RNA) and add 80 .mu.L RT
reaction mix from step 5.2.3. Mix by pipetting up and down.
[0196] 5 Incubate sample at room temperature for 10 minutes.
[0197] 6 Incubate sample at 37.degree. C. for 1 hour.
[0198] 7 Incubate sample at 90.degree. C. for 10 minutes.
[0199] 8 Quick spin samples in microcentrifuge.
[0200] 9 Place sample on ice if doing PCR immediately, otherwise
store sample at -20.degree. C. for future use.
[0201] 10 PCR QC should be run on all RT samples using 18S and
.beta.-actin (see SOP 200-020).
Example 2
[0202] Different inflammatory stimuli give rise to different,
baseline profile data sets so that the calibrated selected profiles
for different agents in the same class of anti-inflammatory result
in different signature profiles.
[0203] FIGS. 11(a) and 11(b) show different inflammatory stimuli
give rise to different, baseline profile data sets that may be used
in determining the calibrated selected profile data sets for the
three anti-inflammatory agents tested, and the resulting different
signature profiles. The different profiles reflect the difference
in the molecular targets and mechanisms of action of the three
agents derived from a single class of therapeutics,
anti-inflammatory agents. FIG. 11(a) also illustrate the
extraordinary range of detection (y-axis) from less than 10 fold
difference from the calibrated profile with respect to some
constituents to a change of 10.sup.13 (10E13) in gene expression of
one constituent (indeed the change for a constituent in FIG. 11(b)
is 10.sup.-22) when compared to the calibrator. Comparison to the
calibrator results in gene expression profiles that are increased,
decreased, or without change from the calibrated set.
[0204] FIG. 11(a) shows relative gene expression (mRNA synthesis)
in heat-killed staphylococci (HKS)-stimulated cells, and the effect
of three different compounds (TPCK, UT-77, and "Dex", or
dexamethasone). Compound TPCK caused a 10-fold decrease in relative
IFN-.gamma. expression, and 100,000-fold decreases in IL-4 and IL-5
expression. Further, compound UT-77 caused even greater magnitude
of increases in relative expression of the gene encoding IL-5, and
more modest increases in IL-1 expression (more than 10-fold) and
IFN-.gamma.. Such effects can be highly significant in disease
etiologies and outcomes, and have predictive value concerning the
usefulness as therapeutic agents of these compounds or similar
chemical entities or chemicals that act similarly. HKS cells may be
used as an in vitro model of Gram-positive bacterial infection.
[0205] FIG. 11(b) displays analyses of expression of the 12 genes
in lipopolysaccharide-(LPS)-treated cells, an in vitro model of
Gram-negative bacterial infection. These data include several
striking contrasts to the data in FIG. 11(a). Thus treatment with
the therapeutic agent Dex caused a striking decrease in expression
of the IL-2 gene in LPS-treated cells, and a striking increase in
IL-2 expression in HKS-treated cells. Strikingly large differences
in gene expression in the differently stimulated cells can be seen
for the IL-4 and the IL-5 genes. Expression of the gene for IFN, in
contrast, responded similarly in cells treated by either of the
stimuli and any of the therapeutic agents.
[0206] By these criteria, expression of the genes for IL-2, IL-4
and IL-5 were observed to be candidate markers or surrogate markers
in cell model systems to distinguish responses of the cells to
Gram-positive and Gram-negative bacterial infection.
Example 3
[0207] A single therapeutic agent for treating a particular
condition can be differentiated from a second therapeutic agent
that also treats the particular condition by a signature profile
for a given selected panel of gene loci.
[0208] FIG. 12 shows a calibrated profile data set for a panel
having 8 constituents that are indicative of a biological condition
that includes inflammation. The profiles are shown for three
different anti-inflammatory agents-methotrexate, meclofenamate and
methylprednisolone. The calibrated profile data sets for each agent
as shown represents a signature profile for that agent. This
signature profile may serve as a device for establishing quality
control for a batch of the agent. Indeed, it is envisaged that
compounds or classes of compounds on the market or in development
may be characterized by a signature profile. The signature profile
may be represented in a graphical format, more particularly as a
bar graph as provided in FIG. 12. For FIG. 12, an ex vivo sample
was tested. A sample of blood was taken from the subject. Aliquots
of the sample were subjected to lipopolysaccharide (LPS) ex vivo.
After 30 minutes, the anti-inflammatory agent as indicated was
added to an aliquot of the sample of blood and after about another
4 hours, the expression of the panel of genes (Il-1.alpha., Il-2,
Il-8, Il-10, Il-12p35, Il-12p40, IL-15, IFN-Gamma and TNF-.alpha.)
was determined. Although the calibrated profile of methotrexate and
meclofenamate were similar, the calibrated profile of
methylprednisolone was substantially different. Differences may be
reflective of the differences of the mechanisms or target(s) of
action of this agent within the general class of anti-inflammatory
compounds. The baseline is the profile data set for
lipopolysaccharide absent any additional agents.
Example 4
[0209] There is relatively low variability with respect to the
profile within a single individual over time when the calibrated
selected profile is determined from the measurement of gene
expression across many gene loci that have been appropriately
induced.
[0210] FIGS. 13(a), 13(b), and 13(c) show graphical representations
of calibrated selected profile data sets for two different samples
of whole blood. Heparinized whole blood from a single normal
healthy volunteer was collected on two separate occasions of more
than 2 weeks apart. FIG. 13a, for sample 991116, and FIG. 13b, for
sample 991028, reflect the biological condition of the tested cells
from the single donor using a selected panel (i.e., the
inflammation selected panel) of 24 members, in response to
stimulation with one of three different agents. The baseline in
this example is derived from untreated cells obtained from the same
individual. The calibrated profiles are shown for cells exposed for
4 to 6 hours to lipopolysaccharide (LPS), heat-killed Stapylococci
(HKS), and phytohemagglutinin (PHA). FIG. 13c shows a direct
comparison of LPS-stimulated blood sample 99116 with respect to
blood sample 991028, i.e., 991028 is used as the calibrator or
baseline profile data set. The messenger RNA levels measured on
Oct. 28, 1999 were used to compare the levels of messenger RNA
measured on Nov. 16, 1999. A perfect identity of RNA levels would
be represented by a flat line at unity. These data show that for
baseline gene expression, there can be as much as an 8 fold
difference (c-jun) in messenger RNA levels. However, for most of
the genes measured, when there is no known substantial
physiological change in the subject, the levels of messenger RNA
measured on one day are similar to those measured on a different
day. Changes in gene expression, whether mRNA or protein, in excess
of 10-20% may be reflective of biological changes in the subject
even though traditional clinical measurements may not identify such
changes. FIG. 13(d) is similar to FIG. 13(c) except that the cells
were not stimulated with LPS.
[0211] FIGS. 13(a) through 13(d) document the relatively low
variability with respect to the profile within a single individual
over time in similar physiological conditions when the calibrated
selected profile is determined from the measurement of gene
expression across many gene loci that have been appropriately
induced. The figures illustrate (1) the class-specific effects
(generally inflammatory as determined by the effect on
pro-inflammatory gene loci, e.g. TNF-alpha, IL-1 alpha and IL-1
beta), (2) the agent-specific effects quantitative differences
between each of the agents at the same gene loci (e.g., IL-2) and
(3) reproducible and therefore predictable effects on the subject
population, TK (FIG. 13c).
Example 5
[0212] Similarities and differences in the effect of a single agent
on cell populations differing in their biological condition.
[0213] Ex-vivo gene expression analysis can be performed by
obtaining the blood of a subject for example by drawing the blood
into a vacutainer tube with sodium heparin as an anticoagulant. An
anti-inflammatory such as 3-methyl-prednisolone at a final
concentration of 10 micromolar was added to blood in a
polypropylene tube, incubated for 30 minutes at 37 C in 5%
CO.sub.2. After 30 minutes a stimuli such as LPS at 10 ng/mL or
heat killed staphlococcus (HKS) at 1:100 dilution was added to the
drug treated whole blood. Incubation continued at 37.degree. C. in
5% CO.sub.2 for 6 hours unless otherwise indicated. Erythrocytes
were lysed in RBC lysis solution (Ambion) and remaining cells were
lysed according to the Ambion RNAqueous-Blood module (catalog #
1913). RNA was eluted in Ambion elution solution. RNA was DNAsed
treated with 1 unit of DNAse I (Ambion #2222) in 1.times.DNAse
buffer at 37.degree. C. for 30 minutes. In this example, first
strand synthesis was performed using the Applied Biosystems TaqMan
Reverse Transcriptase kit with MultiScribe reverse transcriptase
(catalog # N.sub.8O.sub.8-0234). Quality check of RT reactions were
performed with Taqman PCR chemistry using the 18S rRNA
pre-developed assay reagents (PDAR) from Applied Biosystems (part
#4310893E). PCR assay of Source Selected Profiles were performed on
6 to 24 genes in four replicates on the Applied Biosystems 7700.
PCR assays were performed according to specifications outlined with
the PDAR product. Relative quantitation of the gene of interest was
calibrated against 18S rRNA expression as described in Applied
Biosystems product User Bulletin 2 (1997) and elaborated in
Hirayama, et al (Blood 92, 1998:46-52) using 18S instead of
GAPDH.
[0214] Relative quantitation of the mRNA was measured by the
difference in threshold cycles between 18S and the gene of
interest. This delta CT was then compared to the normalizing
condition, either subject before treatment, or stimuli without drug
in an ex-vivo assay to measure "fold induction" represented in the
bar graphs (FIG. 14). For example, in the above graph, IFN-levels
are {fraction (1/50)} less on day 3 than before treatment.
Example 6
[0215] In Vivo and Ex vivo samples provide comparable signature
profiles.
[0216] FIG. 15 shows the calibrated profile data set for two
subjects (Subject 1 and Subject 2) who have been treated over a
three day period with a standard dose of the corticosteroids,
dexamethasone. Blood from each subjects was obtained 72 hours later
and a quantitative measure of the amount of RNA corresponding to
the panel constituents was determined. Although, the calibrated
profile data set for each subject was similar for most gene loci,
some notable differences were also detected, for example for Il-2,
Il-10, Il-6 and GM-CSF. A calibrated profile data set is also shown
for comparison for an ex vivo sample of blood from sample 1 prior
to treatment with corticosteroid where the ex vivo sample is
subjected to an equivalent amount of corticosteroid in vitro as
calculated to be the plasma level in the subject. The similarity in
the calibrated profile data set for ex vivo samples when compared
to in vivo samples provides support for an in vitro assay that will
predict the in vivo action of the compound. We have observed a
similar comparable effect between in vivo and ex vivo samples
infected with an infectious agent, more particularly bacterial or
viral agents. We have concluded therefore that the ex vivo samples
provide an effective method of determining the effect of a single
compound or multiple compounds on a patient, where the multiple
compounds may be either used in combination, in parallel or
sequentially to optimize the selection of an agent for a biological
condition for the subject.
Example 7
[0217] Demonstration of reproducibility of an in vitro response
with an approved anti-inflammatory on 5 different donor
subjects.
[0218] Comparison and analysis of the FIGS. 18a through 18e
demonstrate the consistency of effect of the stimulus and in vitro
treatment with an approved anti-inflammatory on 5 different donors
(each figure representing a unique donor). The use of a known and
tested stimulus results in a highly reproducible gene response in
vitro that may be correlated with a predictable in vivo
response.
[0219] FIGS. 18a-18e provide the results of analysis of 5 donors
from which a blood sample has been taken. The blood samples were
exposed to a therapeutic agent at various concentrations ranging
from 0.1 .mu.M to 5 .mu.M, more particularly 0.1 .mu.M, 0.3 .mu.M,
1 .mu.M, 3 .mu.M and 5 .mu.M, for a 4 hour period. Different
concentrations of the drug resulted in a calibrated profile data
set for an inflammation panel at each concentration that was
qualitatively different from the next. FIG. 18a corresponds to
donor 1, FIG. 18b corresponds to donor 2, FIG. 18c corresponds to
donor 3, FIG. 18d corresponds to donor 4, and FIG. 18e corresponds
to donor 5. Each individual varied from the other and also provided
a variable profile for a different concentration. This set of
figures illustrates the high level of information obtainable by
calibrated profile data sets.
Example 8
[0220] A calibrated profile data set may provide a signature
profile for a complex mixture of compounds.
[0221] FIG. 21 illustrates the effect of three different
anti-inflammatory herbs on a selected panel of constituents
including constituents of an Inflammation Selected Panel
(TNF-.alpha., Il-1b, ICAM, Il-8, Il-10, Il-12p40, ICE, cox-2, cox-1
and mmp-3) a cell growth and differentiation selected panel (c-fos,
c-jun and STAT3), a toxicity selected panel (SOD-1, TACE, GR,
HSP70, GST, c-fos, c-jun, INOS) and a liver metabolism selected
panel (INOS, cyp-a and u-pa). The cells assayed in FIG. 21 are
aliquots of blood from a subject that are exposed ex vivo to
lipopolysaccharide and to Echinacea (SPM9910214) Arnica
(SPM9910076) and Siberian Ginseng (SPM9910074), each of the
nutraceuticals being applied to the blood sample at the same
concentration of 200 ug/mL. The baseline is cell sample with
lipopolysaccharide in the absence of a nutraceutical. Each
nutraceutical (formed from a complex mixture) has a characteristic
signature profile just as did the single compound pharmaceutical
anti-inflammatory agents. The signature profile may be provided in
a graphic form that can be use to identify a herbal while providing
information concerning its properties and its efficacy for a single
subject or for an average population of subjects.
Example 9
[0222] A quality control assay for Echinacea brands using
calibrated profile data sets.
[0223] FIG. 24 shows a graphic representation of the calibrated
profile data sets for four different commercial brands of
Echinacea. Brands using an Inflammation Selected Panel. As
expected, SPM007 and SPM003 gave the signature, calibrated profiles
similar to authentic Echinacea. Samples SPM010 and SPM 016,
although labeled and sold as Echinacea when tested using the system
described in FIG. 14, resulted in signature calibrated profiles
that were substantially similar to the profile obtained with
lipopolysaccharide alone. Echinacea samples SPM010 and SPM016 were
found to have elevated, highly biologically active levels of
endotoxin while the LPS levels in SP007 and SP003 were
undetectable. A stored signature profile for active Echinacea
obtained from a selected panel designed to test efficacy and mode
of action, e.g., the inflammation panel, permits evaluation of new
batches of Echinacea, differentiation of existing or new brands of
Echinacea, guide the isolation and development of new compounds
with different or similar activities from a complex compound like
Echinacea or may be used in the development of quality assurance in
the production, analysis and sale of new or previously marketed
compounds. In the example cited, two of the brands of Echinacea
SP010 and SP016 result in calibrated profiles that are
characteristic of authentic Echinacea.
Example 10
[0224] Comparison of three herbal preparations using an indicator
cell line.
[0225] FIGS. 25(a) through 25(c) provide calibrated profile data
sets for three herbal preparations with respect to an indicator
cell line (THP-1) rather than a blood sample from a subject. In
FIG. 25(a), the baseline is the profile data set for THP-1 cells
absent the herbal while the histograms represent the calibrated
profile data sets for the same herbal from three different
manufacturing sources of the same herb at 250 ug/ml. Gene
expression results are shown on a log scale. Similar to the
observation in FIG. 14, these demonstrate that similarly labeled
compounds obtained from different sources have demonstrable and
quantifiable differences in calibrated profiles using a specific
panel, e.g., the inflammation selected panel designed to obtain
information about the expression of gene products related to
inflammation and infection. This suggests that the compounds likely
have different efficacies when used for specific purposes.
[0226] FIG. 25(b) provides a comparison of the calibrated profile
of a single herb at three concentrations using the indicator cell
line of THP-1. The baseline profile data set is untreated THP-1
cells. Analysis of the data suggests a concentration-dependent
response in the indicator cell lines which, although demonstrated
here, may be indicative of a similar response in subjects.
[0227] FIG. 25(c) provides a comparison of four commercial
Echinacea brands used at the same concentration and tested against
a panel of constituents using a THP-1 cell line as an indicator
cell population. Differential expression, as revealed, for example,
by inspection or calculation of differences in the calibrated
profiles, allows direct comparisons of complex compounds to be
made. For example, analysis of the differences in the calibrated
profiles may be used to guide compound isolation and development,
product differentiation in the marketplace, or used by the consumer
or health professional to guide the individualized choice of a
single compound from a class of similar compounds that may be
suited for a particular biological condition.
Example 11
[0228] Set up of a 24-gene Human Selected Panel for
Inflammation
[0229] Materials
[0230] 1 20.times. Primer/Probe Mix for each gene of interest.
[0231] 2 20.times. Primer/Probe Mix for 18S endogenous control.
[0232] 3 2.times. Taqman Universal PCR Master Mix
[0233] 4 cDNA transcribed from RNA extracted from cells
[0234] 5 Applied Biosystems 96-Well Optical Reaction Plates
[0235] 6 Applied Biosystems Optical Caps, or optical-clear film
[0236] 7 AB Prism 7700 Sequence Detector
[0237] Methods
[0238] 1 Make stocks of each Primer/Probe mix containing the
Primer/Probe for the gene of interest, Primer/Probe for 18S
endogenous control, and 2.times.PCR Master Mix as follows. Make
sufficient excess to allow for pipetting error e.g. approximately
10% excess. The following example illustrates a typical set up for
one gene with quadruplicate samples testing two conditions (2
plates).
2 1X(1 well) 9X(2 plates worth) 2X Master Mix 12.50 112.50 20X 18S
Primer/Probe Mix 1.25 11.25 20X Gene of interest Primer/Probe 1.25
11.25 Mix Total 15.00 135.00
[0239] 2 Make stocks of cDNA targets by diluting 95 .mu.l of cDNA
into 2000 .mu.l of water. The amount of cDNA to be adjusted to give
Ct values between 10 and 18.
[0240] 3 Pipette 15 .mu.l of Primer/Probe mix into the appropriate
wells of a Applied Biosystems 96-Well Optical Reaction Plate.
[0241] 4 Pipette 10 .mu.l of cDNA stock solution into each well of
the Applied Biosystems 96-Well Optical Reaction Plate.
[0242] 5 Seal the plate with Applied Biosystems Optical Caps, or
optical-clear film.
[0243] 6 Analyze the plate on the AB Prism 7700 Sequence
Detector.
[0244] In Examples 12 through 19 below, procedures analogous to
those of Examples 1 and 11 were followed to determine relative mRNA
expression.
Example 12
[0245] Calibrated profile data sets, using a subset of the
Inflammation Selected Panel, show the effect of administration of a
steroid.
[0246] In FIGS. 26(a) and 26(c), subjects 1 and 2 respectively have
been subjected to a course of administration of methylprednisone
twice a day for three consecutive days. In FIGS. 26(b) and 26(d),
two other individuals, identified as control 1 and control 2
respectively, were administered a placebo over a corresponding
period. In each case, the blood samples of the individuals were
taken prior to the course of administration and immediately
following the course of administration. The graphs of these each of
these figures show the relative concentration of each constituent
of a subset of the Inflammation Selected Panel, the subset being
chosen for its ability to discriminate as to the effect of
anti-inflammatory agents. For each constituent, the
post-administration concentration is shown as a ratio in relation
to its pre-administration concentration; hence the baseline of 1 is
indicative of the same concentration of the constituent before and
after administration, and the presence of a bar below the baseline
indicates a post-administration drop in concentration of the
constituent. FIGS. 26(a) and 26(c) show that the response of
subjects 1 and 2 to the administration of the steroid are
qualitatively and quantitatively similar-indeed, strikingly
similar. Moreover, and in contrast, the responses of controls 1 and
2 to the placebo are markedly distinct from the responses of
subjects 1 and 2.
Example 13
[0247] Calibrated profile data sets, using a subset of the
Inflammation Selected Panel, provide a comparison of the effects of
administration of methylprednisone and Ibuprofen. In this example,
FIGS. 27(a) and 27(c) are identical to FIGS. 26(a) and 26(c), and
show the responses of subjects 1 and 2 respectively to the
administration of methylprednisone. FIGS. 27(b) and 27(d) show the
responses of the same subjects, namely subjects 1 and 2
respectively, to the administration of high-dose Ibuprofen (800 mg
administered three times per day over a three-day period). (FIGS.
27(b) and 27(d) use the same conventions as FIGS. 27(a) and 27(c)
in showing post-administration concentration of constituents
relative to pre-administration concentration.) It can be seen from
these figures that the responses of the subjects to Ibuprofen are
qualitatively and quantitatively similar-again, strikingly similar.
Moreover, the responses of the subjects to Ibuprofen are distinct
from the responses of the subjects to methylprednisone. In fact,
the distinct pattern of response to Ibuprofen correlates with other
information known about Ibuprofen. For example, IL-1-.beta. is
known to be of importance in responding to joint destruction, and
Ibuprofen here is shown as not raising the level (and in the case
of subject 2, lowering the level) of IL-1-.beta. expression. And
Ibuprofen is known to be not very effective in treating joint
destruction. Similarly, IL-10 activity is associated with
anti-inflammatory activity and is useful in addressing bowel
inflammatory disease; Ibuprofen here is shown as in fact depressing
the level of IL-10 expression. These phenomena are consistent with
the fact that Ibuprofen is known to be ineffective in addressing
bowel inflammatory disease.
Example 14
[0248] Calibrated profile data sets, using a subset of the
Inflammation Selected Panel, identify chronic obstructive pulmonary
disease (COPD) patients.
[0249] The graphs of these each of FIGS. 28(a) through 28(d) show
the relative concentration of each constituent of a subset of the
Inflammation Selected Panel, the subset being chosen for its
ability to discriminate as to the presence of COPD. For each
constituent, the concentration is shown as a ratio in relation to a
concentration that is normative of the concentrations of the
constituent in a population of healthy subjects. Thus for any
constituent, a baseline level of 1 corresponds to a normal
concentration. FIGS. 28(a) and 28(c) show the relative
concentrations of constituents in COPD patients 1 and 2
respectively, while FIGS. 28(b) and 28(d) show the relative
concentrations of constituents in two healthy individuals
identified as control 1 and control 2 respectively. Indeed, FIGS.
28(b) and 28(d) show levels of constituents in controls 1 and 2 as
being close to population normals, whereas FIGS. 28(a) and 28(c)
show levels of constituents in COPD patients 1 and 2 as being
dramatically different from normal levels.
Example 15
[0250] Evaluations of the effects of drug exposure performed in
vitro correspond closely with evaluations performed in vivo,
employing in each case calibrated profile data sets, using a subset
of the Inflammation Selected Panel.
[0251] FIGS. 29(a) and 29(b) present graphs showing response to the
administration of methylprednisone. The response shows the relative
concentration of each constituent of a subset of the Inflammation
Selected Panel, the subset being chosen for its ability to
discriminate as to the effect of anti-inflammatory agents. For each
constituent illustrated, the darker bar (on the right) shows the in
vivo response of a subject to a course of administration of
methylprednisone twice a day for three consecutive days. In the in
vivo cases, a blood sample of the subject was taken prior to the
course of administration and immediately following the course of
administration, the constituents were measured, and the responses
are shown in FIGS. 29(a) and 29(b). The post-administration
concentration of each constituent is shown as a ratio in relation
to its pre-administration concentration; hence the baseline of 0.1
is indicative of the same concentration post-administration as
pre-administration. The procedures of FIGS. 29(a) and 29(b) were
conducted on two different occasions a year apart. At the same time
that each in vivo procedure was conducted, methylprednisone was
also administered in vitro to a sample of the blood of the same
subject. For each constituent illustrated in each of FIGS. 29(a)
and 29(b), the lighter bar (on the left) shows the in vitro
response of the sample to the administration of the drug. Again,
the post-administration concentration of each constituent is shown
as a ratio in relation to its pre-administration concentration.
[0252] What is remarkable about the results shown in FIGS. 29(a)
and 29(b) is that in each procedure, the in vitro response is
strikingly similar to the in vivo response, in most cases even
where the results in vivo differed over time. This result shows the
value of in vitro modeling for the evaluation of the effect of the
administration of agents using Selected Panels.
Example 16
[0253] The effect of different agents is evaluated using a subset
of the Selected Prostate Panel.
[0254] In FIG. 30 is shown the response of five different cell
lines to the administration of various agents, using a subset of
the Selected Prostate Panel (listed in Table 5). This figure also
shows broad functions of constituents of the panel.
Example 17
[0255] The use of a rat liver metabolism selected panel to measure
the effect of a pharmaceutical agent, clofibrate, Male rats were
treated with 400 mg/kg/day of clofibrate administered by mouth and
the levels of gene expression were measured in liver tissue.
Clofibrate is used here because its metabolism in the rat and human
liver is well described. As expected, clofibrate induces gene
expression at the cyp 1A1 locus, but the agent also induces
expression at a number of other metabolic loci in the selected
panel as measured in this cohort of in-bred Spraque-Dawley rats.
The ratio of the concentration of each constituent for the
clofibrate treated rats is measured with reference to a control
(baseline) which is a set of rats treated only with the carrier
compound. The resultant selected profile is provided in FIG.
31.
Example 18
[0256] The ability of the rat metabolism selected panel to
differentiate drug responses
[0257] Male rats were treated with 400 mg/kg/day of clofibrate or
benzo[a]pyrene administered by mouth and the levels of gene
expression were measured in liver tissue. The response to
clofibrate and benzo[a]pyrene was determined in Spraque-Dawley rats
using a rat metabolism panel. The results are shown in FIG. 32.
Each drug gives a characteristic and distinct pattern of gene
induction across the selected panel. As expected from the prior
art, benzo[a]pyrene specifically induces gene expression at the
loci for cyp 4A1 and HD. The control (baseline) is a set of rats
treated only with the carrier control.
Example 19
[0258] The effect of administration of a stimulant is measured by a
skin-epithelial/vascular/inflammation selected panel.
[0259] FIG. 33 illustrates the response of a subject to the
administration of a stimulant (TNF-alpha, 10 ng/ml), as measured by
a skin-epithelial, vascular/inflammation selected panel. In this
example, the selected panel is created from constituents that are
also found in other panels which have been here selected for
purposes of best establishing an effect resulting from the
stimulant.
Example 20
Use of a human liver selected panel for determining the metabolic
properties of cryopreserved human hepatocytes.
[0260] Time/dose response experiments utilizing compounds with
well-described toxicities and mechanisms of action are an early
step in the biological validation of the selected panel. FIG. 34
shows the gene expression profile resulting from a timed study at
constant dose of benzo(a)pyrene when administered to cryopreserved
human hepatocytes.
Example 21
[0261] Response of human umbilical vein endothelial cells to
TNF-.alpha..
[0262] FIG. 35 illustrates how endothelial cells respond to the
inflammatory TNF.alpha. by the induction of expression of a number
of gene loci, notably the adhesion molecules ELAM, ICAM, and VCAM.
The cells were exposed for 24 hours. Both 5 ng/ml and 10 ng/ml are
high doses of the immunostimulant TNF.alpha. and, as expected, no
clear concentration response is observed at this dose level.
Example 22
[0263] This example is one illustration of the wide embodiments of
the selected panels. In this example the effect of a compound in
solution (NAC) is compared directly to the effect of an
environmental stimulus (UVB) and the combined effect is read out as
differential gene expression. FIG. 19 illustrates an similar effect
of NAC only in blood obtained smokers and non-smokers. Dose and
time experiments were conducted prior to this illustrated
experiment.
Example 23
[0264] Gene expression profiles provide information on the effect
of an environmental stimulus on cells.
[0265] This example is one illustration of the wide embodiments of
the selected panels. In this example (see FIG. 36) the effect of a
compound in solution (N-acetylcysteine) is compared directly to the
effect of an environmental stimulus (UVB) and the combined effect
is read out as differential gene expression. FIG. 19 illustrates an
similar effect of NAC only in blood obtained smokers and
non-smokers. Dose and time experiments were conducted prior to this
illustrated experiment.
3TABLE 1 Inflammation Selected Panel IL-1.alpha. Interleukin-1
alpha IL-1.beta. Interleukin-1 Beta IL-2 Interleukin-2 IL-4
Interleukin-4 IL-6 Interleukin-6 IL-7 Interleukin-7 IL-8
Interleukin-8 IL-10 Interleukin-10 IL-12p40 Interleukin-12p40 IL-15
Interleukin-15 IL-18 Interleukin-18 GM-CSF Granulocyte colony
stimulating factor IFN-.gamma. Interferon gamma TGF-.alpha. Tumor
growth factor alpha TNF-.alpha. Tumor necrosis factor alpha
TNF-.beta. Tumor necrosis factor beta Cox 2
Cyclooxygenase/prostaglandin-endoperoxide synthase 2 ICE
Interleukin-1 converting enzyme c-jun MKK7, MAP2K7 mmp9 Matrix
metalloproteinase UPA Urokinase plasminogen activator HSP70 Heat
Shock Protein 70 kDa CRE cAMP Response Element ICAM Intercellular
Adhesion Molecule
[0266]
4TABLE 2 Cell Growth and Differentiation Selected Panel BIRC5
(Survivin) Apoptosis Inhibitor NFKB1 NF-kappaB CDKN2A (P16) Cell
cycle inhibitor TP53 (P53) Tumor suppressor TNF-.alpha. Tumor
Necrosis Factor alpha TERT Telomerase Catalytic Subunit BCL2
Represses Apoptosis BAX Promotes Apoptosis CASP1 (ICE) Interleukin
Converting Enzyme GADD45A Growth arrest protein TNFRSF11A Receptor
activator of NFkB (RANK) PDCD8 (AIF) Apoptosis Inducing Factor
Apaf-1 Apoptotic protease activating factor 1 DFFB (DFF40) Caspase
activated DNAse BAIAP3 (IAP1, Inhibitor of apoptosis protein 1
(BAI-associated BIRC3) protein 3) BIRC2 (IAP2) Inhibitor of
apoptosis protein 2 Bik BCL2 interacting killer BCL2L1 (BCL-X)
BCL2-Like 1 DAD1 Defender against cell death 1 MADD MAP Kinase
activating death domain MAP3K14 Mitogen-activated protein kinase
kinase kinase 14 PTEN Protein tyrosine phosphatase k-alpha-1 Alpha
tubulin (housekeeping, high abundance) TOSO Anti-fas induced
apoptosis cdk2 Cyclin dependent kinase 2 cdk4 Cyclin dependent
kinase 4 CASP 3 Apoptosis-related cysteine protease, 3 CASP 9
Apoptosis-related cysteine protease, 9 RAD52 DNA ds break repair
XRCC5 (Ku80) X-ray repair complementing defective repair in Chinese
hamster cells 5 PNKP Polynucleotide kinase phosphatase MRE11A
Meiotic recombination 11 homolog A CCND1 (cyclin D1) PRAD1:
parathyroid adenomatosis 1 CCND3 (cyclin D3) Cyclin D3 CCNE1
(cyclin E) Cyclin E CCNA2 (cyclin A) Cyclin A CCNB1 (cyclin B)
Cyclin B CDKN2B (p15) Cyclin-dependent kinase inhibitor 2B (p15,
inhibits CDK4) CDKN1A (p21) Cyclin-dependent kinase inhibitor 1A
(p21, Cip1) RB1 Retinoblastoma BID BH3 interacting domain death
agonist BAK1 BCL2-antagonist/killer 1 BAD BCL2-antagonist of cell
death SMAC Second mitochondria-derived activator of caspase VDAC1
Voltage-dependent anion channel 1 CHEK1 Checkpoint, S. pombe,
homolog of, 1 ABL1 v-abl Abelson murine leukemia viral oncogene
homolog 1 TNFRSF12 Tumor necrosis factor receptor superfamily,
member 12 TRADD TNFRSF1A-associated via death domain FADD Fas
(TNFRSF6) - associated via death domain TRAF1 TNFR associated
factor 1 TRAF2 TNFR associated factor 2
[0267]
5TABLE 3 Human Liver Metabolism and Toxicity Selected Panel (See
FIG. 34) CYP1A2 Polycyclic aromatic hydrocarbon (PAH) metabolism;
induced by smoking Catalyzes formation of toxic APAP metabolite
CYP2A6 Catalyzes oxidation in some pharmaceuticals, procarcinogens,
and smoke consituents; upregulated in vitro after exposure to
barbiturates or dex. CYP2E1 Converts many small organic Compounds
(i.e. EtOH, APAP, CC14) into reactive intermediatesInduced in
alcoholics and in fatt phenotype. CYP2D6 Broad catalytic activities
for over 30 therapeutic drugs. CYP3A4 Metabolism for a wide variety
of drug types UGT2B7 UDP glycosyltransferase 2B7 UGT2B15 UDP
glycosyltransferase 2B15 EPHX1 Microsomal epoxide hydrolase
Multiple tissue-specific splicing variants GSTA1 Glutathione
S-transferase alpha 1 GSTA2 Glutathione S-transferase alpha 2 UCP-2
Mitochondrial uncoupling protein 2 TNF-.alpha. Local inflammation
Endothelial activation Released by PBMCs, Kupffer cells and
activated tissue macrophages in the liver TGF-.beta. Transforming
growth factor beta 1 iNOS Inducible nitric oxide synthase; SCF Stem
cell factor, released by activated hepatic stellate cells (HSC*);
recruits mast cells (MC) to the liver IFN-.gamma. Activation of
macrophages Galectin-3 .beta.-galactoside-binding lectin associated
with cell growth, tumor transformation, and metastasis. FAP
Fibroblast activation protein; membrane protease expressed at sites
of tissue remodelling. Procollagen C-proteinase Extracellular
matrix protein (aka BMP1) (bone morphogenetic protein 1) required
for cartilage formation Collagen I Extracellular matrix (ECM)
component Collagen III ECM component Collagen IV ECM component
Laminin ECM component Fibronectin ECM component
[0268]
6TABLE 4 Skin Response Selected Panel CRABP2 Cellular retinoic
acid-binding protein 2 KRT14 Keratin 14 KRT5 Keratin 5 KRT16
Keratin 16 FGF7 Fibroblast growth factor (KGF) Keratinocyte growth
factor FN1 Fibronectin 1 IVL Involucrin COL7A1 Type VII collagen,
alpha 1 CTGF Connective tissue growth factor IL-1.alpha.
Interleukin-1.alpha. IL-8 Interleukin 8 GRO1 Melanoma growth
stimulatory activity (MGSA) PTGS2 Prostaglandin-endoperoxid- e
synthase 2 (COX2) Cyclooxygenase 2 TNF-.alpha. Tumor necrosis
factor alpha TGF-.beta.1 Transforming growth factor beta 1 PI3
Proteinase inhibitor 3 (SKALP) Skin-derived antileukoproteinase BSG
Basignin (EMMPRIN) Extracellular MMP inducer MMP1 Matrix
metalloproteinase 1 (interstitial collagenase) MMP2 Matrix
metalloproteinase 2 (72 kD gelatinase) MMP3 Matrix
Metalloproteinase 3 (Stromelysin 1) TIMP1 Tissue inhibitor of
matrix metalloproteinase HMOX1 Heme oxygenase1 GADD45A Growth
arrest and DNA- damage-inducible alpha PCNA Proliferating cell
nuclear antigen DUSP1 Dual specificity phosphatase(CL100) MAPK8
Mitogen activated protein kinase TP53 Tumor protein p53 (p53) Bc12
B-cell CLL/lymphoma 2 Bax Bcl2-associated X protein JUN c-jun FOS
c-fos NR1I2 Nuclear receptor subfamily 1, group I, member 2 (PAR2)
Protease activated receptor 2 S100A7 S100 calcium-binding protein
A7 (PSOR1) psoriasin 1 TNSF6 Tumor necrosis factor (ligand)
superfamily, member 6 (FASL) Fas ligand
[0269]
7TABLE 5 Prostate Selected Panel (See FIG. 30) PSA Prostate
Specific Antigen DD3 Prostate cancer antigen 3 Survivin Apoptosis
Inhibitor 4 PSMA Prostate Specific Membrane Antigen Folate
Hydrolase 1 TERT Telomerase Reverse Transcriptase Telomerase
Catalytic Subunit KLK2 Human Kallikrein 2 PDEF Prostate-Derived Ets
Factor PSCA Prostate Stem Cell Antigen POV1 Prostate Cancer
Overexpressed Gene 1 PART-1 Prostate Androgen-Regulated Transcript
1 MYC c-myc NRP1 Neurophilin 1 KAI1 Human Metastasis Suppressor
Gene LGALS8 Galectin 8 p16 Cyclin-Dependent Kinase 2A GSTT1
Glutathione-S-Transferase theta 1 PAI1 Plasminogen Activator
Inhibitor 1 bcl-2 B-cell CLL/Lymphoma 2 STAT3 Transcriptional
activator IL-6 Interleukin 6 u-pa Urokinase-Type Plasminogen
Activator KRT-5 Keratin 5 TGF-.beta. Transforming Growth Factor
Beta IL-8 Interleukin 8 VEGF Vascular Endothelia Growth Factor ACPP
Acid phosphatase, prostate KRT-19 Keratin 19 CK-8 Cytokeratin 8
Maspin Protease Inhibitor 5 HMG-I/Y Non-histone chromosomal protein
IGFR1 Insulin Growth Factor Receptor 1 HUPAP Human
Prostate-Associated Protease P53 Tumor suprressor COX-2
Cyclooxygenase 2 E-CAD e-cadherin N-CAD n-cadherin CTNNA1 .alpha.-1
catenin PCANAP7 Prostate cancer associated gene 7 MRP1 Multiple
Drug Resistance Protein 1 HSP-70 Heat shock protein TNF-.alpha.
Tumor Necrosis Factor
[0270]
8TABLE 6 Vascular Selected Panel VEGF Vascular Endothelial Growth
Factor NF kappa B Nuclear Factor kappa B TEK/TIE2 Tyrosine kinase,
endothelial ERK2 MAPK1: mitogen-activated protein kinase 1 SELE
selectin E (endothelial adhesion molecule 1) Flt-1 fms-related
tyrosine kinase 1 (vascular endothelial growth factor/vascular
perme-ability factor receptor) PTX3 pentaxin-related gene, rapidly
induced by IL-1 beta HMOX-1 HMOX1 = heme oxygenase (decycling) 1
HIF-1 Hypoxia-inducible factor 1, alpha subunit GRD1 Glutathione
Reductase 1 iNOS Inducible nitric oxide synthase ET-1 Endothelin 1
ECE-1 endothelin converting enzyme 1 PLAT plasminogen activator,
tissue ADAMTS1 a disintegrin-like and metalloprotease (reprolysin
type) with thrombospondin type 1 motif, 1 PTGIS prostaglandin I2
(prostacyclin) synthase COX-2 prostaglandin-endoperoxide synthase 2
(prostaglandin G/H synthase and cyclooxygenase) VCAM vascular cell
adhesion molecule 1 IL-8 Interleukin 8 I1-1.beta. Interleukin-1
beta IGFBP3 Insulin-like growth factor binding protein 3 GJA1 gap
junction protein, alpha 1, 43 kD ICAM-1 intercellular adhesion
molecule 1
[0271]
9TABLE 7 Rat Liver Metabolism and Toxicity Panel (See FIGS. 31 and
32) 1 ALDH 2 aldose reductase 3 ARG 4 CYP1A1 5 CYP1B1 6 CYP2A2 7
CYP2B2 8 CYP2C11 9 CYP2D2 10 CYP2E1 11 CYP2A1 12 CYP3A1 13 CYP1A2
14 CYP4A1 15 CYP4A3 16 CYP4F1 17 cytochrome P450 oxidoreductase 18
epoxide hydrolase 19 HD 20 MAO-B 21 quinone reductase 22
alpha-1-AGP 23 PPAR-alpha 24 GGT
* * * * *