U.S. patent application number 14/106416 was filed with the patent office on 2014-09-25 for methods for drug discovery, disease treatment, and diagnosis using metabolomics.
This patent application is currently assigned to CORNELL RESEARCH FOUNDATION, INC.. The applicant listed for this patent is Cornell Research Foundation, Inc., Metabolon, Inc.. Invention is credited to Rima Kaddurah-Daouk, Bruce S. Kristal.
Application Number | 20140287936 14/106416 |
Document ID | / |
Family ID | 46301673 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287936 |
Kind Code |
A1 |
Kaddurah-Daouk; Rima ; et
al. |
September 25, 2014 |
METHODS FOR DRUG DISCOVERY, DISEASE TREATMENT, AND DIAGNOSIS USING
METABOLOMICS
Abstract
The small molecule profiles of cells are compared to identify
small molecules which are modulated in altered states. Cellular
small molecule libraries, methods of identifying tissue sources,
methods for treating genetic and non-genetic diseases, and methods
for predicting the efficacy of drugs are also discussed.
Inventors: |
Kaddurah-Daouk; Rima;
(Belmont, MA) ; Kristal; Bruce S.; (White Plains,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell Research Foundation, Inc.
Metabolon, Inc. |
Ithaca
Durham |
NY
NC |
US
US |
|
|
Assignee: |
CORNELL RESEARCH FOUNDATION,
INC.
Ithaca
NY
METABOLON, INC.
Durham
NC
|
Family ID: |
46301673 |
Appl. No.: |
14/106416 |
Filed: |
December 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12182828 |
Jul 30, 2008 |
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14106416 |
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11405033 |
Apr 14, 2006 |
7550258 |
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12182828 |
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10695265 |
Oct 27, 2003 |
7329489 |
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11405033 |
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09835119 |
Apr 13, 2001 |
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10695265 |
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60421226 |
Oct 25, 2002 |
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60239340 |
Oct 11, 2000 |
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60239541 |
Oct 10, 2000 |
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60197117 |
Apr 14, 2000 |
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60197085 |
Apr 14, 2000 |
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Current U.S.
Class: |
506/6 ; 506/10;
506/12 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/57 20180101; G01N 33/5038 20130101; G01N 33/6893 20130101;
G01N 2800/304 20130101; G01N 33/5011 20130101; G01N 33/5079
20130101; Y02A 90/10 20180101; G01N 33/4833 20130101; Y02A 90/26
20180101; G01N 2800/52 20130101; G01N 33/5091 20130101 |
Class at
Publication: |
506/6 ; 506/12;
506/10 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/483 20060101 G01N033/483 |
Claims
1. A method for identifying small molecules relevant to a disease
state, the method comprising: obtaining a small molecule profile of
a sample from a subject suffering from said disease state using two
or more techniques that detect 50% or more of the small molecules
in said sample, said small molecule profile comprising information
regarding the presence of two or more types of small molecules
selected from the group consisting of: sugars, fatty acids, amino
acids, nucleotides, metabolites, and products of catabolism; and
comparing the small molecule profile from said subject to a
standard small molecule profile, thereby identifying the small
molecules relevant to said disease state using said small molecule
profile.
2. The method of claim 1, wherein said subject is a human.
3. The method of claim 1, wherein said subject's small molecule
profile is obtained from said subject's tissue or biological
fluids.
4. The method of claim 3, wherein said tissue is stomach, colon,
skin, breast, muscle, lung, kidney, or liver tissue.
5. The method of claim 3, wherein said tissue comprises a
tumor.
6. The method of claim 5, wherein said tumor is malignant.
7. The method of claim 1, wherein said small molecule profile is
obtained from said subject's blood, spinal fluid, serum, cells,
tissue, or cellular organelles.
8. The method of claim 1, wherein said subject's small molecule
profile and said standard small molecule profile are obtained using
two or more of the following: HPLC, TLC, electrochemical analysis,
mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violet
spectroscopy (UV), fluorescence analysis, radiochemical analysis,
Near-Infra Red spectroscopy (Near-IR), Nuclear Magnetic Resonance
(NMR) and Light Scattering analysis (LS).
9. The method of claim 1, wherein said disease state is an
immunological, neurological, metabolic, oncological, viral, or
cardiovascular disorder, or a bacterial infection.
10. The method of claim 1, wherein said disease state is a nervous
system disorder.
11. The method of claim 10, wherein said nervous system disorder is
a neuropathy, Alzheimer disease, Parkinson's disease, Huntington's
disease, amyotrophic lateral sclerosis, motor neuron disease,
traumatic nerve injury, multiple sclerosis, acute disseminated
encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis,
dysmyelination disease, mitochondrial disease, migrainous disorder,
bacterial infection, fungal infection, stroke, aging, dementia,
peripheral nervous system diseases, or a mental disorder.
12. The method of claim 1, wherein said disease state is an
oncological disorder.
13. The method of claim 12, wherein said oncological disorder is
leukemia, brain cancer, pancreatic cancer, prostate cancer, liver
cancer, stomach cancer, colon cancer, throat cancer, breast cancer,
ovarian cancer, skin cancer, melanoma, lymphoma, or a cancerous
tumor.
14. The method of claim 12, wherein said oncological disorder is
prostate cancer.
15. The method of claim 1, wherein said disease state is a
metabolic disorder.
16. The method of claim 15, wherein said metabolic disorder is
diabetes.
17. The method of claim 16, wherein said diabetes is
insulin-dependent diabetes.
18. The method of claim 15, wherein said metabolic disorder is a
body weight disorder.
19. The method of claim 18, wherein said body weight disorder is
obesity, cachexia, or anorexia.
20. A method for identifying small molecules affected by an agent,
the method comprising: generating a small molecule profile of a
cellular compartment treated with an agent using one or more
techniques that detect 50% or more of the small molecules in said
sample, said small molecule profile comprising information
regarding the presence of two or more types of small molecules
selected from the group consisting of: sugars, fatty acids,
nucleotides, metabolites, products of catabolism, and amino acids,
wherein said cellular compartment is obtained from a patient;
wherein said cellular compartment is obtained from a patient; and
comparing said small molecule profile to a standard small molecule
profile, thereby identifying the small molecules affected by said
agent in said cellular compartment.
21. The method of claim 20, wherein said agent is a toxin.
22. The method of claim 20, wherein said agent is a therapeutic
agent.
23. The method of claim 22, wherein said therapeutic agent is a
therapeutic agent used for the treatment of a metabolic,
immunological, neurological, oncological, viral, or other
disorder.
24. The method of claim 20, wherein said patient is suffering from
a metabolic, immunological, neurological, oncological, viral, or
other disorder.
25. The method of claim 20, wherein said cellular compartment is a
cell.
26. The method of claim 20, wherein said cellular compartment is
mitochondria.
27. The method of claim 20, wherein said cellular compartment is a
nucleus.
28. The method of claim 20, wherein said small molecule profile of
a cellular compartment and said standard small molecule profile are
obtained using one or more of the following: gas chromatography
(GC), HPLC, TLC, electrochemical analysis, mass spectrometry, mass
spectroscopy, refractive index spectroscopy (RI), Ultra-Violet
spectroscopy (UV), fluorescent analysis, radiochemical analysis,
Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonance
spectroscopy (NMR), and Light Scattering analysis (LS).
29. A method for identifying small molecules affected by an agent
comprising: generating a small molecule profile of a sample from a
human subject treated with an agent using one or more techniques
that detect 50% or more of the small molecules in said sample, said
small molecule profile comprising information regarding the
presence of two or more types of small molecules selected from the
group consisting of: sugars, fatty acids, nucleotides, metabolites,
products of catabolism and amino acids; and comparing said small
molecule profile to a standard small molecule profile, thereby
identifying the small molecules affected by said agent.
30. The method of claim 29, wherein said standard small molecule
profile is a profile of said sample from said subject prior to
administration of said agent.
31. The method of claim 30, wherein said small molecule profile is
obtained from a cell culture.
32. The method of claim 30, wherein said small molecule profile is
obtained from a biological fluid of said subject.
33. The method of claim 32, wherein said biological fluid is blood,
serum, spinal fluid or saliva.
34. A method for metabolomically predicting a human's response to a
therapeutic agent, the method comprising: generating a small
molecule profile of a sample from said human using one or more
techniques that detect 50% or more of the small molecules in said
sample, said small molecule profile comprising information
regarding the presence of two or more types of small molecules
selected from the group consisting of sugars, fatty acids,
nucleotides, metabolites, products of catabolism and amino acids;
and comparing said small molecule profile of said sample from said
human to a known standard established for the therapeutic agent as
an indication of whether the human would benefit from treatment
with the therapeutic agent, said known standard obtained from one
or more humans treated with the agent, thereby predicting said
human's response to said therapeutic agent.
35. The method of claim 34, wherein said human is suffering or
suspected of suffering from a disease state.
36. The method of claim 35, wherein said disease state is an
immunological disorder, a neurological disorder, a metabolic
disorder, an oncological disorder, a viral disorder, a
cardiovascular disorder, or a bacterial infection.
37. The method of claim 35, wherein said disease state is a nervous
system disorder.
38. The method of claim 37, wherein said nervous system disorder is
a neuropathy, Alzheimer disease, Parkinson's disease, Huntington's
disease, amyotrophic lateral sclerosis, motor neuron disease,
traumatic nerve injury, multiple sclerosis, acute disseminated
encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis,
dysmyelination disease, mitochondrial disease, migrainous disorder,
bacterial infection, fungal infection, stroke, aging, dementia,
peripheral nervous system diseases, or a mental disorder.
39. The method of claim 34, wherein said human's small molecule
profile is obtained from said human's tissue or biological
fluids.
40. The method of claim 34, wherein said human's small molecule
profile is obtained from said human's blood, spinal fluid, serum,
cells, cellular organelles, urine, interstitial fluid, or
saliva.
41. The method of claim 34, wherein said small molecule profile of
said sample from said human and said known standard are obtained
using one or more of the following: HPLC, TLC, electrochemical
analysis, mass spectroscopy, refractive index spectroscopy (RI),
Ultra-Violet spectroscopy (UV), fluorescent analysis, radiochemical
analysis, Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic
Resonance spectroscopy (NMR), gas chromatography (GC), and Light
Scattering analysis (LS).
42. The method of claim 34, wherein said therapeutic agent is an
anti-tumor drug.
43. The method of claim 34, wherein said therapeutic agent is
administered during clinical trials.
44. The method of claim 34, wherein said therapeutic agent is a
peptidomimetic, a protein, a peptide, a small molecule, or a drug
candidate.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/182,828, filed on Jul. 30, 2008, which is a
continuation of U.S. patent application Ser. No. 11/405,033, filed
on Apr. 14, 2006, which is a continuation of U.S. patent
application Ser. No. 10/695,265, filed on Oct. 27, 2003, now U.S.
Pat. No. 7,329,489; which claims priority to U.S. Provisional
Patent Application Ser. No. 60/421,226, filed on Oct. 25, 2002; and
which is also a continuation-in-part of U.S. patent application
Ser. No. 09/835,119, filed on Apr. 13, 2001; which claims priority
to U.S. Provisional Application Ser. No. 60/239,340, filed on Oct.
11, 2000; U.S. Provisional Application Ser. No. 60/239,541, filed
on Oct. 10, 2000; U.S. Provisional Application Ser. No. 60/197,117,
filed on Apr. 14, 2000; and U.S. Provisional Application Ser. No.
60/197,085, filed on Apr. 14, 2000. The entire contents of each of
the aforementioned applications and patents are hereby incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Living organisms are autonomous chemical systems which
include diverse sets of small molecules. Small molecules found in
living systems include, for example, sugars, fatty acids, amino
acids, nucleotides, and intermediates of metabolic and signaling
pathways. Sugars are a primary source of chemical energy for cells.
The cells break the sugars down through a series of oxidative
reactions to small sugar derivatives and, ultimately, CO.sub.2 and
H.sub.2O. Fatty acids used for both energy storage and as major
components of cellular membranes. Amino acids are the building
blocks of proteins. Nucleotides are involved in intracellular
signaling, energy transfer, and as the monomers of the information
macromolecules, RNA and DNA.
[0003] The cellular small molecules are, generally, composed of six
elements (C, H, N, O, P, S). If water is excluded, carbon compounds
comprise a large majority of the cellular small molecules. The
cellular small molecules repeatedly use certain distinctive
chemical groups, such as methyl (CH.sub.3), carboxyl (COOH) and
amino (NH.sub.2) groups.
[0004] Generally, most cellular small molecules are synthesized
from and broken down to the same basic compounds. Synthesis and
metabolism occurs through sequences of controlled chemical
reactions, catalyzed by enzymes. Most of the metabolic reactions of
the cell occur in the cytoplasm, which contains many distinctive
organelles. For example, the mitochondria are responsible for
respiration and energy production. Mitochondria are the "power
plants" of eukaryotic cells, harnessing energy contained by
combining oxygen with metabolites to make ATP. Other organelles of
the cell include the Golgi apparatus, a system of stacked, membrane
bound, flattened sacs involved in modifying, sorting and packaging
of macromolecules for secretion or for delivery to other
organelles. The endoplasmic reticulum (ER) is a series of flattened
sheets, sacks, and tubes of membrane extending throughout the
cytoplasm of eukaryotic cells. The ER membrane is in structural
continuity with the outer membrane of the nuclear envelope and
specializes in the synthesis and transport of lipids and membrane
proteins.
SUMMARY OF THE INVENTION
[0005] In recent years, scientists have attempted to study cells
and living systems through the cataloging of the entire genome of
an organism (e.g., genomics). Genomics is a powerful tool, useful
for identifying and interrogating the entire inventory of genes of
a living system. Recently, scientists have also attempted to
identify and interrogate all the proteins present in the cell or
organism through proteomics. However, most pharmaceutical companies
who study genomics and proteomics realize that many of their
anticipated products are not proteins nor genes but small
molecules.
[0006] For example, once a novel gene or target is discovered by
genomics, the investigators must first validate the target using
expensive and time consuming procedures which are far removed from
the actual disease state. Examples of typical validation procedures
include expression profiling, generating knock-out mice or
transgenic mice, in situ hybridization, etc. Once a target is
validated, the investigators typically screen enormous random small
molecule libraries to identify molecules which interact with the
protein targets. The identified small molecules typically optimized
through chemical synthesis in order to obtain a marketable
product.
[0007] The invention pertains, at least in part, to the generation
and the analysis of small molecule profiles of cells, cellular
compartments, and specific organelles (e.g., mitochondria, Golgi,
endoplasmic reticulum, cytoplasm, nucleus, etc.) Small molecule
profiles allow for the identification and interrogation of
inventories of small molecules (e.g., the metabolome) to find, for
example, disease-relevant small molecules as well as potential
targets for drug design.
[0008] Small molecule profiles of cells and organelles can be used
directly to identify drug candidates. Unlike genomics, small
molecule profiling can either eliminate entirely or accelerate the
process of identifying genes and proteins associated with a disease
state. In one embodiment of the invention, the methods of the
invention include, for example, comparing small molecule profiles
of diseased cells, cellular compartments, and organelles to
standard profiles of a healthy cells, cellular compartments, and
organelles. Therefore, if a particular diseased cell, cellular
compartment, or organelle was found to be deficient in a particular
compound, the deficiency may be overcome by simply administering
the compound or an analogue thereof. Metabolomics offers a new
route to the identification of potentially therapeutic agents and
targets.
[0009] Metabolomics eliminates much of the guess work surrounding
genomics. For example, small molecule profiling allows one to
investigate the very biochemical pathway (e.g., cellular
metabolites) involved in the disease state by comparing small
molecule profiles of cells, cellular compartments, or organelles
with those of cells, cellular compartments, or organelles treated
with toxins, chemical agents or other therapeutic agent (or derived
from an organism treated with the agent or drug).
[0010] The invention also includes methods for identifying
potential cell drug targets (e.g., cellular components which
interact with the labeled small molecules). This method is
particularly useful because it can identify components which are
known to interact with disease relevant small molecules. Therefore,
targets identified through this method are "pre-validated," and
some of the guess work surrounding the choice of target is
eliminated. In a further embodiment, this method can be used in
conjunction with conventional genomics as a further validation step
to identify targets for further research.
[0011] Unlike genomics, small molecule profiling is not limited to
disease states with a genetic component. Many disease states are
not genetically determined and genomics offers little to those
suffering or at risk of suffering from non-genetic linked disease
states. Therefore, there is a need for a comprehensive method to
study the effects of nongenetic factors on cells and living
systems.
[0012] Small molecule profiling of cells, organelles, or
extracellular material can be used to study both genetic and
non-genetically linked disease states. For example, methods of the
invention can be used to identifying small molecules associated
with, for example, body weight disorders, central nervous system
disorders, cardiovascular disorders, immunological disorders,
oncological disorders, etc.
[0013] In addition, metabolomics can be used in tandem with
genomics and/or proteomics. For example, small molecule profiles
can be used to identify small molecules regulated, modulated, or
associated with genetic modification or alterations of cells, both
engineered and naturally occurring.
[0014] In addition, metabolomics can also be applied to the field
of predictive medicine. For example, the invention pertains to
diagnostic assays, prognostic assays, pharmacometabolomics, and the
monitoring clinical trails which are used for prognostic
(predictive) purposes to treat an individual prophylactically,
based on an individual's "metaboprint." Unlike pharmacogenetics,
which is limited to genetic factors, pharmacometabolomics is able
to predict an individual's response to a drug based not only on
genetic factors, but also non-genetic factors, such as other drugs
in the patient's body, the patient's current state of health, etc.
Pharmacometabolomics allows for the use of a subject's small
molecule profile (or "metaboprint") to deliver the right drug to
the right patient. Subjects respond differently to drugs based on
their small molecule profiles.
[0015] The small molecule profiling of cells, cellular
compartments, particular organelles, and/or extracellular material
of the present invention can also be used to identify individuals
from minute biological samples. The method includes taking one or
more samples from a subject and determining the small molecule
profiles of the samples; taking a sample from a unknown source and
determining its small molecule profile; and comparing the two small
molecule profiles to determine whether the small molecule profiles
are from the same individual.
[0016] The invention also pertains, for example, to pharmaceutical
compositions comprised of compounds identified by the methods of
the invention and a pharmaceutical carrier.
[0017] In another embodiment of the invention, the invention
includes a method for the identification of insecticides,
herbicides, and other compositions for agricultural use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a graph which shows markers in subjects with
amylotropic lateral sclerosis (ALS) who are not taking
Riluzole.
[0019] FIG. 2 is a graph which shows markers in subjects with
amylotropic lateral sclerosis (ALS) who are taking Riluzole.
[0020] FIG. 3 is a three dimensional graph showing the statistical
separation of subjects. The population, ALS1, are suffering from
ALS and are undergoing Riluzole treatment, while the population
ALS2 are suffering from ALS and not undergoing Riluzole
treatment.
[0021] FIG. 4 is a partial least squares-discriminant analysis
(PLS-DA) which separates ALS patients from their control
counterparts.
[0022] FIG. 5 is shows three chromatograms generated using the
HPLC-CEAS for subjects suffering from ALS.
[0023] FIGS. 6A, 6B, and 6C are based on an HPLC analysis of the
metabolome. In FIG. 6A, 39 compounds are reduced. In FIG. 6B, it is
shown that 17 compounds are increased in patients on Riluzole (RZ).
FIG. 6B also shows that 17 other compounds are increased in ALS
patients with prominent lower motor neuron signs and slow course.
In FIG. 6C, partial least squares-discriminant analysis was used to
differentiate controls, subjects suffering from ALS on Riluzole,
subjects suffering from ALS not on Riluzole and subjects suffering
from lower motor neuron signs and slow course.
DETAILED DESCRIPTION OF THE INVENTION
1. Small Molecule Profiles of Cells, Cellular Compartments, and
Organelles
[0024] The invention pertains, at least in part, to the generation
of small molecule profiles of samples, cells, and cellular
compartments. Small molecule profiles "fingerprint" the cell or
cellular compartment and identify the presence, absence or relative
quantity of small molecules. The small molecule profiles of the
cells or cellular compartments may be obtained through, for
example, a single technique or a combination of techniques for
separating and/or identifying small molecules known in the art.
Examples of separation and analytical techniques which can be used
to separate and identify the compounds of the small molecule
profiles include: HPLC, TLC, electrochemical analysis, mass
spectroscopy, refractive index spectroscopy (RI), Ultra-Violet
spectroscopy (UV), fluorescent analysis, radiochemical analysis,
Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonance
spectroscopy (NMR), Light Scattering analysis (LS) and other
methods known in the art. Preferably, the methods of the invention
detect both electrically neutral as well as electrochemically
active compounds. Detection and analytical techniques can be
arranged in parallel to optimize the number of molecules
identified.
[0025] The term "sample" include cellular extracts from which a
small molecule profile of the extract can be obtained. In one
embodiment, the samples are substantially free of macromolecules
(e.g., large proteins and polynucleotides with molecular weights of
greater than 10,000). The sample may be obtained from the entire
cell or from specific cellular compartments. Examples of specific
cellular compartments include the cytoplasm, the mitochondria, the
Golgi apparatus, the endoplasmic reticulum, the nucleus, the
chloroplasts, the cytosol, etc. The term "samples" includes both
isolated small molecules and mixtures of small molecules.
[0026] The term "cells" includes prokaryotic cells, eukaryotic
cells, yeast cells, bacterial cells, plant cells, animal cells,
such as, reptilian cells, bird cells, fish cells, mammalian cells.
Preferred cells include those derived from humans, dogs, cats,
horses, cattle, sheep, pigs, llamas, gerbils, squirrels, goats,
bears, chimpanzees, mice, rats, rabbits, etc. The term cells
includes transgenic cells from cultures or from transgenic
organisms. The cells may be from a specific tissue, body fluid,
organ (e.g., brain tissue, nervous tissue, muscle tissue, retina
tissue, kidney tissue, liver tissue, etc.), or any derivative
fraction thereof. The term includes healthy cells, transgenic
cells, cells affected by internal or exterior stimuli, cells
suffering from a disease state or a disorder, cells undergoing
transition (e.g., mitosis, meiosis, apoptosis, etc.), etc.
[0027] In a further embodiment, the samples are obtained from a
specific cellular compartment. The term "cellular compartment"
includes organelles (such as mitochondria, Golgi apparatus,
centrioles, chloroplasts), the nucleus, the cytoplasm (optionally
including the organelles), and other cellular regions capable of
being isolated. In one embodiment, the cellular compartment is the
entire cell.
[0028] The analysis of a particular cellular compartment has many
advantages over analysis of whole cells, whole cell lysates, body
fluids, etc. For example, often the mechanism of action of a drug,
a toxic compound, etc. is directed to a specific cellular function,
such as, for example, the electron transport chain in the
mitochondria, nucleic acid replication in the nucleus, etc. By
isolating the specific cellular compartment or organelle (e.g.,
mitochondria, nuclei, Golgi apparatus, endoplasmic reticulum,
ribosomes, etc.), it is possible to narrow the focus of the profile
to small molecules involved in the relevant pathway. Previously,
metabolome studies have been complicated by the large number of
chemical species present in a given sample. By narrowing the scope
of the study to the particular organelle, researchers will be able
to study the pathway of interest in more detail without irrelevant
molecules present in interstitial fluid, blood, spinal fluid,
saliva, etc. The term "small molecules" includes organic and
inorganic molecules which are present in the cell, cellular
compartment, or organelle. The term does not include large
macromolecules, such as large proteins (e.g., proteins with
molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,
8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids
with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g.,
polysaccharides with a molecular weights of over 2,000, 3,000,
4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). The small
molecules of the cell are generally found free in solution in the
cytoplasm or in other organelles, such as the mitochondria, where
they form a pool of intermediates which can be metabolized further
or used to generate large molecules, called macromolecules. The
term "small molecules" includes signaling molecules and
intermediates in the chemical reactions that transform energy
derived from food into usable forms. Examples of small molecules
include sugars, fatty acids, amino acids, nucleotides,
intermediates formed during cellular processes, and other small
molecules found within the cell. In one embodiment, the small
molecules of the invention are isolated.
[0029] The term "metabolome" includes all of the small molecules
present in a given organism. The metabolome includes both
metabolites as well as products of catabolism. In one embodiment,
the invention pertains to a small molecule profile of the entire
metabolome of a species. In another embodiment, the invention
pertains to a computer database (as described below) of the entire
metabolome of a species, e.g., an animal, e.g., a mammal, e.g., a
mouse, rat, rabbit, pig, cow, horse, dog, cat, bear, monkey, and,
preferably, a human. In another embodiment, the invention pertains
to a small molecule library of the entire metabolome of an organism
(as described below), e.g., a mammal, e.g., a mouse, rat, rabbit,
pig, cow, horse, dog, cat, bear, monkey, and, preferably, a
human.
[0030] The language "small molecule profile" includes the inventory
of small molecules in tangible form within a targeted cell, tissue,
organ, organism, or any derivative fraction thereof, e.g., cellular
compartment, that is necessary and/or sufficient to provide
information to a user for its intended use within the methods
described herein. The inventory would include the quantity and/or
type of small molecules present. The ordinarily skilled artisan
would know that the information which is necessary and/or
sufficient will vary depending on the intended use of the "small
molecule profile." For example, the "small molecule profile," can
be determined using a single technique for an intended use but may
require the use of several different techniques for another
intended use depending on such factors as the disease state
involved, the types of small molecules present in a particular
targeted cellular compartment, the cellular compartment being
assayed per se., etc.
[0031] The relevant information in a "small molecule profile" also
may vary depending on the intended use of the compiled information,
e.g. spectra. For example for some intended uses, the amounts of a
particular small molecule or a particular class of small molecules
may be relevant, but for other uses the distribution of types of
small molecules may be relevant.
[0032] The ordinarily skilled artisan would be able to determine
the appropriate "small molecule profiles" for each method described
herein by comparing small molecule profiles from diseased and/or
test subjects with standard and/or healthy subjects. These
comparisons can be made by individuals, e.g., visually, or can be
made using software designed to make such comparisons, e.g., a
software program may provide a secondary output which provides
useful information to a user. For example, a software program can
be used to confirm a profile or can be used to provide a readout
when a comparison between profiles is not possible with a "naked
eye". The selection of an appropriate software program, e.g., a
pattern recognition software program, is within the ordinary skill
of the art. An example of such a program is Pirouette. It should be
noted that the comparison of the profiles can be done both
quantitatively and qualitatively.
[0033] The small molecule profiles can be obtained from an organism
suffering from a disease state, genetic alteration, or any of the
models discussed in more detail below. In one embodiment, the small
molecule profile of an organism is determined by using HPLC
(Kristal, et al. Anal. Biochem. 263:18-25 (1998)), thin layer
chromatography (TLC), or electrochemical separation techniques
(see, WO 99/27361, WO 92/13273, U.S. Pat. No. 5,290,420, U.S. Pat.
No. 5,284,567, U.S. Pat. No. 5,104,639, U.S. Pat. No. 4,863,873,
and U.S. RE32,920). Other techniques for determining the presence
of small molecules or determining the identity of small molecules
of the cell are also included, such as refractive index
spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent
analysis, radiochemical analysis, Near-InfraRed spectroscopy
(Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light
Scattering analysis (LS) and other methods known in the art. The
small molecule profiles of the invention may also be referred to as
"metaboprints." The exact combination of techniques used to
determine the small molecule profiles can be determined by
[0034] In one embodiment, the invention pertains to small molecule
profiles generated by several methods, e.g., HPLC, TLC,
electrochemical analysis, mass spectroscopy, refractive index
spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent
analysis, radiochemical analysis, Near-InfraRed spectroscopy
(Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light
Scattering analysis (LS) and other methods known in the art.
[0035] The methods of the invention have several advantages over
methods which rely only on a single mode of analysis, such as
electrochemical separation. While electrochemical separation works
only for "electrochemically" active compounds, it does not
effectively separate neutral molecules. The invention here relates
to the use in tandem and in parallel of a multitude of these
detectors. This will result in the identification of a more
comprehensive database. The detectors are usually attached to the
HPLC columns where they can detect and emit a response due to the
eluting sample and subsequently signal a peak on a chromatogram.
The bandwidth and height of the peaks may usually be adjusted using
the coarse and fine tuning controls and the detection and
sensitivity parameters may also be controlled. There are many
detectors that can be used with the HPLC. Some detectors which can
be used in the methods of the invention include: Refractive Index
(RI), Ultra-Violet (UV), Fluorescent, Radiochemical,
Electrochemical, Near-InfraRed (Near-IR), Mass Spectroscopy (MS),
Nuclear Magnetic Resonance (NMR), Light Scattering (LS) among
others.
[0036] The methods of the invention can be used to detect both
electrochemically active molecules as well as electrochemically
neutral molecules. In a further embodiment, the invention pertains
to methods which detect about 50% or more, about 60% or more, about
70% or more, about 75% or more, about 77.5% or more, about 80% or
more, about 82.5% or more, about 85% or more, about 86% or more,
about 87% or more, about 88% or more, about 89% or more, about 90%
or more, about 91% or more, about 92% or more, about 93% or more,
about 94% or more, about 95% or more, about 96% or more, about 97%
or more, about 98% or more, about 99% or more of the small
molecules of a cellular compartment (e.g., mitochondria,
chloroplast, endoplasmic reticulum, nuclei, Golgi apparatus,
cytosol, etc.).
[0037] In one embodiment, HPLC columns equipped with coulometric
array technology can be used to analyze the samples, separate the
compounds, and/or create a small molecule profiles of the samples.
Such HPLC columns have been used extensively in the past for serum,
urine and tissue analysis and are suitable for small molecule
analysis (Acworth et al., 300; Beal et al., J. Neurochem. 55,
1327-1339, 1990; Matson et al., Life Sci. 41, 905-908, 1987; Matson
et al., Basic, Clinical and Therapeutic Aspects of Alzheimer's and
Parkinson's Diseases, vol II, pp. 513-516, Plenum, New York 1990;
LeWitt et al., Neurology 42, 2111-2117, 1992; Milbury et al., J.
Wildlife Manag., 1998; Ogawa et al., Neurology 42, 1702-1706, 1992;
Beal et al., J. Neurol. Sci 108, 80-87, 1992, Matson et al., Clin.
Chem. 30, 1477-1488, 1984; Milbury et al., Coulometric Electrode
Array Detectors for HPLC, pp. 125-141, VSP International Science
Publication; Acworth et al., Am. Lab 28, 33-38, 1996). HPLC columns
equipped with coulometric arrays have been used for the
simultaneous analysis of the majority of low-molecule weight,
redox-active compounds in mitochondria. (Anal. Biochem. 263, 18-25,
1998).
[0038] For the detection and characterization of the small
molecules in an effort to create a comprehensive small molecule
profiles, a multitude of detection methods can be used. These
methods are described in more detail below.
A. Mass Spectroscopy (MS) Detectors:
[0039] The sample compound or molecule is ionized, it is passed
through a mass analyzer, and the ion current is detected. There
various methods for ionization. Examples of these methods of
ionization include electron impact (EI) where an electric current
or beam created under high electric potential is used to ionize the
sample migrating off the column, chemical ionization utilizes
ionized gas to remove electrons from the compounds eluting from the
column; and fast atom bombardment where Xenon atoms are propelled
at high speed in order to ionize the eluents from the column
B. Pyrolysis Mass Spectrometry:
[0040] Pyrolysis is the thermal degradation of complex material in
an inert atmosphere or vacuum. It causes molecules to cleave at
their weakest points to produce smaller, volatile fragments called
pyrolysate (Irwin 1982). Curie-point pyrolysis is a particularly
reproducible and straightforward version of the technique, in which
the sample, dried onto an appropriate metal is rapidly heated to
the Curie-point of the metal. A mass spectrometer can then be used
to separate the components of the pyrolysate on the basis of their
mass-to-charge ratio to produce a pyrolysis mass spectrum
(Meuzelaar et al 1982) which can then be used as a "chemical
profile" or fingerprint of the complex material analyzed. The
combined technique is known as pyrolysis mass spectrometry
(PyMS).
C. Nuclear Magnetic Resonance (NMR) Detectors:
[0041] Certain nuclei with odd-numbered masses, including H and
.sup.13C, spin about an axis in a random fashion. When they are
placed between poles of a strong magnet, the spins are aligned
either parallel or anti-parallel to the magnetic field, with
parallel orientation favored since it is slightly lower energy. The
nuclei are then irradiated with electromagnetic radiation which is
absorbed and places the parallel nuclei into a higher energy state
where they become in resonance with radiation. Different spectra
will be produced depending on the location of the H or .sup.13C and
on adjacent molecules or elements in the compound because all
nuclei in molecules are surrounded by electron clouds which change
the encompassing magnetic field and thereby alter the absorption
frequency.
D. Refractive Index (RI):
[0042] In this method, detectors measure the ability of samples to
bend or refract light. This property for each compound is called
refractive index. For most RI detectors, light proceeds through a
bi-modular flow to a photodetector. One channel of the flow-cell
directs the mobile phase passing through the column while the other
directs only the other directs only the mobile phase. Detection
occurs when the light is bent due to samples eluting from the
column, and is read as a disparity between the two channels. Laser
based RI detectors have also become available.
E. Ultra-Violet (UV) Detectors:
[0043] In this method, detectors measure the ability of a sample to
absorb light. This could be accomplished at a fixed wavelength
usually 254 nm, or at variable wavelengths where one wavelength is
measured at a time and a wide range is covered, alternatively Diode
Array are capable of measuring a spectrum of wavelengths
simultaneously. Sensitivity is in the 10.sup.-8 to 10.sup.-9 gm/ml
range. Laser based absorbance or Fourier Transform methods have
also been developed.
F. Fluorescent Detectors:
[0044] This method measure the ability of a compound to absorb then
re-emit light at given wavelengths. Each compound has a
characteristic fluorescence. Each compound has a characteristic
fluorescence. The excitation source passes through the flow-cell to
a photodetector while a monochromator measures the emission
wavelengths. Sensitivity is in the 10.sup.-9 to 10.sup.-11 gm/ml.
Laser based fluorescence detectors are also available.
G. Radiochemical Detection:
[0045] This method involves the use of radiolabeled material, for
example, tritium (.sup.3H) or carbon 14 (.sup.14C). It operates by
detection of fluorescence associated with beta-particle ionization,
and it is most popular in metabolite research. The detector types
include homogeneous method where addition of scintillation fluid to
column effluent causes fluorescence, or heterogeneous detection
where lithium silicate and fluorescence by caused by beta-particle
emission interact with the detector cell. Sensitivity is 10.sup.-9
to 10.sup.-10 gm/ml.
H. Electrochemical Detection:
[0046] Detectors measure compounds that undergo oxidation or
reduction reactions. Usually accomplished by measuring gains or
loss of electrons from migration samples as they pass between
electrodes at a given difference in electrical potential.
Sensitivity of 10.sup.-12 to 10.sup.-13 gms/ml.
I. Light Scattering (LS) Detectors:
[0047] This method involves a source which emits a parallel beam of
light. The beam of light strikes particles in solution, and some
light is then reflected, absorbed, transmitted, or scattered. Two
forms of LS detection may be used to measure transmission and
scattering.
[0048] Nephelometry, defined as the measurement of light scattered
by a particular solution. This method enables the detection of the
portion of light scattered at a multitude of angles. The
sensitivity depends on the absence of background light or scatter
since the detection occurs at a black or null background.
Turbidimetry, defined as the measure of the reduction of light
transmitted due to particles in solution. It measures the light
scatter as a decrease in the light that is transmitted through
particulate solution. Therefore, it quantifies the residual light
transmitted. Sensitivity of this method depends on the sensitivity
of the machine employed, which can range from a simple
spectrophotometer to a sophisticated discrete analyzer. Thus, the
measurement of a decrease in transmitted light from a large signal
of transmitted light is limited to the photometric accuracy and
limitations of the instrument employed.
[0049] Near Infrared scattering detectors operate by scanning
compounds in a spectrum from 700-1100 nm. Stretching and bending
vibrations of particular chemical bonds in each molecule are
detected at certain wavelengths. This is a fast growing method
which offers several advantages; speed, simplicity of preparation
of sample, multiple analyses from single spectrum and
nonconsumption of the sample (McClure, 1994).
J. Fourier Transform Infrared Spectroscopy (FT-IR):
[0050] This method measures dominantly vibrations of functional
groups and highly polar bonds. The generated fingerprints are made
up of the vibrational features of all the sample components
(Griffiths 1986). FT-IR spectrometers record the interaction of IR
radiation with experimental samples, measuring the frequencies at
which the sample absorbs the radiation and the intensities of the
absorptions. Determining these frequencies allows identification of
the samples chemical makeup, since chemical functional groups are
known to absorb light at specific frequencies. Both quantitative
and qualitative analysis are possible using the FT-IR detection
method.
K. Dispersive Raman Spectroscopy:
[0051] Dispersive Raman Spectroscopy is a vibrational signature of
a molecule or complex system. The origin of dispersive raman
spectroscopy lies in the inelastic collisions between the molecules
composing say the liquid and photons, which are the particles of
light composing a light beam. The collision between the molecules
and the photons leads to an exchange of energy with consequent
change in energy and hence wavelength of the photon.
[0052] To create a small molecule profile (or "Metaboprint")
organs, cells, cellular compartments, or organelles are homogenized
in standard ways know for those skilled in the art. Different
fractionation procedures can be used to enrich the fractions for
small molecules. The small molecules obtained will then be passed
over several fractionation columns. The fractionation columns will
employ a variety of detectors used in tandem or parallel to
generate the small molecule profile for the organ, cell, cellular
compartment, or organelle.
[0053] For example, to generate a small molecule profile of water
soluble molecules, the cell, cellular compartment, or organelle
extracts will be fractionated on HPLC columns with a water soluble
array. The water soluble small molecules can then be detected using
fluorescence or UV detectors to generate the small molecule
profiles. Alternatively, electrochemical detectors can be used with
diads to pick up redox active compounds and the absorbance of
active compounds. For generating detecting non water soluble
molecules, hydrophobic columns can also be used to generate small
molecule profiles. In addition, gas chromatography combined with
mass spectroscopy, liquid chromatography combined with mass
spectroscopy, MALDI combined with mass spectroscopy, ion spray
spectroscopy combined with mass spectroscopy, capillary
electrophoresis, NMR and IR detection are among the many other
combinations of separation and detection tools which can be used to
generate small molecule profiles.
[0054] These small molecule profiles (or "metaboprints") will be
able to define and characterize organs, cells, cellular
compartments, and organelles by their small molecule content in
both health and disease states. The information generated by the
small molecule profiles will be both qualitative and
quantitative.
2. Methods of Identification of Disease-Relevant Small
Molecules
[0055] In another embodiment, the invention includes a method of
identifying disease-relevant small molecules. The method includes
comparing small molecule profiles of diseased cells, cellular
compartments, or organelles to a standard profile of a healthy
cell, cellular compartment, or organelle. The method also involves
identifying the small molecules which are present in aberrant
amounts in the diseased small molecule profile. The small molecules
present in aberrant amounts in the diseased cells are
"disease-relevant small molecules."
[0056] The language "disease-relevant small molecules" includes
both small molecules present in aberrant amount in diseased small
molecule profiles and, in addition, small molecules which are
potentially involved in disease initiation, progression or
prediction. The term also includes small molecules which are
identified using the assays for particular diseases given below, as
well as, compounds which are identified as being associated with
particular genes of interest, also given below. The term also may
include small molecules which when modulated, result in the
lessening or curing of at least one symptom of a disease. The
disease relevant small molecules are ideal drug candidates in the
screening assays discussed elsewhere in the application.
[0057] For example, identified disease relevant small molecules may
be screened using in vitro or in vivo assays known in the art to
determine biological activity. The biological activity of disease
relevant small molecules can also be pinpointed by using screening
assays against protein targets which have been implicated in the
disease state. In another embodiment, the biological activity of
disease relevant small molecules can be determined using cell-based
assays, e.g., tumor cell assays (Lillie et al. Cancer Res.
53(13):3172-8 (1993)). The disease relevant small molecules can
also be tested for neuronal protection activity by exposing primary
or cultured neurons to the compounds and toxic agents, such as
glutamate, and identifying the compounds which protect the neurons
from death Animal models can also be used to further identify the
biological activity of disease relevant small molecules. For
example, animal models of Huntington's Disease, Parkinson's
disease, and ALS can be used to identify small molecules useful as
neuroprotective agents. (Kilvenyi, Nature Med. 5:347-350 (1999);
Mathews et al, Experimental Neurology 157:142-149 (1999)). In a
further embodiment, the disease relevant small molecules can be
chemically modified to further enhance their pharmaceutical or
nutriceutical properties.
[0058] The term "disease" or "disease state" includes all disease
which result or could potentially cause a change of the small
molecule profile of a cell, cellular compartment, or organelle in
an organism afflicted with said disease. Examples of diseases
include metabolic diseases (e.g., obesity, cachexia, diabetes,
anorexia, etc.), cardiovascular diseases (e.g., atherosclerosis,
ischemia/reperfusion, hypertension, restenosis, arterial
inflammation, etc.), immunological disorders (e.g., chronic
inflammatory diseases and disorders, such as Crohn's disease,
reactive arthritis, including Lyme disease, insulin-dependent
diabetes, organ-specific autoimmunity, including multiple
sclerosis, Hashimoto's thyroiditis and Grave's disease, contact
dermatitis, psoriasis, graft rejection, graft versus host disease,
sarcoidosis, atopic conditions, such as asthma and allergy,
including allergic rhinitis, gastrointestinal allergies, including
food allergies, eosinophilia, conjunctivitis, glomerular nephritis,
certain pathogen susceptibilities such as helminthic (e.g.,
leishmaniasis) and certain viral infections, including HIV, and
bacterial infections, including tuberculosis and lepromatous
leprosy, etc.), nervous system disorders (e.g., neuropathies,
Alzheimer disease, Parkinson's disease, Huntington's disease,
amyotropic lateral sclerosis, motor neuron disease, traumatic nerve
injury, multiple sclerosis, acute disseminated encephalomyelitis,
acute necrotizing hemorrhagic leukoencephalitis, dysmyelination
disease, mitochondrial disease, migrainous disorder, bacterial
infection, fungal infection, stroke, aging, dementia, peripheral
nervous system diseases and mental disorders such as depression and
schizophrenia, etc.), oncological disorders (e.g., leukemia, brain
cancer, pancreatic cancer, prostate cancer, liver cancer, stomach
cancer, colon cancer, throat cancer, breast cancer, ovarian cancer,
skin cancer, melanoma, etc.). The term also include disorders which
result from oxidative stress.
[0059] The language "aberrant levels" includes any level, amount,
or concentration of a small molecule in a cell, cellular
compartment, or organelle which is different from the level of the
small molecule of a standard sample.
[0060] The term "standard profile" includes profiles derived from
healthy cells, advantageously from a similar origin as the source.
In one embodiment, the standard profile is an average of many
samples of a certain cell type and/or a certain cellular
compartment. In another embodiment, the standard profile may be
derived from a patient prior to the onset of the disease state or
from cells not affected by the disease state. Or, in another
embodiment the standard profile can be an average of the profiles
obtained from numerous sources, e.g., the standard profile may be
an average of small molecule profiles obtained from 2 or more
subjects. The standard profile can be a small molecule profile of a
certain cellular compartment or from a certain subset of cells. In
one embodiment, the invention pertains to the standard profile of
healthy cells. Advantageously, the small molecules with aberrant
levels in the sample are identified, e.g., HPLC, TLC,
electrochemical analysis, mass spectroscopy, refractive index
spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent
analysis, radiochemical analysis, Near-InfraRed spectroscopy
(Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light
Scattering analysis (LS) and other methods known in the art. In one
embodiment, the small molecule profile of the sample, cell, or
cellular compartment, is compared to the standard profile by using
subtracting one profile from the other. The compounds which are
present in aberrant amounts can then be used in drug design to
identify deregulated cellular components. Standard profiles can
also be made of the effects of certain agents (e.g., drugs,
therapeutic agents, toxins, etc.) on both healthy and diseased
cells (e.g., cells diseased with the type of disease treated by the
therapeutic agent).
[0061] Furthermore the language "standard profile" includes
information regarding the small molecules of the profile that is
necessary and/or sufficient to provide information to a user for
its intended use within the methods described herein. The standard
profile would include the quantity and/or type of small molecules
present. The ordinarily skilled artisan would know that the
information which is necessary and/or sufficient will vary
depending on the intended use of the "standard profile." For
example, the "standard profile," can be determined using a single
technique for an intended use but may require the use of several
different techniques for another intended use depending on such
factors as the types of small molecules present in a particular
targeted cellular compartment, the cellular compartment being
assayed per se., etc.
[0062] The relevant information in a "standard profile" also may
vary depending on the intended use of the compiled information,
e.g. spectra. For example for some intended uses, the amounts of a
particular small molecule or a particular class of small molecules
of the standard profile may be relevant, but for other uses the
distribution of types of small molecules small molecules of the
standard profile may be relevant.
[0063] Furthermore, comparison of the standard profiles to profiles
from diseased cells can be used to identify small molecules
deregulated in the disease state. The small molecules identified
can be used to guide the drug discovery effort. For example, the
small molecules present in aberrant levels in the sample cells, can
be identified and used as pharmaceutical or nutricutical agents.
For example, if a patient is suffering from a disease state
associated with a aberrantly low level of a certain compound, the
compound or a precursor thereof may be tested in an assay that
mimics the disease state. In another embodiment, the small
molecules present in aberrant amounts may be used as targets for
drug design to develop agents with enhanced activity, e.g.,
enhanced activity to treat the disease state associated with the
aberrant levels of the small molecule. Additionally libraries of
small molecules based on the structures of the small molecules
present in aberrant amounts can be used to develop more potent
therapeutics. The cellular targets and pathways could also be used
to guide drug design.
[0064] In a further embodiment, the invention pertains to a method
for treating a patient with a deficiency in certain disease
relevant small molecules. The method includes obtaining cells from
the patient, obtaining the small molecule profile of either a
particular organelle (e.g., mitochondria, nucleus, cytoplasm, Golgi
apparatus, endoplasmic reticulum, etc.) or a cell, comparing the
small molecule profile with a standard profile, determining a
deficiency in the patient's small molecule profile of a certain
disease relevant small molecule, and administering the disease
relevant small molecule to the patient.
[0065] In a further embodiment, the invention features diagnostic
assays for the detection of disease states. For example, the method
includes identifying a small molecule which is present in aberrant
amounts in a particular disease state, e.g., by comparing standard
profiles of diseased cells or cellular compartments with healthy
cells or cellular compartments to identify compounds which are
present in aberrant amounts in the diseased cell or cellular
compartment. The method also involves designing a reagent that
specifically reacts with the compound present in aberrant amounts
to indicate the presence or absence of the compound, and therefore,
the presence or the absence of the disease. The invention also
pertains to kits which include the reagent and instructions for its
use to diagnose the disease.
3. Methods of Identifying the Effect of Chemical Agents on Small
Molecule Profiles of Cells, Cellular Compartments, Organelles, and
Extracellular Material
[0066] In another aspect, the invention pertains to the comparison
of small molecule profiles of cells, cellular compartments,
organelles, or extracellular material with those of cells, cellular
compartments, organelles, or extracellular material treated with
toxins, chemical agents or therapeutic agent (or derived from an
organism treated with the agent or drug). In one embodiment, the
cells, cellular compartments, organelles, or extracellular material
are diseased (or derived from a diseased organism) and are treated
with a therapeutic agent which is known to modify or treat that
disease. For example, the small molecule profile of a cell treated
with a therapeutic agent, chemical agent, or toxin, can be compared
the small molecule profile of a normal cell, e.g., a healthy cell
of similar lineage, or a diseased cell of similar lineage which was
not treated with the therapeutic agent, chemical agent, or toxin.
Examples of toxins include bacterial toxins such as endotoxins and
exotoxins, such as cholera toxin, diptheria toxin, verotoxin,
enterotoxin, etc. In a further embodiment, the cells are
genetically altered.
[0067] Extracellular material include blood, sera, spinal fluid,
brain fluid, saliva, urine, semen, mucosal excretions, etc. Small
molecule profiles of these extracellular materials of a particular
organism may be obtained in a similar fashion to small molecule
profiles of cells, cellular compartments and organelles.
[0068] In addition, subtraction profiles can be obtained by
subtracting the non-treated profile or a standard profile with the
small molecule profile from a treated cell, cellular compartment,
organelle, or extracellular fluid. The subtraction profiles can
then be used to identify certain small molecules the presence or
the absence of which may indicate the efficacy or the toxicity of
the compound. The subtraction profiles can be made using, for
example, computer programs known to those of skill in the art,
e.g., pattern recognition software program. An example of such a
program is Pirouette. It should be noted that the comparison of the
profiles can be done both quantitatively and qualitatively.
[0069] In a further embodiment, the invention pertains to certain
small molecules which indicate the efficacy or the toxicity of the
compound. The invention also applies to assays which can be
developed to indicate the presence or absence of these certain
small molecules. For example, if the presence of a certain small
molecule is essential for the efficacy of a particular therapeutic
compound, then an assay can be developed to quickly determine the
presence or absence of this certain small molecule in cell samples
treated with test compounds. This can be both an effective and
inexpensive method to determine the potential efficacy of
compounds. It can be used alone or in combination with traditional
drug screening assays such as, for example, binding assays and
other enzymatic assays.
[0070] For example, in search of molecules with anti-tumor
activity, small molecule profiles could be taken of cells at
certain intervals after being treated with a known anti-tumor drug
(e.g., taxol, cis platin, adriamycin, etc.). Comparison of the
small molecule profiles of these cells could lead to the
identification of small molecules regulated by these drug. The
identified small molecules could then be used to guide drug
discovery by pointing to pathways which could be targeted for drug
design or by using them as therapeutic or nutriceutical agents.
Furthermore, both the targets and the identified small molecules
can be used in assays of the invention described in detail in later
sections.
[0071] The invention also includes a method for determining the
toxicity of a test compound, e.g., a compound in development as a
therapeutic agent. The method includes culturing cells, contacting
a portion of the cells with the test compound, taking small
molecule profiles of both the cells contacted with the test
compound, taking the small molecule profiles of cells not contacted
with the test compound, and comparing the profiles to either each
other or profiles from cells contacted with a known therapeutic
agent or cells contacted with a known toxin. The method also can
include a step of purifying a particular organelle of interest from
the cells and obtaining the small molecule profile of the
particular organelle of interest (e.g., nuclei, mitochondria, Golgi
apparatus, endoplasmic reticulum, ribosome, etc.). Extracellular
material also may be monitored in a similar fashion.
[0072] In a further embodiment, the invention pertains to a method
for reducing side effects of drugs under development. For example,
cells can be cultured, contacted with the test compound, the small
molecule profile can be generated, and compared to the profiles of
known toxins and therapeutic agents. Changes then can be made to
the structure of the test compound to reduce the side effects. For
example, in order to test for liver toxicity, the compound may be
incubated the in a liver cell culture to mimic the
biotransformation that occurs in the liver. The small molecule
profiles of cells and organelles in the treated and untreated liver
cultures can be compared to the small molecule profiles of known
toxins. Both the total cellular small molecule profile could be
compared or the small molecule profile of a particular organelle,
e.g., mitochondria, Golgi apparatus, nuclei, ribosomes, endoplasmic
reticulum, etc.
[0073] The methods of the invention are particularly useful because
they offer a quick and relatively inexpensive method to determine
whether a certain test compound is likely toxic to a body organ,
such as the liver. This allows for pharmaceutical companies to
quickly screen and identify compounds which are toxic and to direct
their research towards non-toxic compounds.
[0074] The methods and small molecule profiles of the invention may
also be used to rescue drugs, e.g., drugs which fail a particular
step in the clinical or pre-clinical trial procedure. The failed
drug can be exposed to cells or a test organism and small molecule
profiles of the cells, cellular compartments, organelles,
extracellular fluid, etc. can be taken and compared to those of
known toxins, known therapeutic agents, etc. to pinpoint the reason
for failure of the drug. Small molecule profiles of various organs
can also be taken if it is advantageous for the study (e.g., small
molecule profiles can be taken from muscle, brain, retinal, nerve,
heart, lung, stomach, colon, skin, breast, fatty tissue, blood,
etc.) Then the drug can be redesigned to avoid the its previous
adverse effects.
[0075] The methods and small molecule profiles of the invention can
also be used to "reposition" drugs.
[0076] The term "reposition" refers to discovering new uses for an
agent. In one embodiment, a dose of an agent is administered to a
subject (e.g., a human or other animal, healthy or diseased) and
small molecule profiles are then taken from various organs,
tissues, cells, cellular compartments, organelles, and/or
extracellular fluid of the subject to determine what tissues,
organs, cells, cellular compartments, organelles, and/or
extracellular fluids are being affected by the administration of
the agent.
4. Methods of Identifying Small Molecules Associated with Body
Weight Disorders
[0077] The invention also pertains to methods for identifying small
molecules associated with, for example, body weight disorders such
as obesity. Examples of methods for identifying small molecules
associated with body weight disorders are described below. The
following experiment are directed to the identification of small
molecules associated with short term appetite control. These
experiments can be used to identify small molecules involved in
signaling hunger and satiety.
[0078] In one embodiment, test subjects, preferably mice, will be
fed normally prior to the initiation of the experiment, and then
divided into one control and two experimental groups. The control
group will then be maintained on ad lib nourishment, while the
first experimental group ("fasted group") will be fasted, and the
second experimental group ("fasted-refed group") will initially be
fasted, and will then be offered a highly palatable meal shortly
before the collection of tissue samples. Each test animal will be
weighed immediately prior to and immediately after the experiment.
Small molecule profiles will be taken of each mouse from each
group. The profiles of each group will be averaged and compared to
those of different groups. Example 2, below, demonstrates the use
of such short term appetite experiments to identify small molecules
which are present in different amounts in control versus fasting
and versus refed animals.
[0079] Other experiments which may be used for the identification
of cellular small molecules involved in, for example, body weight
disorders, are experiments designed to analyze small molecules
which may be involved genetic obesity. In the case of mice, for
example, such experiments may identify small molecules regulated by
the ob, db, and/or tub gene products. In the case of rats, for
example, such paradigms may identify small molecules regulated by
the fatty (fa) gene product.
[0080] In one embodiment of such an experiment, test subjects may
include ob/ob, db/db, and/or tub/tub experimental mice and lean
littermate control animals. The animals would be offered normal
nourishment for a given period, after which tissue samples would be
collected for analysis. Example 2, below, demonstrates the use of
such genetic obesity paradigms in identifying small molecules which
are present in different amounts in the small molecule profiles of
obese versus lean animals.
[0081] In additional experiments, ob/ob, db/db, and/or tub/tub
experimental mice and lean control animals may be used as part of
the short term appetite control experiments discussed above, or in
other experiments discussed herein, such as set-point experiments
and drug related experiments.
[0082] Experiments which may be used for the identification of
small molecules involved in body weight disorders may include
experiments designed to identify those small molecules which may be
regulated in response to changes in body weight, e.g., "set point
experiments".
[0083] In one experiment, test subjects, preferably mice, will be
fed normally prior to the initiation of the experiment, and then
divided into one control and two experimental groups. The control
group will then be maintained on an ad lib diet of normal
nourishment in order to calculate daily food intake. The first
experimental group ("underweight group") will then be underfed by
receiving some fraction of normal food intake, 60-90% of normal,
for example, so as to reduce and maintain the group's body weight
to some percentage, for example 80%, of the control group. The
second experimental group ("overweight group") will be overfed by
receiving a diet which would bring the group to some level above
that of the control, for example 125% of the control group. Tissue
samples will then be obtained for analysis to identify small
molecules which are present in different amounts in control versus
overweight and/or underweight conditions.
[0084] Additionally, human subjects may be used for the
identification of obesity-associated small molecules. In one
embodiment of such an experiment, tissue samples may be obtained
from obese and lean human subjects and analyzed for the presence of
small molecules which are present in different amounts in the
tissue, cells, or cellular organelles of one group as opposed to
another (e.g. differentially present in lean versus obese
subjects). In another embodiment, obese human subjects may be
studied over the course of a period of weight loss, achieved
through food restriction. Tissue from these previously obese
subjects may be analyzed for differing amounts of small molecules
relative to tissue obtained from control (lean, non-previously
obese) and obese subjects.
[0085] Experiments may also be designed to identify of small
molecules involved in body weight disorders and may also may
include experiments designed to identify small molecules associated
with body weight disorders induced by some physical manipulation to
the test subject, such as, for example, hypothalamic lesion-induced
body weight disorders. For example, bilateral lesions in the
ventromedial hypothalamus (VMH) of rodents may be utilized to
induce hyperphagia and gross obesity in test subjects, while
bilateral lesions in the ventrolateral hypothalamus (VLH) of
rodents may be used to induce aphagia in test subjects. In such
experiments, tissue from hypothalamic-lesioned test subjects and
from control subjects would be analyzed for the identification of
small molecules which are present in different amounts in control
versus lesioned animals.
[0086] Drugs known to affect (e.g., ameliorate) human or animal
body weight and/or appetite (such as short term appetite) may be
incorporated into the experiments designed to identify small
molecules which are involved in body weight disorders and/or body
weight or appetite regulation. These compounds may include known
therapeutics, as well as compounds that are not useful as
therapeutics due to, for example, their harmful side effects. Among
the categories of control and test subjects which may be used in
such experiments are, for example, lean subjects, obese subjects,
and obese subjects which have received the drug of interest. In
variations of the experiment, subjects such as these may be fed a
normal ad lib diet, a caloric restriction maintained diet, or a
caloric restriction ad lib diet. Control and test subjects may
additionally be pairfed i.e., the control and test subjects may be
fed via a coupled feeding device such that both control and test
subjects receive identical amounts and types of food).
5. Methods of Identifying Small Molecules Associated with
Immunological Diseases
[0087] The invention also pertains to methods for identifying small
molecules associated with, for example, normal and abnormal immune
responses. Examples of methods for identifying small molecules
associated with immune responses are described below. The following
experiment are directed to the identification of small molecules
which are differentially present within and among TH cell
subpopulations, including but not limited to TH1 and TH2
subpopulations. Such small molecules can be involved in, for
example, TH cell subpopulation differentiation, maintenance, and/or
effector function, and in TH cell subpopulation-related disorders.
For example, TH cells can be induced to differentiate into either
TH1 or TH2 states, can be stimulated with, for example, a foreign
antigen, and can be collected at various points during the
procedure for analysis of their small molecule profiles. This
example is merely meant to be illustrate several experiments which
can be done using small molecule profiles to determine small
molecules associated with immunological disorders. This example is
not intended to limit the invention to the specific types of cells
or subjects discussed in this section.
[0088] In one experiment, transgenic animals, preferably mice, will
be used which have been engineered to express a particular T cell
receptor, such that the predominant T cell population of the immune
system of such a transgenic animal recognizes only one antigen.
Such a system will be used because it provides a source for a large
population of identical T cells whose naivete can be assured, and
because its response to the single antigen it recognizes is also
assured. T helper cells can be isolated from such a transgenic
animal can then be induced, in vitro, to differentiate into TH cell
subpopulations such as TH1, TH2, or TH0 cell subpopulations. In one
embodiment, one T helper cell group (the TH1 group) is exposed to
IL-12, a cytokine known to induce differentiation into the TH1
state, a second T helper cell group (the TH2 group) is exposed to
IL-4, a cytokine known to induce differentiation into the TH2
state, and a third group is allowed, by a lack of cytokine-mediated
induction, to enter a TH-undirected state. Small molecule profiles
of each type of cells can then be taken and compared.
[0089] In another experiment, mature TH cell clones can be used,
such as TH1 and TH2 and TH1-like and TH2-like cell lines,
preferably human cell lines. Such TH cell lines can include, but
are not limited to the following well known murine cell lines:
Doris, AE7, D10.G4, DAX, D1.1 and CDC25. Such T cell lines can be
derived from normal individuals as well as individuals exhibiting
TH cell subpopulation-related disorders, such as, for example,
chronic inflammatory diseases and disorders, such as Crohn's
disease, reactive arthritis, including Lyme disease,
insulin-dependent diabetes, organ-specific autoimmunity, including
multiple sclerosis, Hashimoto's thyroiditis and Grave's disease,
contact dermatitis, psoriasis, graft rejection, graft versus host
disease, sarcoidosis, atopic conditions, such as asthma and
allergy, including allergic rhinitis, gastrointestinal allergies,
including food allergies, eosinophilia, conjunctivitis, glomerular
nephritis, certain pathogen susceptibilities such as helminthic
(e.g., leishmaniasis) and certain viral infections, including HIV,
and bacterial infections, including tuberculosis and lepromatous
leprosy.
[0090] The TH cell clones can be stimulated in a variety of ways.
Such stimulation methods include, but are not limited to,
pharmacological methods, such as exposure to phorbol esters,
calcium ionophores, or lectins (e.g., Concanavalin A), by treatment
with antibodies directed against T-cell receptor epitopes (e.g.,
anti-CD3 antibodies) or exposure, in the presence of an appropriate
antigen presenting cell (APC), to an antigen that the particular TH
cells are known to recognize. Following such primary stimulation,
the cells can be maintained in culture without stimulation and, for
example, in the presence of IL-2, utilizing standard techniques
well known to those of skill in the art. The cells can then be
exposed to one or more additional cycles of stimulation and
maintenance. The small molecule profiles of the cells o cellular
compartments can be taken at any time during the process of the
stimulation in this experiment.
[0091] A third experiment can also be used to discover determine
small molecules present in different amounts. In vivo stimulation
of animal models forms the basis for this experiment. The in vivo
nature of the stimulation can prove to be especially predictive of
the analogous responses in living patients. Stimulation can be
accomplished via a variety of methods. For example, animals, such
as transgenic animals described earlier, can be injected with
appropriate antigen and appropriate cytokine to drive the desired
TH cell differentiation. Draining lymph nodes can then be harvested
at various time points after stimulation. Lymph nodes from, for
example, TH1-directed animals can be compared to those of
TH2-directed animals. A wide range of animal models, representing
both models of normal immune differentiation and function as well
as those representing immune disorders can be utilized for this in
vivo experiment.
[0092] Cell or organelle samples can be collected during any point
of such a procedure for small molecule profiling. For example,
cells or organelles can be obtained following any stimulation
period and/or any maintenance period. Additionally, the cells or
organelles can be collected during various points during the TH
cell differentiation process. The small molecule profiles of the
cells or organelles can be compared using the methods outlined in
the Examples. For example, small molecule profiles from TH0, TH1
and TH2 groups isolated at a given time point can then be analyzed
and compared. Additionally, small molecule profiles from stimulated
and non-stimulated cells within a given TH cell group can also be
compared and analyzed. Further, small molecule profiles from
undifferentiated TH cells can be compared to small molecule
profiles from cells at various stages during the differentiative
process which ultimately yields TH cell subpopulations.
6. Methods of Identifying Small Molecules Associated with
Cardiovascular Disorders
[0093] The small molecule profiles of the invention can be used to
identify small molecules which are relevant to cardiovascular
disease.
[0094] According to the invention, profiles are generated for small
molecules present in endothelial cells or endolethial cell
organelles subject to fluid shear stress in vitro. Shear stress may
be responsible for the prevalence of atherosclerotic lesions in
areas of unusual circulatory flow.
[0095] Cell cultures are exposed to fluid shear stress which is
thought to be responsible for the prevalence of atherosclerotic
lesions in areas of unusual circulatory flow. Unusual blood flow
also plays a role in the harmful effects of ischemia/reperfusion,
wherein an organ receiving inadequate blood supply is suddenly
reperfused with an overabundance of blood when the obstruction is
overcome.
[0096] Cultured HUVEC monolayers are exposed to laminar shear
stress by rotating the culture in a specialized apparatus
containing liquid culture medium (Nagel et al., 1994, J. Clin.
Invest. 94: 885-891). Static cultures grown in the same medium
serve as controls. After a certain period of exposure to shear
stress, experimental and control cells will be harvested,
organelles isolated and small molecule profiles will be generated
to identify molecules which are present in exposed versus control
cells.
[0097] In experiments designed to identify small molecules which
are involved in cardiovascular disease, compounds such as drugs
known to have an ameliorative effect on the disease symptoms may be
incorporated into the experimental system. Such compounds may
include known therapeutics, as well as compounds that are not
useful as therapeutics due to their harmful side effects. Test
cells that are cultured, for example, may be exposed to one of
these compounds and analyzed for different small molecule profiles
with respect to untreated cells, according to the methods described
below in the Examples. In principle, according to the particular
experiment, any cell type involved in the disease may be treated at
any stage of the disease process by these therapeutic
compounds.
[0098] Test cells may also be compared to unrelated cells (e.g.,
fibroblasts) that are also treated with the compound, in order to
screen out generic effects on small molecule profiles that may not
be related to the disease. Such generic effects might be manifest
by changes in small molecule profiles that are common to the test
cells and the unrelated cells upon treatment with the compound.
[0099] By these methods, the small molecules upon which these
compounds affect can be identified and used in the assays described
below to identify novel therapeutic compounds for the treatment of
cardiovascular disease.
[0100] In another experiment, small molecules are identified from
monocytes from human subjects. This experiment involves
differential treatment of human subjects through the dietary
control of lipid consumption. The human subjects are held on a low
fat/low cholesterol diet for three weeks, at which time blood is
drawn, monocytes are isolated according to the methods routinely
practiced in the art, organelles, such as mitochondria, nuclei, and
the cytosol, are isolated and profiles are generated. These same
patients are subsequently switched to a high fat/high cholesterol
diet and monocyte organelles are purified again. The patients may
also be fed a third, combination diet containing high fat/low
cholesterol and monocyte organelles may be purified once again. The
order in which patients receive the diets may be varied. The small
molecules of the organelles derived from patients maintained on two
of the diets, or on all three diets, may then be compared and
analyzed.
[0101] In addition to the detection of different small molecule
profiles in monocytes, paradigms focusing on endothelial cells may
be used to detect small molecules involved in cardiovascular
disease. In one experiment, human umbilical vein endothelial cells
(HUVEC's) are grown in vitro. Experimental cultures will then be
treated with human IL-1.beta., a factor known to be involved in the
inflammatory response, in order to mimic the physiologic conditions
involved in the atherosclerotic state. Alternatively experimental
HUVEC cultures may be treated with lysophosphatidylcholine, a major
phospholipid component of atherogenic lipoproteins or oxidized
human LDL. Control cultures are grown in the absence of these
compounds. After a certain period of treatment, experimental and
control cells will be harvested and small molecule profiles will be
taken of the cells and/or organelles and analyzed.
7. Methods of Identifying of Small Molecules Associated with
Central Nervous System and Other Neurological and Neurodegenerative
Disorders
[0102] The small molecule profiles of the invention can be used to
identify small molecules which are relevant to central nervous
system and other neurological and neurodegenerative disorders.
Examples of such disorders include, for example, neuropathies,
Alzheimer disease, Parkinson's disease, Huntington's disease,
amyotropic lateral sclerosis, motor neuron disease, traumatic nerve
injury, multiple sclerosis, acute disseminated encephalomyelitis,
acute necrotizing hemorrhagic leukoencephalitis, dysmyelination
disease, mitochondrial disease, migrainous disorder, bacterial
infection, fungal infection, stroke, aging, dementia, peripheral
nervous system diseases and mental disorders such as depression and
schizophrenia, etc.
[0103] One method for identifying small molecules which are
relevant to central nervous system and other neurological and
neurodegenerative disorders, is to compare the small molecule
profiles of a diseased cell, cellular compartment or organelle of a
diseased organism to a small molecule profile of a healthy cell,
cellular compartment, or organelle (e.g., a standard small molecule
profile) For example, the cells can be derived from the subjects'
brain, muscle, retinal, nerve tissue, spinal fluid, blood, etc.
[0104] The diseased organism can be either a human or animal
patient suffering from a neurological disorder or from an animal
model of such a disorder. In certain embodiments, the invention
pertains to the small molecules which are found in aberrant amounts
in the small molecule profiles of diseased cells. In other
embodiments, the invention pertains to the small molecule
subtraction profiles of particular neurological disorders (e.g.,
subtraction profiles of the diseased small molecule profile
compared to the standard small molecule profile, etc.).
8. Methods of Identifying Small Molecules Associated with
Oncological Disorders
[0105] In one embodiment, the invention pertains to methods of
identifying small molecules associated with oncological disorders,
e.g., cancerous tumors, leukemia, lymphoma, etc.
[0106] In one embodiment, small molecules associated with an
oncological disorder are identified by comparing small molecule
profiles of cancerous tissue with normal tissue. In a further
embodiment, the tissue is from the same individual, e.g., normal
and malignant prostate tissues are excised from a mammalian
subject, e.g., mouse, rat, or human. Small molecule profiles of
cells, cellular compartments, or organelles of the normal tissue is
compared with the corresponding small molecule profiles of the
malignant tissue. When the small molecule profiles are compared,
certain small molecules may appear to be present in aberrant
amounts in the cancerous tissue.
[0107] The invention also pertains to methods for detecting
aberrant amounts of the identified compound in other tissue, e.g.,
the methods of the invention can be used to develop a reagent that
specifically reacts with cancerous tissue.
9. Methods of Identifying Small Molecules Regulated by Genes of
Interest
[0108] In another embodiment, the invention pertains to methods of
identifying small molecules regulated, modulated, or associated
with genetic modification or alterations of cells, both engineered
and naturally occurring. The identified small molecules can be
used, for example, to determine the function of unknown genes in
functional genomics. For example, the comparison of the small
molecules found in genetically altered cells can be used to
elucidate the function of any given gene. For example, the
invention pertains to a method for identifying small molecules
associated with expression vectors of interest by comparing the
small molecules of host cells expressing an expression vector to
the small molecules of host cells not expressing the expression
vector. In one embodiment, the expression vector comprises a
portion or fragment of the genome, e.g., human genome. In another
embodiment, the expression vector may be known to be associated
with a particular disease state. The small molecules of the cells
with and with out the expression vector expressed can be used to
identify small molecules of interest, pathways of interest, and
targets for drug design and/or future study.
[0109] In a further embodiment, the small molecules of the cells
are identified by through separation techniques such as HPLC, mass
spectroscopy and coulometric array technology to create small
molecule profiles (see, for example, Kristal, B. S. et al. Anal.
Biochem. 263:18-25 (1998)). The resulting small molecule profile
can then be compared to the small molecule profile of other cells,
e.g., cells not genetically modified.
[0110] The term "vector" includes nucleic acid molecules capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "expression vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. In the present specification, "plasmid" and
"vector" can be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0111] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc.
[0112] The recombinant expression vectors of the invention can be
designed for expression in prokaryotic or, preferably, eukaryotic
host cells. For example, the vectors can be expressed in bacterial
cells such as E. coli, insect cells (using baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are
discussed further in Goeddel, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. (1990).
[0113] Expression of vectors in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Examples of inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[0114] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San
Diego, Calif.).
[0115] Alternatively, the vector can be expressed in insect cells
using baculovirus expression vectors. Baculovirus vectors available
for expression of vectors in cultured insect cells (e.g., Sf 9
cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol.
3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39).
[0116] In a preferred embodiment, a nucleic acid of the interest is
expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
(1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
6:187-195). When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989.
[0117] The terms "host cell" and "recombinant host cell" are used
interchangeably. These cells include not only the particular
subject cell but to the progeny or potential progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0118] For this method, a host cell can be any prokaryotic or
eukaryotic cell. For example, a protein of interest can be
expressed in bacterial cells such as E. coli, insect cells, yeast
or, preferably, mammalian cells (such as Chinese hamster ovary
cells (CHO) or COS cells). Other suitable host cells are known to
those skilled in the art.
[0119] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
The terms "transformation" and "transfection" include a variety of
art-recognized techniques for introducing foreign nucleic acid
(e.g., DNA) into a host cell, including calcium phosphate or
calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Suitable methods for
transforming or transfecting host cells can be found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989), and other laboratory manuals.
[0120] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as the
gene or a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0121] Furthermore, in yet another embodiment, the invention also
pertains to methods for identifying small molecules regulated by a
gene expressed in a particular host cell. In this embodiment, the
gene is removed, functionally disrupted, otherwise not expressed in
the cell and the small molecules of the cell are compared to those
of a similar cell wherein the gene is expressed. The small
molecules which are regulated, modulated or associated with this
gene can then be identified by the comparison of the small
molecules of the cells with and without the gene expressed. The
small molecules which are present in aberrant amounts can then be
used to identify pathways, targets, and other small molecules
associated with this gene, using methods of the invention.
[0122] To functionally disrupt a gene of a cell, a vector is
prepared which contains at least a portion of a gene of interest
into which a deletion, addition or substitution has been introduced
to thereby alter, e.g., functionally disrupt, the gene of interest.
The gene of interest can be a human gene, or a non-human homologue
of a human gene. In an embodiment, the vector is designed such
that, upon homologous recombination, the endogenous gene of
interest is functionally disrupted (i.e., no longer encodes a
functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous gene of interest is
mutated or otherwise altered but still encodes, for example, a
functional protein (e.g., the upstream regulatory region can be
altered to thereby alter the expression of the endogenous protein).
In the homologous recombination vector, the altered portion of the
gene of interest is flanked at its 5' and 3' ends by additional
nucleic acid sequence of the gene of interest to allow for
homologous recombination to occur between the exogenous gene of
interest carried by the vector and an endogenous gene of interest
in a cell. The additional flanking nucleic acid sequence should be
of sufficient length for successful homologous recombination with
the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a
description of homologous recombination vectors). The vector is
introduced into a cell line (e.g., by electroporation) and cells in
which the introduced gene of interest has homologously recombined
with the endogenous gene of interest are selected (see e.g., Li, E.
et al. (1992) Cell 69:915). The small molecules of the cells can
then be compared to cells with out the gene of interest disrupted,
thus identifying small molecules associated with the gene of
interest.
10. Assays for Identifying Potential Cell Drug Targets Using
Labeled Disease Relevant Small Molecules
[0123] In another embodiment, the invention also pertains to
methods for identifying potential cell drug targets (e.g., cellular
components which interact with the labeled small molecules). This
method is particularly useful because it can identify components
which are known to interact with disease relevant small molecules.
Therefore, targets identified through this method are
"pre-validated," and some of the guess work surrounding the choice
of target is eliminated. In a further embodiment, this method can
be used in conjunction with conventional genomics as a further
validation step to identify targets for further research.
[0124] The method includes obtaining a cell from a source,
obtaining samples of small molecules from the cell; testing the
samples for biological activity; identifying the biologically
active small molecules of the samples; labeling the biologically
active small molecules; contacting the labeled small molecules with
cellular components; and identifying interactions between cellular
components and said labeled small molecules. The invention includes
the identified cell drug targets as well as the identified
biologically active small molecules.
[0125] In another embodiment, the invention includes a method for
identifying potential cell drug targets. The method includes
contacting a labeled disease relevant small molecule with cellular
components; and identifying interactions between said cell
components and the labeled disease-relevant small molecule.
[0126] The labeled small molecules also include labeled
"disease-relevant small molecules," identified by any of the
techniques described herein (e.g., comparison of small molecule
profiles in healthy and diseased cells, etc.). In another
embodiment, the method includes contacting a labeled disease
relevant small molecule with cellular components, and identifying
the interactions between the cellular components and the labeled
disease relevant small molecule.
[0127] The term "label" includes any moieties or molecules which
enhance the ability of the labeled small molecules to be detected.
Examples of suitable labels are well known in the art. radiolabels
and fluorescent labels. The term "label" includes direct labeling
of the small molecule by radiolabeling, coupling (i.e., physically
linking) a detectable substance (e.g., a fluorescent moiety) to the
small molecule, and indirect labeling of the small molecule by
reacting the small molecule with another reagent that is directly
labeled. Examples of indirect labeling include detection of a small
molecules by labeling it with biotin such that it can be detected
with fluorescently labeled streptavidin. In one embodiment, the
small molecules are fluorescently labeled or radiolabeled.
[0128] The term "cellular components" includes material derived
from cells. The cellular components can be purified or crude
cellular extracts. The cellular components can be derived from one
type of cell, or even a specific cellular compartment such as an
organelle (e.g., mitochondria, nucleus, cytoplasm). Furthermore,
the term includes both natural proteins found within biological
systems and chimeric and other engineered proteins. In one
embodiment, the term "cellular component" includes cellular
receptors. The term also includes natural and unnatural
polysaccharides and nucleic acids. In one embodiment, the term
"cellular component" is a crude cellular extract from a human cell.
The term "cellular component" includes "targets."
[0129] Samples of the invention that bind to cellular components
can be identified by preparing a reaction mixture of the cellular
components and the samples under conditions and for a time
sufficient to allow the components and the sample to interact and
bind, thus forming a complex which can be removed and/or detected
in the reaction mixture. The cellular components used can vary
depending upon the goal of the screening assay. In one embodiment,
the sample of the invention is an isolated, labeled small molecule,
e.g., a disease relevant small molecule, a small molecule with
biological activity or another small molecules which is present in
aberrant levels in disease states. The assay can be used to
determine which cellular components the small molecule interacts
with. The identified cellular components which interact with the
small molecule can then be used for drug design.
[0130] In a further embodiment, the cellular components are a
nucleic acid array. High density arrays of nucleic acids (such as
cDNA's and synthetic oligonucleotides) allow for a high degree of
automation, repetitive analysis and duplication at minimal cost
(Fraser, Electrophoresis, 18:1207-1215 (1997)). The development of
recent technology has provided methods for making very large arrays
of oligonucleotide probes in very small areas (see, for example,
U.S. Pat. No. 5,143,854, WO 90/15070 and WO 92/10092, each of which
is incorporated herein by reference). In one embodiment, the
nucleic acids of the array are human genes. Examples of nucleic
acid arrays include those mentioned in U.S. Pat. No. 6,027,880 and
U.S. Pat. No. 5,861,242. The nucleic acids also can be
representative of RNA molecules present in a cell, tissue or organ
(e.g., the "transcriptome", see Hoheisel, J. et al. Trends
Biotechnol. 15:465-469 (1997); Velculescu, Cell, 88:243-251
(1997)). In one embodiment, the nucleic acids are in array.
[0131] In another further embodiment, the cellular components are a
protein array. Examples of protein arrays include those employing
conventional protein separation techniques, such as 2-dimensional
gel electrophoresis, chromatographic procedures (e.g., FPLC, SMART
by Pharmacia, Uppsala, Sweden), capillary electrophoretic
techniques and mass spectrometry. In another embodiment, the
protein array is a soup of proteins that contains a significant
portion of the diversity encoded by a genome (see WO 99/39210).
[0132] In a further embodiment, the cellular components are a 2D
protein gel. The 2D protein gel may be a complete or an incomplete
set of the protein molecules present in a cell, tissue or organ
(e.g., the proteome, see Sagliocco, et al. Yeast 12, 1519-1534
(1996); Shevalanko, et al. Porch. Nat. Acad. Sci. 93, 14440-14445
(1996)). Labeled biologically active small molecules previously
identified through methods of the invention can then be contacted
with the 2D gels and interactions between the labeled small
molecules and the protein of the 2D gel can be detected.
[0133] The proteins identified through this method can then be
further tested for biological activity, e.g., biological activity
relating to that of the small molecule, e.g., through knock-out
mice, inhibition studies, and other techniques known in the art.
Furthermore, the identified proteins can then be used in drug
design to identify other molecules (either naturally occurring or
chemically synthesized) which bind or interact with the protein
which may have advantageous characteristics (e.g., enhanced
biological activity, less toxic side effects).
11. Predictive Medicine and Pharmacometabolomics
[0134] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacometabolomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining small molecule
profiles, in the context of a biological sample (e.g., blood,
serum, cells, tissue, cellular organelles) to thereby determine
whether an individual is afflicted with a disease or disorder, or
is at risk of developing a disorder, associated with aberrant
levels of small molecules. The invention also provides for
prognostic (or predictive) assays for determining whether an
individual is at risk of developing a disorder associated with
relevant small molecules. For example, aberrant levels of small
molecules can be profiled from a biological sample. Such assays can
be used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with a relevant small
molecule.
[0135] Another aspect of the invention provides methods for
determining small molecule profiles of an individual to thereby
select appropriate therapeutic or prophylactic agents for that
individual (referred to herein as "pharmacometabolomics").
Pharmacometabolomics allows for the selection of agents (e.g.,
drugs) for therapeutic or prophylactic treatment of an individual
based on the small molecule profile of the individual (i.e., the
individual's "metaboprint"). The metaboprint of the individual is
examined to predict what the person's reaction to a particular
therapeutic compound will be. Yet another aspect of the invention
pertains to monitoring the influence of agents (e.g., drugs or
other compounds) on the small molecule profiles of the patients in
clinical trials.
[0136] Pharmacometabolomics is similar to pharmacogenomics but it
is also able to taken in to account environmental and other
non-genetic factors (e.g., other drugs, etc.) which may affect an
individual's response to a particular therapeutic compound.
Pharmacometabolomics can be used alone or in combination with
pharmacogenomics to predict an individual's reaction to a
particular drug based upon their metaboprint (e.g., small molecule
profile) and/or their genotype.
[0137] Pharmacometabolomics is particularly useful because it
provides an early warning sign, due to its capability of detecting
aberrant small molecules long before any disease symptoms or
predisposed phenotypes are noticed.
A. Diagnostic Assays
[0138] In one embodiment, the invention pertains to a method for
facilitating the diagnosis of a disease state of a subject. The
method includes obtaining a small molecule profile from a subject
suspected of having and/or having a disease state, and comparing
the small molecule profile from the subject to a standard small
molecule profile.
[0139] The invention provides a method of assessing small molecule
profiles, especially aberrant small molecule profiles. Aberrant
small molecule profiles (e.g., excessive amounts of a particular
molecule, deficient amounts of a particular molecule, the presence
of a small molecule not usually present, etc.) may indicate the
presence of a disease state. More generally, aberrant small
molecule profiles may indicate the occurrence of a deleterious or
disease-associated metaboprint contributed by small molecules
present in aberrant amounts.
[0140] The standard small molecule profile can be obtained from
healthy subjects or subjects afflicted with the disease state which
is the subject is suspected of having. The small molecule profiles
can be taken from a particular organ, tissue, or combinations or
organs or tissues. The small molecule profiles can also be taken of
cells, cellular compartments, particular organelles, or
extracellular material.
[0141] The term "disease state" includes any states which are
capable of being detected metabolomically by comparing small
molecule profiles of a subject having the disease to a standard
small molecule profile. Examples of disease states include, but are
not limited to, include metabolic diseases (e.g., obesity,
cachexia, diabetes, anorexia, etc.), cardiovascular diseases (e.g.,
atherosclerosis, ischemia/reperfusion, hypertension, restenosis,
arterial inflammation, etc.), immunological disorders (e.g.,
chronic inflammatory diseases and disorders, such as Crohn's
disease, reactive arthritis, including Lyme disease,
insulin-dependent diabetes, organ-specific autoimmunity, including
multiple sclerosis, Hashimoto's thyroiditis and Grave's disease,
contact dermatitis, psoriasis, graft rejection, graft versus host
disease, sarcoidosis, atopic conditions, such as asthma and
allergy, including allergic rhinitis, gastrointestinal allergies,
including food allergies, eosinophilia, conjunctivitis, glomerular
nephritis, certain pathogen susceptibilities such as helminthic
(e.g., leishmaniasis) and certain viral infections, including HIV,
and bacterial infections, including tuberculosis and lepromatous
leprosy, etc.), nervous system disorders (e.g., neuropathies,
Alzheimer disease, Parkinson's disease, Huntington's disease,
amyotropic lateral sclerosis, motor neuron disease, traumatic nerve
injury, multiple sclerosis, acute disseminated encephalomyelitis,
acute necrotizing hemorrhagic leukoencephalitis, dysmyelination
disease, mitochondrial disease, migrainous disorder, bacterial
infection, fungal infection, stroke, aging, dementia, peripheral
nervous system diseases and mental disorders such as depression and
schizophrenia, etc.), oncological disorders (e.g., leukemia, brain
cancer, pancreatic cancer, prostate cancer, liver cancer, stomach
cancer, colon cancer, throat cancer, breast cancer, ovarian cancer,
skin cancer, melanoma, etc.). The term also include disorders which
result from oxidative stress.
[0142] The term "subject" includes humans, animals, and plants. In
one embodiment, the subject is a human suffering from or at risk of
suffering from a disease state.
[0143] The invention also encompasses kits for detecting the
presence of a particular relevant small molecule in a biological
sample (a test sample). Such kits can be used to determine if a
subject is suffering from or is at increased risk of developing a
disorder associated with the relevant small molecule (e.g., drug
resistance). For example, the kit can comprise a labeled compound
or agent capable of detecting the relevant small molecule in a
biological sample and means for determining the amount of the
relevant small molecule in the sample (e.g., an antibody against
the relevant small molecule another molecular or chemical sensor).
Kits may also include instruction for observing that the tested
subject is suffering from or is at risk of developing a disorder
associated with the relevant small molecule if the amount of the
relevant small molecule is above or below a normal level.
[0144] The kit may also comprise, e.g., a buffering agent, a
preservative, or a stabilizing agent. The kit may also comprise
components necessary for detecting the detectable agent (e.g., a
substrate). The kit may also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample contained. Each component of the kit is usually enclosed
within an individual container and all of the various containers
are within a single package along with instructions for observing
whether the tested subject is suffering from or is at risk of
developing a disorder associated with the relevant small
molecule.
B. Prognostic Assays
[0145] The invention also pertains to a method for predicting
whether a subject is prediposed to having a disease state. The
method includes obtaining a small molecule profile from the
subject; and comparing the small molecule profile from the subject
to a standard small molecule profile, thereby predicting whether a
subject is prediposed to having a disease state.
[0146] The methods described herein can furthermore be used as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with aberrant
small molecule profiles. For example, the assays described herein,
such as the preceding diagnostic assays or the following assays,
can be utilized to identify a subject having or at risk of
developing a disorder associated with an aberrant small molecule
profile, such as drug resistance of tumor cells. Alternatively, the
prognostic assays can be utilized to identify a subject having or
at risk for developing such a disease or disorder. Thus, the
present invention provides a method in which a test sample is
obtained from a subject and a small molecule profile is taken,
wherein an aberrant small molecule profile is diagnostic for a
subject having or at risk of developing a disease or disorder
associated with an aberrant small molecule profile. The term "test
sample" is a biological sample obtained from a subject of interest.
For example, a test sample can be a biological fluid (e.g., serum,
blood, saliva, etc.), cell sample, or tissue. Advantageously, the
test sample may consist of cells, extracellular material, or
individual organelles, e.g., mitochondria, nuclei, Golgi apparatus,
endoplasmic reticulum, ribosomes, chloroplasts, etc.
[0147] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate) to treat a disease or disorder
associated with an aberrant small molecule profile. For example,
such methods can be used to determine whether a subject can be
effectively treated with a specific agent or class of agents (e.g.,
agents of a type which effect the small molecule profile in
particular ways). Thus, the present invention provides methods for
determining whether a subject can be effectively treated with an
agent for a disorder associated with an aberrant small molecule
profile in which a test sample is obtained and an aberrant small
molecule profile is detected (e.g., wherein the presence or
relative quantity of particular relevant small molecules is
diagnostic for a subject that can be administered the agent to
treat a disorder associated with the aberrant small molecule
profile). In some embodiments, the foregoing methods provide
information useful in prognostication, staging and management of
particular states that are characterized by altered small molecule
profiles and thus by a particular metaboprint. The information more
specifically assists the clinician in designing treatment regimes
to eradicate such particular states from the body of an afflicted
subject.
[0148] The methods of the invention can also be used to detect the
presence or absence of relevant small molecules, thereby
determining if a subject is at risk for a disorder associated with
this relevant small molecule. For example, the presence or absence
of relevant small molecules, may indicate whether the process of
developing a disease state has been initiated or is likely to arise
in the tested cells. In preferred embodiments, the methods include
detecting the presence or absence of the relevant small molecule,
in a sample of cells or extracellular material from the subject,
the presence or absence of a disease state. Preferably the sample
of cells or extracellular material is obtained from a body tissue
suspected of comprising diseased cells. Thus, the present method
provides information relevant to diagnosis of the presence of a
disease state. In one embodiment, the sample of cells is comprised
mainly of a particular cellular organelle, e.g., mitochondria,
Golgi apparatus, nuclei, etc.
[0149] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
reagent for detecting a relevant small molecule, which may be
conveniently used, e.g., in clinical settings to diagnose patients
exhibiting symptoms or family history of a disease or illness
involving a relevant small molecule.
C. Pharmacometabolomics
[0150] The invention also pertains to a method for predicting a
subject's response to a therapeutic agent. The method includes
obtaining a small molecule profile from the subject, and comparing
the small molecule profile of the subject to a known standard
established for the therapeutic agent as an indication of whether
the subject would benefit from treatment with the therapeutic
agent.
[0151] Agents, or modulators which alter levels of particular
relevant small molecules, as identified by a screening assay
described herein can be administered to individuals to treat
(prophylactically or therapeutically) disorders associated with the
relevant small molecules. In conjunction with such treatment, the
pharmacometabolomics (i.e., the study of the relationship between
an individual's metaboprint and that individual's response to a
foreign compound or drug) of the individual may be considered.
Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, the pharmacometabolomics of the individual permits the
selection of effective agents (e.g., drugs) for prophylactic or
therapeutic treatments based on a consideration of the individual's
metaboprint. Such pharmacometabolomics can further be used to
determine appropriate dosages and therapeutic regimens.
Accordingly, the small molecule profile of an individual can be
determined to thereby select appropriate agent(s) for therapeutic
or prophylactic treatment of the individual.
[0152] The known standard can be obtained from subjects who
benefited from the agent, e.g., patients who were treated with the
agent and were cured, maintained their health, or prevented or
slowed the deterioration of health. The known standard can be taken
from a particular tissue, organ. It can also be taken from any
organelle, cell, or cellular compartment during any point during
the beneficial treatment. It can be derived from a single patient
or from an average of more than one patient who were treated
successfully with the agent. In addition, the known standard can
also be derived using other techniques.
[0153] Pharmacometabolomics deals with clinically significant
hereditary and non-hereditary variations in the response to drugs
due to altered drug disposition and abnormal action in affected
persons. In general, several types of pharmacometabolomic
conditions can be differentiated. For example, certain
pharmacometabolomic conditions may be the result of genetic
conditions. The genetic conditions may be transmitted as a single
factor altering the way drugs act on the body (altered drug action)
or genetic conditions transmitted as single factors altering the
way the body acts on drugs (altered drug metabolism). These
pharmacometabolomic conditions can occur either as rare defects or
as polymorphisms. For example, glucose-6-phosphate dehydrogenase
deficiency (G6PD) is a common inherited enzymopathy in which the
main clinical complication is haemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans. Examples of non-hereditary conditions
which may affect the way drugs act on the body or the way the body
acts on the drugs include the ingestion of certain drugs, the
substance dependence of the patient, the diet of the patient,
non-hereditary medical conditions of the patient, etc.
[0154] The small molecule profile and metaboprint an individual can
be determined to select appropriate agent(s) for therapeutic or
prophylactic treatment of the individual. In addition,
pharmacometabolomic studies can be used to identify an individual's
drug responsiveness metaboprint. This knowledge, when applied to
dosing or drug selection, can avoid adverse reactions or
therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when treating a subject with an agent, such as an agent
identified by one of the exemplary screening assays known in the
art.
D. Monitoring of Effects During Clinical Trials
[0155] The invention also pertains to a method for metabolomically
monitoring the effectiveness of a therapeutic agent in clinical
trials. The method includes obtaining a small molecule profile from
a subject in a clinical trial being treated with a therapeutic
agent, and monitoring changes in the small molecule profile of the
subject as an indication of the effectiveness of the therapeutic
agent in the subject. In one embodiment, the small molecule profile
of the subject can be compared to a predetermined standard.
[0156] Monitoring the influence of agents (e.g., drugs, therapeutic
compounds) on the small molecule profile can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase levels of certain relevant small
molecules, can be monitored in clinical trails of subjects
exhibiting decreased levels of certain small molecules.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease levels of a certain relevant small
molecule, can be monitored in clinical trails of subjects
exhibiting increased levels of the certain relevant small molecule.
In such clinical trials, the level of the certain small molecule
and, preferably, the remainder of the small molecule profile can be
used as a "read out" of the disease state of the particular
cell.
[0157] For example, and not by way of limitation, small molecules
that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) can be identified in screening
assays. The effect of agents on cellular proliferation disorders,
for example, can be studied in a clinical trial. For example, cells
can be isolated and small molecule profiles of either whole cells
or particular organelles can be taken. In this way, the small
molecule profile can serve as a marker, indicative of the
physiological response of the cells to the agent. Accordingly, this
response state may be determined before, and at various points
during, treatment of the individual with the agent.
[0158] In an embodiment, the present invention provides a method
for monitoring the effectiveness of treatment of a subject with an
agent (e.g., peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate identified by the screening
assays described herein) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the small molecule profile of the
preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
small molecule profile of the post-administration samples; (v)
comparing the small molecule profile of the pre-administration
sample with the small molecule profile of the post administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased
administration of the agent may be desirable to increase the level
of certain relevant small molecules to higher levels than detected,
i.e., to increase the effectiveness of the agent. Alternatively,
decreased administration of the agent may be desirable to decrease
the level of certain relevant small molecules to lower levels than
detected, i.e., to decrease the effectiveness of the agent.
12. Identification of Compounds which Protect Mitochondrial
Function
[0159] In a further embodiment, the samples are obtained from a
specific organelle, e.g., mitochondria, nuclei, ribosomes, Golgi
apparatus, endoplasmic reticulum, etc. The small molecule profiles
obtained from the organelles can be used to identify small
molecules which are of particular relevance to the particular
organelle, in both health and disease states. In one embodiment,
the invention pertains to standard small molecule profiles of
particular organelles, e.g., nuclei, mitochondria, chloroplasts,
Golgi apparatus, endoplasmic reticulum, ribosomes, etc.
[0160] The term "standard small molecule profiles of particular
organelles" includes the averaged profiles of particular
organelles. The standard profiles can be averaged over more than
one profile from a particular individual, or from a population.
Furthermore, the profiles can all be from the same type of cells
(e.g., liver cells, muscle cells, nerve cells, brain tissue cells,
blood cells, immune system cells, etc.) or different types of
cells. The standard profiles can be taken of any organelle of
interest. The organelle of interest can be obtained, generally,
through methods known in the art, such as fractional
centrifugation. Examples of organelles which can be analyzed
through this method include, for example, nuclei, mitochondria,
chloroplasts, centrioles, ribosomes, Golgi apparatus, endoplasmic
reticulum, etc.
[0161] In one embodiment, the organelles are mitochondria.
Mitochondrial dysfunction has been implicated in a wide range of
physiological conditions, such as neurodegenerative diseases, aging
processes, diabetes, and cancer. Mitochondria buffer intracellular
calcium, are responsible for production of ATP, and play a key role
in cell death pathways, such as apoptosis (Green and Reed, Science
281: 1309-1312, 1998; Susin et al., Biochim. Et. Biophys. Acta
1366: 151-165, 1998). Due to their central role in a wide array of
cellular respiratory, oxidative and metabolic processes, defects in
mitochondrial activity impact the rate of ATP production, calcium
homeostasis, free radical production and release of apoptosis
inducing factors. (Ernster and Schatz, J. Cell Biol. 91:227s-255,
1981).
[0162] Therefore, in one embodiment, the invention pertains to a
method for identifying compounds relevant to mitochondrial related
disorders. In one embodiment, the invention pertains to small
molecule profiles of mitochondria which identify about 70% or more
of the small molecules, about 75% or more of the small molecules,
about 80% or more of the small molecules, about 85% or more of the
small molecules, about 90% or more of the small molecules, about
91% or more of the small molecules, about 92% or more of the small
molecules, about 93% or more of the small molecules, about 94% or
more of the small molecules, about 95% or more of the small
molecules, about 96% or more of the small molecules, about 98% or
more of the small molecules, about 99% or more of the small
molecules of the mitochondria. In another embodiment, the invention
pertains to methods for identifying compounds which are present in
aberrant amounts when standard small molecule profiles of
mitochondria are compared to small molecule profiles of
mitochondria afflicted with a mitochondrial related disorder. The
invention pertains to both the standard mitochondria profile and
the compounds identified to be relevant to mitochondria related
disorders.
[0163] The term "mitochondrial related disorders" include disorders
associated with processes associated with the mitochondria.
Mitochondrial related disorders include neurodegenerative diseases,
aging, diabetes, and cancer. Further more mitochondrial related
diseases included those related to the production of ATP,
intracellular calcium, free radicals, and apopotosis.
[0164] In another embodiment, the compounds identified from the
small molecule profile of the mitochondria are assayed for
biologically active small molecules which protect mitochondrial
function. Examples of assays that can be used for evaluating
mitochondrial function include assays which evaluate the inhibition
of production of reactive oxygen species (e.g., assays using
dichlorofluorescin diacetate), assays for mitochondrial
permeability transition (MPT) (e.g., assays using dyes such as 2-4
dimethylaminostyryl-N-Methlypyridinim); mitochodrial
electrochemical potential gradients; and cell death assays with
signals which can be used to induce insult, measure or release
apoptogenic molecules, structural changes in cells, DNA changes,
activation of caspases, and translocation of membrane components.
Assays that evaluate effects of compounds on electron transport
chain are also included (Parker et al., Neurology 44: 1090-96,
1994; Miller et al., J. Neurochem. 67: 1897, 1996). In another
embodiment, the compounds which are determined to be present in
aberrant amounts in mitochondrial related disorders are assayed for
protecting function. The invention also pertains to a
pharmaceutical composition comprising any compound identified by
the assays described herein and a pharmaceutically acceptable
carrier.
[0165] In a further embodiment, the biologically active small
molecules are chemically modified to enhance their biological
activity. It is known in the art that through chemical
modifications, one can enhance the biological activity, stability,
or otherwise modify a molecule to make it more suitable as a
therapeutic or nutriceutical agent.
13. Small Molecules Libraries and Methods of Use
[0166] In one embodiment, the invention pertains to the creation of
small molecule libraries from cells, cellular compartments, and
organelles, e.g., cells, cellular compartments, and organelles in
health, diseased, and altered states. The small molecule libraries
can be derived from the same or different animal organs. For
example, the small molecule libraries can be derived from cells of
the heart, brain, kidney, liver, done, blood, gastrointestinal
tract, and/or muscle. In addition, the small molecule libraries can
be derived from individuals suffering from a particular disease
state, e.g., cardiovascular diseases, neurodegenerative diseases,
diabetes, obesity, immunological disorders, etc.
[0167] The creation of the libraries involves fractionating cell
components, preparing cellular extracts, and fractionating the
small molecules by methods such as HPLC, thin layer chromatography
(TLC), electrochemical techniques and other methods known in the
art for separating such compounds. The compounds can be also
separated using their charge, mass, hydrophilicity, and
hydrophobicity. Furthermore, the compounds can be characterized
using methods such as Mass Spectroscopy, NMR, IR, and other
techniques known in the art for identifying organic compounds.
[0168] The term "library" includes searchable populations of small
molecules or mixtures of molecules. In one embodiment, the library
is comprised of samples or test fractions (either mixtures of small
molecules or isolated small molecules) which are capable of being
screened for activity. For example, the samples could be added to
wells in a manner suitable for high throughput screening assays. In
a further embodiment, the library could be screened for binding
compounds by contacting the library with a target of interest,
e.g., a protein or a nucleic acid.
[0169] In further embodiment, the invention pertains to a library
of compounds from cells on a solid support, e.g., a solid support
suitable for screening assays, e.g., a solid support suitable for
high throughput assays. The invention also pertains to a cellular
small molecule library packaged in a container comprising the small
molecule library, a solid support, and a label identifying the
contents.
[0170] In yet another embodiment, the small molecule libraries are
derived from specific cellular compartments, e.g., the cytoplasm,
the nucleus, the mitochondria, the choloroplast.
[0171] In one embodiment, the samples are screened for activity as
a library. Within the last decade, small molecule libraries have
been generated using combinatorial chemistry techniques to identify
biologically active molecules. For example, libraries of compounds
have been screened for biological activity using high throughput
assays. For example, anti-tumor assays involve adding compounds to
cancer cells in plastic wells and monitoring the effects of the
compounds on cell survival. The compounds which effect cell
survival, are identified as potential lead molecules.
[0172] Libraries can be screened to determine whether any samples
of the library have a desired activity, and, if so, to identify the
active compound or sample. Methods of screening libraries have been
described (see, e.g., Gordon et al., J. Med. Chem.). Soluble small
molecule libraries can be screened by affinity chromatography with
an appropriate receptor to isolate ligands for the receptor,
followed by identification of the isolated ligands by conventional
techniques (e.g., mass spectrometry, NMR, and the like).
Immobilized samples can be screened by contacting the samples with
a soluble receptor; preferably, the soluble receptor is conjugated
to a label (e.g., fluorophores, colorimetric enzymes,
radioisotopes, luminescent compounds, and the like) that can be
detected to indicate ligand binding. Alternatively, immobilized
samples can be selectively released and allowed to diffuse through
a membrane to interact with a receptor.
[0173] In vitro systems may be designed to identify the samples of
the libraries of the invention capable of interacting with targets
of interest. The identified samples may contain useful compounds
which may, for example, modulate the activity of the target; be
useful in elaborating the biological function of the target; be
utilized in screens for identifying additional compounds that
disrupt the normal interactions of the target; or be useful
themselves as disrupters of such interactions.
[0174] The term "target" includes proteins and mixtures of proteins
(e.g., naturally occurring proteins, polypeptides, peptidomimitics,
mutant proteins, and recombinant proteins). The term "target" also
includes nucleic acid and mixtures of nucleic acids (e.g., RNA and
DNA, both naturally occurring nucleic acids, synthesized nucleic
acids, mutant nucleic acids, and recombinant nucleic acids) or
lipids (e.g., membranes or membrane fragments). In one embodiment,
the target is involved in a disease state of interest. Furthermore,
it may be necessary to vary the conditions such that the target is
able to maintain its cellular configuration.
[0175] The screening assays can be conducted in a variety of ways.
For example, one method for identifying small molecules that
interact with a target or targets involves anchoring a target onto
a solid phase, contacting it with samples of small molecules, and
detecting target/sample complexes anchored on the solid phase at
the end of the reaction.
[0176] For example, microtiter plates may be used as the solid
phase. The anchored component may be immobilized by non-covalent or
covalent attachments. Non-covalent attachment may be accomplished
by simply coating the solid surface with a solution of the sample
or the target and drying. The surfaces may be prepared in advance
and stored. Covalent attachments include, for example, chemically
linking the compound or target to the plate.
[0177] In one method of conducting the assay, the nonimmobilized
component is added to the coated surface containing the anchored
component. After the reaction is complete, unreacted components are
removed under conditions such that any target-sample complexes
formed are capable of being detected. The complexes may be anchored
on to the solid surface. The detection of complexes anchored on the
solid surface can be accomplished in a number of ways. Where the
previously nonimmobilized component is pre-labeled, the detection
of label immobilized on the surface indicates that complexes were
formed. Where the previously nonimmobilized component is not
pre-labeled, an indirect label can be used to detect complexes
anchored on the surface; e.g., using a labeled antibody specific
for the previously nonimmobilized component (the antibody, in turn,
may be directly labeled or indirectly labeled with a labeled
anti-Ig antibody).
[0178] In another embodiment, the invention provides a method (also
referred to herein as a "screening assay") for identifying targets
which bind to the samples of the library. It also includes methods
for identifying samples of the library which have a stimulatory or
inhibitory effect on targets, for example, or target activity.
[0179] In an embodiment, the invention provides assays for
screening libraries of the invention to identify samples which bind
to or modulate the activity of a target. Libraries of samples may
be presented in solution (e.g., Houghten (1992) Biotechniques
13:412-421), on beads (Lam (1991) Nature 354:82-84), or chips
(Fodor (1993) Nature 364:555-556).
[0180] For example, in one embodiment, the samples are prepared
appropriately for a interaction with a specific target using a high
thoroughput screen. The high throughput screen then is used to
identify which of the samples, bind or otherwise interact with the
target.
[0181] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a target (e.g., a protein of interest or a
biologically active portion thereof) is contacted with a sample of
the library and the ability of the sample to modulate the target's
activity is determined. Determining the ability of the sample to
modulate the target's activity can be accomplished by methods
suitable for the particular target. Determining the ability of the
sample to modulate the ability of a target to bind to its substrate
can be accomplished, for example, by coupling the substrate with a
radioisotope or enzymatic label such that binding of the target to
its substrate can be determined by detecting the labeled substrate
in a complex with the target. For example, substrates can be
labeled with .sup.125I .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, substrates can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0182] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a target substrate with the
samples of the invention and determining the ability of the samples
to modulate (e.g., stimulate or inhibit) the activity of the
target.
[0183] Determining the ability of a target to bind to or interact
with a target substrate can be accomplished by one of the methods
described above for determining direct binding. In an embodiment,
determining the ability of the target to bind to or interact with
its substrate can be accomplished by determining the activity of
the substrate. For example, the activity of the substrate can be
determined by detecting induction of a cellular second messenger of
the target, detecting catalytic/enzymatic activity of the target or
its substrate, detecting the induction of a reporter gene
(comprising a target-responsive regulatory element operatively
linked to a nucleic acid encoding a detectable marker, e.g.,
luciferase), or detecting a target-regulated cellular response.
[0184] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a target (e.g., a protein, a
polypeptide, or a nucleic acid) is contacted with a sample of the
invention and the ability of the samples to bind to the target is
determined Binding of a sample to the target can be determined
either directly or indirectly as described above. In a further
embodiment, the assay includes contacting the target with a
compound which is known to bind to the target to form an assay
mixture, contacting the assay mixture with a sample of the
invention, and determining the ability of the sample to interact
with the target, wherein determining the ability of the sample to
interact with the target comprises determining the ability of the
sample to preferentially bind to the target as compared to the
known compound.
[0185] In another embodiment, the assay is a cell-free assay in
which a target is contacted with a library of compounds of the
invention and the ability of the compounds to modulate (e.g.,
stimulate or inhibit) the activity of the target is determined
Determining the ability of the test compound to modulate the
activity of the target can be accomplished, for example, by
determining the ability of the target to bind to another molecule
by one of the methods described above for determining direct
binding. Determining the ability of the target to bind to another
molecule can also be accomplished using a technology such as
real-time Biomolecular Interaction Analysis (BIA). Sjolander, S,
and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al.
(1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA"
is a technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the optical phenomenon of surface plasmon resonance (SPR) can be
used as an indication of real-time reactions between biological
molecules.
[0186] In yet another embodiment, the cell-free assay involves
contacting the target with a compound which is known to bind to the
target to form an assay mixture, contacting the assay mixture with
a sample of the invention, and determining the ability of the
sample to interact with the target, wherein determining the ability
of the sample to interact with the target comprises determining the
ability of the sample to preferentially bind to or modulate the
activity of the target.
[0187] The libraries of compounds of the invention can also be
screened using combinatorial methods such as those described in WO
99/31267 for the simultaneous identification of novel biological
targets and lead structures for drug development.
14. Small Molecules Databases and Methods of Use
[0188] In one embodiment, the invention pertains to the creation of
small molecule databases containing information regarding the
metabolome of cells, cellular compartments, and organelles, e.g.,
cells, cellular compartments, and organelles in health, diseased,
and altered states. The information regarding the small molecules
of each cell, cellular compartment, or organelle can be found using
the separation and analytical techniques described elsewhere in the
application. The small molecule databases can include compounds
derived from the same or different animal organs. For example, the
small molecule databases can include compounds obtained from cells
of specific organs such as a heart, brain, kidney, liver, done,
blood, gastrointestinal tract, and/or muscle. In addition, the
small molecule databases can include information regarding
compounds obtained from individuals suffering from a particular
disease state, e.g., cardiovascular diseases, neurodegenerative
diseases, diabetes, obesity, immunological disorders, etc.
[0189] The databases can be made based on information obtained from
the techniques described elsewhere in the application to determine
the identity and presence of various small molecules in cells,
cellular compartments, and organelles. The databases may include
information regarding the compounds found, such as structure,
molecular weight, amounts found in particular organelles in a
particular state of health, and any other information that a person
of skill in the art would consider relevant and useful to be
contained in the database. For example, information regarding known
biochemical pathways involving the particular compound may also be
included as well as other such information.
[0190] In one embodiment, the databases of the invention contain
information on the compounds of the metabolome of a particular
organelle of a particular species in a particular state of health
from a particular organ (e.g., one database may include compounds
of the metabolome of the mitochondria of a healthy human heart). In
other embodiments, the databases may include information regarding
the metabolome of a variety of organelles (e.g., mitochondria,
nuclei, Golgi apparatus, endoplasmic reticulum, ribosomes, cytosol,
chloroplasts, etc.) or cells from a particular species from a
particular organ in a particular state of health. In another
embodiment, the databases may include information regarding either
specific organelles or cells from a variety of tissues (e.g., fatty
tissue, muscle tissue, nerve tissue, brain tissue, heart tissue,
bone tissue, blood, connective tissue, retinal tissue, etc.) from
an organism in a health or diseased stated (e.g., the tissue can be
from an organism suffering from any disorder known to afflict it).
Examples of disorders include neurological disorders, central
nervous system disorders, metabolic disorders, cardiovascular
disorders, immunological disorders, oncological disorders. In a
further embodiment, a database may comprise information regarding
compounds of the entire metabolome of a particular species, e.g.,
human, rat, mouse, dog, cat, etc.
[0191] If the database is in electronic form, the program used to
organize the database can be any program known in the art which is
capable of storing the information in a useful format.
[0192] The databases of the invention can be organized in such a
way that they can be licensed to companies, such as pharmaceutical
companies. The databases can then be used for many purposes, such
as drug discovery, design, etc.
14. Methods of Identifying Biologically Active or Disease Relevant
Small Molecules from Cell Samples
[0193] In one embodiment, the invention pertains to yet another
method for identifying biologically active small molecules. This
method includes obtaining cells from a tissue culture, an animal
source or extracellular material from a subject; obtaining samples
of small molecules; testing the samples for biological activity;
and identifying samples which have biological activity. The samples
that are found to have biological activity can then be further
fractionated and profiled by methods known in the art, such as
HPLC, thin layer chromatography (TLC), and electrochemical methods.
The resulting compounds or fractions can then be retested for
biological activity. If desired, the fractionation can continue
until the individual compounds or mixtures of compounds with
biological activity are identified. These biologically active
compounds can then be used, for example, as lead compounds for drug
design, used as pharmaceutical or nutriceutical agents, labeled and
used to identify other components of pathways associated it, or
used in other advantageous capacities.
[0194] The term "tissue culture or source" includes subjects, such
as plants, bacteria, prokaryotes, eukaryotes, animals (e.g., yeast,
mammals, e.g., rats, mice, dogs, cats, primates (e.g., humans,
chimpanzees, monkeys), horses, cattle, and bears). The subjects may
be healthy, suffering from a disease state, or at risk of suffering
from a disease state. Examples of disease states include those
which alter the amounts of various small molecules of the cell or
cellular compartment (e.g., diabetes, cancer, AIDS,
neurodegenerative disorders, etc.). The language "tissue culture or
source" includes cell lines such as can be found deposited with
ATCC (e.g., cell lines corresponding to disease states, bacterial
cell lines, animal cell lines, etc.). If necessary, the cells can
be cultured according to methods and techniques known in the art
(see, for example, Ausubel et al. Current Protocols in Molecular
Biology (New York: John Wiley & Sons). Other examples of
sources include, for example, cells from CAP23, Hela, human cell
cultures, human placenta, lymphoblasts, mammalian muscle biopsies,
rat brain, rat liver, and yeast.
[0195] In one embodiment, the samples are test fractions,
fractionated by techniques known in the art, such as, for example,
HPLC (Kristal, et al. Anal. Biochem. 263:18-25 (1998)), thin layer
chromatography (TLC), or other methods known in the art (Methods of
Enzymology). The test fractions may be separated based on molecular
weight. The test fractions can then be screened for biological
activity, before, after, or without further purification. The
biologically active samples or test fractions can then be further
fractionated and characterized using methods such as, for example,
mass spectroscopy (Teusink, B. et al. Methods Microbiol., 26:
297-336 (1998)), infra-red spectroscopy, and nuclear magnetic
resource (Brindle, K. et al. J. Mol. Recog. 10:182-187 (1997)) to
identify biologically active small molecules.
[0196] The term "biologically active small molecules" include small
molecules which modulate the activity of a biological system or
pathway. For example, biologically active small molecules may be
identified using in vitro or in vivo assays known in the art.
Biologically active small molecules can also be identified by
screening assays against protein targets which have been implicated
in a disease state. In another embodiment, biologically active
small molecules can be identified using cell-based assays. For
example, biologically active small molecules with anti-tumor
activity can be identified, for example, by their effect on the
growth of a panel of tumor cell lines (Lillie et al. Cancer Res.
53(13):3172-8 (1993)). Similarly, biologically active small
molecules with neuronal protection activity may be identified by
exposing primary or cultured neurons to the compounds and toxic
agents, such as glutamate, and identifying the compounds which
protect the neurons from death Animal models can also be used to
identify biologically active small molecules. For example, animal
models of Huntington's Disease, Parkinson's disease, and ALS can be
used to identify biologically active small molecules useful as
neuroprotective agents. (Kilvenyi, Nature Med. 5:347-350 (1999);
Mathews et al, Experimental Neurology 157:142-149 (1999)). In a
further embodiment, the identified biologically active small
molecules can then be chemically modified to further enhance their
pharmaceutical or nutriceutical properties.
[0197] The term "isolated" includes molecules (e.g., small
molecules) which are separated from other molecules which are
present in the natural source of the molecules. In an embodiment,
an "isolated" small molecule is free of other molecules (both other
small molecules and macromolecules) which naturally are present of
the organism from which the small molecules are derived.
15. Use of Small Molecule Profiles for Tissue Typing and Forensic
Science
[0198] The small molecule profiling of cells, cellular
compartments, particular organelles, and/or extracellular material
of the present invention can also be used to identify individuals,
populations, or species from minute biological samples. The method
includes taking one or more samples from a subject, population, or
species and determining the small molecule profiles of the samples;
taking a sample from a unknown source and determining its small
molecule profile; and comparing the two small molecule profiles to
determine whether the small molecule profiles are from the same
individual, population or species.
[0199] It is expected that certain small molecules will be present
in unique amounts in each person's cells, cellular compartments,
organelles, or extracellular material. It is also expected that
certain small molecules may be present in unique amounts in a
particular population or species' cells, cellular compartments,
organelles, or extracellular material. By using several of these
compounds as markers, one could determine whether or not a sample
was or was not obtained from the same individual, population, or
species as a reference sample. This method of tissue typing can be
used alone or in combination with more conventional techniques for
determining the source of a tissue sample. Examples of conventional
techniques include RFLP (restriction fragment length polymorphism,
U.S. Pat. No. 5,272,057), DNA analysis (e.g., PCR), blood typing,
etc.
[0200] In addition, a database can be created out of numerous
individuals', populations', or species' small molecule profile,
thus enabling the positive identification of even a small tissue
sample whose small molecule profile is registered with the
database.
[0201] Small molecule profiling of cells, cellular organelles, and
extracellular material can also be used in forensics. Forensic
biology is a scientific field employing, for example, genetic
typing of biological evidence found at a crime scene as a means for
positively identifying a perpetrator of a crime. In traditional
DNA-based forensics, PCR technology is used to amplify DNA
sequences taken from very small biological samples such as tissues,
e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen
found at a crime scene. The amplified sequence can then be compared
to a standard, thereby allowing identification of the origin of the
biological sample.
[0202] It is believed that the small molecule profile of each
person's body fluids, cells, or cellular organelles, should contain
unique amounts of various small molecule. Therefore, a small
molecule profile of a given sample should yield a unique
"fingerprint" of the perpetrator of the crime. Unlike conventional
techniques, the present invention allows for a quick profile of the
sample without the time consuming task of PCR. PCR is dependent on
a multitude of repeated copies of the perpetrator's DNA, and
therefore, its reliability is somewhat uncertain. The invention
also encompasses methods for using the claimed small molecule
profiles in combination with more conventional techniques, such as
PCR, for enhanced sensitivity.
[0203] The small molecule profiles of the invention can also be
used to develop small molecule reagents to identify particular
tissue types. For example, certain tissues (e.g., muscle, blood,
urine, spinal fluid, interstial fluid, nervous tissue, fatty
tissue, etc.) should have unique small molecule profiles as
compared to other body tissues. These tissues will have enhanced
concentrations of certain key small molecules and have diminished
concentrations of others. The identity of these tissue specific
small molecules may be consistent over a subset of the population
or the entire species as a whole.
[0204] Therefore, the invention pertains to the use of small
molecule reagents that specifically react with key small molecules
identified as being localized to a specific tissue over a subset of
the population. The small molecule reagents can then be used to
identify the identity of a tissue of unknown origin (e.g., brain,
blood, urine, spinal fluid, interstial fluid, muscle, fatty tissue,
etc.).
16. Pharmaceutical Compositions
[0205] In another embodiment, the invention pertains to
pharmaceutical compositions comprising a biologically active small
molecule, disease relevant, or another molecule obtained through
using the methods of the invention and a pharmaceutically
acceptable carrier. In another embodiment, the invention includes
nutriceutical preparations of biologically active small molecules
of the invention.
[0206] The biologically active small molecules may be chemically
modified to enhance their biological activity. It is known in the
art that through chemical modifications, one can enhance the
biological activity, stability, or otherwise modify a molecule to
make it more suitable as a pharmaceutical or nutriceutical
agent.
[0207] The language "pharmaceutical composition" includes
preparations suitable for administration to mammals, e.g., humans.
When the compounds of the present invention are administered as
pharmaceuticals to mammals, e.g., humans, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0208] The phrase "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material,
composition or vehicle, suitable for administering compounds of the
present invention to mammals. The carriers include liquid or solid
filler, diluent, excipient, solvent or encapsulating material,
involved in carrying or transporting the subject agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations.
[0209] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0210] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0211] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0212] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0213] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0214] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0215] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0216] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0217] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluent commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0218] Besides inert dilutents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0219] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0220] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0221] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0222] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0223] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0224] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0225] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active compound in a polymer
matrix or gel.
[0226] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0227] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0228] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0229] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0230] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0231] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0232] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administration is
preferred.
[0233] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0234] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0235] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0236] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0237] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0238] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0239] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0240] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. If
desired, the effective daily dose of the active compound may be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms.
[0241] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition.
[0242] As set out above, certain embodiments of the present
compounds can contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically acceptable salts" is art recognized and includes
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or by separately reacting a purified compound of
the invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm. Sci. 66:1-19).
[0243] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances includes relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like.
[0244] The term "pharmaceutically acceptable esters" refers to the
relatively non-toxic, esterified products of the compounds of the
present invention. These esters can be prepared in situ during the
final isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent. Carboxylic acids can be
converted into esters via treatment with an alcohol in the presence
of a catalyst. Hydroxyls can be converted into esters via treatment
with an esterifying agent such as alkanoyl halides. The term also
includes lower hydrocarbon groups capable of being solvated under
physiological conditions, e.g., alkyl esters, methyl, ethyl and
propyl esters. (See, for example, Berge et al., supra.)
[0245] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated by
reference.
17. Agricultural Methods of the Invention
[0246] In another embodiment of the invention, the invention
includes a method for the identification of agents useful for
agriculture, such as for example, insecticides, pesticides,
herbicides, and fertilizers.
[0247] Plants are an excellent source of small molecules. Many
plant small molecules have been shown to have therapeutic benefit.
Therefore, in one embodiment, the invention pertains to a library
of the small molecules from plant extracts (e.g., extracts from a
particular plant or part of plant (e.g., seeds, flowers, berries,
roots, sap, leaves, etc.), cells from the plant, organelles (e.g.,
mitochondria, chloroplasts, nuclei, Golgi apparatus, etc.),
cellular compartments, etc. These libraries can also be screened
for biologically active molecules using the methods described in
previous sections. Furthermore, the plants also can be analyzed
using any of the separation or analytical techniques described
herein, e.g., HPLC, TLC, electrochemical analysis, mass
spectroscopy, refractive index spectroscopy (RI), Ultra-Violet
spectroscopy (UV), fluorescent analysis, radiochemical analysis,
Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonance
spectroscopy (NMR), Light Scattering analysis (LS) and other
methods known in the art.
[0248] Furthermore, comparison of plant small molecule profiles
could lead to the identification of compounds which are relevant to
the plant's resistance of certain diseases or environmental
conditions.
[0249] In addition, the method also pertains to small molecule
profiles and small molecule libraries of plants. For example, the
small molecule profiles can be used to determine plant deficiencies
of certain compounds, and analyze plant diseases in a method
analogous to the comparison of animal small molecule profiles. For
example, a small molecule profile can be determined of a specific
plant cell, cell compartment or organelle (e.g., chloroplast,
mitochondria, endoplasmic reticulum, Golgi apparatus, etc.).
Standard plant cell profiles can also be generated. These can be
compared to plants in particular disease states to determine which
small molecules are present in aberrant amounts in the diseased
cells.
[0250] In one method of the invention, small molecule profiles of
insect cells, cellular compartments, or specific organelles are
compared to small molecule profiles of insect cells, cellular
compartments, or organelles treated with a known insecticide. The
small molecule profiles can be compared to identify compounds which
are relevant to the insecticide activity. The compounds which are
identified as relevant can then be identified to further optimize
the insecticidal activity of the compounds.
[0251] The term "insecticides" include compounds which kill or
other wise limit the reproductive capacity of organisms from the
order Isopoda (e.g., Oniscus asellus, Armadillidium vulgare and
Porcellio scaber, the order Diplopoda (e.g., Blaniulus guttulatus),
the order Chilopoda (e.g., Geophilus carpophagus, Scutigera spec,
etc.), the order Symphyla (e.g., Scutigerella immaculata, etc.),
the order Thysanura (e.g., Lepisma saccharina, the order Collembola
(e.g., Onychiurus armatus), the order Orthoptera (e.g., Blatta
orientalis, Periplaneta americana, Leucophaea maderae, Blattella
germanica, Acheta domesticus, Gryllotalpa spp., Locusta migratoria
migratorioides, Melanoplus differentialis and Schistocerca
gregaria, etc.), the order Dermaptera (e.g., Forficula auricularia,
etc.), the order Isoptera (e.g., Reticulitermes spp, etc.), the
order Anoplura (e.g., Pediculus humanus corporis, Haematopinus
spp., Linognathus spp. etc.), the order Mallophaga (e.g.,
Trichodectes spp. Damalinea spp., etc.), the order Thysanoptera
(e.g., Hercinothrips femoralis, Thrips tabaci), the order
Heteroptera (Eurygaster spp., Dysdercus intermedius, Piesma
quadrata, Cimex lectularius, Rhodnius prolixus and Triatoma spp.,
etc.), the order Homoptera (e.g., Aleurodes brassicae, Bemisia
tabaci, Trialeurodes vaporariorum, Aphis gossypii, Brevicoryne
brassicae, Cryptomyzus ribis, Doralis fabae, Doralis pomi, Eriosoma
lanigerum, Hyalopterus arundinis, Macrosiphum avenae, Myzus spp.,
Phorodon humuli, Rhopalosiphum padi, Phylloxera vastatrix,
Pemphigus spp., Empoasca spp., Euscelis bilobatus, Nephotettix
cincticeps, Lecanium corni, Saissetia oleae, Laodelphax
striatellus, Nilaparvata lugens, Aonidiella aurantii, Aspidiotus
hederae, Pseudococcus spp., Psylla spp., etc.), the order
Lepidoptera, (e.g., Pectinophora gossypiella, Bupalus piniarius,
Chematobia brumata, Lithocolletis blancardelia, Hyponomeuta
padella, Plutella maculipennis, Malacosoma neustria, Euproctis
chrysorrhoea, Lymantria spp. Bucculatrix thurberiella,
Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltia spp.,
Earias insulana, Heliothis spp., Laphygma exigua, Mamestra
brassicae, Panolis flammea, Prodenia litura, Spodoptera spp.,
Trichoplusia ni, Carpocapsa pomonella, Pieris spp., Chilo spp.,
Pyrausta nubilalis, Ephestia kuehniella, Galleria mellonella,
Tineola bisselliella, Tinea pellionella, Hofmannophila
pseudospretella, Cacoecia podana, Capua reticulana, Choristoneura
fumiferana, Clysia ambiguella, Homona magnanima, Tortrix viridana,
etc.), the order Coleoptera (e.g., Anobium punctatum, Rhizopertha
dominica, Bruchidius obtectus, Acanthoscelides obtectus, Hylotrupes
bajulus, Agelastica alni, Leptinotarsa decemlineata, Phaedon
cochleariae, Diabrotica spp., Psylliodes chrysocephala, Epilachna
varivestis, Atomaria spp., Oryzaephilus surinamensis, Anthonomus
spp., Sitophilus spp., Otiorrhynchus sulcatus, Cosmopolites
sordidus, Ceuthorrhynchus assimilis, Hypera postica, Dermestes
spp., Trogoderma spp., Anthrenus spp., Attagenus spp., Lyctus spp.,
Meligethes aeneus, Ptinus spp., Niptus hololeucus, Gibbium
psylloides, Tribolium spp., Tenebrio molitor, Agriotes spp.,
Conoderus spp., Melolontha melolontha, Amphimallon solstitialis
Costelytra zealandica, etc.), the order Hymenoptera, (Diprion spp.,
Hoplocampa spp., Lasius spp., Monomorium pharaonis, Vespa spp.,
etc.), the order of the Diptera (e.g., Aedes spp., Anopheles spp.,
Culex spp., Drosophila melanogaster, Musca spp., Fannia spp.,
Calliphora erythrocephala, Lucilia spp., Chrysomyia spp., Cuterebra
spp., Gastrophilus spp., Hyppobosca spp., Stomoxys spp., Oestrus
spp., Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus,
Oscinella frit, Phorbia spp., Pegomyia hyoscyami, Ceratitis
capitata, Dacus oleae, Tipula paludosa, etc.), the order
Siphonaptera (e.g., Xenopsylla cheopis and Ceratophyllus spp.,
etc.), the order Arachnida (e.g., Scorpio maurus, Latrodectus
mactans, etc.), the order Acarina (e.g., Acarus siro, Argas spp.,
Ornithodoros spp., Dermanyssus gallinae, Eriophyes ribis,
Phyllocoptruta oleivora, Boophilus spp., Rhipicephalus spp.,
Amblyomma spp., Hyalomma spp., Ixodes spp., Psoroptes spp.,
Chorioptes spp., Sarcoptes spp., Tarsonemus spp., Bryobia
praetiosa, Panonychus spp., Tetranychus spp, etc.), Pratylenchus
spp., Radopholus similis, Ditylenchus dips aci, Tylenchulus
semipenetrans, Heterodera spp., Meloidogyne spp., Aphelenchoides
spp., Longidorus spp., Xiphinema spp., and Trichodorus spp.
[0252] In another embodiment, small molecule profiles of insect
cells treated with a test compound can be compared to small
molecule profiles of insect cells treated with a known insecticide
to determine whether the test compound may be an active
insecticide.
[0253] The invention also pertains to insecticides comprising one
or more insecticides identified by the methods of the invention. In
one embodiment, the insecticides of the invention are non-toxic to
humans.
[0254] The insecticide compositions of the invention, both solids
and liquids, may be applied to insect infestations or insect
populations by spraying. The methods and equipment needed for a
given treatment may be determined by one skilled in the art.
Furthermore, methods of the invention described herein may be used
to treat insect infestations or populations in dry, moist, or
aquatic systems (e.g., the insect-infested area is a flowing or a
standing body of water). An aquatic system which is treated with
methods of the present invention may be either fresh water or salt
water. Furthermore, the insect control compositions of the
invention may be applied directly onto a host (e.g., an
agricultural crop, a turfgrass).
EXEMPLIFICATION OF THE INVENTION
Example 1
Method for Obtaining a Small Molecule Profile of a Cellular
Compartment
Method for Obtaining a Small Molecule Profile of Mitochondria Using
HPLC
[0255] The following method demonstrates how small molecules are
isolated from mitochondria for methods as described herein.
Mitochondrial Isolation
[0256] Mitochondria from a mammalian source are isolated by
differential centrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4.
Following the final wash, mitochondria are resuspended in the same
buffer and aliquots are quick frozen in liquid nitrogen. Protein
determinations are carried out by Lowry using the Sigma Protein
Assay Kit P5656).
[0257] Other mitochondrial samples are purified using a modified
version of a published protocol (Rigobello et al. (1995) Arch.
Biochem. Biophys. 319, 225-230). Mammalian liver mitochondria are
obtained after decapitation of the subjects. The livers are
dissected out and are placed in an ice-cold solution containing 250
mM mannitol, 75 mM sucrose, 100 .mu.M EDTA, 500 .mu.M EGTA, and 10
mM Hepes (pH 7.4). The livers are homogenized with a motor-driven
Teflon pestle and the homogenate is centrifuged at 1000 g for 10
min Supernatants are removed and centrifuged at 10,000 g for 15
min. The pellets are washed in 250 mM mannitol, 75 mM sucrose, 100
.mu.M EDTA, 500 .mu.M EGTA, and 10 mM Hepes (pH 7.4) with 0.5%
bovine serum albumin (BSA) (Sigma A-6003). Following
centrifugation, the pellets are then washed twice more in 250 mM
mannitol, 75 mM sucrose, 30 .mu.M EDTA, and 10 mM Hepes (pH 7.4)
with 0.5% BSA. Following the final wash, mitochondria are
resuspended in the 5 ml of the final buffer without BSA. An aliquot
is removed, pelleted in a microfuge, washed once with 160 mM KC1,
repelleted, and dry-frozen at -80.degree. C. Samples are analyzed
by HPLC within 1 week of isolation.
HPLC Standards
[0258] Basic HPLC and Coulometric array methodology has been
previously described with regard to their use for serum, urine, and
tissue analysis (Beal et al. (1990) J. Neurochem. 55:1327; Matson
et al. (1987) Life Sci. 41:905; LeWitt et al. (1992) Neurology
42:2111; Ogawa et al. (1992) Neurology 42:1702; Beal et al. (1992)
J. Neurol. Sci. 108:80). Standards for stock solutions are obtained
from Sigma and stored at -80.degree. C. as 1 mg/ml stocks in 20%
MeOH containing 1% phosphoric acid. Subsequent dilution to working
strength is made into 0.1 M NaCl. The assay sequence is: standard,
8 samples, sample pool, standard, etc. Within-run precision is
derived from the prevision of the repeated pool assays. Precision
varies primarily as a function of the level of the analyte and
secondarily as the complexity of the region in which it occurs.
Typically at 5 pg precision is .+-.20%, at 500 pg .+-.5%, and at 1
ng .+-.3%.
Sample Preparation
[0259] Mitochondrial samples, including approximately 5 mg of
mitochondrial protein, are precipitated and extracted in 4 vol of
acetonitrile, 4% acetic acid at -20.degree. C. One milliliter of
centrifuged supernatant is removed, evaporated to dryness under
vacuum, and reconstituted in 200 .mu.l of mobile phase A (11
g/liter of pentane sulfonic acid at pH 3.00 with acetic acid).
Recoveries, verified by sequential extractions and comparisons of
entire patterns, ranged from 93 to 100% for all compounds resolved.
This protocol conserves reactive species such as ascorbate and
homogentistic acid at 1 ng/ml concentrations. Reconstituted extract
equivalent to 2 mg of mitochondrial protein is placed in an auto
sample vial and immediately analyzed. Remaining extract is frozen
at -80.degree. C. for future confirmation analysis. Immediately
prior to injection, samples are maintained in an autoinjector at
0-1.degree. C.
Chromatographic Methods
[0260] To retain stability of retention times and response
potentials, a mobile phase combination of mobile phase A (above)
and mobile phase B (0.1 M Li-acetate at pH 3.00 with acetic acid in
80/10/10 methanol/acetonitrile/isopropanol) is used. The
chromatographic method involves a 120-min complex gradient from 0%
B to 100% B, with flow rate adjusted to compensate for aziotropic
viscosity effects, and has been previously described in detail
(Milbury et al. (1997) in Progress in HPLC, Coulometic Electrode
Array Detectors for HPLC, pp. 125-141, VSP International Science
Publications). The mixed gradient is delivered from a peak
suppressor/gradient mixer to a PEEK-lined pulse damper prior to
flowing through the auto sampler injector and on to two series C18
columns [META250, 5-.mu.m ODS, 250.times.4.6 mm I.D., ESA,
Inc.].
[0261] The small molecules are detected using a 16-channel
coulometric electrode array (ESA, Inc., Model 5600 CEAS gradient
system equipped with a Kontron Model 460 autosampler) incremented
from 0 to 900 mV in 60-mV steps. Peak suppressor/gradient mixer,
pulse damper, columns, and detectors are contained within a
temperature-controlled enclosure maintained at 35.degree. C. System
functions are controlled by 5600-CEAS software installed on a 386
microcomputer.
[0262] The detected small molecules can then be analyzed on a
computer to create a small molecule profile. The small molecule
profile can then be compared, e.g., via subtraction, with small
molecule profiles of other samples. The isolated small molecules
can then be used in assays known in the art to determine biological
activity.
Example 2
Method for Analyzing Metabolic Disorders Using Small Molecule
Profiles
[0263] Method for Analyzing Differences in Small Molecule Profiles
of Genetically Altered Mice after Short Term Diet Variations
[0264] 15 female C57B1/6J ob/ob mice and lean littermate controls
(15 female C57B1/6J ?/+) and 15 male C57B1/Ks db/db mice and lean
littermate controls (15 male C57B 1/ks +/+) are obtained from
Jackson labs at 4.5 weeks of age, and are housed individually on
normal mouse chow (West, D. B., 1992, Am. J. Physiol.
262:R1025-R1032) for 1 week prior to the initiation of the study.
The four groups of 15 mice each are then sacrificed by CO.sub.2
euthanasia and tissues are then collected. Body weight (grams) of
the four groups of mice at the time of sacrifice are measured.
Small molecule profiles from cells from the hypothallumus are then
obtained from each group of mice and compared.
[0265] The mice (normal, lean, ob/ob, db/db, and/or tub/tub) are
fed normally prior to the initiation of the experiment, and then
they are divided into one control and two experimental groups. The
control group are then maintained on ad lib nourishment, while the
first experimental group ("fasted group") is fasted, and the second
experimental group ("fasted-refed group") is initially fasted, and
then offered a highly palatable meal shortly before the collection
of tissue samples for small molecule profiling. Each test animal is
weighed immediately prior to and immediately after the experiment.
Small molecule profiles are taken of each mouse from each group
before and after the experiment. The profiles of each group are
averaged and compared to those of different groups.
Method for Comparing Small Molecule Profiles of Mice after Long
Term Diet Variations
[0266] Mice are fed normally prior to the initiation of the
experiment, and then are divided into one control and two
experimental groups. The control group is then maintained on an ad
lib diet of normal nourishment in order to calculate daily food
intake. The first experimental group ("underweight group") is then
underfed by receiving some fraction of normal food intake, 60-90%
of normal, for example, so as to reduce and maintain the group's
body weight to some percentage, for example 80%, of the control
group. The second experimental group ("overweight group") is
overfed by receiving a diet which would bring the group to some
level above that of the control, for example 125% of the control
group. Tissue samples are obtained for small molecule profiles to
determine compounds which are present in different amounts in
control versus overweight and/or underweight conditions.
Example 3
Comparison of Small Molecule Profiles of Different Types of Immune
Cells
Method for Comparing the Small Molecule Profiles of TH1 and TH2
Cells
[0267] The transgenic T cell example is used to identify cellular
small molecules present in TH2 cells. The identified small
molecules are be present in different amounts in TH2 cells compared
to TH1 cells.
Transgenic Mice
[0268] Naive CD4.sup.+ cells are obtained from the spleens and/or
lymph nodes of unprimed transgenic mouse strains harboring a T cell
receptor (TCR) recognizing ovalbumin (Murphy et al., 1990, Science
250:1720).
Ova-Specific Transgenic T Cells
[0269] Suspensions of ova-specific T cells are co-cultured with
stimulatory peptide antigen and antigen presenting cells
essentially as described in Murphy et al. (Murphy et al., 1990,
Science 250:1720). Briefly, 2-4.times.10.sup.6 T cells are
incubated with approximately twice as many TA3 antigen presenting
cells in the presence of 0.3 .mu.M Ova peptide. TH1 cultures may
contain approximately 10 ng/ml recombinant mIL-12. Conversely, TH2
cells received IL-4 (1000 .mu./ml). Cultures are harvested at
various time points after initiation of culture. T cells are
purified of TA3 cells using anti-CD4 coated magnetic beads (Dynal,
Inc.). T cells are then pelleted by gentle centrifugation and
lysed.
Tissue Collection:
[0270] Cells are then quick frozen on dry ice. Samples are
homogenized together with a mortar and pestle under liquid
nitrogen.
Mitochondrial Isolation and Generation of Small Molecule
Profiles
[0271] Cellular mitochondria are isolated and small molecule
profiles are generated using the procedure given in Example 1.
Method for Comparing Small Molecule Profiles of Different TH Cell
Subpopulations
[0272] In this Example, the generation of small molecule profiles
representing small molecules which are present in different amounts
in TH cell subpopulations and/or during the differentiation of such
subpopulations are described.
[0273] TH cell clones such as, D10.G4 (TH2), AE7 (TH1) and D1.1
(TH1), are used. Prior to stimulation, cell cultures are enriched
for live cells by centrifugation through a Ficoll gradient.
Recovered cells are then counted and their viability is examined
using trypan blue exclusion. Cells are replated into either T25 or
T75 flasks at approximately 5.times.10.sup.6 cells in 5 mLs or
1.5.times.10.sup.6 cells in 10 mLs of culture medium,
respectively.
[0274] Coating is then performed, generally, according to Current
Protocols in Immunology, 1992, Coligan, J. E. et al., John Wiley
& Sons, NY, pp 3.12.4-3.12.6). Specifically, prior to plating,
the flasks are coated with anti-CD3-.epsilon. antibodies (hybridoma
supernatant from the 145-C11 hybridoma; Parmingen, Inc., San Diego
Calif.). For coating, antibodies are resuspended in PBS at 1-2
.mu.g/ml at a volume sufficient to coat the bottom of the flasks.
Coating solution is incubated on the flasks for at least one hour
at 37.degree. C.
[0275] After incubation, the antibody coating solution is removed
by aspiration and cells will be immediately added. Flasks will then
be placed in a 37.degree. C. incubator for 6 hours. Cells are
harvested by, for example, removal of supernatant from the culture.
The mitochondria are removed from the cells by the procedure given
above. Small molecule profiles of each type of TH cell can then be
done and analyzed to determine differences and similarities between
the subpopulations.
Example 5
Method of Identifying Cardiovascular Disease Relevant Small
Molecules
Method Using an Endolethial Cell Shear Stress Models to Obtain
Small Molecule Profiles and Identify Disease Relevant Small
Molecules
Cell Culture
[0276] Primary cultures of HUVEC's are established from normal term
umbilical cords as described (In Progress in Hemostasis and
Thrombosis, Vol. 3, P. Spaet, editor, Grune & Stratton Inc.,
New York, 1-28). Cells are grown in 20% fetal calf serum complete
media (1989, J. Immunol. 142: 2257-2263) and passaged 1-3 times
before shear stress induction.
[0277] For induction, second passage HUVEC's are plated on tissue
culture-treated polystyrene and subjected to 10 dyn/cm.sup.2
laminar flow for 1 and 6 hr. as described (1994, J. Clin. Invest.
94: 885-891) or 3-10 dyn/cm.sup.2 turbulent flow as previously
described (1986 Proc. Natl. Acad. Sci. U.S.A. 83: 2114-2117).
Mitochondrial Isolation
[0278] Mitochondria from the HUVEC's are isolated by differential
centrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4. Following the
final wash, mitochondria are resuspended in the same buffer and
aliquots are quick frozen in liquid nitrogen. Protein
determinations are carried out by Lowry using the Sigma Protein
Assay Kit P5656).
Sample Preparation
[0279] Mitochondrial samples, including approximately 5 mg of
mitochondrial protein, are precipitated and extracted in 4 vol of
acetonitrile, 4% acetic acid at -20.degree. C. One milliliter of
centrifuged supernatant is removed, evaporated to dryness under
vacuum, and reconstituted in 200 .mu.l of mobile phase A (11
g/liter of pentane sulfonic acid at pH 3.00 with acetic acid).
Reconstituted extract equivalent to 2 mg of mitochondrial protein
is placed in an auto sample vial and immediately analyzed.
Immediately prior to injection, samples are maintained in an
autoinjector at 0-1.degree. C.
Chromatographic Methods
[0280] To retain stability of retention times and response
potentials, a mobile phase combination of mobile phase A (above)
and mobile phase B (0.1 M Li-acetate at pH 3.00 with acetic acid in
80/10/10 methanol/acetonitrile/isopropanol) is used. The
chromatographic method involves a 120-min complex gradient from 0%
B to 100% B, with flow rate adjusted to compensate for aziotropic
viscosity effects, and has been previously described in detail
(Milbury et al. (1997) in Progress in HPLC, Coulometic Electrode
Array Detectors for HPLC, pp. 125-141, VSP International Science
Publications). The mixed gradient is delivered from a peak
suppressor/gradient mixer to a PEEK-lined pulse damper prior to
flowing through the auto sampler injector and on to two series C18
columns [META250, 5-.mu.m ODS, 250.times.4.6 mm I.D., ESA,
Inc.].
[0281] The small molecules are detected using a 16-channel
coulometric electrode array (ESA, Inc., Model 5600 CEAS gradient
system equipped with a Kontron Model 460 autosampler) incremented
from 0 to 900 mV in 60-mV steps. Peak suppressor/gradient mixer,
pulse damper, columns, and detectors are contained within a
temperature-controlled enclosure maintained at 35.degree. C. System
functions are controlled by 5600-CEAS software installed on a 386
microcomputer.
[0282] The detected small molecules can then be analyzed on a
computer to create a small molecule profiles. The small molecule
profiles of the cells are then compared to those not subjected to
the turbulent flow. Small molecule present in aberrant amounts in
the sample subjected to the turbulent flow are identified for
further investigation.
Example 6
Identification of Disease Relevant Small Molecules in Human Cell
Samples
Human Tumor Example
[0283] In this example, a cell sample is taken from a malignant
tumor in a human subject. Normal tissue is also collected from the
subject from the same or similar tissue as the tumor (e.g., normal
breast tissue and breast tumor tissue; normal prostate tissue and
prostate tumor tissue, etc.). Normal tissue is also collected from
a healthy subject from an analogous tissue location.
[0284] The tissue samples are the homogenized and the mitochondria
are isolated.
Mitochondrial Isolation
[0285] Mitochondria from a each tissue source are isolated by
differential centrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4.
Following the final wash, mitochondria are resuspended in the same
buffer and aliquots are quick frozen in liquid nitrogen. Protein
determinations are carried out by Lowry using the Sigma Protein
Assay Kit P5656).
Sample Preparation
[0286] The mitochondrial samples, including approximately 5 mg of
mitochondrial protein, are precipitated and extracted in 4 vol of
acetonitrile, 4% acetic acid at -20.degree. C. One milliliter of
centrifuged supernatant is removed, evaporated to dryness under
vacuum, and reconstituted in 200 .mu.l of mobile phase A (11
g/liter of pentane sulfonic acid at pH 3.00 with acetic acid).
Reconstituted extract equivalent to 2 mg of mitochondrial protein
is placed in an auto sample vial and immediately analyzed.
Immediately prior to injection, samples are maintained in an
autoinjector at 0-1.degree. C.
Chromatographic Methods
[0287] To retain stability of retention times and response
potentials, a mobile phase combination of mobile phase A (above)
and mobile phase B (0.1 M Li-acetate at pH 3.00 with acetic acid in
80/10/10 methanol/acetonitrile/isopropanol) is used. The
chromatographic method involves a 120-min complex gradient from 0%
B to 100% B, with flow rate adjusted to compensate for aziotropic
viscosity effects, and has been previously described in detail
(Milbury et al. (1997) in Progress in HPLC, Coulometic Electrode
Array Detectors for HPLC, pp. 125-141, VSP International Science
Publications). The mixed gradient is delivered from a peak
suppressor/gradient mixer to a PEEK-lined pulse damper prior to
flowing through the auto sampler injector and on to two series C18
columns [META250, 5-.mu.m ODS, 250.times.4.6 mm I.D., ESA,
Inc.].
[0288] The small molecules are detected using a 16-channel
coulometric electrode array (ESA, Inc., Model 5600 CEAS gradient
system equipped with a Kontron Model 460 autosampler) incremented
from 0 to 900 mV in 60-mV steps. Peak suppressor/gradient mixer,
pulse damper, columns, and detectors are contained within a
temperature-controlled enclosure maintained at 35.degree. C. System
functions are controlled by 5600-CEAS software installed on a 386
microcomputer.
Analysis
[0289] The small molecule profiles of the healthy subject are
compared to the small molecule profiles from the tumor tissue and
the non-tumor tissue of the cancer patient. Small molecules which
are present in aberrant amount in the tumor tissue are identified
by comparing the profiles.
Example 7
Identification of ALS Relevant Small Molecules in Human Samples
[0290] The sample set consisted of about 50 blinded sera of an
undisclosed number of control and diagnosed amlotroptic lateral
sclerosis (ALS) subjects. Within the control population of
subjects, some subjects were suffering from other central nervous
system disease states. The population of ALS subjects included
subjects who were newly discovered to severely-impaired. In
addition, the majority of ALS subjects were taking Riluzole as
their primary medication. All of the subjects were on complex drug
regimens. All of the samples were examined for their total chemical
constituents, each constituent was scored for concentration and the
final results were databased.
[0291] At this point, half of the sera were unblinded as to their
clinical nature (control/non-control, age, gender, therapies,
etc.). Using this half as reference, the database was examined for
possible markers. General markers of the class of ALS subjects were
identified as well as sub-class markers within the population
taking the drug Riluzole. FIG. 1 shows markers which were
identified in subjects with ALS who are not taking Riluzole. FIG. 2
shows markers which were identified in subjects with ALS who were
taking Riluzole.
[0292] These markers were then used to predict the population
affiliation of the remaining unblinded samples. The correct
population affiliation was determined for each of the samples using
the markers. The metabolic significance of the markers we
identified is currently under investigation. It is believed that
the most prominent of these markers are not drug metabolites, but
represent the successful metabolic response of the subject to the
drug.
[0293] In this example, it was shown that the use of metabolomics
can be used to determine biochemical response of a subject to a
drug as well as the unique biochemical characteristics of ALS
subjects. FIG. 3 is a three dimensional graph showing the
statistical separation of subjects. The population, ALS1, are
suffering from ALS and are undergoing Riluzole treatment, while the
population ALS2 are suffering from ALS and not undergoing Riluzole
treatment.
Example 8
Use of Databases Containing Metabolites and Other ALS Relevant
Small Molecules
[0294] This example shows that databases can be created using
metabolomics to successfully determine whether or not a subject is
suffering from a particular disorder, such as, for example,
ALS.
[0295] Databases of metabolites in the plasma of ALS patients and
in controls were generated. Metabolites in the plasma were
separated using different HPLC methods and detected by CEAS
(coulometric electrode array system), LC/MS (liquid
chromatograph/mass spectrometry) and/or GC/MS (gas
chromatograph/mass spectrometry). Samples from controls and ALS
patients were profiled and data on each metabolite was extracted
and stored in the respective database.
[0296] Once the samples were collected, they were maintained in a
frozen state. Each sample was thawed and immediately aliquoted into
100 .mu.l portions, accessioned into LIMS and refrozen at -80.
Subsequently, a known mass was homogenized in an equal volume of
acidic acetone slush with Polyethylene Glycol 100 (PEG100) and
Dithiothreitol (DTT). Aliquots were divided between each analysis
platform and each preparation is diluted (1:1) with a reference
compound solution (RCS).
[0297] For LC sample preparation, the reference compound solution
is composed of 20 reference compounds that are present at
predefined concentrations. These reference compounds are all
fluorescently-derivatized compounds of varying lipophilicity. The
reference compounds are synthetically pure compounds, chosen for
their chemical stability. In the case of GC sample preparations,
the RCS comprises straight chain hydrocarbons. The chromatographic
fluorescence profiles (LC) and paraffin profiles (GC) are used as
the basis for a post-separation quality control (QC) on the
chromatography. Furthermore, specific information from these
profiles is used in the interpretation of the data files produced
by other instrumentation.
[0298] A Surveyor HPLC fitted with a fluorescence detector and a
bar-code reader is used. The effluent is split three ways with 2%,
2% and 96% of the stream being directed to two Thermo-Finnigan
Mat-95 XP mass spectrometers and an ESA 16 channel Coularray
electrochemical detector, respectively. A single Thermo-Finnigan
LTQ-FT mass spectrometer, which has an ion-trap (IT) front end and
a Fourier-Transform (FT) backend, is set for monitoring both
positive and negative ions respectively. The electrochemical
detector allows one to see most electrochemically-active species
with extreme sensitivity. Some compounds may be redundantly
visualized across more than one of these machines. By using a
combination of detectors, the vast majority of metabolites are
detected.
[0299] The samples destined for GC are dried under vacuum
desiccation for a minimum of 24 hours, before being derivatized
under dried argon using Bistrimethyl-silyl-trifluoroacetamide
(BSTFA) catalyzed with derivatization reagent Trimethylsulfonyl
chloride (TMSC1). A 5% phenyl column with a temperature range from
40.degree. to 300.degree. C. is used. Samples are analyzed on a
Thermo-Finnigan Mat-95 XP using Electron Impact ionization, and
high resolution. The resulting spectra are used for analysis of
elemental composition and identification.
[0300] Samples for induced coupling plasma/mass spectroscopy
(ICP/MS) are acid digested for 24 hours, filtered and separated by
ion chromatography prior to the introduction of the column effluent
into the plasma of the ICP/MS.
[0301] The methods described in this example identify, quantify,
and store for statistical analysis, peaks representative of unknown
compounds in addition to the known compounds/peaks. Where a peak is
identified as statistically significant, i.e. a biomarker to a
definable population or sub-population, it is subjected to a
chemical identification process using many of the instruments used
in the original analysis. For example, if the unknown peak is seen
in the LC, the ion-trap (IT) portion is used to do a detailed
fragmentation analysis of the molecule. If needed, repeated
fragmentation cycles in the IT will be conducted. If the unknown
peak is seen in the GC, additional fragment analyses are done at a
higher resolution than normal (profiling resolution .about.20,000,
compound identification resolution .about.100,000).
[0302] Several mathematical tools were employed to differentiate
between the databases of data generated for the ALS and control
groups. Mathematical tools used for data analysis included partial
least squares-discriminant analysis (PLS-DA), "relative class
association/weighted voting", and scatter analysis (Multivariate
Statistical Methods: A Primer: Bryan Manley, CRC Press; Kennedy R
et al. Solving data Mining Problems through Pattern Recognition,
Prentice Hall PTR; Erikson I et al. Multi and Megavariate Analysis:
Principles and Applications. Umetrics, Umea, Sweden, 2001 edition).
These analytical approaches were used to separate ALS patients from
controls and derive an initial metabolic signature for ALS based on
significant differences in their metabolomes. These signatures
highlight metabolites that are found at significantly higher or
lower concentrations in ALS, marking the disease.
[0303] Partial least square discriminate analysis (PLS-DA) is
similar to the more widely known principal components analysis.
However, rather than finding independent components that best
explain overall variance in a data set, PLS-DA finds components
that best explain the differences between two classes. These
components can then be used to predict the class membership of new
examples.
[0304] Relative class association/weighted voting was first used to
distinguish two types of leukemia based on gene expression profiles
(Golub T, et al. Science 1999; 286:531). This method begins by
computing a "relative class association" for each compound, which
is a measure of the degree to which the concentration of the
compound is associated with one of two classes of interest. The
method then determines by a permutation test which compounds have
significant relative class associations, and uses these compounds
to "vote" for membership in one of the two classes, and may also
use cross-validation on the training set to further prune the
compounds considered. The vote of each compound is weighted by its
relative class association. The sample is assigned to one of the
classes according to the sum of the votes.
[0305] Scatter analysis first detects compounds that have means
that are significantly higher or lower among a target class (for
example ALS) than among controls. The cutoff is usually 5 standard
deviations in the control distribution above or below the mean
concentration for controls. For prediction, the presence of one of
these compounds with a concentration that is highly different from
the mean in controls is taken as evidence that the source sample is
not in the control class.
[0306] FIG. 4 shows that using PLS-DA, ALS patients were
distinguishable from their control counterparts. Fifteen patients
with SALS had unique features that separated them from the eight
control counterparts.
[0307] The databases generated can also be used to distinguish
between controls, subjects with SALS, and subjects with pure lower
motor neuron disease. In this example, a set of 59 blinded plasma
samples contained an undisclosed number of controls, subjects
diagnosed with SALS, and a few patients with pure lower motor
neuron disease. Each chemical constituent was scored for
concentration and the final results were stored in a database.
There were no significant differences in weight, age, or gender
distribution between the ALS, and the control cohort, although more
subjects with ALS took antioxidants than the control cohort (85%
vs. 30%, p<0.00001). After blinded data analysis, information on
chromatographic peaks was extracted from the raw chromatographic
data files and placed in a database. The clinical origin of 35 of
the 59 samples was unblinded and scatter analysis was used to
determine which compounds were at higher concentrations in ALS
patients than in the controls.
[0308] The three statistical analysis approaches were then employed
to evaluate differences in metabolite levels in ALS versus
controls, and a set of class predictors was constructed from the
unblinded set of samples. Compounds which are significantly
different between ALS and controls were discovered by algorithms
and used to build "class predictors". The class predictors used the
concentrations of these informative compounds to distinguish the
profiles of ALS patients from profiles of individuals in the
control groups. Broadly speaking, this is "supervised learning", a
kind of data mining in which a learning algorithm is presented with
examples of two or more "classes" and then attempts to derive a set
of rules (a class predictor) that will predict the class membership
of new examples. Thus, the supervised learning algorithm searches
the profiles to find the compounds that best let it distinguish ALS
profiles from non-ALS profiles, and base its class predictor on
these compounds.
[0309] Subsequently, the disease status of the rest of the 24
blinded samples was predicted using the methods of the invention.
Assignment of 11/12 of the control samples was done correctly, 8/8
of the ALS patients on Riluzole were correctly assigned and 3/4 of
the ALS patients not taking Riluzole were also correctly assigned.
An illustration of chromatograms generated using the HPLC-CEAS is
shown in FIG. 5.
[0310] After complete unblinding, a full analysis of the data set
was done and the results are shown in FIGS. 6A-6C. Using the peaks
identified as consistently variant (from controls) in one or more
populations (FIGS. 6A and 6B), metabolometric analysis was used to
determine which subjects were suiffering from ALS and which
subjects were on Riluzole therapy. In addition, subjects with pure
lower motor neuron disease and SALS were differentiated. After
unblinding, it was possible to identify four groups: controls
(Black), ALS subjects on Riluzole (Blue), ALS patients off Riluzole
(Red), and atypical ALS (Yellow) (FIG. 6C).
[0311] Three out of the four samples represented under atypical ALS
turned out to be patients with pure lower motor neuron disease. The
fourth had a long disease course (>9 years). A series of
endogenous cellular metabolites induced by the drug Riluzole were
identified. These compounds may be a response to the drug and may
be a part of the mechanism of action of Riluzole or part of its
side effects.
[0312] In this example it was shown that metabolomics can be used
to accurately distinguish between subjects suffering from ALS and
control subjects. The example shows that unique chemical markers
can be discovered by studying the databases using analytical
techniques. This example also shows that the chemical markers
discovered using the analytical techniques can successfully be used
to diagnosis and distinguish between groups of subjects suffering
from or not suffering from a particular disorder, such as ALS.
INCORPORATION BY REFERENCE
[0313] The entire contents of all references and patents cited
herein are hereby incorporated by reference. The entire contents of
U.S. Pat. No. 5,908,609 and all its references also expressly
incorporated herein.
EQUIVALENTS
[0314] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
* * * * *