U.S. patent application number 14/275359 was filed with the patent office on 2014-12-11 for method for determining the stability of organic methyleneamines in the presence of semicarbazide-sensitive amine oxidase.
The applicant listed for this patent is SANOFI-AVENTIS. Invention is credited to Adedayo ADEDOYIN, Michael ANGELASTRO, Julie BICK, Jennifer CAIRNS, Yongqing HUANG, Guyan LIANG, Heng-Keang LIM.
Application Number | 20140363836 14/275359 |
Document ID | / |
Family ID | 39361382 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140363836 |
Kind Code |
A1 |
ADEDOYIN; Adedayo ; et
al. |
December 11, 2014 |
Method for Determining the Stability of Organic Methyleneamines in
the Presence of Semicarbazide-Sensitive Amine Oxidase
Abstract
The present invention provides methods for determining the
stability of methyleneamine, methyleneamine-like compounds or
compounds containing an methyleneamine moiety in the presence of
semicarbazide-sensitive amine oxidase (SSAO) or a biological sample
containing SSAO activity. The disclosed methods may be configured
in an assay format for high throughput screening applications.
Inventors: |
ADEDOYIN; Adedayo;
(Stewartsville, NJ) ; ANGELASTRO; Michael;
(Bridgewater, NJ) ; BICK; Julie; (Easton, PA)
; CAIRNS; Jennifer; (Lebanon, NJ) ; HUANG;
Yongqing; (Flemington, NJ) ; LIANG; Guyan;
(Warren, NJ) ; LIM; Heng-Keang; (Lawrenceville,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SANOFI-AVENTIS |
Paris |
|
FR |
|
|
Family ID: |
39361382 |
Appl. No.: |
14/275359 |
Filed: |
May 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13680068 |
Nov 18, 2012 |
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14275359 |
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12518318 |
Jun 9, 2009 |
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PCT/US2008/050456 |
Jan 8, 2008 |
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13680068 |
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60884263 |
Jan 10, 2007 |
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Current U.S.
Class: |
435/25 |
Current CPC
Class: |
G01N 33/5008 20130101;
C12Q 1/26 20130101; G01N 2500/10 20130101; G01N 2560/00
20130101 |
Class at
Publication: |
435/25 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A method of identifying metabolic stability of a test agent due
to semicarbazide-sensitive amine oxidase (SSAO) catalyzed
metabolism, wherein the test agent is a drug candidate and the
metabolic stability of the test agent due to SSAO catalyzed
metabolism is unknown, the method comprising: a) culturing cells
expressing SSAO; b) adding a test agent to the cells; c) incubating
the test agent with the cells for a predetermined period of time;
d) measuring the amount of the test agent remaining in the presence
of the cells comprising expressed SSAO at the predetermined period
of time; and e) comparing the amount of the test agent measured in
step (d) to a control to determine the metabolic stability of the
test agent in the presence of cells comprising expressed SSAO.
2. The method of claim 1, wherein the cells expressing SSAO are
produced by transient or stable transfection of the cells with DNA
encoding SSAO.
3. The method of claim 1, wherein the cells expressing SSAO are
eukaryotic.
4. The method of claim 1, wherein the amount of test agent is
determined by a technique selected from the group consisting of
mass spectrometry, high-pressure liquid chromatography, liquid
chromatography/mass spectrometry, liquid chromatography/mass
spectrometry/mass spectrometry and liquid chromatography/radiomatic
detection.
5. The method of claim 1, wherein one or more steps are performed
by a robotic device.
6. The method of claim 1, wherein the metabolic stability of the
test agent is determined by measuring one or more metabolites of
the test agent in addition to or in place of measuring the test
agent.
7. The method of claim 1, wherein the control is a condition in
which the test agent is incubated for the predetermined period of
time in the absence of cells expressing SSAO or in cell lysates
obtained from such cells.
8. The method of claim 2, wherein the DNA comprises the nucleotide
sequence of SEQ ID NO: 2.
9. The method of claim 2, wherein the DNA encodes the amino acid
sequence of SEQ ID NO: 1.
Description
[0001] The present invention provides methods for determining the
stability of methyleneamine, methyleneamine-like compounds or
compounds containing an methyleneamine moiety in the presence of
semicarbazide-sensitive amine oxidase (SSAO) or a biological sample
containing SSAO activity. The disclosed methods may be configured
in an assay format for high throughput screening applications.
BACKGROUND OF THE INVENTION
[0002] Semicarbazide-sensitive amine oxidases (SSAO) are widely
distributed in tissues, particularly in blood vessels, suggesting a
role for this enzyme for inactivating circulating methyleneamines
(Lyles, Prog. Brain Res., 106:293-303, 1995). Many investigators
have focused their efforts on finding an endogenous ligand for SSAO
and on looking for inhibitors of this enzyme (Precious et al.,
Biochem. Pharmacol., 37:707-713, 1988; Crosbie and Callingham, J.
Neural Transm. Suppl., 41:427-432, 1994; Elliot et al., Biochem.
Pharmacol. 38:1507-1515, 1989; Boomsma et al., Comp. Biochem.
Physiol. C Toxicol. Pharmacol., 126:69-78, 2000; Yraola et al., J.
Med. Chem. 49:6197-6208; WO 02/066669). Others have searched for
alternative physiological roles of SSAO. For example, SSAO, also
called vascular adhesion protein-1 (VAP-1), has been shown to be
up-regulated under inflammatory conditions and to mediate the
binding of lymphocytes (WO 98/53049). The instant invention
utilizes the enzyme activity of SSAO to measure the metabolic
stability of test agents in cells and biological samples.
[0003] Metabolic stability of test agents has become a critical
factor in drug development, thus, the instant invention solves the
problem of identifying potential test agents that are subject to
metabolism by SSAO. Advances in chemistry, molecular biology and
high-throughput technology have provided drug discovery programs
with the ability to screen an enormous number of compounds against
a large number of targets to identify lead compounds. These leads
then undergo more selection criteria to identify those compounds
with optimal "drug-like" properties (i.e., adequate
physico-chemical stability, solubility, safety, efficacy, in vivo
disposition). Significant efforts are directed toward identifying
and eliminating compounds (or compound classes) that are not likely
to have "drug-like" properties at earlier stages of discovery. The
three main reasons a drug fails during clinical trials are lack of
efficacy, unacceptable adverse effects, and unfavorable ADME
(absorption, distribution, metabolism, excretion) properties.
Therefore the ultimate success of a compound is not only defined by
its biological activity and potency, but also by its ADME/toxicity
properties. As a result, lead optimization programs have
incorporated screens to select drugs with desirable ADME/toxicity
properties to enhance the likelihood that new lead compounds will
have success in the clinic. Metabolic transformation of drug
molecules represents a key process by which drugs are cleared from
the body. Given the wide distribution of SSAO in many bodily
compartments, we believe SSAO may be an important enzyme that
contributes to a drug's potential metabolic liability. Thus, the
instant invention uses methods of determining the metabolic
stability of test agents exposed to SSAO. Methods directed to
determining the metabolism of a drug or other test agent by enzymes
involved in biotransformation, in particular SSAO, have important
implications for drug development.
[0004] In in vitro studies, compound A (U.S. Pat. No. 6,977,263 B)
was incubated with plasma of various species including human.
Compound A was observed to be more stable in human plasma in
comparison to sheep, guinea pig, rat, and mouse up to one hour.
Additionally, no significant metabolism or degradation was observed
for the compound in human plasma up to 4 hours with or without
semicarbazide. However, pharmacokinetic data from the "first in
man" study showed that the same compound was rapidly metabolized
and to a far greater extent than that demonstrated in vitro using
plasma from human and from other species. Subsequent studies
confirmed that membrane-bound SSAO was primarily responsible for
the metabolism of the compound in man. These data indicate that
soluble SSAO found in human plasma does not possess the same level
of enzyme activity and/or substrate specificity as the membrane
bound SSAO under physiological conditions. Therefore, as
illustrated by compound A, measuring human plasma stability is not
predictive of SSAO stability in man and thus is of limited utility
in selecting pharmaceutical agents as clinical candidates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 shows the amino acid sequence of human SSAO (Genbank
Accession No. Q16853; SEQ ID NO: 1).
[0006] FIG. 2 shows the nucleotide sequence of human SSAO (Genbank
Accession No. NM.sub.--00374; SEQ ID NO: 2).
[0007] FIG. 3 shows the chemical structure of compound A.
DETAILED DESCRIPTION OF THE INVENTION
[0008] All publications cited herein are hereby incorporated by
reference. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this invention
pertains.
[0009] The terminology used in this specification and the appended
claims is for the purpose of describing particular embodiments only
and the use in the specification is not intended to be limiting of
the invention. The singular forms of a word are intended to include
the plural forms unless the context clearly indicates otherwise.
For example, the singular forms of "a", "an" and "the" are intended
to include the plural forms as well. Further, reference to an agent
may include a mixture of two or more agents. Thus, the term "an
agent" includes a plurality of agents, including mixtures and/or
enantiomers thereof. It should also be noted that the term "or" is
generally employed in its sense including "and/or" unless the
content clearly dictates otherwise. It will be further understood
that the terms "comprises" and/or "comprising," when used in this
specification, specify the presence of stated features, steps,
elements, and/or components, but do not preclude the presence or
addition of one or more other features, steps, elements,
components, and/or groups thereof.
[0010] Furthermore, in accordance with the present invention there
may be employed conventional molecular biology, microbiology,
recombinant DNA and analytical techniques within the skill of the
art. Such techniques are explained fully in the literature. See,
e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A
Laboratory Manual, Second Edition (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et
al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II
(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.
1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins
eds. (1985)]; Transcription and Translation [B. D. Hames & S.
J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed.
(1986)]; Immobilized Cells and Enzymes [IRL Press, (1986)]; B.
Perbal, A Practical Guide to Molecular Cloning (1984); F. M.
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. (1994); A Handbook of Bioanalysis and Drug
Metabolism, G. Evans (2004).
[0011] An embodiment of the present invention is a method of
identifying metabolic stability of a test agent due to
semicarbazide-sensitive amine oxidase (SSAO) catalyzed metabolism,
the method comprising: (a) culturing cells expressing SSAO; (b)
adding a test agent to the cells; (c) incubating the test agent
with the cells for a predetermined period of time; (d) measuring
the amount of the test agent remaining in the presence of the cells
comprising expressed SSAO at the predetermined period of time; and
(e) comparing the amount of the test agent at the predetermined
period of time to the amount of test agent in a control to
determine a value wherein the value identifies the metabolic
stability of the test agent in the presence of cells comprising
expressed SSAO.
[0012] One skilled in the art will recognize that the method may be
carried out using the cell lysate instead of the cells. Thus, an
embodiment of the present invention is a method of identifying the
metabolic stability of a test agent due to semicarbazide-sensitive
amine oxidase (SSAO) catalyzed metabolism, the method comprising:
(a) culturing cells expressing SSAO; (b) lysing the cells to form a
cell lysate; (c) adding a test agent to the cell lysate; (d)
incubating the test agent with the cell lysate for a predetermined
period of time; (e) measuring the amount of the test agent
remaining in the presence of the cell lysate comprising expressed
SSAO at the predetermined period of time; and (f) comparing the
amount of the test agent at the predetermined period of time to the
amount of test agent in a control to determine a value wherein the
value identifies the metabolic stability of the test agent in the
presence of cell lysate comprising expressed SSAO. Another
embodiment combines steps (b) and (c) into a single step.
[0013] A further embodiment of the present invention is a method of
identifying the metabolic stability of a test agent due to SSAO
catalyzed metabolism in a biological sample, the method comprising:
(a) obtaining a biological sample comprising SSAO; (b) adding a
test agent to the biological sample; (c) incubating the test agent
with the biological sample for a predetermined period of time; (d)
measuring the amount of the test agent at the predetermined period
of time; and (e) comparing the amount of the test agent at the
predetermined period of time to the amount of test agent in a
control to determine a value wherein the value identifies the
metabolic stability of the test agent in the presence of a
biological sample comprising SSAO.
[0014] An embodiment of the present invention uses primary cell
culture or cell lines that are commercially available. As a
non-limiting example, cells that can be used are available from the
American Tissue Culture Company. In one embodiment, CHO cells are
used. Cells may be prokaryotic or eukaryotic. The scope of the
invention is not limited by the type of cells used.
[0015] A biological sample may include, but is not limited to,
tissue or fluids, sections of tissues such as biopsy and autopsy
samples, and frozen sections taken for histologic purposes. Such
samples include blood, sputum, tissue, cultured cells, (e.g.,
primary cultures, explants, and transformed cells), parts of or
whole organs (e.g., liver, lung, ileum, artery, umbilical cord),
stool, urine, etc. A biological sample can be obtained from an
eukaryotic organism, including from mammals such as a primate,
e.g., chimpanzee, macaque or human, cow, dog, cat, a rodent, e.g.,
guinea pig, rat, mouse, rabbit, or a bird, reptile, or fish. A
non-limiting example of one embodiment of the instant invention is
to investigate whether a particular test agent or compound has
greater risk for metabolic liability or conversely is metabolically
stable in one tissue compartment versus other tissue compartments,
e.g., whether a compound has greater metabolic stability in liver
compared to lung. Further, a biological sample can be processed to
provide a suspension of its cellular components or used for primary
culture of the cellular components.
[0016] An embodiment of the invention is to use a biological sample
from a human or non-human subject to quantitate the metabolic
stability of a test agent. A biological sample may be an organ
sample derived from one or more organs of non-human animals or
humans, a tissue sample derived from one or more tissues of
non-human animals or humans, as well as cell samples, derived from
one or more cells of non-human animals or humans or from cell
cultures. For animal experimentation, biological samples may
comprise target organ tissues obtained, for example, after necropsy
or biopsy or may be body fluids, such as blood. For clinical use,
samples may comprise body fluids, like blood, sera, plasma, urine,
synovial fluid, spinal fluid, cerebrospinal fluid, semen or lymph,
as well as body tissues obtained by biopsy. A reference or control
is understood by one skilled in the art. A reference or control can
include, but is not limited to, a biological sample from a
non-diseased subject wherein the subject is a non-human animal or
human. Further, a reference or control can be a biological sample
from a non-treated subject. Alternatively, a reference or control
can be from the same subject before, during and after treatment. A
reference or control can be from the same subject but is a
different cell, tissue or organ sample than the cell, tissue or
organ source used to identify the metabolic stability of the test
agent. A reference or control does not have to be a biological
sample but can be a sample with a known amount of SSAO activity. A
reference or control may be an agent that is not metabolized by
SSAO or that is metabolized by SSAO with a known amount of SSAO
liability.
[0017] The present invention provides methods for identifying the
metabolic stability or metabolic liability of test agents. The term
"test agent" as used herein describes any molecule, e.g. protein,
non-protein organic compound or pharmaceutical, with the capability
of being affected by or affecting the enzyme activity of SSAO.
There are no particular restrictions as to the test agents that can
be assayed. In an embodiment of the invention, a test agent is a
compound. Examples of test agents include single agents or
libraries of small, medium or high molecular weight chemical
molecules. An agent can be in the form of a library of test agents,
such as a combinatorial or randomized library that provides a
sufficient range of diversity or conversely are limited to similar
structures or features. Agents can be optionally linked to a fusion
partner, e.g., targeting compounds, rescue compounds, dimerization
compounds, stabilizing compounds, addressable compounds, and other
functional moieties. Conventionally, new chemical entities with
useful properties are generated by identifying a test agent (called
a "lead compound" or a "lead") with some desirable property or
activity, e.g., inhibiting activity or modulating activity. The
lead compound is then used as a scaffold to create variants of the
lead compound, and further evaluate the property and activity of
those variant compounds. One skilled in the art will appreciate the
utility of using the instant invention to optimize compound
selection by identifying metabolic stability and thus the metabolic
liability of potential lead compounds and identifying or selecting
those compounds with minimal liability to SSAO metabolism.
[0018] SSAO of the present invention is full-length SSAO or a
fragment thereof that retains total or partial enzyme activity.
Partial enzyme activity may be 30% of the total enzyme activity or
greater, for example, 40% or 50%. A non-limiting example is an SSAO
isolated from a species other than human. Non-limiting examples of
other species include rodents such as rats, mice and guinea pigs or
non-human primates such as monkeys or chimpanzees. A further
non-limiting example is full-length human SSAO as disclosed in SEQ
ID NO: 1 or fragments thereof having methyleneamine oxidase
activity.
[0019] Metabolic stability or metabolic liability are terms of art
understood by the skilled artisan. Metabolic stability refers to
the susceptibility or extent to which a test agent or a drug
molecule undergoes metabolism under a given condition. Thus, the
higher the extent of metabolism, the lower the metabolic stability.
Metabolic stability is one of several major determinants in
defining the oral bioavailability and systemic clearance of a drug,
compound or test agent. As a non-limiting illustration, after a
drug is administered orally, it first encounters metabolic enzymes
in the gastrointestinal lumen as well as in the intestinal
epithelium. After it is absorbed into the bloodstream through the
intestinal epithelium, it is delivered to the liver via the portal
vein. A drug can be effectively cleared by intestinal or hepatic
metabolism before it reaches systemic circulation, a process known
as first pass metabolism. The stability or liability of a drug to
metabolism within the liver as well as extra-hepatic tissues will
ultimately determine the concentration of drug found in the
systemic circulation and affect its half-life and residence time
within the body. The type of biotransformations typically referred
to as Phase I metabolism include oxidation, reduction, and
hydrolysis which primarily serve to increase the hydrophilicity and
enhance the excretion of a drug by unveiling or incorporating a
polar functional group into the molecule (OH, SH, NH.sub.2, or
CO.sub.2H). Phase II reactions or conjugation reactions further
increase the polarity of a drug by modifying a functional group to
form O- or N-glucuronides, sulfate esters, alpha-carboxyamides and
glutathionyl adducts. An embodiment of the instant invention is the
metabolic stability or metabolic liability of a drug, compound or
test agent to metabolism by SSAO.
[0020] An embodiment of the invention selects the most relevant
biological samples from human and other species to examine
metabolic stability due to SSAO metabolism. For instance,
liver-based metabolism is responsible for metabolic clearance of
most drugs so concern in first pass metabolism may focus on hepatic
and sometimes intestinal metabolism. The ubiquitous presence of
SSAO in tissues and biological fluids and its particularly high
activity in many tissues, such as lung and blood vessels, makes it
imperative to use the appropriate biological samples in addition to
or in place of liver to examine a test agent's metabolic stability
in the presence of SSAO. In addition to intestinal and hepatic
first-pass metabolism, the exposure of a test agent subject to SSAO
may be limited by first pass metabolism at sites other than
intestinal and hepatic tissues, such as portal veins, blood
vessels, and lungs. A test agent may demonstrate different
metabolic stability against different forms of SSAO. Different
biological samples may contain different forms of SSAO. A
non-limiting example is that a test agent may show relative
metabolic stability against the soluble form of SSAO in human
plasma but much higher metabolic instability against membrane-bound
SSAO in tissues. Additionally, an animal species may show much
higher or lower soluble SSAO activity in plasma than the level of
soluble SSAO activity found in human, under comparable assay
conditions.
[0021] An embodiment of the invention uses various chemical
inhibitors to inhibit various forms of amine oxidase activities and
attributes the observed metabolic instability as either SSAO
catalyzed or monoamine oxidase (MAO)-A or MAO-B catalyzed
metabolism. A non-limiting example is the addition of hydralazine
as a non-discriminating amine oxidase inhibitor, clorgyline as a
specific MAO-A inhibitor, pargyline as a mixed MAO-A and MAO-B
inhibitor, semicarbazide and bromoethylamine as specific SSAO
inhibitors.
[0022] Another non-limiting example of the invention is to
determine the metabolic stability of a test agent in individuals
treated with a drug or a combination of drugs to determine if there
is a change in the metabolic stability of the test agent in such
individuals compared to control subjects. One skilled in the art
will appreciate the usefulness of obtaining biological samples from
human or non-human subjects that suffer from one or more diseases
or have been manipulated to induce one or more disease states,
surgically or genetically altered or pretreated with a drug,
compound or test agent. A non-limiting example includes using the
invention to determine the metabolic stability of a test agent in
organs that may be compromised by disease compared to non-diseased
organs. As an additional non-limiting example, the metabolic
stability of a compound can be assessed in lung tissue obtained
from a human subject suffering from asthma and compared to the
metabolic stability of the compound in healthy lung tissue from an
age-matched or control subject. Another non-limiting example would
be to study the metabolic stability of a compound in a biological
sample such as in "young" liver compared to "old" or "aged" liver
where "young", "old" and "aged" are defined by the particular
species under investigation.
[0023] An embodiment of the invention uses a homogeneous cell
population or one biological sample. An alternative embodiment of
the invention uses a heterogeneous cell population or a combination
of more than one biological sample. The cells or biological sample
can be of any type and in any proportion to complete the methods of
the invention.
[0024] An embodiment of the invention uses a recombinant cell
expressing SSAO. A recombinant expression vector of the invention
comprises a nucleic acid molecule in a form suitable for expression
of the nucleic acid in a host cell. Thus, a recombinant expression
vector of the present invention can include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operably linked to the nucleic acid 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 that
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 include 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 Vol. 185, Academic
Press, San Diego, Calif. (1990). Regulatory sequences include those
that direct constitutive expression of the nucleotide sequence in
many types of 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 host cell to be transformed, the level of expression
of protein desired, etc. The expression vectors of the invention
can be introduced into host cells to produce proteins or peptides
encoded by nucleic acids as described herein.
[0025] The term "overexpression" as used herein, refers to the
expression of a polypeptide at a level that is greater than the
normal level of expression of the polypeptide in a cell that
normally expresses the polypeptide or in a cell that does not
normally express the polypeptide. For example, expression of the
polypeptide may by 10%, 20%, 30%, 40%, 50%, 60%, 70, 80%, 90%,
100%, or more as compared to expression of the polypeptide in a
wild-type cell that normally expresses the polypeptide. Mutants,
variants, or analogs of the polypeptide of interest may be
overexpressed.
[0026] As used herein, the term "transient" expression refers to
expression of exogenous nucleic acid molecule(s) which are separate
from the chromosomes of the cell. Transient expression generally
reaches its maximum 2-3 days after introduction of the exogenous
nucleic acid and subsequently declines.
[0027] As used here, the term "stable" expression refers to
expression of exogenous nucleic acid molecule(s) that have become
an integrated part of the chromosome(s) of the cell. In general,
vectors for stable expression of genes include one or more
selection markers.
[0028] Cell culturing techniques for transformed, non-transformed,
primary culture and biological samples are well known in the art.
Biological samples or cultured cells can be stored until required
for use. The media used for culturing can be specifically designed
or purchased from commercial sources.
[0029] An embodiment of the invention involves lysing the cells.
Cells can be lysed by the addition of a detergent containing lysis
buffer. However, the invention is not limited to the use of
detergent in the lysis buffer but may include any method that is
appropriate for lysing cells. For example, cells may be lysed by
exposure to hypertonic buffer, sonication or freeze/thaw. These and
other methods of lysing cells are well known to those skilled in
the art.
[0030] An embodiment of the invention uses a control. A control is
a term of art well understood by skilled artisans. An appropriate
control may be dependent on the assay parameters utilized or the
experimental question under investigation. A control may be a
particular set of assay conditions or the addition or elimination
of a particular compound to the assay. Therefore, a non-limiting
example of a control is a condition where a test agent is incubated
in the absence of cells or cell lysates expressing SSAO. A control
may be considered a positive control in that the assay conditions
or control compound added brings about the anticipated response.
For example, if the agent under investigation is expected to be
metabolized, a positive control would be a compound that is
metabolized by SSAO. Further, a positive control may be a compound
that is metabolized by SSAO into known metabolic by-products. A
non-limiting example of a positive control is the addition of
benzylamine. A control may also be a negative control. A negative
control may be a particular set of assay conditions or the addition
or elimination of a particular compound to the assay that would not
bring about the anticipated response. For example, if the agent
under investigation is expected to be metabolized, then a negative
control would be expected not to be metabolized. A non-limiting
example of a negative control is the addition of benzyoic acid that
does not have a methyleneamine group and is not metabolized by
SSAO. A non-limiting example of a particular set of assay
conditions as a negative control may be the addition of an
inhibitor targeting SSAO enzyme activity, such as hydralazine,
wherein the SSAO activity is inhibited. A control may be a
"vehicle" control. For example, if the test agent is dissolved in
DMSO then the vehicle control would be DMSO without test agent. A
control may simply be the use of historical data.
[0031] An embodiment of the present invention is measuring the
amount of the test agent incubated in the presence of SSAO at the
predetermined period of time and comparing the amount of the test
agent at the predetermined period of time to the amount of test
agent in a control to determine the metabolic stability of the test
agent in the presence of SSAO. It is readily apparent to one
skilled in the art that metabolic stability of test agent can be
measured by the disappearance of the test agent or alternatively by
measuring the appearance of one or more metabolites of the test
agent. A further embodiment of the present invention is to measure
the appearance of one or more metabolites of the test agent in
addition to or in place of measuring the amount of test agent.
[0032] The amount of test agent or the appearance of one or more
metabolites of the test agent can be measured by any number of
techniques available to one skilled in the art. Non-limiting
examples include mass spectrometry, high-pressure liquid
chromatography (HPLC), liquid chromatography/mass spectrometry,
liquid chromatography/mass spectrometry/mass spectrometry and
liquid chromatography/radiomatic detection. Further, the amount of
test agent or the appearance of one or more metabolites of the test
agent may be measured by the use of indicator molecules such as
radioisotopes, fluorescent dyes or antibodies. The instant
invention is not limited by the method of measuring the test agent
or any one or more metabolites of the test agent.
[0033] The test agent may be labeled with a radioisotope, such as,
but not limited to .sup.3H or .sup.14C. Thus, an embodiment of the
invention is a method of identifying the metabolic stability of a
radiolabeled test agent due to semicarbazide-sensitive amine
oxidase (SSAO) catalyzed metabolism, the method comprising: (a)
culturing cells expressing SSAO; (b) adding a radiolabeled test
agent to the cells; (c) incubating the radiolabeled test agent with
the cells for a predetermined period of time; and (d) measuring the
amount of the radiolabeled test agent remaining in the presence of
the cells comprising expressed SSAO at the predetermined period of
time wherein the percentage of radiolabeled test agent that has not
been metabolized identifies the metabolic stability of the
radiolabeled test agent in the presence of cells comprising
expressed SSAO. One skilled in the art will recognize the method
may be carried out using cell lysates instead of the cells.
Further, the instant invention is not limited by the use of
radioisotopes but may use any means available in the art to label
the test agent.
[0034] A further embodiment of the present invention is a method of
identifying metabolic stability of a radiolabeled test agent due to
SSAO catalyzed metabolism in a biological sample, the method
comprising: (a) obtaining a biological sample comprising SSAO; (b)
adding a radiolabeled test agent to the biological sample; (c)
incubating the radiolabeled test agent with the biological sample
for a predetermined period of time; and (d) measuring the amount of
the radiolabeled test agent at the predetermined period of time
wherein the percentage of radiolabeled test agent that has not been
metabolized identifies the metabolic stability of the test agent in
the presence of a biological sample comprising SSAO.
[0035] The skilled artisan can appreciate the usefulness of
comparing the metabolic stability of a test agent in cells or cell
lysates to the metabolic stability of the test agent in biological
samples. Thus an embodiment of the present invention includes a
method of determining the metabolic stability profile of a test
agent, the method comprising (a) obtaining the value of metabolic
stability of the test agent incubated in the presence of cells or
cell lysate comprising expressed SSAO and (b) obtaining the value
of metabolic stability of the test agent incubated in the presence
of a biological sample comprising expressed SSAO wherein the value
of metabolic stability of the test agent in the presence of the
cells or cell lysate comprising expressed SSAO is compared to the
value of metabolic stability of the test agent in the presence of
the biological sample comprising SSAO providing the metabolic
stability profile of the test agent.
[0036] Refinements such as using the present invention with high
throughput screening (HTS) methods are well within the knowledge
and capability of the skilled artisan and are considered
embodiments of the invention. An embodiment of the invention is use
in high throughput screening (HTS) methods. HTS is the automated,
simultaneous testing of thousands of distinct chemical compounds in
assays designed to model biological mechanisms or aspects of
disease pathologies. More than one compound, e.g., a plurality of
compounds, can be tested simultaneously. In one embodiment, the
term HTS screening method refers to assays which test the metabolic
stability of one compound in a plurality of biological samples or a
plurality of compounds in one or more biological samples.
[0037] An embodiment of the present invention comprises an array of
receptacles that can receive cells, cell lysates, bodily samples or
other materials such as a methyleneamine or methyleneamine-like
test agent under investigation. An array of receptacles can be any
number of receptacles from at least one or more than one receptacle
suitable for holding materials within the scope of the invention.
Examples include but are not limited to flasks, culture dishes,
slides, tubes such as 1.5 mL tubes, 12 well plates, 96 well plates,
384 well plates and miniaturized microtiter plates with perhaps
4000 receptacles (U.S. Patent Application 20050255580). The array
of receptacles may be amendable to the addition of a protective
covering thus preventing against entry of contaminants or
evaporation of contents.
[0038] A further characteristic of the receptacles is that the
receptacle may allow for analysis. Non-limiting examples include
analysis by mass spectrometry or HPLC. However, there is not a
limitation to receptacles that can be used within the scope of the
present invention given that samples can be transferred to a
suitable container for further analysis. A non-limiting example is
to modify the method such that the method further comprises
providing a second array of receptacles wherein one or more steps
are performed in the second array of receptacles.
[0039] Liquid handling systems, analytical equipment such as
fluorescence readers or scintillation counters and robotics for
cell culture and sample manipulation are well known in the art.
Mechanical systems such as robotic arms or "cherry-picking" devices
are available to the skilled artisan. Commercial plate readers are
available to analyze conventional 96-well or 384-well plates.
Single sample, multiple sample or plate sample readers are
available that analyze predetermined wells and generate raw data
reports. The raw data can be transformed and presented in a variety
of ways.
[0040] A further embodiment of the present invention is a kit
comprising at least one element of an assay system to perform the
methods disclosed herein and instructions for use. Thus, the
components of the assay system may be provided separately or to may
be provided together in such a kit. Components of the assay system
may be prepared and included in a kit according to methods that
maximize the stability of the individual components. Such methods
are familiar to those persons skilled in the art. For example,
cells of the assay system may be provided as a suspension or the
cells may be frozen or lyophilized. Additional components of the
assay system may also be included such as buffers, containers for
mixing the assay components such as microtiter plates or test
tubes. The assay system can be provided in the form of a kit that
includes instructions for performing the assay and instructions for
data handling and interpretation.
[0041] The present invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims. While the invention has been described and
exemplified in sufficient detail for those skilled in this art to
produce and use, various alternatives, modifications, and
improvements should be apparent without departing from the spirit
and scope of the invention. One skilled in the art readily
appreciates that the present invention is well adapted to carry out
the objective and obtain the ends and advantages mentioned, as well
as those inherent therein. The examples that follow are
descriptions of embodiments and are not intended as limitations on
the scope of the invention. Modifications therein and other uses
will occur to those skilled in the art. These modifications are
encompassed within the spirit of the invention and are defined by
the scope of the claims.
[0042] The present invention illustratively described herein may be
practiced in the absence of any element or elements, limitation or
limitations, which are not specifically disclosed herein. The terms
and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by embodiments and optional features,
modification and variation of the concepts herein disclosed may be
made by those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
Example 1
A. Cloning and Expression of Human SSAO
[0043] 1) in Order to Facilitate Expression Construct Production,
an Initial pDONR221-Full-Length SSAO Plasmid was Generated as
Follows:
[0044] A first PCR was performed using gene-specific primers shown
below. The forward primer has half of the att site (Invitrogen) and
a Kozak sequence shown in bold. The reverse primer has the
remaining half of the att site shown in bold. PCR was performed
using human umbilical cord as the DNA template (prepared from RNA
purchased from Biochain and Invitrogen's RT-PCR superscript II
kit):
Initial Cloning Primers:
TABLE-US-00001 [0045] Forward (SEQ ID NO: 3):
5'-AAAAGCAGGCTTAGGAATGAACCAGAAGACAATCCTC-3' Reverse (SEQ ID NO: 4):
5'-CAAGAAAGCTGGGTCCTAGTTGTGAGAGAAGCCCC-3'
[0046] A second PCR was then carried out using the universal
primers shown below. The forward primer and the reverse primer
included att site sequences (shown in bold) and vector
sequences.
Universal Primer Sequences:
TABLE-US-00002 [0047] Forward (SEQ ID NO: 5):
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGGA-3' Reverse (SEQ ID NO: 6):
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTC-3'
[0048] This second PCR product was cloned into pDONR221 using the
Invitrogen BP reaction. Using this entry vector as template, six
variations of human SSAO expression constructs were prepared; three
forms (one full-length and two truncated) without tags and three
with carboxyl-terminal 6.times.HIS tags to facilitate purification
of the enzyme.
2) the Full-Length SSAO:
[0049] Sequences shown in bold on the forward primer and reverse
primers indicate att site sequence.
Primer Sequences for Cloning Untagged, Full-Length SSAO:
TABLE-US-00003 [0050] Forward (SEQ ID NO: 7):
5'-AAAAGCAGGCTTAGGAATGAACCAGAAGACAATCCTC-3' Reverse (SEQ ID NO: 8):
5'-CAAGAAAGCTGGGTCCTAGTTGTGAGAGAAGCCCCCGTGG-3'
To add 3' 6.times.HIS before the stop codon the following primers
were used:
[0051] Sequences shown in bold on the forward primer and the
reverse primer indicate att site sequence, underlined sequences on
the reverse primer indicate the 6.times.HIS tag.
Primer Sequences for Cloning Full-Length SSAO with C-Terminal
6.times.HIS Tag:
TABLE-US-00004 Forward (SEQ ID NO: 9):
5'-AAAAGCAGGCTTAGGAATGAACCAGAAGACAATCCTC-3' Reverse (SEQ ID NO:
10): 5'-CAAGAAAGCTGGGTCCTAATGGTGATGGTGATGGTGGTTGTGAGAG
AAGCCCCCGTGG-3'
3) T.sub.1 (Gly27-Asn763) Representing the Enzyme Lacking the
Membrane Anchoring Region:
[0052] Sequences shown in bold on the forward primer and the
reverse primer indicate att site sequence, underlined sequences on
the forward primer indicate additional N-terminal Met.
Primer Sequences for Cloning N-Terminal Truncated, Untagged
SSAO:
TABLE-US-00005 [0053] Forward (SEQ ID NO: 11):
5'-AAAAGCAGGCTTAGGAATGGGCAGGGGTGGAGATGGGGGTG-3' Reverse (SEQ ID NO:
12): 5'-CAAGAAAGCTGGGTCCTAGTTGTGAGAGAAGCCCC-3'
To add 3' 6.times.HIS before the stop codon the following primers
were used:
[0054] Sequences shown in bold on the forward primer and the
reverse primer indicate att site sequence, underlined sequences on
the forward primer indicate additional N-terminal Met, and
underlined sequences on the reverse primer indicate the 6.times.HIS
tag.
Primer Sequences for Cloning N-Terminal Truncated, C-Terminal
6.times.HIS Tag SSAO:
TABLE-US-00006 [0055] Forward (SEQ ID NO: 13):
5'-AAAAGCAGGCTTAGGAATGGGCAGGGGTGGAGATGGGGGTG-3' Reverse (SEQ ID NO:
14): 5'-CAAGAAAGCTGGGTCCTAATGGTGATGGTGATGGTGGTTGTGAGAG
AAGCCCCCGTGG-3'
4) T.sub.2 (Met211-Asn763) Representing the Putative Catalytic
Domain.
[0056] Sequences shown in bold on the forward primer and the
reverse primer indicate att site sequence, and underlined sequences
on the forward primer indicate additional N-terminal Met.
Primer Sequences for Cloning Catalytic Domain of Untagged SSAO:
TABLE-US-00007 [0057] Forward (SEQ ID NO: 15):
5'-AAAAGCAGGCTTAGGAATGACCACGGCTCCCCGTGGTC-3' Reverse (SEQ ID NO:
16): 5'-CAAGAAAGCTGGGTCCTAGTTGTGAGAGAAGCCCC-3'
To add 3' 6.times.HIS before stop codon the following primers were
used:
[0058] Sequences shown in bold on the forward primer and the
reverse primer indicate att site sequence, underlined sequences on
the forward primer indicate additional N-terminal Met, underlined
sequences on the reverse primer indicate the 6.times.HIS tag.
Primer Sequences for Cloning Catalytic Domain of SSAO with
C-Terminal 6.times.HIS Tag:
TABLE-US-00008 Forward (SEQ ID NO: 17):
5'-AAAAGCAGGCTTAGGAATGACCACGGCTCCCCGTGGTC-3' Reverse (SEQ ID NO:
18): 5'-CAAGAAAGCTGGGTCCTAATGGTGATGGTGATGGTGGTTGTGAGAG
AAGCCCCCGTGG-3'
[0059] The expression constructs were prepared by cloning these PCR
products into the pcDNA5-FRT-To-DEST vector (Invitrogen, Gateway
System.TM.). The forward and reverse DNA sequence of each construct
was confirmed including expressed sequences and at least 100 base
pairs either side of this. Stable cell lines for Flp-In CHO, Flp-In
CHO T-Rex, and Flp-In HEK 293 (Invitrogen, Flp-In System.TM.) were
generated using these 6 constructs according to manufacturer's
instructions. Cells were co-transfected with the regulatory vector,
pOG44, and each of the pcDNA5-FRT-To-DEST constructs using
Lipfectamine (Invitrogen) according to manufacturer's instructions.
Thus, 18 cell lines were generated: CHO Flp-In full-length SSAO;
CHO Flp-In full-length SSAO with C-terminal 6.times.HIS tag; CHO
Flp-In SSAO; CHO Flp-In T.sub.1 SSAO with C-terminal 6.times.HIS
tag; CHO Flp-In T.sub.2 SSAO; CHO Flp-In T.sub.2 SSAO with
C-terminal 6.times.HIS tag; CHO T-Rex Flp-In full-length SSAO; CHO
T-Rex Flp-In full-length SSAO with C-terminal 6.times.HIS tag; CHO
T-Rex Flp-In T.sub.1 SSAO; CHO T-Rex Flp-In T.sub.1 SSAO with
C-terminal 6.times.HIS tag; CHO T-Rex Flp-In T.sub.2 SSAO; CHO
Flp-In T.sub.2 SSAO with C-terminal 6.times.HIS tag; HEK 293 Flp-In
full-length SSAO; HEK 293 Flp-In full-length SSAO with C-terminal
6.times.HIS tag; HEK 293 Flp-In T.sub.1 SSAO; HEK 293 Flp-In
T.sub.1 SSAO with C-terminal 6.times.HIS tag; HEK 293 Flp-In
T.sub.2 SSAO; HEK 293 Flp-In T.sub.2 SSAO with C-terminal
6.times.HIS tag.
[0060] Adherent CHO cells were cultured at 37.degree. C., 5%
CO.sub.2 in media containing Ham's F12, 10% fetal bovine serum, 2
mM L-glutamine, 1% Penicillin/Streptomycin, 300 .mu.M hygromycin B.
Adherent HEK 293 cells were cultured at 37.degree. C., 5% CO.sub.2
in D-MEM (high glucose), 10% fetal bovine serum, 2 mM L-glutamine,
and 200 .mu.M hygromycin B. Stable transformants were identified by
loss of zeocin resistance. Once the cells were 80% confluent, SSAO
expression was induced by the addition of Tetracycline to 1
.mu.g/mL. Following 18 hours, the cells were harvested using a
trypsin treatment of 3 minutes, at room temperature. Cells were
washed 3 times in phosphate buffered saline before flash freezing
in liquid nitrogen.
[0061] For the preparation of cell lysate for activity testing,
frozen cell pellets were resuspended in lysate buffer containing 10
mM Tris-HCl (pH 7.2), 150 mM NaCl, 1.5 mM MgCl.sub.2, 1% v/v NP-40
and incubated for 15 minutes on ice. Lysate was cleared by
centrifugation 800.times.g for 10 minutes, 4.degree. C. and stored
in aliquots at -80.degree. C. until use. Proteins were determined
using Pierce Coomassie Protein Reagent, according to manufacturer's
instructions.
B. Testing the Recombinant Protein for Enzymatic Activity
(i).Sample Preparation.
[0062] For each assay run, 1 mL of cell lysate (1.5 mg/mL protein)
was mixed with benzylamine to 2000 ng/mL final concentration. At
each corresponding time point, 20 .mu.L of the incubation mixture
was transferred to a microcentrifuge tube (1.7 mL) and mixed with
50 .mu.L of acetonitrile. The tubes were vortex mixed briefly to
ensure complete mixing and then centrifuged at 10,000.times.g for 5
minutes. The supernatant was removed and 50 .mu.L was transferred
to an autosampler vial and mixed with 50 .mu.L of water before
LC/MS/MS analysis. Benzylamine remaining in the lysate was analyzed
with LC/MS/MS method.
(ii). Analysis
[0063] LC/MS/MS analysis was performed with a PE Sciex API 3000
mass spectrometer under the following conditions listed below and
in Tables 1 and 2:
[0064] Analytical column: Jupiter C-4, 5 .mu.M, 50.times.2.1 mm
[0065] Column temperature: ambient
[0066] Flow rate: 0.2 mL/min
[0067] Injection volume: 15 .mu.L
TABLE-US-00009 TABLE 1 Mobile Phase Gradient Time (min) SolutionA
(%) SolutionB (%) 0 40 60 0.1 95 5 2.9 95 5 3.0 40 60 6.0 40 60
SolutionA was 90% methanol in water, SolutionB was 10 mM ammonium
acetate buffer, pH 7.0 Divert valve: 0-1.5 min to waste, 1.5-5 min
to MS detector
TABLE-US-00010 TABLE 2 LC/MS/MS Compound RT (min) Mass transition
Benzylamine 2.74 108.1 to 65.1 +Ve
(iii) Results of Cell Lysate LC/MS/MS Analysis are Shown in Table
3.
TABLE-US-00011 TABLE 3 SSAO Enzymatic Activity in Cell Lines
Expressing Full-length or Truncated SSAO Benzylamine Source of Cell
SSAO Remaining (%) Lysate Expressed Tag 0 min 10 min 2 h Control
CHO none None 100 111 110 Control CHO none None 100 104 99 Flp-In
CHO Flp-In Full- None 100 1 1 length CHO Fip-In Full- C-terminal
100 101 73 length 6xHIS CHO Flp-In T1 None 100 109 103 CHO Flp-In
T1 C-terminal 100 101 98 6xHIS CHO Flp-In T2 None 100 105 98 CHO
Flp-In T2 C-terminal 100 108 102 6xHIS CHO Flp-In None None 100 104
99 T-Rex CHO Flp-In Full- None 100 6 0 T-Rex length CHO Flp-In
Full- C-terminal 100 94 59 T-Rex length 6xHIS CHO Flp-In T1 One 100
104 92 T-Rex CHO Flp-In T1 C-terminal 100 95 93 T-Rex 6xHIS CHO
Flp-In T2 None 100 97 83 T-Rex CHO Flp-In T2 C-terminal 100 100 97
T-Rex 6xHIS HEK293 control none None 100 106 123 HEK293 Full- None
100 58 2 length HEK293 Full- C-terminal 100 105 98 length 6xHIS
HEK293 T1 None 100 105 102 HEK293 T1 C-terminal 100 111 103 6xHIS
HEK293 T2 None 100 113 101 HEK293 T2 C-terminal 100 100 117
6xHIS
[0068] The active lysates from CHO Flp-In, CHO Flp-In T-Rex or
HEK293 Flp-In expressing full-length SSAO (untagged) were
subsequently tested and activity confirmed (Table 4). Since the
most active SSAO cell lines was the CHO Flp-In expressing the
full-length (untagged) SSAO, this preparation was used for
subsequent activity testing.
TABLE-US-00012 TABLE 4 SSAO Enzymatic Activity in Cell Lines
Expressing Full-length SSAO Cell Type Expressing Full- Benzylamine
Remaining (%) length SSAO (untagged) 0 min 10 min 30 min HEK293
Flp-In 100 50 9 CHO Flp-In 100 9 1 CHO Flp-In T-Rex 100 16 1
Example 2
Optimising LCMS/MS Assay Conditions
[0069] The Amplex Red assay (a commercially available assay), was
used initially to measure the specific activity of the recombinant
human SSAO. Amplex Red is a colorless and nonfluorescent derivative
of dihydroresorufin. In the presence of amine oxidases and SSAO,
the Amplex Red reacts with H.sub.2O.sub.2 to produce the highly
fluorescent product, resorufin. Resorufin has an excitation maximum
at 563 nm and emission maximum at 587 nm and the assay can be
quantified by reading the microtitre plate at 587 nm. The specific
activity of recombinant SSAO was determined to be 280 pmol/min/mg
protein and this was determined to be equivalent to the specific
activity of SSAO found in human lung tissue. For subsequent assays
to profile compounds in the LC/MS/MS assay, recombinant human SSAO
(rhSSAO) was used at this defined specific activity.
[0070] The LC/MS/MS assay as described above was used to evaluate
the susceptibility of compound A to metabolism by rhSSAO. The
chemical structure of compound A is shown in FIG. 3 (U.S. Pat. No.
6,977,263 B2 generically encompasses molecules and methods of
making the same). Compound A shown in Table 5 was incubated at
37.degree. C. with rhSSAO (having a defined specific activity that
is compatible with that found in human tissues as described above)
for 4 hours initially or for 24 hours. At time zero and time 4
hours (or 24 hours), an aliquot of the sample was removed and
quenched with acetonitrile. The samples were then analyzed by
LC/MS/MS to measure the amount of parent compound remaining at 24
hours compared to that present at time zero.
[0071] The values shown in Table 5 are percent of parent compound
remaining at the various times indicated following incubation with
rhSSAO.
TABLE-US-00013 TABLE 5 Percent of Parent Compound Remaining
Following Incubation with rhSSAO % Parent Compound Remaining
Compound 0 h (%) 4 h (%) 24 h (%) A 100 25 3.2
Example 3
Testing of Metabolic Stability of Compound a in Supernatant
Preparations of Human and Animal Tissue Homogenates
(i) Tissues Used
[0072] The following tissues were used: lung, liver, and ileum from
guinea pig, cynomolgus monkey, and human; artery from guinea pig
and monkey; and umbilical cord from human.
(ii) Tissue Preparation
[0073] Tissues were processed to obtain supernatants. Briefly, the
excised tissues were rinsed immediately with copious amounts of
physiological saline (prechilled to 4.degree. C.) to remove blood.
Pooled tissues were homogenized in chilled 0.01 M sodium phosphate
buffer at pH 7.4 (10 mL per g tissue) with a Polytron. The
homogenate was treated with Triton X-100 at an added concentration
of ca. 1% under continuous gentle shaking for 2 hours at 4.degree.
C. and then centrifuged (4.degree. C., 15 minutes, 3000.times.g).
The supernatant was carefully separated and aliquoted into 2 mL
portions and frozen at approximately -80.degree. C. until use.
(iii) Optimization of Incubation Conditions with Test Compound
[0074] All incubations were conducted at 37.degree. C. using a
prewarmed water bath. .sup.14C-radiolabeled test compound
M..quadrature.were incubated at 1 or 10 Preliminary incubations
were performed for each tissue/compound combination to select the
appropriate incubation durations. Incubation durations of 1.5 to 6
hours were then used. Sufficient incubation volume was used to
allow for at least five serial samples during the incubation
duration. Individual aliquots (1 mL) taken from the incubation
mixture were immediately quenched with two volumes of acetonitrile
and the quenched mixtures were freeze-dried. The freeze-dried
samples were either reconstituted immediately for sample analysis
or stored at approximately -80.degree. C. until analysis.
Reconstitution was performed with a 500 L.quadrature. mixture (90%
20 mM ammonium acetate, pH 4.0 and 10% methanol (v/v)). L of
the.quadrature.After centrifugation, 100 reconstituted sample was
injected for high performance liquid chromatographic (HPLC)
separation followed by radiomatic detection.
(iv) Data Analysis and Calculations
[0075] Reconstituted samples from incubations were analyzed by HPLC
followed by radiomatic detection. The identity of the test compound
and respective benzylaldehyde and acid metabolites were verified by
matching the retention times with the reference standards of the
parent compound and their respective metabolites. The amount of the
test compound remaining is calculated as the percent of the
radioactivity of the parent peak in the total radioactivity of the
radiochromatogram of a reconstituted sample. The percent of test
compound remaining versus incubation time showed first-order
kinetics when plotted on a log-linear scale. The first-order rate
constant was calculated for individual incubations as the slope
between the logarithm of the test compound remaining versus time
based on least square linear regression analysis.
(v) Results of Test Compound Metabolic Stability in Tissue
Preparations
[0076] The first-order rate constant (1/h) for the metabolic
degradation of the test compound is listed in Table 6. Assuming
that the metabolism followed Michaelis-Menten kinetics, the
approximation to first-order kinetics M implied that
K.quadrature.for up to 10 .sub.m>>M for the enzymatic
reaction..quadrature.10
TABLE-US-00014 TABLE 6 Metabolic Stability of Compound A in Tissue
Preparations Compound A Species Tissue M.quadrature.1
M.quadrature.10 Human Liver 0.418 0.365 Ileum 0.352 0.353 Lung
0.675 0.706 Umbilical cord 0.688 0.697 Guinea Liver 0.318 0.305 pig
Ileum 0.0775 0.0782 Lung 0.747 0.709 Artery 0.127 0.111 Monkey
Liver 2.05 2.64 Ileum 0.328 0.277 Lung 0.177 0.208 Artery 0.835
0.673
[0077] As shown Table 6, the present invention demonstrates a good
correlation between the tissue data and the observed clinical
metabolic profile of compound A. Compared to using only human
plasma stability measurements, the present invention can better
reflect human SSAO stability of a pharmaceutical agent under
physiological conditions and is a more relevant predictive tool for
guiding the selection of and the pharmaceutical development of
clinical candidates.
Sequence CWU 1
1
181763PRTHomo sapiens 1Met Asn Gln Lys Thr Ile Leu Val Leu Leu Ile
Leu Ala Val Ile Thr 1 5 10 15 Ile Phe Ala Leu Val Cys Val Leu Leu
Val Gly Arg Gly Gly Asp Gly 20 25 30 Gly Glu Pro Ser Gln Leu Pro
His Cys Pro Ser Val Ser Pro Ser Ala 35 40 45 Gln Pro Trp Thr His
Pro Gly Gln Ser Gln Leu Phe Ala Asp Leu Ser 50 55 60 Arg Glu Glu
Leu Thr Ala Val Met Arg Phe Leu Thr Gln Arg Leu Gly 65 70 75 80 Pro
Gly Leu Val Asp Ala Ala Gln Ala Arg Pro Ser Asp Asn Cys Val 85 90
95 Phe Ser Val Glu Leu Gln Leu Pro Pro Lys Ala Ala Ala Leu Ala His
100 105 110 Leu Asp Arg Gly Ser Pro Pro Pro Ala Arg Glu Ala Leu Ala
Ile Val 115 120 125 Phe Phe Gly Arg Gln Pro Gln Pro Asn Val Ser Glu
Leu Val Val Gly 130 135 140 Pro Leu Pro His Pro Ser Tyr Met Arg Asp
Val Thr Val Glu Arg His 145 150 155 160 Gly Gly Pro Leu Pro Tyr His
Arg Arg Pro Val Leu Phe Gln Glu Tyr 165 170 175 Leu Asp Ile Asp Gln
Met Ile Phe Asn Arg Glu Leu Pro Gln Ala Ser 180 185 190 Gly Leu Leu
His His Cys Cys Phe Tyr Lys His Arg Gly Arg Asn Leu 195 200 205 Val
Thr Met Thr Thr Ala Pro Arg Gly Leu Gln Ser Gly Asp Arg Ala 210 215
220 Thr Trp Phe Gly Leu Tyr Tyr Asn Ile Ser Gly Ala Gly Phe Phe Leu
225 230 235 240 His His Val Gly Leu Glu Leu Leu Val Asn His Lys Ala
Leu Asp Pro 245 250 255 Ala Arg Trp Thr Ile Gln Lys Val Phe Tyr Gln
Gly Arg Tyr Tyr Asp 260 265 270 Ser Leu Ala Gln Leu Glu Ala Gln Phe
Glu Ala Gly Leu Val Asn Val 275 280 285 Val Leu Ile Pro Asp Asn Gly
Thr Gly Gly Ser Trp Ser Leu Lys Ser 290 295 300 Pro Val Pro Pro Gly
Pro Ala Pro Pro Leu Gln Phe Tyr Pro Gln Gly 305 310 315 320 Pro Arg
Phe Ser Val Gln Gly Ser Arg Val Ala Ser Ser Leu Trp Thr 325 330 335
Phe Ser Phe Gly Leu Gly Ala Phe Ser Gly Pro Arg Ile Phe Asp Val 340
345 350 Arg Phe Gln Gly Glu Arg Leu Val Tyr Glu Ile Ser Leu Gln Glu
Ala 355 360 365 Leu Ala Ile Tyr Gly Gly Asn Ser Pro Ala Ala Met Thr
Thr Arg Tyr 370 375 380 Val Asp Gly Gly Phe Gly Met Gly Lys Tyr Thr
Thr Pro Leu Thr Arg 385 390 395 400 Gly Val Asp Cys Pro Tyr Leu Ala
Thr Tyr Val Asp Trp His Phe Leu 405 410 415 Leu Glu Ser Gln Ala Pro
Lys Thr Ile Arg Asp Ala Phe Cys Val Phe 420 425 430 Glu Gln Asn Gln
Gly Leu Pro Leu Arg Arg His His Ser Asp Leu Tyr 435 440 445 Ser His
Tyr Phe Gly Gly Leu Ala Glu Thr Val Leu Val Val Arg Ser 450 455 460
Met Ser Thr Leu Leu Asn Tyr Asp Tyr Val Trp Asp Thr Val Phe His 465
470 475 480 Pro Ser Gly Ala Ile Glu Ile Arg Phe Tyr Ala Thr Gly Tyr
Ile Ser 485 490 495 Ser Ala Phe Leu Phe Gly Ala Thr Gly Lys Tyr Gly
Asn Gln Val Ser 500 505 510 Glu His Thr Leu Gly Thr Val His Thr His
Ser Ala His Phe Lys Val 515 520 525 Asp Leu Asp Val Ala Gly Leu Glu
Asn Trp Val Trp Ala Glu Asp Met 530 535 540 Val Phe Val Pro Met Ala
Val Pro Trp Ser Pro Glu His Gln Leu Gln 545 550 555 560 Arg Leu Gln
Val Thr Arg Lys Leu Leu Glu Met Glu Glu Gln Ala Ala 565 570 575 Phe
Leu Val Gly Ser Ala Thr Pro Arg Tyr Leu Tyr Leu Ala Ser Asn 580 585
590 His Ser Asn Lys Trp Gly His Pro Arg Gly Tyr Arg Ile Gln Met Leu
595 600 605 Ser Phe Ala Gly Glu Pro Leu Pro Gln Asn Ser Ser Met Ala
Arg Gly 610 615 620 Phe Ser Trp Glu Arg Tyr Gln Leu Ala Val Thr Gln
Arg Lys Glu Glu 625 630 635 640 Glu Pro Ser Ser Ser Ser Val Phe Asn
Gln Asn Asp Pro Trp Ala Pro 645 650 655 Thr Val Asp Phe Ser Asp Phe
Ile Asn Asn Glu Thr Ile Ala Gly Lys 660 665 670 Asp Leu Val Ala Trp
Val Thr Ala Gly Phe Leu His Ile Pro His Ala 675 680 685 Glu Asp Ile
Pro Asn Thr Val Thr Val Gly Asn Gly Val Gly Phe Phe 690 695 700 Leu
Arg Pro Tyr Asn Phe Phe Asp Glu Asp Pro Ser Phe Tyr Ser Ala 705 710
715 720 Asp Ser Ile Tyr Phe Arg Gly Asp Gln Asp Ala Gly Ala Cys Glu
Val 725 730 735 Asn Pro Leu Ala Cys Leu Pro Gln Ala Ala Ala Cys Ala
Pro Asp Leu 740 745 750 Pro Ala Phe Ser His Gly Gly Phe Ser His Asn
755 760 22292DNAHomo sapiens 2atgaaccaga agacaatcct cgtgctcctc
attctggccg tcatcaccat ctttgccttg 60gtttgtgtcc tgctggtggg caggggtgga
gatgggggtg aacccagcca gcttccccat 120tgcccctctg tatctcccag
tgcccagcct tggacacacc ctggccagag ccagctgttt 180gcagacctga
gccgagagga gctgacggct gtgatgcgct ttctgaccca gcggctgggg
240ccagggctgg tggatgcagc ccaggcccgg ccctcggaca actgtgtctt
ctcagtggag 300ttgcagctgc ctcccaaggc tgcagccctg gctcacttgg
acagggggag ccccccacct 360gcccgggagg cactggccat cgtcttcttt
ggcaggcaac cccagcccaa cgtgagtgag 420ctggtggtgg ggccactgcc
tcacccctcc tacatgcggg acgtgactgt ggagcgtcat 480ggaggccccc
tgccctatca ccgacgcccc gtgctgttcc aagagtacct ggacatagac
540cagatgatct tcaacagaga gctgccccag gcttctgggc ttctccacca
ctgttgcttc 600tacaagcacc ggggacggaa cctggtgaca atgaccacgg
ctccccgtgg tctgcaatca 660ggggaccggg ccacctggtt tggcctctac
tacaacatct cgggcgctgg gttcttcctg 720caccacgtgg gcttggagct
gctagtgaac cacaaggccc ttgaccctgc ccgctggact 780atccagaagg
tgttctatca aggccgctac tacgacagcc tggcccagct ggaggcccag
840tttgaggccg gcctggtgaa tgtggtgctg atcccagaca atggcacagg
tgggtcctgg 900tccctgaagt cccctgtgcc cccgggtcca gctccccctc
tacagttcta tccccaaggc 960ccccgcttca gtgtccaggg aagtcgagtg
gcctcctcac tgtggacttt ctcctttggc 1020ctcggagcat tcagtggccc
aaggatcttt gacgttcgct tccaaggaga aagactagtt 1080tatgagataa
gcctccaaga ggccttggcc atctatggtg gaaattcccc agcagcaatg
1140acgacccgct atgtggatgg aggctttggc atgggcaagt acaccacgcc
cctgacccgt 1200ggggtggact gcccctactt ggccacctac gtggactggc
acttcctttt ggagtcccag 1260gcccccaaga caatacgtga tgccttttgt
gtgtttgaac agaaccaggg cctccccctg 1320cggcgacacc actcagatct
ctactcgcac tactttgggg gtcttgcgga aacggtgctg 1380gtcgtcagat
ctatgtccac cttgctcaac tatgactatg tgtgggatac ggtcttccac
1440cccagtgggg ccatagaaat acgattctat gccacgggct acatcagctc
ggcattcctc 1500tttggtgcta ctgggaagta cgggaaccaa gtgtcagagc
acaccctggg cacggtccac 1560acccacagcg cccacttcaa ggtggatctg
gatgtagcag gactggagaa ctgggtctgg 1620gccgaggata tggtctttgt
ccccatggct gtgccctgga gccctgagca ccagctgcag 1680aggctgcagg
tgacccggaa gctgctggag atggaggagc aggccgcctt cctcgtggga
1740agcgccaccc ctcgctacct gtacctggcc agcaaccaca gcaacaagtg
gggtcacccc 1800cggggctacc gcatccagat gctcagcttt gctggagagc
cgctgcccca aaacagctcc 1860atggcgagag gcttcagctg ggagaggtac
cagctggctg tgacccagcg gaaggaggag 1920gagcccagta gcagcagcgt
tttcaatcag aatgaccctt gggcccccac tgtggatttc 1980agtgacttca
tcaacaatga gaccattgct ggaaaggatt tggtggcctg ggtgacagct
2040ggttttctgc atatcccaca tgcagaggac attcctaaca cagtgactgt
ggggaacggc 2100gtgggcttct tcctccgacc ctataacttc tttgacgaag
acccctcctt ctactctgcc 2160gactccatct acttccgagg ggaccaggat
gctggggcct gcgaggtcaa ccccctagct 2220tgcctgcccc aggctgctgc
ctgtgccccc gacctccctg ccttctccca cgggggcttc 2280tctcacaact ag
2292337DNAArtificialinitial cloning primer - forward 3aaaagcaggc
ttaggaatga accagaagac aatcctc 37435DNAArtificialinitial cloning
primer - reverse 4caagaaagct gggtcctagt tgtgagagaa gcccc
35534DNAArtificialuniversal primer - forward 5ggggacaagt ttgtacaaaa
aagcaggctt agga 34630DNAArtificialuniversal primer - reverse
6ggggaccact ttgtacaaga aagctgggtc 30737DNAArtificialforward primer
sequence for cloning untagged full-length SSAO 7aaaagcaggc
ttaggaatga accagaagac aatcctc 37840DNAArtificialreverse primer
sequence for cloning untagged full-length SSAO 8caagaaagct
gggtcctagt tgtgagagaa gcccccgtgg 40937DNAArtificialforward primer
sequence for cloning full-length SSAO with C-terminal 6xHIS tag
9aaaagcaggc ttaggaatga accagaagac aatcctc
371058DNAArtificialreverse primer sequences for cloning full-length
SSAO with C-terminal 6xHIS tag 10caagaaagct gggtcctaat ggtgatggtg
atggtggttg tgagagaagc ccccgtgg 581141DNAArtificialforward primer
sequences for cloning N-terminal truncated untagged SSAO
11aaaagcaggc ttaggaatgg gcaggggtgg agatgggggt g
411235DNAArtificialreverse primer sequences for cloning N-terminal
truncated untagged SSAO 12caagaaagct gggtcctagt tgtgagagaa gcccc
351341DNAArtificialforward primer sequences for cloning N-terminal
truncated C-terminal 6xHIS tag SSAO 13aaaagcaggc ttaggaatgg
gcaggggtgg agatgggggt g 411458DNAArtificialreverse primer sequences
for cloning N-terminal truncated C-terminal 6xHIS tag SSAO
14caagaaagct gggtcctaat ggtgatggtg atggtggttg tgagagaagc ccccgtgg
581538DNAArtificialforward primer sequences for cloning catalytic
domain of untagged SSAO 15aaaagcaggc ttaggaatga ccacggctcc ccgtggtc
381635DNAArtificialreverse primer sequences for cloning catalytic
domain of untagged SSAO 16caagaaagct gggtcctagt tgtgagagaa gcccc
351738DNAArtificialforward primer sequences for cloning catalytic
domain of SSAO with C-terminal 6xHIS tag 17aaaagcaggc ttaggaatga
ccacggctcc ccgtggtc 381858DNAArtificialreverse primer sequences for
cloning catalytic domain of SSAO with C-terminal 6xHIS tag
18caagaaagct gggtcctaat ggtgatggtg atggtggttg tgagagaagc ccccgtgg
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