U.S. patent application number 13/246502 was filed with the patent office on 2012-02-02 for protein kinase c gamma as a biomarker for neuropsychological and cognitive functions in the central nervous system.
Invention is credited to Kathleen S. Madden.
Application Number | 20120028274 13/246502 |
Document ID | / |
Family ID | 39645195 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120028274 |
Kind Code |
A1 |
Madden; Kathleen S. |
February 2, 2012 |
PROTEIN KINASE C GAMMA AS A BIOMARKER FOR NEUROPSYCHOLOGICAL AND
COGNITIVE FUNCTIONS IN THE CENTRAL NERVOUS SYSTEM
Abstract
Embodiments of the present invention are directed to a
biological marker, the gamma isoform of protein kinase C (PKCg),
which surprisingly allows rapid identification of compromised
cognitive, behavioral, and neuropsychological functions under
conditions associated with acute, transient hypoxia in humans. It
was surprisingly discovered that PKCg is released from neural cells
and can be detected in peripheral blood after hypoxic events
unrelated to the reduction or elimination of blood flow through
affected tissues. Embodiments of this invention are also directed
to a broad range of clinical applications, particularly in
emergency medicine. Other embodiments are related to compositions
and methods for distinguishing between hypoxic encephalopathies and
conditions arising from neuroanatomical/structural anomalies and/or
incidental pathologies, for example, alcohol intoxication.
Inventors: |
Madden; Kathleen S.;
(Bethesda, MD) |
Family ID: |
39645195 |
Appl. No.: |
13/246502 |
Filed: |
September 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12020348 |
Jan 25, 2008 |
8026077 |
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13246502 |
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60886532 |
Jan 25, 2007 |
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Current U.S.
Class: |
435/7.4 |
Current CPC
Class: |
G01N 2800/28 20130101;
G01N 2333/9121 20130101; G01N 33/6893 20130101 |
Class at
Publication: |
435/7.4 |
International
Class: |
G01N 33/573 20060101
G01N033/573 |
Claims
1-13. (canceled)
14. A method for assessing the potential for impairment of a
neuropsychological or cognitive function of the central nervous
system of a patient comprising: contacting a sample obtained from
the patient with a binding partner capable of forming a binding
complex with protein kinase C gamma (PKCg) to form a PKCg/binding
partner complex; determining the quantity of PKCg/binding partner
complex formed; and assessing the potential for impairment of the
neuropsychological or cognitive function from the quantity of
PKCg/binding partner complex formed, the impairment of the
neuropsychological or cognitive function being associated with
hypoxia and not connected with an ischemic event or the restriction
of blood flow to the central nervous system.
15. The method of claim 14, further comprising comparing the amount
of PKCg/binding partner complex formed with an amount of
PKCg/binding partner complex formed with a control sample, wherein
said control sample is indicative of a known impairment of a
neuropsychological or cognitive function of the central nervous
system.
16. The method of claim 14, further comprising monitoring the
amount of PKCg/binding partner complex formed over a period of
time.
17. A method for diagnosing a disorder of a neuropsychological or
cognitive function of the central nervous system of a patient
comprising: contacting a sample obtained from said patient with a
binding partner capable of forming a binding complex with PKCg to
form a PKCg/PKCg binding partner complex, wherein detection of a
PKCg/PKCg binding partner complex is diagnostic of said disorder,
the disorder being associated with hypoxia and not connected with
an ischemic event or the restriction of blood flow to the central
nervous system.
18. A kit for diagnosis of a disorder of the neuropsychological or
cognitive function of the central nervous system of a patient
comprising: a first substrate having immobilized thereon an
anti-PKCg antibody or antibody fragment for contact with a sample
obtained from the patient; a detectable label that is capable of
binding to a PKCg/PKCg antibody complex; a second substrate which
has bound thereto a PKCg/binding partner complex which is
detectably labeled at a detection level that is representative of a
disorder of a neuropsychological or cognitive function of the
central nervous system of a similar patient, the disorder being
associated with hypoxia and not connected with an ischemic event or
the restriction of blood flow to the central nervous system.
19. The kit of claim 18, further comprising a second anti-PKCg
antibody reactive with another epitope of PKCg.
20. The kit of claim 18, wherein said first antibody is immobilized
on a solid support.
21. The kit of claim 20, wherein the solid support is selected from
the group consisting of a plastic multiwall plate, plastic or glass
beads or rods, and a plastic or glass film.
22. The kit of claim 18, wherein the antibody or antibody fragment
is labeled.
23. The kit of claim 18, further comprising one or more additional
substrates each of which has bound thereto a PKCg/binding partner
complex which is detectably labeled at a detection level that is
representative of another disorder of a neuropsychological or
cognitive function of the central nervous system of a similar
patient.
24-26. (canceled)
27. The method of claim 14, wherein the patient's blood has a
PaO.sub.2 of between about 25 mm Hg and 80 mm Hg.
28. The method of claim 27, wherein the hypoxia is associated with
a condition selected from the group consisting of: altitude
sickness HAPE, HACE, retinal encephalopathy, and hypoxic
encephalopathy.
29. The method of claim 27, wherein the hypoxia is associated with
one selected from the group consisting of: a breathing disorder,
apnea, a pulmonary disorder, a surgical procedure, administration
of medication, a change in altitude and a change in barometric
pressure.
30. The method of claim 17, wherein the patient's blood has a
PaO.sub.2 of between about 25 mm Hg and 80 mm Hg.
31. The method of claim 30, wherein the hypoxia is associated with
a condition selected from the group consisting of: altitude
sickness, HAPE, HACE, retinal encephalopathy, and hypoxic
encephalopathy.
32. The method of claim 30, wherein the hypoxia is associated with
one selected from the group consisting of: a breathing disorder,
apnea, a pulmonary disorder, a surgical procedure, administration
of medication, a change in altitude and a change in barometric
pressure.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/886,532 entitled "Protein Kinase C Gamma as a
Biomarker for Neuropsychological and Cognitive Functions in the
Central Nervous System," filed Jan. 25, 2007, the entirety of which
is specifically and entirely incorporated by reference.
BACKGROUND
[0002] 1. Field of Invention
[0003] This invention is directed to kits, compositions and methods
of identifying neurological, neuropsychological, neuropsychiatric,
behavioral and cognitive functions in the central nervous system,
PKCg biomarkers directed to non-ischemic events.
[0004] 2. Description of the Background
[0005] The events of ischemia include hypoxia and hypoglycemia due
to the reduction or elimination of blood flow through affected
tissues. However, hypoxia also occurs under conditions of normal or
even increased cerebral blood flow.
[0006] The effects of hypoxia on central nervous system functions,
such as short-term memory loss and the impairment of cognition and
performance tasks have been studied through a variety of
experimental approaches and models. Neurological,
neuropsychological and psychometric tests have been used to assess
central nervous system function. Additionally, dysfunctions of the
cerebellum include cognitive affective syndrome, ataxia, and
dysmetria of thought. Considerable efforts are underway to clarify
the cellular and molecular mechanisms contributing to these
processes. The gamma isoform of protein kinase C (PKCg) is unique
to the central nervous, including the eye. Although broadly
distributed throughout the brain, PKCg is concentrated in the
hippocampus and cerebellum, structures associated with information
processing such as learning and cognition. In ischemia, PKCg has
been found in samples of peripheral blood (see U.S. Pat. No.
6,268,223); however, hypoxia occurring under conditions of normal
or even increased cerebral blood flow has not heretofore been
studied in detail.
[0007] Acute mountain sickness is among the most prominent
pathophysiological conditions related to hypoxia at high altitudes
and is characterized by a range of neurological impairments. Table
1 depicts physiological variables in healthy subjects at sea level
and at high altitude. If not effectively treated, high altitude
cerebral encephalopathies (HACE), a potentially lethal condition,
ensue.
TABLE-US-00001 TABLE 1 Central Hemodynamic, Blood Gases, and
Transcranial Doppler Variables in 35 Healthy Subjects at Sea Level
and at High Altitude. (Cf. Van Osta) Sea Level Altitude P HR, bpm
76 .+-. 20 85 .+-. 12 <0.05 SaO.sub.2 97 .+-. 1 79 .+-. 7
<0.001 PaO.sub.2, mm Hg 90 .+-. 11 41 .+-. 6 <0.001
PaCO.sub.2, mm Hg 40 .+-. 2 30 .+-. 2 <0.001 Systolic BP, mm Hg
137 .+-. 20 137 .+-. 26 NS Diastolic BP, mm Hg 81 .+-. 12 82 .+-.
17 NS Mean BP, mm Hg 100 .+-. 14 100 .+-. 18 NS Systolic Vmca, cm/s
69 .+-. 11 72 .+-. 18 NS Diastolic Vmca, cm/s 34 .+-. 7 37 .+-. 13
NS Mean Vmca, cm/s 49 .+-. 8 52 .+-. 14 NS ARI 4.44 .+-. .086 4.55
.+-. 1.16 NS Data are given as mean .+-. SD.
[0008] Table 2 depicts hypoxic thresholds for central nervous
system dysfunctions at sea level and simulated altitudes causing
hypoxia.
TABLE-US-00002 TABLE 2 Hypoxic Thresholds for CNS Dysfunction
(Values derived from younger volunteers subjected to acute
(minutes) decompression hypoxia. F.sub.iO.sub.2, fractional
percentage of ambient oxygen. (Cf. Bailey; Butterworth; Huff;
Kirkham & Datta) Simulated altitude (ft) F.sub.iO.sub.2 (%)
PaO.sub.2 (mm Hg) Neurological status Sea level 21 90 Normal 5,000
17 80 Impaired dark vision 8,000-10,000 15-14 55-45 Impaired
short-term memory, difficulty learning complex tasks 15,000-20,000
11-9 40-30 Loss of judgment, euphoria, obtundation >20,000 <9
<25 Coma
[0009] Retinal encephalopathies also occur at these altitudes and
include torturous, leaky vasculature consistent with established
physical symptoms of HACE. These are clear examples of hypoxic
encephalopathies at partial pressure of oxygen; PKCg is present in
samples of peripheral blood during hypoxic encephalopathies. These
same oxygen pressures elicit short-term memory impairment,
difficulty learning complex tasks, loss of judgment, euphoria, and
obtundation.
SUMMARY OF THE INVENTION
[0010] The present invention overcomes the problems and
disadvantages associated with existing neurological and
psychometric assessments of cognitive, neuropsychological, and
neuropsychiatric functions.
[0011] One embodiment of the invention is directed to a method for
assessing an abnormality of a neurological or cognitive function of
the central nervous system of a patient comprising: contacting a
sample obtained from said patient with a binding partner capable of
forming a complex with protein kinase C gamma (PKCg) to form a
PKCg/binding partner complex; and detecting PKCg/binding partner
complex formed and determining the presence of the abnormality in
the neurological or cognitive function assessed.
[0012] Another embodiment is directed to a method wherein the
abnormality assessed is selected from the group consisting of an
abnormal behavioral, neuropsychological, or neuropsychiatric
function, a movement disorder, a drug-induced or toxin-induced
disorder, short-term memory loss, an encephalopathy, and
combinations thereof.
[0013] Another embodiment is directed to a method wherein the
abnormal neurological or cognitive function assessed is associated
with chronic hypoxia. Another embodiment is directed to a method
wherein the chronic hypoxia is associated with a breathing
disorder, apnea, a pulmonary disorder, a surgical procedure,
administration of a medication, a change in altitude or barometric
pressure or a combination thereof.
[0014] Another embodiment is directed to a method wherein the
abnormality assessed is not connected with an ischemic event or the
restriction of blood flow to the central nervous system.
[0015] Another embodiment is directed to a method wherein the
patient is a mammal. In another embodiment, the mammal is a
human.
[0016] Another embodiment is directed to a method wherein the
sample is a sample of peripheral blood. Another embodiment is
directed to a method wherein the sample is a sample of retinal
fluid.
[0017] Another embodiment is directed to a method wherein the
binding partner is an anti-PKCg antibody or antibody fragment. In
another embodiment, the anti-PKCg antibody is detectably
labeled.
[0018] Another embodiment is directed to a method further
comprising quantitatively determining the amount of PKCg/binding
partner complex formed.
[0019] Another embodiment is directed to a method further
comprising determining the relative amount of PKCg/binding partner
complex formed as compared with the amount of PKCg/binding partner
complex of a control sample indicative of a normal (non-abnormal)
neurological or cognitive function.
[0020] Another embodiment is directed to a method for assessing the
potential for impairment of a neurological or cognitive function of
the central nervous system of a patient comprising: contacting a
sample obtained from the patient with a binding partner capable of
forming a binding complex with protein kinase C gamma (PKCg) to
form a PKCg/binding partner complex; determining the quantity of
PKCg/binding partner complex formed; and assessing the potential
for impairment of the neurological or cognitive function from the
quantity of PKCg/binding partner complex formed.
[0021] Another embodiment is directed to a method further
comprising comparing the amount of PKCg/binding partner complex
formed with an amount of PKCg/binding partner complex formed with a
control sample, wherein said control sample is indicative of a
known impairment of a neurological or cognitive function of the
central nervous system.
[0022] Another embodiment is directed to a method further
comprising monitoring the amount of PKCg/binding partner complex
formed over a period of time.
[0023] Another embodiment is directed to a method for diagnosing a
disorder of a neurological or cognitive function of the central
nervous system of a patient comprising: contacting a sample
obtained from said patient with a binding partner capable of
forming a binding complex with PKCg to form a PKCg/PKCg binding
partner complex, wherein detection of a PKCg/PKCg binding partner
complex is diagnostic of said disorder.
[0024] Another embodiment is directed to a kit for diagnosis of a
disorder of the neurological or cognitive function of the central
nervous system of a patient comprising: a first substrate having
immobilized thereon an anti-PKCg antibody or antibody fragment for
contact with a sample obtained from the patient; a detectable label
capable of binding to a PKCg/PKCg antibody complex; a second
substrate which has bound thereto a PKCg/binding partner complex
which is detectably labeled at a detection level that is
representative of a disorder of a neurological or cognitive
function of the central nervous system of a similar patient.
[0025] Another embodiment is directed to a kit further comprising a
second anti-PKCg antibody reactive with another epitope of
PKCg.
[0026] Another embodiment is directed to a kit wherein said first
antibody is immobilized on a solid support.
[0027] Another embodiment is directed to a kit wherein the solid
support is selected from the group consisting of a plastic
multiwall plate, plastic or glass beads or rods, and a plastic or
glass film.
[0028] Another embodiment is directed to a kit wherein the antibody
or antibody fragment is labeled.
[0029] Another embodiment is directed to a kit further comprising
one or more additional substrates each of which has bound thereto a
PKCg/binding partner complex which is detectably labeled at a
detection level that is representative of another disorder of a
neurological or cognitive function of the central nervous system of
a similar patient.
[0030] Other embodiments and advantages of the invention are set
forth in part in the description, which follows, and in part, may
be obvious from this description, or may be learned from the
practice of the invention.
DESCRIPTION OF THE INVENTION
[0031] PKCg is used as a biomarker for assessing and diagnosing
events related to insults resulting in interrupted cranial blood
flow, such as stroke. It was surprisingly discovered that PKCg
release from neural cells can be detected in peripheral blood after
hypoxic events unrelated to the reduction or elimination of blood
flow through affected tissues. Thus, hypoxia is a surprising and
unique stimulus for the movement of PKCg from the cytoplasm to
membranes in neural cells. Embodiments of this invention are
generally directed to a broad range of clinical applications
related to the treatment and identification of neurological
functions and conditions, particularly in emergency medicine. The
methods of the present invention are especially contemplated to
benefit human subjects but are suitable for any mammalian
subject.
[0032] One embodiment of the present invention is directed to a
PKCg biological marker, which allows rapid assessment and/or
identification of compromised cognitive, behavioral,
neuropsychological, and/or neuropsychiatric (collectively referred
to throughout as "neurological") functions under conditions
associated with acute, transient hypoxia in humans.
[0033] Another embodiment is directed to assessments of cognitive,
neuropsychological and/or neuropsychiatric functions employing a
biological marker of hypoxia. In certain embodiments, the
assessments are neurological and psychometric. Preferably, the
biological marker of hypoxia is PKCg. More preferably, a
diagnostically effective amount of PKCg is detected indicating,
identifying, and/or distinguishing among functions of the central
nervous system. In one embodiment, a sample of blood is assessed
for a diagnostically effective amount of PKCg. In another
embodiment, a sample of cells, preferably lysed, is assessed
according to embodiments of the present invention for a
diagnostically effective amount of PKCg.
[0034] Yet another embodiment is directed to a biological molecular
marker, preferably one comprising PKCg, which rapidly identifies
compromised neurological functions. Preferably, these functions are
associated with hypoxia in humans and other mammals.
[0035] Without wishing to be bound by theory, the neurological
functions, preferably related to hypoxic events, provide a specific
stimulus for the movement of PKCg from the cytoplasm to membranes
in neural cells. The release of PKCg from neural cells indicates a
loss of normal cellular and molecular functions. This loss of
normal and molecular functions underlies short- and long-term
memory acquisition such as, for example, synthesis, release and
metabolism of glutamate and other neurotransmitter substances as
well as the regulation of gap junctions in endothelial cells of
vascular tissues.
[0036] Additional embodiments facilitate categorization of and
distinguishing between hypoxic encephalopathies and conditions
arising from neuroanatomical/structural anomalies and/or metabolic
pathologies. Such pathologies are characterized by focal metabolic
changes in basal ganglia and cerebellar structures and result in
disorders of movement control and coordination. Examples include
the ingestion of toxic substances (e.g., alcohol), vitamin
deficiencies, or adverse effects of drugs and treatments that act
on central nervous system tissues.
[0037] Encephalopathies, or brain illnesses, are a significant
clinical problem characterized by diffuse and complex arrays of
cognitive, neuropsychological and/or psychiatric symptoms. Studies
to gain insight into the underlying pathophysiology are complicated
by a general lack of access to central nervous system tissues that
are encased in bone and membranous structures. Embodiments of the
present invention distinguish between hypoxic encephalopathies and
certain metabolic encephalopathies. Other embodiments of this
invention distinguish between conditions arising from
neuroanatomical/structural anomalies and/or incidental pathologies,
for example, alcohol intoxication or adverse psychoactive drug
effects. Additional embodiments are directed to detection and
treatment of retinal encephalopathies, which afford a unique window
on neurological functions, preferably functions as affected by
conditions associated with hypoxia.
[0038] In another embodiment, PKCg is a molecular biomarker
correlate for neurological functions as a result of conditions
associated with acute, transient hypoxia. A diagnostically
effective amount of PKCg is detected according to embodiments of
the present invention, indicating an abnormal neurological function
and/or a hypoxic event. Hypoxia can be a consequence of certain
metabolic encephalopathies that result in generalized depression of
cerebral function, including consciousness which may result from
diminished neurocortical and other forebrain activity involved in
cognition. Arousal of these structures is mediated by the ascending
reticular activating system (ARAS) through thalamic synaptic relays
to the neocortex. Metabolic encephalopathies result from
alterations of brain chemistry at both neocortical and brainstem
ARAS centers. The typical decrease in respiration would result in
hypoxia. Other breathing disorders include apnea and/or chronic
obstructive pulmonary disorders, surgical procedures, medications,
and/or extremes of altitude and barometric pressure. The sequelae
to these types of conditions are characterized by cognitive and
neuropsychological dysfunctions and ataxia. A variety of treatments
and established clinical protocols are presently used as
interventions. The current technology for assessing short-term
memory, cognitive functions or various performance tasks is based
on batteries of neurological, neuropsychological and/or
psychometric tests using numerous instruments. The ability to
monitor the effects of established as well as novel therapeutic
interventions using PKCg as a biomarker according to embodiments of
the present invention facilitates diagnoses and allows for better,
more effective treatments to be provided.
[0039] Surprisingly, PKCg is implicated in the underlying
mechanisms of conditions characterized by hypoxia. The activation
of PKCg in neural cells is additionally implicated in the molecular
mechanisms of learning and behavior. In one embodiment of the
present invention, determining whether PKCg in samples of
peripheral blood is elevated during and/or after adverse events
associated with the onset and/or recovery from acute hypoxia
provides a means to assess brain function that is based on a
physical, biological marker.
[0040] Oxygen is a critical consideration in the central nervous
system due to the high metabolic rate, limited intrinsic energy
stores and critical dependence on aerobic metabolism of the brain.
Consequently, hypoxia is a well-established contributor to
oxidative stress including an oxygen sensing signal cascade
influenced by reactive oxygen species. The activation of
phospholipases by ionic and neurotransmitter disturbances leads to
the release of fatty acid and lipid co-factors that activate PKCg
and facilitate its calcium-dependent translocation from the cytosol
to cell membranes. Activated PKCg is implicated in the molecular
processes in membranes including the regulation of tight/gap
junctions and of glutamate receptors.
[0041] Arachidonic acid, alone, has been shown to activate PKCg
from bovine cerebellum. The multi-enzymic oxygenase pathways
mediating the synthesis of prostaglandins and other biologically
active derivatives of certain essential fatty acids require
molecular oxygen as they interact with other enzyme systems that
act to regulate membrane integrity and mitochondrial functions that
require molecular oxygen to produce the "energy currency" of cells,
ATP. In addition to phospholipases and oxygenases, these include
acyl-transferases, acyl-carrier proteins, elongases and desaturases
as well as alpha-, beta-, and omega-degradation pathways. Certain
of these components of the pathways metabolizing arachidonic and
specific other essential fatty acids also produce substances with
the established ability to increase the permeability of endothelial
cells lining the microvasculature which would contribute to
high-altitude cerebral edema, high-altitude pulmonary edema and
high altitude retinal encephalopathies.
[0042] Other biomarkers of ischemia have been identified, such as
S-100 and neuron-specific enolase (NSE). While a number of proteins
are known to require arachidonic, diacylglycerol and/or other
lipids for activation, PKCg is the only one presently identified
that is unique to the central nervous system. In studies related to
ischemic events, it was observed that when the blood brain barrier
is compromised as a result of an ischemic event, PKCg appeared in
peripheral blood. The presence of PKCg in peripheral blood is
detectable in peripheral blood almost immediately following an
ischemic event, and, most importantly, within the critical time
window in which diagnosis and treatment of ischemic injury can
prevent permanent damage to CNS tissue. PKCg is activated by a
calcium- and arachidonic acid dependent association with cell
membranes. PKCg slips across membranes that are compromised by
diminished oxygen supplies and membranes that are leaky. Although
PKCg release into the peripheral blood has been linked to ischemic
events (i.e. events in which there is a restriction in blood
supply), it was surprisingly discovered that PKCg can be used as a
biomarker for events occurring during normal or even increased
cerebral blood flow.
[0043] Performing one or more tests for ischemia using non-PKCg
biomarkers in addition to assessing or detecting levels of PKCg in
a sample from a subject indicates whether an ischemic event has
occurred or not. A positive non-PKCg ischemic event biomarker
result indicates the occurrence of an ischemic event, whereas a
negative non-PKCg ischemic event biomarker result in conjunction
with detection of a diagnostically effective amount of PKCg
according to various embodiments of the present invention indicates
a neurological function abnormality, disorder or impairment that is
not associated with a transient, acute ischemic event.
[0044] Additional embodiments are directed to assessing PKCg levels
in a patient at repeated time intervals; preferably, if a
diagnostically effective amount of PKCg is detected, the test is
repeated at 2 hour, 4 hour, 8 hour, 12 hour, 16 hour, 24 hour,
and/or weekly intervals. Repeated detection of a diagnostically
effective amount of PKCg is correlated to chronic hypoxia; an
encephalopathy; a metabolic pathology; a metabolic encephalopathy;
a neuroanatomical or structural anomaly; an impairment that is not
associated with an ischemic event; a neurological dysfunction; an
abnormal behavioral, neuropsychological, or neuropsychiatric
function; a movement disorder; a drug-induced or toxin-induced
disorder; a breathing disorder; apnea; a pulmonary disorder; a
condition associated to a surgical procedure or administration of a
medication; an acute and transient hypoxic event that is not
connected with an ischemic event or the restriction of blood flow
to the central nervous system; or combinations thereof.
[0045] Using a model of ischemic insult, middle cerebral artery
occlusion (MCAO) in the rat, earlier studies have indicated that
PKCg is detectable in peripheral blood samples and that the
quantity directly correlates to the severity of neurological
impairment in the central nervous system of the rat. Data
substantiating these observations are presented in U.S. Pat. No.
6,268,223 ("Assay for Detecting Damage to the Central Nervous
System" issued on Jul. 31, 2001), which is incorporated herein by
reference in its entirety. Additionally, a 66% decrease in the
partial pressure of oxygen in tissues before and during MCAO in the
rat is associated with the presence of PKCg in peripheral blood
samples. A pilot study with human samples has also been completed,
and the results are consistent with the MCAO data from the rat.
[0046] Dysfunctions (such as those shown in Table 2, infra) of the
central nervous system are consistent with an acute hypoxic state.
Embodiments of this invention are directed to detecting PKCg, whose
release from neural structures in the central nervous system,
particularly the hippocampus and cerebellum, is triggered during
hypoxia. Released PKCg increases in samples of peripheral blood and
can be detected. Embodiments of this invention provide an index of
the oxygenation of neural tissues in the central nervous system as
well as the means to estimate whether cognitive,
neuropsychological, neuropsychiatric and/or motor functions
associated with neural tissues are adversely affected. As the
technology for measuring PKCg in peripheral blood samples becomes
more accessible, sensitive and miniaturized, online analysis can
lead to immediate remedial efforts that can reduce or even block
the impairment of central nervous system functions, particularly
short-term memory loss and the anxiety of associated behavioral
changes.
[0047] Embodiments of this invention have broad applications in
clinical practice, particularly by emergency medical personnel.
[0048] PKCg is a specific marker for central nervous system tissue
that is activated by certain fatty acid moieties such as
arachidonic acid (AA). In addition, AA is the fully competent
precursor of the oxygenase pathways. In the CNS, noteworthy
pathways are cyclooxygenase, producing prostaglandins, thromboxanes
and prostacyclins, and lipoxygenases (including 5-HETE, 12-HETE,
LTC.sub.4, LxA). PKCg specific for the central nervous system is
not normally found in peripheral blood; however PKCg is activated
during hypoxic events and/or during events related to neurological
function impairment not necessarily associated with ischemia.
[0049] The amount of PKCg detected in a peripheral blood sample is
proportional to the degree of neurological dysfunction; one
embodiment is thus directed to quantitative assay of the PKCg in a
sample to indicate the extent of impairment of or abnormality in a
neurological function.
[0050] In ischemic studies, the disruption of cranial blood flow
has been explained as triggering a common pathway of cell mediated
damage which originates with the activation and eventual release of
PKCg from neural tissues. PKCg is normally found only in the CNS
and is not known to be localized to any other tissues. In the event
of ischemic damage, an accompanying breakdown of the blood brain
barrier has been observed, which has been explained as resulting in
the release of PKCg from its normal location in the brain into
venous blood. The present invention, in contrast, is directed to
PKCg release during events of normal or increased blood flow. In
one embodiment, the PKCg release is linked to underlying central
nervous system functions; in another embodiment, PKCg release is
linked to hypoxia.
[0051] Any method of detection for a diagnostically effective
amount of PKCg marker is suitable for embodiments of this
invention, and any known methods of detecting a specific protein in
a sample may be employed. Preferably, PKCg is detected in a sample
of blood, preferably in a diagnostically effective amount of PKCg,
from a mammalian subject, by contacting the sample with a binding
partner for PKCg, that is, a peptide, immunoglobulin, small
molecule or other moiety capable of forming an association complex
with PKCg. Most preferably, a diagnostically effective amount of
PKCg in a sample is detected using antibodies specific for PKCg.
Several such antibodies are known, and monoclonal antibodies
recognizing different epitopes of PKCg are available commercially,
making simple sandwich assays readily practicable. (See U.S. Pat.
No. 6,268,223.)
[0052] For detection or measurement of PKCg levels in a sample,
fluorescently labeled antibodies are most preferred. Many other
methods of detecting PKCg directly or detecting a complex of PKCg
with another moiety are known, including gas chromatography mass
spectroscopy, thin layer chromatography, hydroxyl apatite
chromatography, high pressure liquid chromatography, colloidal gold
immunolabeling read by electron microscopy, enzyme-linked
immunosorbent assays, radioactively labeled tags or antibodies
specific for PKCg read using a scintillation counter,
bioluminescently labeled antibodies read on a colorimeter, etc.;
however, most of these methods require several hours or even days
for sample preparation and/or measurement of the signal, making
them inferior to sensitive fluorescence-based assays such as
described in U.S. Pat. No. 6,268,223. Also, a typical detection
apparatus in some cases (e.g., mass spectrophotometer, electron
microscope) would not fit inside an ambulance, making performance
of the assays by emergency medical personnel before a patient is
brought to a hospital impossible.
[0053] In additional embodiments of the invention, the materials
for detection of a diagnostically effective amount of PKCg in a
sample of venous blood are conveniently assembled into a kit, so
that personnel treating or transporting a patient can identify,
treat and/or categorize the type of neuropsychological, behavioral
and/or cognitive functions occurring in a patient's central nervous
system. One kit useful for such diagnoses is based on PKCg binding
and is capable of providing multiple levels of detection and
quantitation. The level of detection provides quantitative
assessment of neurological function based on calibration of
fluorescently tagged antibodies to PKCg detected in venous
blood.
[0054] Another embodiment is directed to correlating retinal
physiology with central nervous system functions. Preferably,
assessment of retinal vasculature is performed via retinography or
similar technologies; more preferably, such assessment is
correlated with the release of PKCg from the central nervous
system. Such release of PKCg is correlated to a hypoxic event, a
metabolic encephalopathy, a central nervous system anomaly, or
combinations thereof.
[0055] In addition to providing rapid diagnosis of a hypoxic event
by emergency and medical personnel, the methods and kits described
herein also may be used to detect a diagnostically effective amount
of PKCg and to monitor PKCg levels as part of a routine checkup
procedure or to monitor general functions and conditions of the
central nervous system. In one embodiment, a PKCg monitor functions
similarly to a glucometer.
[0056] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All references
cited herein, including U.S. Provisional Application No. 60/886,532
to which the present application claims the benefit of priority,
all publications, U.S. and foreign patents and patent applications,
are specifically and entirely incorporated by reference. The term
"comprising" as used throughout this application includes the more
limiting terms and phrases "consisting essentially of" and
"consisting." It is intended that the specification and examples be
considered exemplary only with the true scope and spirit of the
invention indicated by the following claims.
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