U.S. patent application number 13/645854 was filed with the patent office on 2013-04-11 for prophylactic and post-acute use of progesterone to better outcomes associated with concussion.
This patent application is currently assigned to FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION. The applicant listed for this patent is FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Michael Lewandowski, Vedrana Marin, John Suber, Jacob W. Vanlandingham.
Application Number | 20130090315 13/645854 |
Document ID | / |
Family ID | 48042460 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130090315 |
Kind Code |
A1 |
Vanlandingham; Jacob W. ; et
al. |
April 11, 2013 |
PROPHYLACTIC AND POST-ACUTE USE OF PROGESTERONE TO BETTER OUTCOMES
ASSOCIATED WITH CONCUSSION
Abstract
Compositions and methods for treating traumatic brain injury
(TBI) and mild traumatic brain injury (mTBI) using progesterone and
ent-progesterone are described.
Inventors: |
Vanlandingham; Jacob W.;
(Tallahassee, FL) ; Suber; John; (Tallahassee,
FL) ; Marin; Vedrana; (Alachua, FL) ;
Lewandowski; Michael; (Odessa, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY RESEARCH FOUNDATION; FLORIDA STATE |
Tallahassee |
FL |
US |
|
|
Assignee: |
FLORIDA STATE UNIVERSITY RESEARCH
FOUNDATION
Tallahassee
FL
|
Family ID: |
48042460 |
Appl. No.: |
13/645854 |
Filed: |
October 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61544502 |
Oct 7, 2011 |
|
|
|
Current U.S.
Class: |
514/177 |
Current CPC
Class: |
A61K 31/57 20130101;
A61P 25/00 20180101; A61K 9/0043 20130101; A61P 25/28 20180101;
A61K 9/06 20130101; A61K 9/08 20130101 |
Class at
Publication: |
514/177 |
International
Class: |
A61K 31/57 20060101
A61K031/57; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method comprising the following step: (a) administering a
composition comprising progesterone to an individual within 15
minutes of the individual suffering a mild traumatic brain injury
(mTBI).
2. The method of claim 1, wherein the composition is an aqueous
solution.
3. The method of claim 1, wherein the composition is a gel.
4. A method comprising the following step: (a) administering a
composition comprising ent-progesterone to an individual within 15
minutes of the individual suffering a mild traumatic brain injury
(mTBI).
5. The method of claim 4, wherein the composition is an aqueous
solution.
6. The method of claim 4, wherein the composition is a gel.
7-27. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S.
Provisional Patent Application No. 61/544,502 to VanLandingham et
al., entitled, "Prophylactic and Post-Acute Use of Progesterone and
Its Enantiomer to Better Outcomes Associated with Concussion,"
filed Oct. 7, 2011, which is incorporated by reference in its
entirety.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The present invention relates to prophylactic and post-acute
treatments of concussions (mild TBI).
[0004] 2. Related Art
[0005] There are many health issues related to traumatic brain
injuries. For example, once an athlete sustains a concussion (one
type of traumatic brain injury (TBI)) the athlete becomes four to
six times more likely to suffer a second head injury. Also, half of
deaths associated with falls in the elderly are caused by a head
injury. In addition, a veteran commits suicide every 80 minutes and
is 25 times more likely to develop Post-Concussion Syndrome (PCS)
following TBI. Furthermore, cerebral plaques in head-injury
patients are similar to cerebral plaques in Alzheimer's disease
patients. One single TBI doubles the risk of Alzheimer's disease in
males, and mild traumatic brain injury (mTBI) is the most common
type of TBI that leads to long-term neurodegenerative disorders.
The estimated annual cost of health care for traumatic brain
injuries is $20 billion.
[0006] There are approximately 1.3 million cases of concussion
reported each year in the United States. It is estimated that an
additional 2 million cases of concussion are unreported. There are
about 600,000 sports-related concussions per year. There have also
been about 360,000 overseas military personnel who have suffered
concussions, often due to the effects of detonation of improvised
explosive devices (IEDs).
[0007] It is estimated that over 1.5 million people suffer
traumatic brain injuries each year. Of these people who suffer
traumatic brain injuries, it is estimated that over 50,000 die and
that another 80,000 become impaired or disabled for life.
SUMMARY
[0008] According to a first broad aspect, the present invention
provides a method comprising the following step: (a) administering
a composition comprising progesterone to an individual within 15
minutes of the individual suffering a mild traumatic brain injury
(mTBI).
[0009] According to a second broad aspect, the present invention
provides a method comprising the following step: (a) administering
a composition comprising ent-progesterone to an individual within
15 minutes of the individual suffering a mild traumatic brain
injury (mTBI).
[0010] According to a third broad aspect, the present invention
provides a method comprising the following step: (a) administering
a composition to an individual by nasal administration, wherein the
composition comprises ent-progesterone and one or more
cyclodextrins, and wherein the molar ratio of ent-progesterone to
the total amount of cyclodextrins in the composition is about
1:1.
[0011] According to a fourth broad aspect, the present invention
provides a method comprising the following step: (a) administering
a composition to an individual by nasal administration, wherein the
composition comprises ent-progesterone and a bioadhesive polymer,
and wherein molar ratio of ent-progesterone to the bioadhesive
polymer in the composition is about 1:1.
[0012] According to a fifth broad aspect, the present invention
provides a composition comprising ent-progesterone and one or more
cyclodextrins, wherein molar ratio of ent-progesterone to the total
amount of cyclodextrins in the composition is about 1:1.
[0013] According to a sixth broad aspect, the present invention
provides a composition comprising ent-progesterone and a
bioadhesive polymer, wherein molar ratio of ent-progesterone to the
bioadhesive polymer in the composition is about 1:1.
[0014] According to a seventh broad aspect, the present invention
provides a method comprising the following step: (a) administering
a composition comprising ent-progesterone to an individual who has
suffered a mild traumatic brain injury (mTBI), wherein step (a) is
repeated at least daily for at least seven days.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings, which are incorporated herein and
constitute part of this specification, illustrate exemplary
embodiments of the invention, and, together with the general
description given above and the detailed description given below,
serve to explain the features of the invention.
[0016] FIG. 1 shows a graph of percent brain water content for rats
suffering from TBI that have been treated with allopregnanolone,
progesterone and ent-progesterone.
[0017] FIG. 2 shows a graph of glutathione reductase activity for
rats suffering from TBI that have been treated with
allopregnanolone, progesterone and ent-progesterone.
[0018] FIG. 3 shows a graph of the binding of ent-progesterone to
the human progesterone receptor.
[0019] FIG. 4 shows a graph showing the effect of progesterone and
ent-progesterone on human pregnane X receptor mediated
transcription.
[0020] FIG. 5 shows a graph showing the effect of progesterone and
ent-progesterone on mouse pregnane X receptor mediated
transcription.
[0021] FIG. 6 shows a graph showing the effect of progesterone and
ent-progesterone on increasing the protein abundance of
P-glycoprotein in the penumbral region of an injured brain 72 hours
after the brain has been injured.
[0022] FIG. 7 shows a graph of latency to platform from Morris
Water Maze Testing for rats that have and have not suffered a
concussion.
[0023] FIG. 8 shows a graph of latency to platform from Morris
Water Maze Testing for rats that have suffered a concussion and
been: (a) untreated, (b) treated with cyclodextrin, (c)
prophylactically treated with progesterone and (d) post
acutely-treated with progesterone and otherwise untreated.
[0024] FIG. 9 is a graph showing the results for a Morris Water
Maze Test for rats.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0025] Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated. For the purposes of
the present invention, a value or property is "based" on a
particular value, property, the satisfaction of a condition, or
other factor, if that value is derived by performing a mathematical
calculation or logical decision using that value, property or other
factor.
[0026] For purposes of the present invention, the term "bioadhesive
polymer" refers to a polymer that can increase bioavailability of
ophthalmic, nasal, buccal, intestinal, rectal and vaginal
formulations. One example of a bioadhesive polymer is carbopol.
[0027] For purposes of the present invention, the term
"ent-progesterone component" and the term "ent-Prog component"
refer to a component of a delivery system that contains
ent-progesterone. The component may be a solution that contains
ent-progesterone.
[0028] For purposes of the present invention, the term "individual"
refers to a mammal. For example, the term "individual" may refer to
a human individual.
[0029] For purposes of the present invention, the term "intranasal
delivery" and term "intranasal administration" are equivalent terms
and refer to the delivery or administration of a chemical
composition through one or both nasal passages of an individual.
Examples of intranasal delivery include actively spraying a
chemical composition containing Prog or ent-Prog into a nasal
passage, having an individual inhale a chemical mist containing
Prog or ent-Prog.
[0030] For purposes of the present invention, the term "mild
traumatic brain injury (mTBI)" refers to nondegenerative, non
congenital insult to the brain from an external mechanical force
that can lead to temporary or permanent cognitive, physical and
psychosocial impairment with an associated diminished or altered
state of consciousness.
[0031] For purposes of the present invention, the term "nasal
spray" refers to a that functions by instilling a fine mist into
the nostril by action of a hand-operated pump.
[0032] For purposes of the present invention, the term
"progesterone component" and the term "Prog component" refer to a
component of a delivery system that contains progesterone. The
component may be a solution that contains progesterone.
[0033] For purposes of the present invention, the term "sham" and
the term "sham control" refers to the members of a control group
that are used to mimic a procedure or treatment without the actual
use of the procedure or test substance.
[0034] For purposes of the present invention, the term "sprayable"
refers to a solution that is turned into a fine mist by action of a
hand operated pump.
[0035] For purposes of the present invention, the term "traumatic
brain injury (TBI)" refers to an injury to the brain caused by an
external mechanical force. A traumatic brain injury may be caused
in various ways such as being exposed to a hit to the head while
playing a sport such as football, hockey, baseball, etc.; being
exposed to an explosion; being in a car accident; hitting one's
head after a fall, etc.
[0036] For purposes of the present invention, the term "vehicle
control" and "vehicle" refers to an animal that is subjected to a
mild TBI and is treated with a drug formulation.
Description
[0037] In the description below, all ratios are by weight, unless
specified otherwise.
[0038] Progesterone (Prog) has thus far been the most promising
neuroprotective drug for the treatment of TBI: it reduces poor
outcomes following injury by inhibiting inflammatory factors
(TNF-.alpha. and IL-.beta.) and subsequently reducing brain edema
(1, 2) Prog-treated rats have demonstrated significant improvements
on a Neurological Severity Score (test for motor and cognitive
functioning) following injury (3). Prog effectively attenuates
edema in both rodent sexes following injury (4). Administering Prog
or its derivative allopregnanolone (ALLO) also results in a
decreased of the presence of the factors of cell death (caspase-3)
and gliosis (GFAP) (5) following injury (6, 7). Formula 1 below
shows the structure of progesterone.
##STR00001##
[0039] Phases I and II of the Prog for TBI clinical trial, ProTECT
conducted at Emory University are complete. Results of this
clinical trial using Prog to treat TBI have shown a 50% reduction
in mortality in moderate to severe TBI patients receiving 72 hours
of continuous intravenous Prog in the emergency room. This clinical
trial has also measured 30 days post-injury outcomes using the
Glasgow Outcome Scale-Extended and Disability Rating Scale scores.
Moderate TBI patients who received Prog had better outcomes
compared to those who did not receive the neurosteroid; there was
little measurable difference in patients with severe brain injuries
(8). However, a clinical study over the longer period of time of
six months demonstrated that severe TBI patients had better
neurological outcomes long-term when treated with Prog (9). The
U.S. National Institutes of Health is now sponsoring nationwide
Phase III clinical trials with Prog for 1200 moderate to severe TBI
patients (10). Prog may extend its benefits by acutely treating
military personnel with moderate to severe type TBIs from IED
blasts.
[0040] Neurosteroids such as Prog have not been tested in an animal
model that represents mTBI seen in military populations, even
though mTBI accounts for more than 80% of all head injuries (11)
and is now recognized as the most common neurological diagnosis
(12). Approximately 360,000, which represent about 25%, of U.S.
veterans of Iraq and; reports that mTBI is a serious yet often
undiagnosed injury: this "silent epidemic" results in non-visible
problems such as memory loss and depression; other symptoms include
problems with concentration, lack of emotional control, headaches,
fatigue, irritability, dizziness, blurred vision and seizures. It
appears that repetitive mTBI may leave veterans at high risks for
longer-term neuropsychological and neurodegenerative disorders
(discussed below), decades after they exit the warzone; one such
outcome is Post-Traumatic Stress Disorder (PTSD) associated with a
high rate of suicide (13, 14).
[0041] With mTBI being the most common type of TBI affecting
military personnel in one embodiment, the present invention
provides a therapy using ent-Prog that may be administered
repetitively and easily as a nasal spray in order to protect troops
from potential harm and promote a safe and quicker return to duty
post-injury.
[0042] The correlation between progesterone (Prog) intake following
moderate to severe traumatic brain injury (TBI) and a reduction in
cerebral inflammation and edema has been well documented (15-17).
Prog also aids in improving short-term cognitive functions and
motor coordination in a rodent model for TBI (5, 16, 18), and it
has been shown to decrease the risk of subjects developing serious
long-term diseases, such as Alzheimer's and Parkinson's (19, 20).
While there has been extensive research on the benefits of
administering Prog during moderate to severe TBI recovery, studies
regarding the impact of Prog on the symptoms of mTBI--commonly
known as concussion--have been extremely limited.
[0043] As with moderate to severe TBI, mild TBI has similar
symptoms of inflammation, edema and decreased motor and cognitive
functioning. Characteristic, yet not necessarily mutually
exclusive, complications of mild TBI include: diffuse axonal
injury; and repeated injury, a condition that causes cumulative
neurological impairments known as chronic traumatic encephalopathy
(CTE) (21). Past research supports acute administration of Prog for
improving outcomes both in rodents and humans who have experienced
moderate to severe TBI, however, Prog has never been used in the
treatment of mild TBI.
[0044] In one embodiment of the present invention, Prog is
administered acutely, within 15 minutes following a mild injury. A
rodent model for concussion has been established (see Research
Design and Methods section of Example 1 below) using memory
impairment as the outcome measure (discussed below). In these
studies Prog is used as a treatment to compare with vehicle
(injured with no treatment) and sham (anesthesia and scalp incision
only) groups of male Sprague Dawley Rats.
[0045] Laboratory and clinical research has demonstrated that
progesterone (Prog) effectively reduces poor outcomes following
traumatic brain injury by inhibiting inflammatory factors and
subsequently reducing brain edema (1, 2)--however, studies in
animals and humans with neuroprotective steroids have been limited
to moderate and severe brain injury (4, 7, 22). Mild traumatic
brain injuries account for more than 80% of all head injuries (10)
and are now the most commonly diagnosed neurological condition
(11). Even a single mild traumatic brain injury (mTBI) may cause
long-term neurological dysfunction and has been determined to
double the risk of developing Alzheimer's Disease (AD) in males
(23). With multiple injuries, such as those endured by football
players, boxers and wrestlers, neuropsychological performance
decreases over time (24, 25); these individuals often suffer from
Post-Concussion Syndrome (PCS) (26)--symptoms of which include
problems with sleep, memory, attention, and cognition that often
last years after injury (27). With about 25% or approximately
360,000 of the U.S. veterans of Iraq and Afghanistan having
sustained mTBI (12), the likelihood of enduring multiple injuries
from improvised explosive device (IED) blast waves is also high.
Unfortunately, there has been little to no research with the use of
neurosteroids in an animal model of mTBI or in humans, which
continues to leave high-risk populations such as athletes and
military personnel susceptible to suffering brain damage.
[0046] In one embodiment, the present invention provides a method
for administering PROG acutely within 15 minutes following the
individual suffering an mTBI.
[0047] Data demonstrates the validity of the above-described mTBI
model and that Prog (prophylactic and post-acute administration)
effectively treats and prevents the motor and cognitive sequelae of
mTBI injury (See FIGS. 7 and 8). However, in order to reduce poor
outcomes following repetitive mTBI and unreported singular mTBI,
the drug must be administered on a repetitive basis to high-risk
populations. Prog is not a sufficient prophylactic treatment for
mTBI; in males it suppresses spermatogenesis, inhibits the
conversion of testosterone to dihydrotestosterone, and reduces the
size of reproductive organs (23, 28, 29). Previous research has
shown that an alternative compound, known as the mirror-image or
enantiomer of Prog (ent-Prog), bind to but does not activate the
traditional progesterone receptor (PR), but operates via the
pregnane x receptor (PXR), decreasing factors of inflammation and
edema with equal efficacy to Prog, and increasing anti-oxidant
activity better than does Prog (30, 31). While ent-Prog should not
have the same sexual side effects as Prog, it does competitively
inhibit the PR (30), so one potential consequence of using ent-Prog
might be preventing pregnancy, or even inducing parturition (early
onset of labor) in pregnant women (32, 33). Conflicting research
has suggested that Prog and related compounds might also increase
hyper-coagulation, thus also increasing a thrombotic risk (34).
Formula I below shows the structure of progesterone.
##STR00002##
[0048] Pre-clinical data from a previous study (8) has shown that
ent-Prog reduces moderate to severe brain injury-induced edema with
equal efficacy to Prog (FIG. 1) and increases glutathione reductase
(anti-oxidant) activity at a significantly higher rate than Prog
(FIG. 2). FIG. 1 shows that ent-Prog (16 mg/kg) normalizes brain
water content (measure of edema) as well as Prog and its
metabolite, alloprenanolone (ALLO) at 72 hours post-injury.
*denotes significance at p<0.05. FIG. 2 shows that ent-Prog
significantly increases the anti-oxidant activity of glutathione
reductase compared to all other groups post-injury at 72 hours
post-injury. **denotes significance at p<0.01.
[0049] Although ent-Prog is correlated with factors associated with
better outcomes following TBI, it does not activate the classical
progesterone receptor (PR); in fact it even inhibits Prog binding
to the PR (31): FIG. 3 depicts PR-binding assays. As the amount of
ent-Prog increases, less Prog binds to the PR. Preliminary research
currently being prepared for publication has identified a potential
mechanism by which ent-Prog treats TBI: by activating a different
receptor, for which Prog is also a ligand, called the pregnane X
receptor (PXR). Both Prog and its enantiomer increased PXR-mediated
transcription with equal efficacy (FIGS. 4 and 5); further, both
neurosteroid treatments elevated the PXR activated P-glycoprotein
(FIG. 6)(35). P-glycoprotein is a membrane channel that exports
intracellular water and thus reduces cytotoxic edema associated
with brain injury (36).
[0050] In FIG. 3 the data represents the percentage of Prog binding
to the classical Human Prog receptor (PR) relative to maximum
binding capacity under conditions of increasing ent-Prog
administration. As shown in FIG. 3, ent-Prog significantly inhibits
Prog binding to the PR FIGS. 4 and 5 show that increasing
concentrations of Prog and ent-Prog increase both human and mouse
PXR-mediated transcription with equal efficiency as measured by a
luciferase gene reporter. As shown in FIG. 6, both Prog and
ent-Prog increase the protein abundance of P-glycoprotein (PGP) in
the penumbral region of the brain injury at 72 hours post-injury
compared to no treatment (vehicle) and sham groups, respectively.
*denotes significance at p<0.05.
[0051] PXR research with ent-Prog demonstrates that activation of
the traditional PR is not necessary in order to effectively treat
TBI. The widespread benefits and potential limitations of the
differences in mechanisms between Prog and ent-Prog in the
treatment of mTBI are discussed below.
[0052] There has been little to no research using therapeutics like
Prog or other neurosteroids in an mTBI animal model. Using a
controlled cortical impactor to induce injury in one mTBI model
produces the data shown in FIG. 7. Rats that experience mTBI have
greater latency to platform in comparison to sham using the Morris
Water Maze swim test. These findings show that this mTBI model
produces acute spatial learning and memory deficits.
[0053] FIG. 7 shows data collected from Morris Water Maze Testing.
These data represent latency to platform in seconds. The points at
-24 hours represent the training period; 0 hours is the time at
which mTBI was induced; the first swim test after injury was
performed at 4 hours. Start section (quadrant) varied day-to-day
and trial-to-trial. *denotes significance at p<0.05.
[0054] The study proposed here is the first of its kind designed to
treat mTBI in an animal model; there has been little research
performed showing the effects of treatments in an mTBI animal model
compared to the quantity of efficacy research performed in moderate
to severe models. One early mTBI animal study correlated
posttraumatic memory scores to neuronal loss; this was the first
study to suggest an association between cognitive deficits
following mTBI and neuropathological changes (37). A later study
demonstrated persistent deficits in cognitive learning abilities
and emergence of depressive-like behavior in injured mice similar
to those reported in human Post-Concussion Syndrome (PCS) (38).
Preliminary data, supported by the literature in this area, show
that this mTBI model is valid.
[0055] Data indicates that this mTBI model can show the beneficial
effects of neurosteroids, particularly Prog. When administered both
prophylactically (15 minutes prior) and post-acutely (15 minutes
post) Prog decreases latency to platform improving memory
performance, in comparison to vehicle-injured rats (FIG. 8).
Because of its similarities to Prog, ent-Prog should react
similarly in this mTBI model.
[0056] FIG. 8 shows data collected from Morris Water Maze Testing.
This data represents latency to platform in seconds. Significant
beneficial differences between progesterone-treated (prophylactic
(1 hour prior) and post-acute (1 hour post) and vehicle rats are
apparent. *denotes significance at p<0.05.
[0057] TBI is characterized by primary mechanical injury followed
by secondary neuronal cell death mediated by increased brain
swelling (22, 39), inflammation (40) and oxidative stress (28).
Both Prog and ent-Prog equivalently reduce factors of cell death,
brain swelling and inflammation; however, ent-Prog has three times
the antioxidant activity than does Prog--which provides potential
mTBI victims with an added benefit. Since ent-Prog does not
activate PR-mediated transcription but does activate the PXR, it
presents a possible therapeutic alternative to Prog following brain
injury (31). Benefits of the alternative compound include fewer
sexual side effects than would be seen with Prog treatment in
males, such as suppression of spermatogenesis; inhibition of the
conversion of testosterone to dihydrotestosterone; and, reduction
in the size of the testes, epididymis and leydig cells (29, 33,
41). ent-Prog competitively inhibits the PR (31), therefore a
potential consequence of using ent-Prog as a TBI therapeutic
involves inducing premature parturition in pregnant women (32, 34),
or preventing pregnancy. There has also been conflicting data
published on whether or not Prog or pregnane derivatives are
associated with an increased thrombotic risk--thus studies may be
designed to test for potential hyper-coagulative effects of the
drugs tested (23).
[0058] Following the successful completion of this study, all the
necessary testing in an animal model to ensure safety of use in
clinical studies may be performed to confirm the safety of using
ent-Prog as nasal inhalant to prophylactically prevent injury, as
well as to post-acutely treat injury. Prog, due to the
aforementioned side effects, potentially should not be used on a
repetitive basis to prevent injury, which is undoubtedly a crucial
factor when considering how to effectively treat repetitive mTBI.
Even a single mTBI has been determined to double the risk of
developing Alzheimer's disease (AD) in males (42)--and repetitive
injuries increase these types of long-term risks and worsen the
severity of symptoms of mTBI patients leading to even poorer
outcomes upon reinjury. The reasons why ent-Prog may significantly
improve long-term outcomes associated with mTBI are covered
below.
[0059] When discussing long-term adverse outcomes following mTBI,
the topic of repetitive injuries is highly relevant because the
impact of each subsequent injury exponentially increases the
severity of an individual's symptoms and the probability of
developing serious neurological disorders. Kane and colleagues
explain how repetitive injury leaves mTBI victims susceptible to
developing PCS and advocates a need for therapeutic options. They
discuss how longer deployment times, increases in the number of
multiple redeployments, as well as improvements in body armor have
led to many soldiers being exposed to numerous blast explosions
and/or non-battlefield injuries, resulting in repetitive mTBIs
(43). Unfortunately, repetitive mTBIs often go undiagnosed and do
not show morphological abnormalities in the brain that can be
detected by standard MRI brain scans (24, 44). Furthermore, recent
findings show that amateur football players have multiple mTBIs
that lead to a decline in neuropsychological performance compared
to individuals with single or no concussions (25, 26, 45). Many
individuals who suffer repetitive mTBIs experience PCS (46, 47).
Unfortunately, the symptoms associated with PCS, which include
disturbances with sleep, memory, attention, and cognition, persist
sometimes for several years, and in other cases may be lifelong
(27, 48-50). The symptoms of PCS are often resistant to current
therapies (51).
[0060] Since PCS is so difficult to treat, it is important to
establish a therapeutic protocol by which to follow in order to
prevent the injury from occurring--or, treating it within a
specified acute window of time post-injury. In a study of 12 Iraq
war veterans with persistent PCS symptoms, veterans with mTBI
exhibited decreased cerebral metabolic rates and also impairments
in verbal fluency, cognitive processing speed, attention and
working memory (52). In one embodiment, the present invention may
prevent PCS from ever occurring using a safe and effective
neurosteroid, ent-Prog. Prevention is also important to the health
of a soldiers because concomitant mTBI and PTSD are associated with
higher rates of other psychological health problems: depression
(53), substance abuse (54), and suicidal behavior (55, 56).
Individuals who have sustained TBI have an 8.1% chance of
attempting suicide; whereas, there is a 1.9% chance in the general
population (57). Among individuals receiving care through the
Veterans Health Administration, compared to individuals without an
injury history, mTBI patients were 1.98 times more likely to die by
suicide; whereas, moderate to severe TBI patients were 1.34 times
more likely to die by suicide. While moderate to severe injuries
are more pathologically damaging at the time of injury, the
psychological effects of mild injuries, especially when repetitive,
may actually lead to more tragic outcomes (58).
[0061] A recent examination of the brains of National Football
League players (59) and wrestlers (60) who have committed suicide
as well as an Iraqi war veteran who committed suicide eight months
after his honorable discharge from the USMC revealed no atrophy,
contusions or hemorrhaging; the only condition noted was brain
swelling. Upon further analysis, the work identified brain tissues
that revealed Chronic Traumatic Encephalopathy (CTE)-changes such
as tau-immunoreactive neurofibrillary tangles (NFTs) and neuritic
threads (61). The tau-NFT pathology of mTBI suicide victims are
very similar to that found in Alzheimer's disease (AD) patients. A
neuroimaging study also discovered similarities between the
pathology of AD and PTSD (62). The pathological similarities
between PTSD and AD as well as AD and mTBI victims may explain why
it is difficult to differentiate symptoms of PTSD and mTBI. With
the same tauopathies that cause AD also contributing to mTBI with
CTE, there has been a lot of research identifying mTBI with CTE
having a higher propensity to cause AD (20, 63). The severity of AD
has been associated with abnormal hyper-phosphorylated protein tau
(hyper-PO.sub.4 tau), containing aggregates of TDP-43 (64).
Repetitive TBI with the development of CTE leads to abnormal TDP-43
expression in about 83% of cases (65). Even a single TBI nearly
doubles the risk for AD in males only (42). The likeliest
explanation for the gender difference in the risk of AD following
head injury is the role of Prog; granted, with multiple head
injuries characterized by CTE pathology, females do not have high
enough levels of Prog to prevent adverse long-term outcomes.
Nonetheless, ent-Prog may be as effective as or even more effective
than Prog in preventing long-term neurodegenerative disorders;
because, one proposed mechanism by which mTBI leads to AD is by a
decrease in function of the PXR and subsequent decrease in PGP
(66).
[0062] It has been shown that by activating PXR, both Prog and
ent-Prog increased PGP by almost three times the levels as in the
sham and vehicle rodents. Several diseases may benefit from an
increase in the function of PGP, such as AD (65-67), Parkinson's
disease (68) and Multiple Sclerosis (69). Prophylactic or acute
treatment that increases activation of the PXR mechanism and
subsequent cerebral PGP will benefit ent-Prog users by preventing
mTBI poor outcomes from ever occurring and therefore eliminate the
risk of future healthcare problems years and even decades later.
With mTBI sometimes being difficult to diagnose--as well as
patients neglecting to seek help for what appears to them to be a
mild injury--in one embodiment of the present invention, veterans
may use the drug as a preventative prophylactic one time per day
when they are on overseas active duty. Of course, ent-Prog may
still be used post-acutely following injuries when they occur but
would be acting against the rapidly proliferating secondary cascade
of the TBI. In order to facilitate ease of use, ent-Prog may be
used as a nasal inhalant. There are also several potential benefits
of using the neurosteroid as a nasal inhalant.
[0063] The nasal route of drug administration continues to receive
increasing attention from pharmaceutical scientists and clinicians
because this route circumvents hepatic first-pass elimination
associated with oral delivery, is easily accessible and suitable
for self-medication (70). Intranasal administration is also
particularly suitable for drugs targeting the brain because certain
drug solutions can bypass the blood-brain barrier (BBB) and reach
the central nervous system (CNS) directly from the nasal
cavity--uptake of these drugs depends on their molecular weight and
lipophilicity (71, 72).
[0064] An example of a successfully developed nasal drug therapy
for treatment of seasickness was previously funded by the Defense
Medical Research and Development. Navy Times staff writer Patricia
Kime describes in her article titled "Nasal spray may prevent onset
of seasickness" a nasal spray that would deliver a preventative
dose of motion sickness medication just before it may be needed.
Kime further reports the nasal spray for seasickness could be
effective with a lower concentration of the active ingredient
scopolamine, which is more easily absorbed in the body
intranasally--the lower concentration will mitigate drug side
effects (73). Lower concentrations of ent-Prog may be needed, which
will cause fewer to no noticeable side effects upon repetitive
administration. Therefore, a prophylactic nasal spray of ent-Prog
may be preferred to other drugs and drug delivery mechanisms.
[0065] Nasal delivery is one of the most attractive non-invasive
routes for therapeutics targeting the central nervous system
because of relatively high permeability of nasal epithelium
membrane, avoidance of hepatic first pass elimination. Nasal
delivery is easy to administer and allows for self-medication by an
individual. Nasal mucociliary clearance is one of the most
important limiting factors to nasal drug delivery. Nasal
mucociliary clearance severely limits the time allowed for drug
absorption to occur and may effectively prevent sustained drug
administration. However, it has been documented that nasal
administration of certain hormones has resulted in a more complete
administration. In one embodiment, the present invention employs
nasal delivery of Prog or ent-Prog for a more local delivery, thus
avoiding any potential side effects of Prog or ent-Prog.
[0066] Cyclodextrins are cyclic oligosaccharides obtained from
enzymatic degradation of starch. Cyclodextrins have been widely
used to improve the delivery of drugs by nasal administration.
Improved nasal delivery has been attributed to changes in nasal
mucosa permeability, alterations in drug solubility and in a change
in the metabolism rate of the drugs at the site of delivery.
Suitable cyclodextrins for use in compositions of the present
invention include hydroxypropyl-.beta.-CD (HP.beta.-CD),
hydroxypropyl-.gamma.-CD (HP.gamma.-CD), permethyl-.beta.-CD
(PM.beta.-CD), and sulfobutylether-.beta.-CD (SBE.beta.-CD). In one
embodiment of the present invention, a composition suitable for
nasal administration, the ratio of cyclodextrin to Prog or ent-Prog
may be about 1:1. In one embodiment of the present invention, a
composition suitable for nasal administration, the ratio of
cylclodextrin to Prog or ent-Prog may be about 2:1.
[0067] In one embodiment of the present invention, a composition
containing Prog or ent-Prog that is suitable for nasal
administration may include one or more bioadhesive polymers. Some
polymers such as carbopol, can adhere onto the nasal mucosa for
reasonably prolonged periods, preventing rapid nasal clearance.
Bioadhesive polymers that may be used in the compositions of the
present invention include 934, 940, 941, 942, 980 and 981. In one
embodiment of the present invention, a composition suitable for
nasal administration, the percentage of bioadhesive polymer in a
suitable solution of Prog and ent-Prog may be 0.1%. In one
embodiment of the present invention, a composition suitable for
nasal administration, the percent of bioadhesive polymer in a
suitable solution of Prog and ent-Prog may be 0.5%. In one
embodiment of the present invention, a composition suitable for
nasal administration, the percentage of bioadhesive polymer in a
suitable solution of Prog and ent-Prog may be 1%.
[0068] In one embodiment of the present invention, a composition
containing Prog or ent-Prog that is suitable for nasal
administration may include one or more surfactants. Surfactants
that may be used in the compositions of the present invention
include different polyethylene glycols (PEGS). In one embodiment of
the present invention, a composition suitable for nasal
administration, the percent of surfactant in a suitable solution of
Prog or ent-Prog may be 1%. In one embodiment of the present
invention, a composition suitable for nasal administration, the
percent of surfactant in a suitable solution of Prog or ent-Prog
may be 2%. In one embodiment of the present invention, a
composition suitable for nasal administration, the percent of
surfactant in a suitable solution of Prog or ent-Prog may be
5%.
[0069] In one embodiment of the present invention, a composition
containing Prog or ent-Prog that is suitable for nasal
administration may include one or more buffering agents for
controlling the pH of the composition. Buffering agents that may be
used in the compositions of the present invention include citric
acid and sodium citrate dihydrate. In one embodiment of the present
invention, a composition suitable for nasal administration, the
percent of buffering agent in a suitable solution of Prog or
ent-Prog may be 0.001%. In one embodiment of the present invention,
a composition suitable for nasal administration, the percent of
buffering agent in a suitable solution of Prog or ent-Prog may be
0.005%.
[0070] The osmolarity of a composition of the present invention may
be controlled by propylene glycol.
[0071] When a composition of the present invention is a gel, the
composition may include a gelling agent such as hydroxylpropyl
cellulose, carbopols, carboxymethylcellulose. And
ethylcellulose
[0072] A composition of the present invention may include a
preservative such as ethylenediaminetetraacetic acid (EDTA) and
benzalkonium chloride.
[0073] Suitable solvents for compositions of the present invention
include water, vegetable oil and ethanol.
[0074] Past and preliminary data show that the novel neurosteroid,
ent-Prog has the potential to be a viable prophylactic treatment
for mTBI. The use of a nasal inhalant should reduce the
concentration required to mitigate poor outcomes associated with
mTBI and prevent unwanted side effects. Furthermore, nasal
administration is a more practical means of delivery in a military
setting.
[0075] In one embodiment, the present invention provides a method
for improving the standard of care for military personnel with mild
traumatic brain injury (mTBI) in the areas of prevention and
treatment via a prophylactic and post-acute intranasal therapeutic.
In one embodiment of the present invention, the active ingredients
the therapeutic ent-Prog. Prog has been effective in the treatment
of patients with moderate to severe brain injury. Ent-Prog may be a
viable clinical alternative to Prog in treating mTBI with
potentially fewer side effects. Since ent-Prog reduces adverse
outcomes to injury as well as Prog does in an animal model of
moderate to severe TBI, ent-Prog may be as effective as Prog in a
mild injury model as well. There are a plethora of benefits to
using ent-Prog in the treatment of mTBI--namely, the drug may be
used prophylactically and regularly without having adverse side
effects, compared to natural Prog. In one embodiment of the present
invention, ent-Prog may be part of a formulation that is delivered
intranasally to facilitate ease of access and use in the field and
to minimize the dose required further limiting side effects. Using
ent-Prog as a therapeutic may reduce poor outcomes following
injury, especially neuropsychological and neurodegenerative
disorders including Chronic Traumatic Encephalopathy (CTE) and
Post-Traumatic Stress Disorder (PTSD) linked to repetitive brain
injuries, an increasing concern for today's military personnel.
[0076] Despite the relatedness of Prog and ent-Prog, they are not
identical compounds. For example, in contrast to Prog which may
have various side effects when administered, ent-Prog may provide a
safe treatment for the prevention of mTBI in high-risk populations
with very few side effects.
[0077] Expected benefits of Prog treatment include: reduced
cerebral inflammation, improved behavioral functioning, and reduced
diffuse axonal injury.
[0078] In one embodiment, the present invention provides a method
to quickly treat concussed patients, especially athletes who endure
sports-related and military-related injuries and risk experiencing
repeated injury with compounded effects. According to some
researchers, the majority of people do not recognize that a
concussion is a brain injury, the consequences of which are also
often misunderstood (74). The rapid administration of Prog
following a mild head trauma may provide emergency medicine
personnel a protocol to follow in the field; and, by increasing the
percentage of mild TBIs treated, greater public awareness will
follow. In FIG. 9 data points represent the latency to platform in
seconds. Day 0 is the day of the hit. Day 0 through 4 are all post
impacts. Start section (quadrant) varied day-to-day and
trial-to-trial. In FIG. 9, "*" denotes significance at
p<0.05.
[0079] In one embodiment, the present invention provides
compositions that are nasal drops, eye drops and nasal sprays. For
the nasal application, a solution or suspension may be used which
is applied as spray, i.e., in the form of a fine dispersion in air
or by means of a conventional pump.
[0080] In one embodiment, the present invention provides
compositions comprising Prog or ent-Prog in the form of eye drops,
nasal drops, or nasal spray. The nasal spray can, for example, be
formed by the use of a conventional spray-squeeze bottle or a
pump.
[0081] Suitable nontoxic pharmaceutically acceptable carriers for
use in a drug delivery system for intranasal administration of Prog
or ent-Prog may include carriers used for nasal pharmaceutical
formulations for other steroids, such as estrogen. The choice of
suitable carriers will depend on the exact nature of the particular
nasal dosage form desired, e.g., whether Prog or ent-Prog is to be
formulated into a nasal solution (for use as drops or as a
spray).
[0082] Formulations of the present invention may contain a
preservative and/or stabilizer. These include, for example:
ethylene diamine tetraacetic acid (EDTA) and its alkali salts (for
example dialkali salts such as disodium salt, calcium salt,
calcium-sodium salt), lower alkyl p-hydroxybenzoates, chlorhexidine
(for example in the form of the acetate or gluconate) and phenyl
mercury borate. Other suitable preservatives are: pharmaceutically
useful quaternary ammonium compounds, for example cetylpyridinium
chloride, tetradecyltrimethyl ammonium bromide, generally known as
"cetrimide",
N-Benzyl-N,N-dimethyl-2-{2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}-
ethanaminium chloride, generally known as "benzethonium chloride"
and myristyl picolinium chloride. Each of these compounds may be
used in a concentration of 0.002 to 0.05%, for example 0.02%
(weight/volume in liquid formulations, otherwise weight/weight).
Preferred preservatives among the quaternary ammonium compounds
are, however, alkylbenzyl dimethyl ammonium chloride and mixtures
thereof, for example, the compounds generally known as
"benzalkonium chloride".
[0083] According to one embodiment of the present invention there
is provided a treatment strategy for athletes who have suffered a
TBI that may not only reduce the time required for safe return to
play but also provide protection from future mild TBIs.
[0084] Administering ent-Prog is a safe prophylactic treatment to
administer before potential mTBIs occur. Intranasal (IN)
administrations may have fewer side effects than intraperitoneal
(IP) administrations due to a shift in pharmaceutical research to
nasal sprays, drops and gels: the nasal route of drug
administration continues to receive increasing attention from
pharmaceutical scientists and clinicians because this route
circumvents hepatic first pass elimination associated with oral
delivery, is easily accessible and suitable for self-medication
(70). Intranasal administration also particularly suits drugs
targeting the brain because certain drug solutions can bypass the
blood-brain barrier (BBB) and reach the central nervous system
(CNS) directly from the nasal cavity--uptake of these drugs depends
on their molecular weight and lipophilicity (71, 75). The
intranasal delivery increases brain levels of the drug while
decreasing systemic concentrations and thus should have less
harmful side effects.
[0085] According to some researchers, the majority of people do not
recognize that a concussion is a brain injury, the consequences of
which are also often misunderstood (74). If ent-Prog is established
as a safe treatment, the repetitive administration of this
neurosteroid will prevent short-term and long-term poor outcomes
and decrease costs to health care (76).
[0086] In one embodiment, the present invention provides a method
of prophylatically administering ent-Prog to individuals who are
involved in activities, such as contact sports or serving in the
armed forces, where there is a possibility of the individuals
suffering mTBI. In one embodiment, the present invention provides a
method for acutely treating individuals who have suffered mTBI. For
populations such athletes and military personnel, who have a high
risk of suffering mTBI, ent-Prog may be made readily available for
treatment when needed. Various delivery systems for ent-Prog
including nasal delivery systems, intravenous (IV) delivery, etc.
may be used to provide effective treatments strategies for
individuals. For acute treatment strategies, nasal administration
of ent-Prog may reduce the time for uptake and increase the
concentration ent-Prog that reaches the brain. An advantage of
ent-Prog over the use of Prog is that ent-Prog has fewer systemic
side effects.
[0087] Having described the many embodiments of the present
invention in detail, it will be apparent that modifications and
variations are possible without departing from the scope of the
invention defined in the appended claims. Furthermore, it should be
appreciated that all examples in the present disclosure, while
illustrating many embodiments of the invention, are provided as
non-limiting examples and are, therefore, not to be taken as
limiting the various aspects so illustrated.
EXAMPLES
Example 1
Specific Aims
[0088] Three specific aims are tested by Magnetic Resonance Imaging
(MRI), behavioral testing and molecular analyses: (1) In order to
evaluate a decrease in diffuse axonal injury, MR images are taken
of the injured brain for Prog treated animals and compared to
vehicle control groups at 24 hours and five days after injury. It
is hypothesized that the lesion size in the right frontal cortex
and diffuse axonal injury of processes connecting the right frontal
cortex to the temporal cortex is reduced in treatment compared to
the vehicle control group. (2) The animals also undergo behavioral
tests for the first five days following injury including: water
maze (learning, memory and anxiety-like behaviors and a balance
beam (motor coordination). It is hypothesized that animals in the
Prog treated group will achieve better post-injury functioning than
the vehicle control group animals. (3) At 24 hours and following
completion of the behavioral testing (five days), the animals are
euthanized and their cerebral tissues analyzed for protein
compositions. In a previous study, Prog down-regulated three
specific genes that promote the inflammatory cascade following TBI:
Bcl-2, IL-1.beta. and Cxcl-10 (50, 77). Animal necropsies will thus
include an evaluation of the proliferation of proteins coded for by
the above genes, as well as the protein phospho-.tau., which is
traditionally up-regulated following TBI (20). It is expected that
the damaged neuronal tissue of animals in the Prog treatment group
to contain lower concentrations of these inflammatory signaling
factors.
[0089] The results of the testing in this example may show that
Prog treatment post-injury is effective at treating mild TBI if
administered acutely.
Research Design and Methods
[0090] Animals and Progesterone Treatment: Sixty male Sprague
Dawley rats are equally divided into three groups (n=20): (1) A
treatment group that receives a mild TBI and Prog; (2) a vehicle
group that undergoes mild TBI; and (3) a sham group that serves as
a surgical control. Animals in the treatment group will receive
intraperitoneal administration of Prog (16 mg/kg) 15 minutes
postsurgery.
[0091] The vehicle treated group receive 2.5% cyclodextrin at the
same 15 minute time point. Shams will not receive an injection.
Thirty animals (n=10/group) undergo five days of behavioral testing
and molecular analysis of inflammatory factors at five days
post-injury. Eighteen animals (n=6/group) are used for 24 hour
post-injury analysis of inflammatory factors. Twelve animals
(n=4/group) are evaluated using MRI at both 24 hours and five days
post-injury.
[0092] Mild Traumatic Brain Injury Model for Concussion: Prior to
surgery, animals are anesthetized with an initial isoflurane
(2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) induction for
four minutes. The animal's scalp is shaved and cleaned with
isopropanol and betadine. During the surgery, anesthesia is
maintained with isoflurane. A medial incision is performed and the
scalp is pulled back over the medial frontal plate. A 6 mm
diameter, 1 mm thick rubber helmet is secured on the skull. The
animal will then be secured bilaterally in the prone position on a
metal platform at the sixth cervical vertebrae. The head of the
animal is placed over a foam pad. An electrically controlled injury
device with a 5 mm metal impactor is positioned over the rubber
helmet. An impact speed of 6.2 m/s at a 11 degree angle from
vertical is used to produce a closed head injury, similar to what
occurs with human concussion where rotation of the head is required
to induce diffuse axonal injury. Three closed head impacts is
performed at the following times: zero, 30 minutes and 60 minutes.
Animals is returned to a heated cage before being re-anesthetized
for successive impacts. Animals in the sham group will receive a
scalp incision with no impact and remain under anesthesia for the
same duration as concussed rats.
Behavioral Testing
[0093] Morris Water Maze: A circular tank with a diameter of 133 cm
is filled with opaque water to a depth of 64 cm. A platform (11
cm.times.11 cm) is submerged to a depth of 2 cm and placed
approximately 28 cm from the wall of the pool in the center of the
northeast quadrant. Each animal is pre-trained and then tested for
five days. At the start of each trial, the rat is placed in the
pool and allowed to swim until it reaches the platform. The
performance of each rat is measured in terms of latency to
platform, length of path to platform, and whether it swam mostly on
the outside rim or near the center of the tank. The water maze test
evaluates spatial learning, memory and anxiety.
[0094] Balance Beam: There are two balance platforms in this test:
a curved platform and a linear platform; both have graded widths
between 4 cm and 1 cm. The animals is tested for five consecutive
days beginning on the first day post-surgery. The time it takes the
rodents to transverse the beam and their number of slips is
recorded. The balance beam evaluates motor control and balance.
[0095] Magnetic Resonance Imaging: A 21 Tesla magnet is used to
examine the morphological effect of injury and treatment in the
animal. Anesthesia induction is performed at 1.0 to 2.5% v/v using
isoflurane gas in a closed induction chamber. Within four to five
minutes the animal is removed from the induction chamber and placed
inside the MRI scanner while maintaining a 1.0 to 1.5% v/v gas flow
to the animal's nose. The typical MR imaging time is approximately
one hour. Diffusion tensor imaging MR software is used to evaluate
diffuse axonal injury by measure of axonal anisotropy (represents
plasma membrane damage).
[0096] Diffusion weighted MR software is used to assess cerebral
edema and lesion size. MR images is taken at 24 hours and five days
following the injury. Effects of Prog treatment is ascertained when
compared to vehicle control images.
[0097] Tissue Collection and Analysis: At 3 and 24 hours following
injury, the animals is euthanized with CO.sub.2 and decapitated for
the collection of brain samples from the penumbra of the injury
site. Analysis of inflammatory protein compositions is performed
using Western Blotting and ELISA.
[0098] Statistical Analyses: The use of one-way ANOVA, t-tests, and
Tukey-Kramer post hoc tests is used to determine significance set
at p<0.05 when comparing all experimental groups.
[0099] The studies described above provide a foundation for the use
of Prog acutely in humans who have sustained a mild TBI. Given that
high risk populations have been identified (athletes and military
personnel) Prog could be made readily available for treatment when
needed. Additionally, nasal delivery method stand to further
improve Prog as a treatment strategy for these high risk
populations, given that this route reduces time for uptake and
specifically increases brain concentrations with less systemic side
effects.
Example 2
Summary
[0100] Prog and ent-Prog are administered both intranasally (IN)
and intraperitoneally (IP) to both male and female Sprague Dawley
Rats. These neurosteroids are used as a daily treatment for two,
four and eight weeks. The outcomes of neurosteroid-treated rats
with vehicle (vehicle-treated) and sham (untreated) rats will be
compared. The sexual side effects in male and female rodents, as
well as the coagulative effects in male rodents will also be
investigated.
Specific Aim 1
[0101] Evaluating Male Sexual Function after Repeated Neurosteroid
Treatments
[0102] In order to evaluate male sexual function following
treatment, rodents are evaluated and compared to vehicle control
groups at the end of 2 and 4 weeks. Male rodents are divided into
20 groups (n=3): daily Prog treated IP and IN at both 16 mg/kg and
32 mg/kg for 2 and 4 weeks; daily ent-Prog-treated IP and IN at 16
mg/kg and 32 mg/kg for 2 and 4 weeks; daily vehicle-treated for 2
and 4 weeks; and sham (untreated) for 2 and 4 weeks. Serum
dihydrotestosterone (DHT) to testosterone ratios are measured,
epididymal sperm concentrations, and relative reproductive organ
weight of the testis, epididymis and prostate. It is crucial to
determine that unlike Prog, ent-Prog does not lower DHT
concentrations: DHT has been shown to be a major predictor of
sexual activity in young men (78); it modulates prostatic function
(79); and low DHT levels correlate with low semen volume and
decreased motility (80). It is hypothesized that sexual functioning
will be significantly better in ent-Prog-treated rodents than
Prog-treated rodents and that the differences in side effects of
these neurosteroids will be exacerbated with both an increase in
concentration as well as length of treatment.
Specific Aim 2
Evaluating Blood Coagulation Post-Treatment
[0103] In order to evaluate coagulative effects of the
neurosteroids, serum samples are taken at the end of each week, 2,
4 and 8. Rodents are divided into the same 20 groups (n=3) that are
listed above in aim one. Blood are evaluated based on clotting
time. The risk of Prog and Prog-related compounds causing
coagulation has been explored mainly in studies involving over the
counter birth control pills: some studies suggest that the risk of
hypercoagulation can be attributed mainly to estrogen, while others
suggest that Prog does increase the risk for those with a genetic
predisposition for the disorder (81, 82). Assuming that Prog will
have some hypercoagulative effects, it is hypothesized that the
time to clot for ent-Prog groups will be greater than it will be
for Prog groups. Furthermore, the differences in time to clot will
increase with both neurosteroid concentration and length of
treatment. It is also expected that IN groups to have fewer side
effects than IP groups.
Collection/Analysis of DHT and Testosterone.
[0104] Plasma DHT and testosterone levels are measured by the ELISA
method using DRG ELISA kits according to the standard protocol
supplied by the kit manufacturer.
Collection and Measurement of Epididymal Sperm Concentration
[0105] The epididymal sperm concentration are determined according
to the modified Turk method (83) cited in R. Bal and colleagues
(84): The right epididymis is finely minced and allowed to incubate
at room temperature for 4 hrs in isotonic saline. The solution is
filtered and drawn into a capillary tube in which the dilution rate
is measured. Then the solution is transferred to a hemocytometer
counting chamber in which the sperm cells are counted with the help
of a light microscope.
Collection and Weighing of Reproductive Organs
[0106] At end of treatment, males are euthanized. Testis,
epididymis and ventral prostate are removed, cleared of adhering
connective tissue and weighed. Relative weight of sexual organs is
calculated by scaling to total body weight.
[0107] Serum for clotting times: Blood is collected following
treatment cessation. Plasma is isolated and assayed for extrinsic
and intrinsic clotting times. A microplate-based blood coagulation
assay described by Pratt and Monroe (85) is performed by adding 30
.mu.L plasma and 304 buffer (20 mM HEPES, 150 mM NaCl, 0.1%
polyethylene glycol, pH 7.4) to wells of microplate which are then
incubated at 25.degree. C. for 2 min. Clotting is initiated by
adding 30 .mu.Ll of 5% thromboplastin in 25 mM CaCl.sub.2 for the
Prothrombin Time Test (extrinsic clotting cascade) and 30 .mu.L
activated partial thromboplastin reagent for the Activated Partial
Thromboplastin Time Test (intrinsic clotting cascade). The increase
in the turbidity of plasma is measured by the change in absorbance
at 405 nm using kinetic microplate reader.
Male and Female Mating
[0108] At the onset of this study, female rodents are 63 days of
age. For the first two weeks, vaginal smears are taken to verify
rodent fertility. On the first day of week three, one untreated
male rodent is paired with each female rodent. Every morning during
the mating period, females are examined for the presence of sperm
plugs or sperm in vaginal smears; if sperm is present, the females
are tested for pregnancy. Females who are not pregnant are returned
to the males. This procedure will continue for six more weeks (86).
Females are monitored daily for signs of miscarriage. Litters born
are examined for signs of viability.
Euthanization
[0109] At the end of treatment, rats are euthanized with
CO.sub.2.
Statistical Analyses
[0110] The use of one way ANOVA, t-tests, and Tukey-Kramer post hoc
tests are used to determine significance set at p<0.05 when
comparing all experimental groups within each design.
Example 3
Specific Aim 1
[0111] Determine the Intraperitoneal Dose and Time at which
Prophylactic and Post-Acute Treatments with Progesterone and its
Enantiomer Improve Responses to Mild Traumatic Brain Injury.
[0112] The efficacy of ent-Prog treatment for mTBI is determined
and compared with natural Prog treatment. Based on past research
with these two neurosteroids in a model of moderate to severe TBI
it is hypothesized that an IP dose of 16 mg/kg at 1 hour after
injury will provide optimal protection following mTBI. With regards
to prophylactic treatment this same dosage will be most beneficial
when given between 1 hour and 15 min prior to induction of mTBI
based on previous research showing this timeframe to offer the
optimal bioavailability of Prog. Furthermore, it is hypothesized
that Prog and ent-Prog will equally improve responses to mTBI with
greater efficacy seen with prophylactic administration. mTBI is
induced in rats using a novel animal model applying closed head
trauma to the right frontal plate of the skull with an electrically
controlled impactor. Rats are injected intraperitoneally (IP, the
standard method used in previous research) with three different
concentrations of Prog or ent-Prog in order to determine the
optimal dosage for prophylactic and post-acute administration. A
window of time--for prophylactic treatment--is established in which
the drugs are effective. Behavioral testing, imaging (edema), serum
and brain drug concentrations, serum biomarker concentrations and
pathological brain protein (markers for cell death, demyelination
and inflammation) abundance are analyzed. The findings from this
specific aim will establish criteria for efficacy as it relates to
dose and time of drug administration before and after injury,
respectively. Preliminary research has demonstrated that IP Prog
improves behavioral outcomes when administered prior to and
following mTBI. The work performed in this aim makes it possible to
determine if ent-Prog is as protective as Prog for the treatment of
mTBI given it stands to have limited to any side effects in
comparison, especially when used on a repetitive basis in humans as
a prophylactic. Drug synthesis, formulation and pharmacokinetic
studies are also outlined in this specific aim.
Specific Aim 2
[0113] Determine the Intranasal Dose in which Prophylactic and
Post-Acute Treatments with Progesterone and its Enantiomer improve
responses to Mild Traumatic Brain Injury.
[0114] The efficacy of the intranasal (IN) administrations of Prog
and ent-Prog is compared to that of IP analyzed in Specific Aim 1
of this example. Previous research has shown that N drug
administration achieves better central nervous system delivery,
crossing the blood brain barrier more efficiently than with IP
administration. It is hypothesized that a significant reduction in
IN dosage will have an equivalent positive response compared to the
optimal IP dosage established in Specific Aim 1 of this example.
Furthermore, it is hypothesized that a 4-fold reduction in dosage
administered IN will show statistically higher efficacy than the
optimal IP dosage established in Specific Aim 1 of this example. As
in Specific Aim 1 it is hypothesized that Prog and ent-Prog will
induce equivalent positive responses and prophylactic
administration will be more advantageous than post-acute treatment.
Rats are administered Prog and ent-Prog before and after the
induction of mTBI as in Specific Aim 1. Prog and ent-Prog are
administered within the prophylactic and post-acute windows of time
for efficacy, outlined in Specific Aim 1. Both neurosteroids are
administered IN at the most effective IP concentration identified
in Specific Aim 1 of this example. Based on bioavailability studies
outlined in Specific Aim 1 of this example, both drugs will also be
administered at two lower concentrations than the most effective IP
concentration. Testing methods identical to those described in
Specific Aim 1 of this example are to establish efficacy following
IN administration in this aim. The ultimate goal is to determine
the optimal dose for IN drug administration that maximizes
absorption from the cerebral circulation and minimizes delivery to
the systemic circulation, while concomitantly reducing brain edema,
cell death and demyelination. This aim is designed to determine if
IN administration more effectively delivers Prog and ent-Prog to
the brain than IP administration and which delivery method is more
protective of the brain when given prior to versus following the
insult. Furthermore as in Specific Aim 1 the experiments of
Specific Aim 2 will aid in determining if ent-Prog and Prog are
equally effective in the treatment of mTBI.
Specific Aim 3
[0115] Determine the Extent to which Prophylactic and Post-Acute
Treatments with Progesterone and its Enantiomer Reduce Pathology
Associated with Repetitive Mild Traumatic Brain Injury
[0116] The efficacy of Prog and ent-Prog in an animal model of
repeated mTBI is determined. It is hypothesized that the
established optimal IN dose in Specific Aim 2 will equally maintain
prophylactic and post-acute protection seen after a single injury
when compared to a 4-injury repetitive mTBI model. Rats receive one
impact weekly for four consecutive weeks. Prog and ent-Prog are
delivered IN at the optimal time and dose determined in Specific
Aim 2. Treatments are administered prophylactically and
post-acutely as discussed in Specific Aims 1 and 2. Rats will
receive varying numbers of treatments for the injury groups: for
only the first injury, the first two injuries, the first three
injuries, or all four injuries. Magnetic resonance imaging will
evaluate the efficacy of treatment in terms of morphological
effects (area of damage and edema), and behavioral testing will
evaluate the efficacy of treatment in terms of motor and cognitive
functioning following each injury. Temporal lobe brain slices are
evaluated for tauopathy and A.beta.-amyloid at 72 hours and 14 days
following the final injury time point. This specific aim is
designed to confirm if Prog and ent-Prog are valid treatment
options and if so which one is more robust in a model of repetitive
mTBI. Drug safety studies with respect to coagulation are also
outlined in this specific aim.
Example 4
Mild Traumatic Brain Injury Model
[0117] Prior to surgery, animals are anesthetized with an initial
isoflurane induction for 4 minutes--the minimum quantity of time
necessary in order to mildly sedate the rodents during surgical
preparation. The animal's scalp is shaved and cleaned with
isopropanol and betadine. During the surgery, anesthesia is
maintained with isoflurane. A medial incision is performed and the
scalp is pulled back over the medial frontal cortex. A 6 mm
diameter, 1 mm thick rubber helmet is placed on the skull and
stabilized with bone wax. The animal is then secured in the prone
position on the heated metal platform of the stereotaxic apparatus
at the cervical 6.sup.th and 7.sup.th vertebrae. The head of the
animal is placed over a foam pad. An electrically controlled injury
device using a 5 mm metal impactor is positioned over the rubber
helmet. An impact speed of 6.2 m/s at a 11.degree. angle from
vertical is used to produce a closed head injury, similar to what
occurs with human concussion. The animal scalp is then sutured and
analgesic applied. The animal is then returned to a heated cage in
order to recover for 30 mins at which time food and water are
provided. Animals in the sham group receive a scalp incision with
no impact and remain under anesthesia for the same duration as
concussed rats.
Specific Aim
[0118] Determine the Intraperitoneal Dose and Time at which
Prophylactic and Post-Acute Treatments with Progesterone and its
Enantiomer Improve Responses to Mild Traumatic Brain Injury
[0119] It is hypothesized that intraperitoneal administration of
the neurosteroids, Prog and ent-Prog are most beneficial when
administered prophylactically in the treatment of a single mTBI.
Past research using a model of moderate to severe TBI has shown
that 16 mg/kg IP of both Prog and ent-Prog is the most beneficial
concentration for reducing edema, inflammation, oxidative stress
and cell death within the penumbral region at 72 hours following
injury (1, 2). mTBI causes less mechanical damage and therefore a
lower pathological response than moderate to severe brain injury.
For these reasons administer 16 mg/kg are administered as the
highest concentration and determine if lower IP concentrations (4
and 8 mg/kg) can achieve the same beneficial effects in a mild
model of TBI. A dose response curve is developed based on drug-dose
efficacy when analyzing cognitive behavior (spatial learning and
memory and anxiety), motoric behavior (balance) and serum and brain
biomarkers of injury.
[0120] Drug stability is tested over a six-month period when stored
at both 4.degree. C. and 25.degree. C. After establishing the
optimal drug formulation a pharmacokinetic profile is created for
the bioavailability of each drug in the brain and serum for each
route of administration.
Example 5
Drug Formulation and Stability
Design
[0121] Three formulations are developed; 2.25% .beta.-cyclodextrin
(.beta.-CD) in dH.sub.2O plus drug (Prog or ent-Prog), Carbopol (1%
in H.sub.2O) plus drugs, 2.25% (.beta.-CD in dH.sub.2O plus
Carbopol plus drugs. Prog-.beta.-CD complex is commercially
available (Sigma-Aldrich, St. Louis, Mo.), and carbopol is a known
nasal absorption enhancer (87-89) thus potentially increasing
bioavailability of Prog and ent-Prog. Carbopol will be added to
water in which the required amount of drug-CD inclusion complex is
dispersed by sonication. These polymeric dispersions are stirred in
a magnetic stirrer for 60 min and the pH will be adjusted to 7.0 by
adding triethanolamine. To determine the most soluble formulation
small aliquots are taken and quantify changes in dynamic light
scattering which represent precipitation/instability of the
solution are quantified. Drug stability of defined formulations
will also be monitored over a 1 year period. Briefly, stock
solutions of each formulation are stored at 4 and 25.degree. C. A
small aliquot (1 mL) are tested every 14 days for changes in
dynamic light scattering indicating precipitation/instability of
the solution.
Example 5
Pharmacokinetic Studies of Bioavailability of Prog and Ent-Prog
Using ELISA Subjects
[0122] To determine the pharmacokinetic parameters of drug delivery
for both Prog and ent-Prog to the brain 27 Sprague-Dawley rats at
280 g each are used (see Table 1). No brain injuries are performed
on animals in this example.
TABLE-US-00001 TABLE 1 Optimal Formulation Group IP IN IV Vehicle 3
3 3 Prog (16 mg/kg) 3 3 3 ent-Prog (16 mg/kg) 3 3 3
Design
[0123] The bioavailability of Prog and ent-Prog is determined by
measuring the serum Prog and ent-Prog abundance using a solid-phase
enzyme immunoassay (ELISA). The optimal formulation determined in
Example 5 above is administered using 16 mg/kg for IP and IN
delivery. One group of rats will receive an intravenous (saphenous
vein) injection of parenteral solution (16 mg/kg) to calculate the
absolute bioavailability. Serial tail vein blood draws are
performed at 30, 60, 120, 240, and 300 minutes after
administration. Plasma is separated by centrifugation at 2,000 rpm
and drug content determined by using an ELISA kit (Diagnostic
Automation, Inc., Calabasas, Calif.) according to the
manufacturer's protocol. Briefly, the assay is based on the
principle of competitive binding between the drug in the test
sample and drug-HRP conjugate. The pharmacokinetic parameters (area
under the concentration-time curve from 0 to 300 min (AUC), peak
concentration (C.sub.max) and time to reach peak concentration
(t.sub.max) following vehicle, Prog and ent-Prog administration is
calculated. The absolute bioavailability following IP or IN
administration is determined by dividing the IP or IN AUC by the
IV. AUC. Refer to Table 1 above for a breakdown of group design.
(Total=27 animals).
Subject
[0124] To determine the optimal effective dose of Prog and ent-Prog
when delivered IP in a model of mTBI 176 Sprague-Dawley rats at 280
grams are used.
Design
[0125] Groups (2 sets) will include sham, vehicle (determined by
formulation studies outlined above), Prog and ent-Prog. Groups from
set 1 will receive a single IP injection at 1 hours prior to mTBI
and set 2 will receive a single IP injection at 1 hour post-mTBI.
An IP injection at 1 hour post has been shown to be beneficial in
moderate to severe TBI when using both Prog and ent-Prog.
Preliminary data shows that Prog is beneficial when administered 1
hour prior to injury. However, an alternative strategy may be
needed here with the addition of animals at a more immediate
prophylactic time point to injury. Bioavailability studies assist
in making changes to the time points of drug administration. Each
set is composed of animals that receive one of three different IP
concentrations of Prog or ent-Prog; 16 mg/kg, 8 mg/kg or 4
mg/kg.
Molecular Studies
Serum Analysis
[0126] Serum is collected at 3, 6, 24 and 48 hours post-injury
using serial draws from the animal tail vein. Protein is processed
and ELISA analysis are performed with the following antibodies:
SBDP-150, and GFAP. Both of these serum biomarkers represent loss
of integrity of the blood brain-barrier (BBB). SBDP-150 and GFAP
also represent loss in neuronal and astrocyte structural integrity,
respectively (90, 91). All serum samples are sent for preparation
and analysis to Banyan Biomarkers, Inc. (Gainesville, Fla., see
Letters of Collaboration). Serum analysis of drug concentration are
performed on each animal from all groups at 1, 6, 24 and 48 hours
following injury using a Prog and ent-Prog ELISA kit (Cayman
Chemical, Ann Arbor, Mich.). No blood is taken prior to injury or
before 1 hour post-injury as mTBI causes mild internal
bleeding.
Brain Tissue Analysis
[0127] Brain tissue from the penumbral region of the impact and the
hippocampus is collected by punch biopsy. Samples are processed for
protein and Western analysis using antibodies for the cell death
marker; caspase 3, demyelination marker; Myelin Basic Protein, and
inflammatory markers; TNF-.alpha. and IL-1.beta.. All markers of
injury are analyzed at 6 and 48 hours post-injury. Briefly, all
samples are collected, homogenized and incubated for 1 hour in TPer
(Tissue protein extraction reagent) with 5% protease cocktail
inhibitor. Protein concentration is determined
spectrophotometrically using NanoDrop at 280 nm. Equal protein
concentration from each sample will separated using SDS PAGE and
transferred onto a PVDF membrane. Appropriate primary and secondary
antibodies are applied to each sample and a Molecular Imager used
for detection and data analysis. Penumbral tissue analysis is
justified to determine the secondary cascade of neuronal death and
inflammation. Retrograde analysis of the hippocampus is required to
determine the degree to which diffuse axonal injury has led to
neuronal death and inflammation when the injury impact is to the
frontal lobe (42, 92). Preliminary data shows that mTBI causes
memory deficits.
[0128] This finding further justifies the analysis of the
hippocampus in these studies.
Imaging Studies
[0129] Sodium Diffusion MRI is performed on animals from each
treatment group at 6, 24 and 48 hours following injury to determine
the level of brain edema. Diffusion Tensor MRI (See Facilities and
Equipment) is performed on animals from each group to quantify
diffuse axonal injury at the same time points. Table 2 below show a
breakdown of the animals used in the Molecular Studies (Total=96
animals).
TABLE-US-00002 TABLE 2 Animal Use: Single mTBI, Molecular Studies
Group Prophylactic at 1 hr Post-Injury at 1 hr ent-Prog n = 4 for
each time point n = 4 for each time point of sacrifice (2 times, 6
(2 times, 6 and 48 hr) and 48 hr) and each and each dose (2) dose
(2). Total: 16 Total: 16 Prog n = 4 for each time point n = 4 for
each time point (2 times, 6 and 48 hr) (2 times, 6 and 48 hr) and
each dose (2). and each dose (2). Total: 16 Total: 16 Vehicle n = 4
for each time point n = 4 for each time point (2 times, 6 and 48
hr) (2 times, 6 and 48 hr) Total: 8 Total: 8 Sham n = 4 for each
time point n = 4 for each time point (2 times, 6 and 48 hr) (2
times, 6 and 48 hr) Total: 8 Total: 8
[0130] The same animals that survive for 48 hours (n=4/group) for
brain tissue analysis are used for serum biomarker studies, serum
drug concentration studies and MRI studies.
Cognitive and Motor Behavioral Studies
[0131] Improvements in functional outcomes associated with Prog and
ent-Prog treatment are determined by analyzing spatial learning and
memory (Morris Water Maze Testing), anxiety-like behaviors
(Elevated Plus Maze) and balance (Beam and Rotorod,). See
Facilities and Equipment page for outline of behavioral tests.
Briefly, 2 pre-trial tests are performed each day starting 48 hours
prior to the induction of mTBI. Following mTBI the testing using
each method is initiated at 4 hours and performed every 24 hours
for 5 days. Table 3 below provide a breakdown of the animals used
in the Behavioral Studies (Total=80 animals).
TABLE-US-00003 TABLE 3 Animal Use: Single mTBI, Behavioral Studies
Group Prophylactic at 1 hr Post-Injury at 1 hr ent-Prog n = 5 for
each dose (3). n = 5 for each dose (3) Total: 15 Total: 15 Prog n =
5 for each dose (3). n = 5 for each dose (3). Total: 15 Total: 15
Vehicle n = 5 n = 5 Sham n = 5 n = 5
Example 8
Specific Aim
[0132] Determine the intranasal dose in which prophylactic and
post-acute treatments with Progesterone and its Enantiomer improve
responses to Mild Traumatic Brain Injury.
Hypothesis
[0133] Prophylactic intranasal administration of the neurosteroids,
Prog and ent-Prog are more beneficial at lower concentrations than
the optimal intraperitoneal dose when treating a single mTBI.
Rationale
[0134] The use of a nasal inhalant for the treatment of TBI is
novel. Prog has been used as a nasal inhalant to investigate
activity of dopaminergic neurons in rats (92) and there has been an
ongoing interest in the nasal delivery of Prog for use in hormone
replacement therapy and contraceptives (93). Nasal administration
of pharmaceuticals has been shown to increase the amount of the
compound available to the brain and reduce the levels in the
systemic circulation while achieving higher concentrations of the
drug in the body in less time. Furthermore, nasal administration
achieves a desired effect at a lower concentration and subsequently
reduces potential harmful side effects systemically associated with
the inhalant compound (72, 73). For these beneficial reasons, in
recent years a significant amount of common use drugs (i.e.
Influenza vaccine and Insulin) have been in development for nasal
administration. In this aim both Prog and ent-Prog are concentrated
into a nasal delivery system that improves bioavailability in the
brain and reduces levels in the systemic circulation. Nasal
inhalants are a much more practical means of a field deliverable
for military personnel and other high risk occupations or
activities (i.e. sporting events).
Experimental Design
[0135] Drug Dose Response with Intranasal Administration
Subjects
[0136] To determine the optimal dose of Prog and ent-Prog when
delivered IN in a model of mTBI 176 Sprague-Dawley rats at 280
grams are used.
Design
[0137] The dose response for IN administration is established using
the same optimal drug formulation, animal groups and treatment time
points outlined in Specific Aim 1. The difference in the drug
concentrations administered IN compared to IP given that past data
has shown that administration of a lower concentration of a given
lipophilic drug given IN is a more efficient means of getting the
drug into the brain than higher concentrations delivered IP,
significantly lower IN concentrations are given IN in this study.
The most efficacious IP dose for each drug established in Specific
Aim 1 is used as the starting point for IN administration. From
this starting concentration both Prog and ent-Prog are diluted to
two lower concentrations based on the results of bioavailability
studies outlined in Specific Aim 1. Statistical comparisons are
made between IN and IP efficacy to determine if an IN deliverable
is advantageous for treating mTBI.
Molecular Studies
[0138] Table 1 above shows a breakdown of the animals used in the
Molecular Studies. Animal use and design is the same as in Example
7.
Serum Analysis
[0139] These studies are carried out with the same design as in
Example 7.
Brain Tissue Analysis
[0140] These studies are carried out with the same design as in
Example 7
Imaging Studies
[0141] These studies are carried out with the same design as
Example 7.
Cognitive and Motor Behavioral Studies
[0142] These studies are carried out with the same design as in
Example 7. Table 2 above shows a breakdown of the animals used in
the Cognitive and Motor Behavioral Studies. Animal use and design
is the same as in Example 7.
Example 9
Specific Aim
[0143] Determine the extent to which prophylactic and post-acute
treatments with Progesterone and its Enantiomer reduce pathology
associated with Repetitive Mild Traumatic Brain Injury.
Hypothesis
[0144] Prophylactic intranasal administration of the neurosteroids,
Prog and ent-Prog prior to each of four repetitive mTBIs will
prevent behavioral abnormalities and reduce pathological markers
associated with poor long term outcomes.
Rationale
Experimental Design
[0145] Neurosteroid Administration for Repetitive mTBI
Neurosteroid Efficacy Studies
[0146] Repetitive mTBI has been associated with initial behavioral
abnormalities including but not limited to; memory impairment,
balance deficits and depression/anxiety disorders (50). Repetitive
mTBI has been postulated to lead to early-onset dementia similar to
that seen in AD patients with increased rates of suicide (54, 57).
Like that seen in the temporal lobes of AD patients, individuals
who have had repetitive mTBI have TDP-43-positive-tauopathy (64)
and elevated levels of A.beta. amyloid (20, 62). The work in this
aim is designed to treat each mTBI either prophylactically or in
the post-acute phase to determine if Prog or ent-Prog can
ameliorate the effects of repetitive TBI on behavior and molecular
pathology.
Subjects
[0147] To determine if IN administration of Prog and ent-Prog are
preventative and or protective in a model of repetitive mTBI 208
Sprague-Dawley rats weighing 280 grams are used.
Design
[0148] Animals are placed into 4 separate groups; treatment
associated with first injury only, treatment associated with first
and second injuries, treatment associated with first, second and
third injuries, treatment associated with four injuries. As in
Specific Aims 1 and 2 some groups will receive only prophylactic
treatment (optimal IN administration time established Specific Aim
2) and others only post-acute treatment. The most efficacious IN
dosage established for both prophylactic and post-acute treatment
in Specific Aim 2 are administered to all animal groups.
Molecular Studies
Serum Analysis
[0149] These studies are carried out with the same design as
Specific Aim 1 and include analysis after each successive
injury.
Brain Tissue Analysis
[0150] Brain tissue from the penumbral region of the impact and the
hippocampus is collected and processed as previously described in
Specific Aim 1. Western analysis is performed using antibodies for
hyper-phosphorylated tau (Thermo Scientific, Rockford, Ill.),
TDP-43 (GenTex, Irvine, Calif.) and A.beta. amyloid (GeneScript,
Piscataway, N.J.). All pathological markers are analyzed at 72
hours (n=4/group) and 14 days (n=4/group) following the 4 wk injury
period.
Imaging Studies
[0151] These studies are carried out with the same design as
Specific Aim 1 and include analysis after each successive
injury.
Cognitive and Motor Behavioral Studies
[0152] The same behavioral studies outlined in the previous aims
and discussed in Facilities and Equipment will be used in this
specific aim. However, behavioral analysis will be ongoing for each
group of animals throughout the 4 week study design no matter how
many injuries the animal received including out to 72 hours
following the designated time point of the 4.sup.th and final
injury. An n=8 for each group is used for behavioral analysis.
These same animals are used for the molecular analysis outlined
above.
Side Effect Studies
[0153] Drugs are administered at multiple times. Given that the
potential daily use of prophylactic ent-Prog for high risk
occupations and that ent-Prog may be a safer deliverable than Prog
due to identifiable side effects associated with the reproductive
system and blood coagulation the work in this aim will determine if
neurosteroids have side effects when administered multiple times
and make a comparison between the Prog and ent-prog. The
experiments in this aim are designed to compare issues of safety in
animal groups that receive 1, 2, 3 or 4 drug treatments over a 4 wk
time frame. Given that the same number of drug injections are
administered as a prophylactic for post-acute treatment, only the
potential harmful side effects are evaluated when treating in a
prophylactic manner. The safety studies in this aim may be used to
establish the effects of the drugs being administered on blood
coagulation. Future work with more animals will have to be
performed for safety characterization of the reproductive system
where tissues can be collected at various time points.
Subjects
[0154] Serum coagulation assays: Blood is collected from half of
the animals in this study at 6, 24 and 48 hrs after each injury for
each treatment group and shams. Plasma is isolated and assayed for
extrinsic and intrinsic clotting times. Plasma clotting times from
all animal groups are compared to determine if neurosteroid
treatments increase coagulation after multiple treatments. Briefly,
A microplate-based blood coagulation assay described by Pratt and
Monroe (85) is performed by adding 30 .mu.l plasma and 30 .mu.l
buffer (20 mM HEPES, 150 mM NaCl, 0.1% polyethylene glycol (PEG),
pH 7.4) to wells of microplate which are then incubated at
25.degree. C. for 2 min. Clotting is initiated by adding 30 .mu.l
of 5% thromboplastin in 25 mM CaCl.sub.2 for the Prothrombin Time
Test (extrinsic clotting cascade) and 30 .mu.l activated partial
thromboplastin reagent for the Activated Partial Thromboplastin
Time Test (intrinsic clotting cascade). The increase in the
turbidity of plasma is measured by the change in absorbance at 405
nm.
[0155] Table 4 below shows a breakdown of the animals used in the
Molecular, Cognitive and Motor Behavioral and Safety Studies in
this aim. (Total=208 animals).
TABLE-US-00004 TABLE 4 Animal Use: Repetitive mTBI Single Two Three
Four Group Injury Injuries Injuries Injuries ent-Prog Prophylactic
n = 8 n = 8 n = 8 n = 8 ent-Prog Post-acute n = 8 n = 8 n = 8 n = 8
Prog Prophylactic n = 8 n = 8 n = 8 n = 8 Prog Post-acute n = 8 n =
8 n = 8 n = 8 Vehicle Prophylactic n = 8 n = 8 n = 8 n = 8 Vehicle
Post-acute n = 8 n = 8 n = 8 n = 8 Sham n = 4 n = 4 n = 4 n = 4
Statistical Techniques for Examples 1, 2, 3, 4, 5, 6, 7, 8 and
9
Alternative Techniques for Examples 1, 2, 3, 4, 5, 6, 7, 8 and
9
Drug Formulation
[0156] Many formulae are available for improving drug solubility
for intranasal administration. Based on past use of cyclodextrin
and carbopol for solubilizing Prog these two compounds should be
useful for solubilizing ent-Prog. However, PEG has the potential to
solubilize Prog and enhance brain uptake through intranasal
delivery. PEG has no reported harmful side effects. Therefore, PEG
may be used as a solvent.
Markers
[0157] The serum biomarkers and brain pathological markers were
chosen for analysis based on past experimental findings primarily
in the laboratories of the Principal and Co-Investigators as well
as collaborators (Banyan Biomarkers, Inc.). However, there are many
other serum and brain markers that may be regulated after mTBI
(i.e. p53, NF.kappa..beta., S-Nitrosocysteine, MAP-2). With regards
to brain pathological markers of Alzheimer's-like pathology in
Specific Aim 3 it appears that these are the best options based on
numerous previously reported findings in this field of
research.
Analysis Technique
[0158] Our model of repetitive mild traumatic brain injury proposes
to evaluate brain pathological markers in animals that get either
1, 2 or 3 mTBIs only at the same time points (72 hours and 14 d)
after those animals that get a 4.sup.th and final mTBI. The problem
with this design is that the time point of analysis following a
given number of injuries is significantly different. An advantage
of this design is that is that it makes it possible to determine if
longer time periods after a given number of injuries shows an
increased abundance in deleterious markers.
Example 10
Nasal Solution
[0159] An example of a nasal solution according to one embodiment
of the present invention is described below. A 1% Tween 80 may be
combined with solution of ent-Prog. That mixture is then combined
with a quantity of isotonic saline sufficient to bring the total
volume to 50 mL. The solution is sterilized by being passed through
a 0.2 micron Millipore filter.
Example 11
Nasal Gel
[0160] An example of a nasal gel according to one embodiment of the
present invention is described below. 250 mL of isotonic saline are
heated to 80.degree. C. and 1.5 g of Methocel are added, with
stirring. The resultant mixture is allowed to stand at room
temperature for 2 hours. Then, 100 mg of ent-progesterone are mixed
together with 1 mg of Tween 80. The ent-progesterone/Tween mixture
and a quantity of isotonic saline sufficient to bring the total
volume to 500 ml are added to the gel and thoroughly mixed.
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[0223] While the present invention has been disclosed with
references to certain embodiments, numerous modification,
alterations, and changes to the described embodiments are possible
without departing from the sphere and scope of the present
invention, as defined in the appended claims. Accordingly, it is
intended that the present invention not be limited to the described
embodiments, but that it has the full scope defined by the language
of the following claims, and equivalents thereof.
* * * * *