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Health Sciences 1I06
The UNSIN Report
Thursday, April 5, 2012
Group Name:
Group Members:
Team Inanna
Priyank Bhatnagar
Nathan Gamble
Alexa Mordhorst
Beatrice Preti
Tul-Zahra Rida
Alex Sapa
Amrit Sandhu
Danusha Vinoraj
Tina Wang
Section 1:
At a Glance
Sin Explored: For the purpose of this report, lust is defined as the intense sexual
desire leading to sex crimes such as sexual assault and child sexual abuse.
Molecular Targets: Serotonin (5-HT) and Dopamine (DA)
- Low levels of 5-HT in the raphe nuclei are correlated with increased sexual desire
- High levels of DA in the hypothalamus, prefrontal cortex (PFC), and ventral tegmental
area (VTA) in the brain are correlated with sexually appetitive behaviour
Proposed intervention: 5-HT precursor and DA autoreceptor agonist: Nadamo
- Nadamo is a multi-target drug which affects the dopaminergic and serotonergic
pathways of lust
- Nadamo has two components: 5-hydroxytryptophan (5-HTP) and a D2 autoreceptor
agonist, patrabandole (PTB)
- The 5-HTP component will increase 5-HT levels in the raphe nuclei via its precursive
involvement in the 5-HT synthesis pathway
- PTB will decrease DA levels in the medial preoptic area (mPOA), mesocorticolimbic
pathway, and nucleus accumbens (NAcc) via activation of the DA transporter (DAT) in
the termination pathway
- Nadamo is administered via the controlled release of 5-HTP and PTB from
microspheres injected into the deltoid muscle
Section 2:
Framing the Problem
Lust is molecularly defined as: 1)
decreased activation of 5-HT1A and 5-HT2A
receptors in the dorsal raphe nuclei
(DRN) and mPOA, due to lower levels of
5-HT, and 2) increased activation of D1-D4
and D2 receptors in the mPOA, PFC, and
the VTA (1).
For the purpose of this report, lust is the
intense sexual desire leading to sex crimes
such as child sexual abuse and sexual
assault. Lust differs from the physical
manifestations of sexual arousal because it
refers to the urge, not the physical ability,
for sexual activity. An excessive response
to erotic stimuli due to decreased sexual
satiety
and
the
activation
of
neuropathways associated with pleasure
characterize lust.
Since 96% of all sex offenders are male,
the demand for an anti-lust treatment is
highest for this demographic (2). By
targeting male sex offenders, who are
known to have a high rate of recidivism, a
decline in overall sexual offences is
expected (2, 3).
Testosterone
Heightened bioavailable testosterone (BT)
increases libido in males by binding to the
androgen-specific receptor NR3A present
in basal epithelial cells (4). Production of
BT is initiated by a cascade of events,
beginning with the testosterone-mediated
release
of
gonadotropin-releasing
hormone from the hypothalamus (5). This
negative feedback loop indicates that
inhibition of testosterone production in
the testes alone is insufficient to attenuate
sexual desire; the brain would compensate
for lowered testosterone levels by
increasing production in the adrenal
cortex (4). Successful inhibition of
physical arousal by targeting testosterone
requires intervention at all locations of
production
which,
without
coprescriptions, would lead to excessive
adverse effects (6). Co-prescription and
long-term homeostatic rebalance were two
significant reasons why testosterone was
not selected as a locus of intervention (7,
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
8). Most importantly, testosterone is
responsible for physical manifestations of
arousal,
not
appetitive
behaviour;
therefore, testosterone is not a viable
target.
Prolactin
Prolactin (PRL) is a polypeptide hormone
synthesised in lactotropic cells of the
anterior pituitary gland (9). High levels of
PRL experienced after orgasm cause
feelings of sexual satiety leading to the
sexual refractory period (10). Low
prolactin levels do not have a significant
impact on sexual desire (11). PRL
regulates itself through a dopaminemodulated negative feedback loop (12). In
sufficient concentrations, PRL binds to
PRL
receptors
on
hypothalamic
dopaminergic neurons, increasing DA
synthesis (12). This increase results in
greater activity on D2 receptors in
lactotrophs,
decreasing
PRL
gene
expression and exocytosis (9, 12).
Additionally, inducing high levels of PRL
will result in gynecomastia; while not a
health risk, the significant aesthetic and
social consequences of this effect would
likely have an appreciable impact on drug
adoption and compliance (13).
Dopamine
DA is a neurotransmitter involved in the
regulation
of
pleasure
pathways,
emotions, cognition, and motor activity.
DA is synthesized from the essential
amino acid tyrosine and transported
through the bloodstream to dopaminergic
neurons, crossing the blood-brain barrier
(BBB) via a low-affinity amino-acidtransport system (14). DA is terminated by
either enzymatic degradation or DAT
reuptake (14). Of the five DA receptors, D1
and D2 are most abundant (14).
Dopaminergic projections from the VTA
and substantia nigra (SN) influence
various aspects of sexual behaviour (15).
Dopaminergic activation of D3 and D4
receptors in the mPOA increases attention
-3-
and subsequent response to erotic stimuli
(1, 15). Increased activation of these
receptors from the mesocorticolimbic
system to the nucleus accumbens (NAcc)
results in hedonic and incentive salient
responses
(15).
The
ventromedial
prefrontal cortex (vPFC) regulates the
control of impulses; decreased D4 activity
in the vPFC results in decreased
motivational
impulsivity
(1).
The
accumulative
effects
of
decreased
dopaminergic activity in each brain region
results in the attenuation of sexually
appetitive behaviour (1, 14).
Serotonin
5-HT plays active roles in both the central
and peripheral nervous systems. Although
it is a neurotransmitter, 5-HT is involved
in haemostasis when absorbed into
platelets and is most concentrated in
enterochromaffin
cells
of
the
gastrointestinal (GI) tract, where it
induces peristalsis (16). The rate-limiting
step in 5-HT synthesis in the brain is the
conversion of the essential amino acid
tryptophan (L-Trp) into 5-HTP by the
enzyme tryptophan hydroxylase (TPH2)
(17, 18). 5-HTP is then converted into 5HT via decarboxylation by the enzyme
aromatic L-amino acid decarboxylase
(AADC) (17). The effects of 5-HT are
terminated
through
reuptake
by
transporters located on the presynaptic
cleft of serotonergic neurons (17).
The effects of 5-HT in the brain are
modulated by serotonergic neurons
concentrated most highly in the DRN of
the midbrain (18). 5-HT1A receptors are
found densely throughout the DRN and
the mPOA, where the presynaptic
autoreceptor variant acts to decrease 5-HT
levels, consequently facilitating sexually
appetitive behaviour (1). However,
agonism of 5-HT1A receptors results in an
overall decrease in sexually appetitive
behaviour by inhibiting adenylyl cyclase
activity and subsequently decreasing
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
cAMP production (19). 5-HT also activates
5-HT2A receptors, attenuating sexual
desire by increasing the activity of
phospholipase C, and subsequently
increasing DAG and IP3 production (20).
Activation of 5-HT2A receptors attenuates
sexual desire by increasing the activity of
phospholipase C and increasing DAG and
IP3 production (20). 5-HT also plays a
role in the physical manifestations of
sexual arousal; decreased agonism of the
5-HT1B receptor facilitates physical
copulatory functions such as penile
tumescence (21).
5-HT increases levels of DA in the mPOA
and mesocorticolimbic system as well as
levels of PRL; activation of 5-HT1A, 5HT2A and 5-HT3 receptors mediate PRL
release in the anterior pituitary (22).
HT and DA does not exist; an increase in
5-HT will result in a compensatory rise in
DA in the mPOA and MCL system. This is
why both 5-HT and DA must be targeted
(23, 24). By increasing levels of synaptic
5-HT, sexual satiety is reduced (23). 5-HT
will act on the 5-HT1A and 5-HT2A
receptors within the DRN and mPOA. By
reducing levels of synaptic DA, sexually
appetitive behaviour will be attenuated
due to decreased activation of D1-D4
receptors in the mPOA, MCL, and NAcc
(15, 23). Simultaneous targeting of DA is
also necessary when treating sexual
offenders in order to avoid incentive
salience underlying the motivation to
reoffend (25). Decreased DA levels in
these areas will also result in lowered
impulsivity toward sexual stimuli (25).
Identification of 5-HT and DA as
Chosen Targets
Potential Loci
Direct introduction of 5-HT into the brain
is not possible because 5-HT does not
cross the BBB; target areas are not
reached
and
peripheral
5-HT
accumulates, leading to a variety of
adverse effects, particularly in the GI tract
(16, 26). Precursors to 5-HT were then
examined. L-Trp was discounted due to its
inability to cross the BBB without the
rate-limiting carrier protein LAT1 (17).
Additionally, excess L-Trp would not
necessarily result in an increase in overall
5-HT; the body would use it in other
processes, such as production pathways,
and convert it to non-serotonergic
metabolic intermediates such as glucose
(27). The enzyme TPH2 is an even less
desirable target, since increasing its
production requires manipulating gene
expression through viral transduction or
artificially programmed cells (28). It is
more effective to bypass the rate-limiting
enzyme TPH2 by directly controlling the
concentrations of 5-HTP, which readily
diffuses across the BBB to form 5-HT in
the DRN (17, 27). 5-HTP was ultimately
chosen as the locus of intervention over
Fig 1. Effects of 5-HT and DA on Lust: An
increase in 5-HT production in the raphe nuclei
will increase activity on 5-HT2A and 5-HT1A
receptors in the PFC and mPOA respectively,
lowering lust. The general increase in 5-HT will
additionally act on 5-HT1A within the VTA and PFC,
inducing increased [DA]. The released DA will act
on both D3 and D4 receptors, given their high
concentrations within the mPOA region, promoting
feelings of lust. An increase of DA via raised levels
of 5-HT should be inhibited to affectively attenuate
sexually appetitive behavior.
Raising intracerebral 5-HT and decreasing
DA levels is the most effective treatment
for lust. A simple opponency between 5-4-
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
specific 5-HT receptor agonism because
multiple receptors are involved in the
mediation of lust. Designing a multi-target
agonist
which
activates
multiple
serotonergic receptors would result in an
increased likelihood of adverse effects due
to the ligand‟s potential affinity for nonserotonergic binding sites (29). The use of
5-HTP to elevate 5-HT concentrations also
allows for agonism of 5-HT1B, which
effectively
attenuates
the
physical
manifestations arising from sexual desire
(21). In addition to attenuating sexually
appetitive behaviour by inducing a
ubiquitous 5-HT increase, activation of 5HT1A receptors in the DRN also results in
decreased depression and anxiety (30).
In the dopaminergic pathway, sexually
appetitive behaviour is mediated by the
activation of D1-D4 receptors, induced by
the overall increase in intracerebral
synaptic DA (15). Intervention in the DA
synthesis pathway may be achieved via a
tyrosine hydroxylase inhibitor (14).
Increasing
concentrations
of
DAdegrading enzymes such as MAO-B, ADH
and COMT could also decrease DA levels;
however, this would require gene therapy
(14). Agonism of the D2 autoreceptor
(D2S) was ultimately chosen as the locus
of intervention due to its ability to
decrease synaptic DA (31).
Section 3:
Contributions, Considerations, JYP
According to Public Safety Canada, 27% of
released sexual offenders are convicted of
another sexual offence within 20 years (3).
This statistic stresses the importance of a
long-term drug approach to counteract
lust. This is why Nadamo will be
administered through controlled-release
microspheres lasting six months and
therefore eliminating the issue of
compliance. Continuity is also essential,
since a break in treatment can result in
regression and return to predation (32).
Furthermore, the use of a constant-release
-5-
drug significantly reduces the likelihood of
relapse.
Mechanism of Action
Nadamo will be delivered through an
intramuscular injection of microspheres
500 μm in diameter into the deltoid.
These microspheres are made from
biodegradable poly(lactic-co-glycolic acid)
(PGLA) polymers and undergo matrix
erosion to allow for the controlled release
of 5-HTP and PTB over the span of 6
months (33).
5-HTP will be released into the
bloodstream and travel to the BBB, which
it crosses via diffusion. 5-HTP will then be
decarboxylated into 5-HT, primarily in the
DRN of the brainstem where the highest
concentration of AADC is present (34). 5HT from the DRN will act on 5-HT1A and
5-HT2A receptors, which are found in
highest abundance in the mPOA and the
prefrontal cortex, respectively, resulting in
reduced libido (1, 35). Although 5-HT1A
autoreceptors,
which
facilitate
the
reuptake of synaptic 5-HT, also exist in
the DRN, the continuous production of 5HT from Nadamo will desensitize these
receptors (36). The produced 5-HT will
also stimulate the 5-HT1B receptor, most
densely found in the frontal cortex,
inhibiting gonadal response to sexual
stimuli (21). Nadamo‟s release of 5-HTP is
such that the level of 5-HT produced will
be maintained between 625-750 ng/g (3739). PTB, a highly hydrophobic molecule,
will be released simultaneously from the
microspheres, travelling through the
bloodstream before diffusing across the
BBB (40, 41). PTB will stimulate the D2S
receptor throughout the brain, initiating
the reuptake of DA via DAT; a decrease of
DA in the mesocorticolimbic and mPOA
regions attenuates sexually appetitive
behaviour by decreasing activation
frequency of D1-D4 receptors (42).
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
Pharmacokinetics
Over a six month period, approximately
73% of Nadamo is absorbed and released,
at a rate of approximately 0.778% per day
(43). The calculated LiPE value (pEC50 logP = 8.12 - 3.038) measures 5.082,
indicating high potency and lipophilicity
(44-46). Nadamo's high potency makes it
effective at low doses, while its high
lipophilicity allows it to readily cross the
BBB (40, 46).
Tests
Team Inanna has made significant
progress in the development of Nadamo.
Evidence of this progress is seen in both in
vivo and in vitro pre-clinical tests. All
animal tests were conducted on male
Sprague-Dawley rats (47).
Test 1: 5-HTP and PTB Dosage
Purpose: To determine the doses of 5HTP and PTB required to achieve 750 ng
of 5-HT/g of brain tissue and 7520 ng of
DA/g of brain tissue (37, 38).
Methods: The method of decapitation
and fluorescence outlined by Hyttel J et al.
and Curzon G et al. was used (48, 49).
Intracerebral concentrations of 5-HT and
DA were measured in rats administered
increasing amounts of 5-HTP and PTB.
Rats (n=100) were divided into groups of
four with each group receiving a specific
concentration of 5-HTP and PTB via
intramuscular injection.
Results: In order to increase levels of
brain 5-HT to 750 ng/g, a mean 5-HTP
dose of 178.75 ng/g of rat brain tissue was
required. This is equivalent to a dose of
429 ng/g of brain tissue per day (37). To
decrease levels of DA to 7520 ng/g of
brain tissue, a mean PTB dose of 4.17 ng/g
was needed after 3 days. This is equivalent
to 10 ng/day (38, 50).
-6-
The administration of varying [5-HTP] to four
groups of rats. The results of brain homogenization
and fluorescence measurement after three days
indicate that an increase in [5-HTP] results in an
increase in [5-HT]. Through extrapolation of the
data, a dosage of 178.75 ng of 5-HTP per gram of
brain tissue was determined to produce the target
[5-HT] of 750 ng/g.
The administration of varying [PTB] in four groups
of rats. The results of brain homogenization and
fluorescence measurement after three days indicate
that an increase in the [PTB] results in a decrease in
[DA]. Through extrapolation of the data, a dosage of
4.00 ng of PTB per gram of brain tissue was
determined to produce the target 5-HT level of 7520
ng/g.
Test 2: Effect of PTB on DA
Receptors and 5-HT Receptors
Purpose: To determine the efficacy of
PTB for human D1, D2L, D2S, D3, D4, D5
sites and 5-HT receptors (5-HT1A, 5-HT1B
and 5-HT2A).
Method: [35S]GTPγS binding assays on
Chinese hamster ovary cells prepared in
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
the method outlined by Newman-Tancredi
A, Cussac D, Audinot V, and Millan MJ
were used (42).
Results:
PTB displayed extremely high efficacy and
selectivity for D2S (max efficacy of 135%).
PTB displayed negligible efficacy for D1,
D2L, D3, D4, D5 sites and 5-HT1A, 5-HT1B
and 5-HT2A sites (42).
The
measured
[5-HT],
through
brain
homogenization, in rats administered with a dose
of 77.2 µg of Nadamo at different time intervals.
The level of 5-HT increased to 750 ng/g during the
first 21 days and was maintained at 750 ng/g until
6 months. The graph only shows results until 10
weeks for simplicity.
The results of brain homogenization and
fluorescence measurement in 4 groups of rats after
three days indicate that an increase in the [PTB]
results in a decrease in [DA]. Through
extrapolation of the data, a dosage of 4.00 ng of
PTB per gram of brain tissue was determined to
produce the target 5-HT level of 7520 ng/g.
Test 3: Long-term Stabilization of
Nadamo
Purpose: To determine the maintenance
of constant levels of serotonin and
dopamine with the administration of
Nadamo.
Method: The method of decapitation and
fluorescence outlined by Hyttel J et al. and
Curzon G et al. was used (37, 48, 49).
Results: Increased brain levels of 5-HT
and DA were measured over the first 14
days, stabilizing close to 750 ng 5-HT/g of
brain tissue and 7520 ng DA/g of brain
tissue (37, 38). The remaining rat groups
sustained these levels for 6 months.
-7-
The
measured
[DA],
through
brain
homogenization, in rats administered with a
dose of 77.2 µg of Nadamo at different time
intervals. The level of DA decreased to 7520 ng/g
during the first 21 days and was maintained at
7520 ng/g until 6 months. The graph only shows
results until 10 weeks for simplicity.
Test 4: Effect of Nadamo on Sexual
Arousal
Purpose: To determine the effectiveness
of Nadamo in reducing sexual arousal in
rats.
Method: Direct copulation tests.
Rats (n=100) underwent an activation
trial as outlined by Harms et al. Following
activation trials, copulatory frequency was
measured and thermography was used to
determine sexual arousal. This data served
as the control (51).
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
A second set of trials was conducted after
a week under constant conditions, only
with a 2 mg dose of Nadamo (1 mg 5HTP, 1 mg PTB) (51).
Results: In trial 1, where Nadamo was
not used, rats exhibited high levels of
sexual desire followed by reproductive
sexual activity, with only 2 males failing to
copulate.
Additionally,
higher
temperatures were measured in the
genital region (52). In trial 2, where
Nadamo was administered, 94 of the 100
males were not receptive to females and
exhibited lower copulatory frequency
during activation trials. Temperatures
lower than those in trial 1 were measured
in the genital area (51).
Test 5: Effects of Nadamo on Mood
Purpose:
To
assess
Nadamo‟s
physiological and behavioural side effects.
Method: Rats (n=100) were divided into
two groups of 50 (53). One group was
administered an intramuscular injection
of microcapsules releasing 1.8 μM
Nadamo/day for 6 months, while the
control group was administered a placebo
injection of microspheres releasing 1.8 μM
saline solution (27).
SHIRPA: To determine the effects of
Nadamo on mood and behaviour in rats, a
primary-stage SHIRPA screening was
conducted.
To
collect
behavioural
observation profiles, the rats were placed
in a transparent viewing arena for five
minutes daily.
Rotarod tests: To assess effects on
balance, the rats were placed on a rod
rotating at increasing speed per minute
for a total of 5 minutes for each speed. The
time and rotation at which the rats fell off
the rotomotor machine were recorded
(54).
Gait analysis: To test gait movements,
the rats were placed on a glass runway.
The runway was lit by a red dim light in a
darkroom monitored by a camera, and the
rats were observed for 3 minutes while
they walked across the runway (55).
-8-
Marble burying test: To test for
anxiety, the rats were placed in a cage and
their burying reflexes to marbles
introduced to the cage were noted (56).
Y-Maze: To test for effects on learning
and memory, especially spatial memory
recognition, the amount of time required
for the rats to cross the Y-maze was
recorded (57-59).
Results:
SHIRPA: Results showed that Nadamo
has nausea-inducing effects.
Rotarod tests: Rats administered
Nadamo fell off the rod at the same rate as
the rats not administered Nadamo,
indicating that the drug had no significant
effects on balance.
Gait analysis: Moderate effects in body
movement and muscle activation were
noticed in 15% of the population
administered Nadamo, showing that
effects on body movement may be a
moderate side effect.
Marble burying test: Frequency of
marble burying decreased in rats
administered Nadamo, indicating that
Nadamo decreased anxiety (56).
Y-Maze: On average, rats administered
Nadama took 12% longer to cross the Ymaze, showing that Nadamo may have
minor negative effects on learning and
memory (60).
Taking Everything into
Consideration
Sexually
appetitive
behaviour
is
modulated by both 5-HT and DA (1).
Therefore, any potential drug must lower
the
compensatory
5-HT1A-induced
increase of DA, which would otherwise
counteract 5-HT‟s inhibitory effects on
lust. This relationship necessitates the coadministration of the D2S agonist, PTB
(1). Additionally, increased levels of
synaptic 5-HT inevitably induce the
activation of 5-HT receptors not
associated with lust and can potentially
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
lead to undesired side effects. By limiting
the rates of release of 5-HTP and PTB, 5HT and DA levels will not deviate
drastically from their respective normal
ranges, thereby reducing the probability of
severe conditions such as dyskinesia and
serotonin syndrome (61).
By designing the drug to release only
enough 5-HTP and PTB to maintain
normal levels, the occurrence of minor
side effects including nausea, hypotension
and urinary hesitancy will also be reduced
(62, 63). However, since Nadamo affects
5-HT and DA throughout the brain, a
SHIRPA behavioural screening was
conducted to verify that Nadamo will have
a minimal effect on other pathways
involving
these
neurotransmitters.
Additionally, adverse GI effects induced
by excess peripheral 5-HT and PTB may
be experienced; however, symptoms may
be mitigated with the use of an AADC
inhibitor (64).
The use of biodegradable microspheres
made from glycolic and lactic acids was
chosen after considering the possibility of
an immune response to foreign substances
forming
a
capsule.
Microsphere
degradation may result in intramuscular
acid accumulation, leading to soreness
(40). However through controlled matrix
erosion such effects should be minimal
(40). An idea for future drug design is the
potential reduction of microsphere size to
allow direct diffusion across the BBB, thus
reducing periphery-related side effects
-9-
while increasing the efficiency of Nadamo
(40).
Justify Your Presence
Sexual crimes pose a serious problem to
government expenditure due to high
recidivism rates; it is estimated that the
cost of a high-risk adult sex offender over
the span of their criminal career in
Canada exceeds CDN $1 million in tax
payer money (65). Existing treatments for
sexual offenders have not been able to
reduce the incidence of sexual crimes
because of their ineffectiveness in the long
term (7, 8).
Nadamo is a long-term multi-target drug
which uniquely induces sexual satiety
while removing the pleasure sexual
predators obtain from their behaviour. It
is a novel approach which targets both the
dopaminergic and serotonergic systems
involved in sexually appetitive behaviour,
and eliminates the motivation behind
these sexual crimes by removing the
feelings of gratification. Nadamo‟s longterm administration method ensures
treatment compliance and simplifies
dosing regimens.
Despite considerable advancements in the
development
of
Nadamo,
funding
assistance is still needed to reach clinical
trials and eventually, consumer markets.
The majority of potential funds would go
towards completing large-animal trials
and phase I, II, and III human trials.
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
1.
Pfaus JG. Pathways of Sexual Desire. J Sex Med [serial on the Internet]. 2009;
6(6): Available from: http://www.ncbi.nlm.nih.gov/pubmed/19453889. Doi:
10.1111/J.1743-6109.2009.01309.X.
This recent review article details the biological mechanisms (i.e. brain pathways,
neurochemicals) involved in sexual desire. This comprehensive review is undertaken
with over 240 references, however, the focus of this paper lies in identifying the
neurological processes involved in normal and hypoactive sexual desire rather than
inhibiting sexual desire in sex offenders. This paper provided an overview of the
excitatory roles of DA, norepinephrine, oxytocin and melanocortins, and the inhibitory
roles of 5-HT, opioids and endocannabinoids. We learned of the unique characteristics
of rat reproductive behaviour, such as lordosis, pacing and multiple intromission
patterns, helping us better understand other studies monitoring copulatory behaviour in
rats. The dopamine component of this review provided an overview of areas of the brain
to which the DA system projects and gave us a basis for further research on the effects of
dopaminergic activation of sexually appetitive behaviour. The serotonin section of this
review detailed the involvement of 5-HT in inducing sexual satiety, which helped us
select this neurotransmitter as a locus of intervention. Additionally, it provided a
thorough explanation of the receptors involved in these pathways and their role in
facilitating feelings of sexual desire. It further clarified the relationship between the
serotonergic and dopaminergic systems which we further investigated. As a result of this
research our drug was designed to specifically take into account the interaction between
the serotonergic and dopaminergic systems. The author of this article is an established
scientist at the accredited Montreal University, adding to the credibility of this review.
2.
Snyder H. Sexual assault of young children as reported to law enforcement:
victim, incident, and offender characteristics. Washington, D.C.: National Center for
Juvenile Justice; 2000.
The following series of reports provided an in-depth look at the demographics behind
sexual assaults in the U.S. in terms of both victims and offenders involved. Our focus
was drawn to the prevalence of male sex offenders over female sex offenders being 96%.
The source is credible due to its publication by the U.S. Department of Justice.
3.
Harris A, Hanson R. Sex offender recidivism2004; 9: Available from:
http://www.publicsafety.gc.ca/res/cor/sum/cprs200407_1-eng.aspx.
According to Public Safety Canada, 27% of released sexual offender are convicted of
another sexual offence within twenty years. This influenced the design of a long-term
release mechanism to cure sex offenders.
4.
Snyder P. Androgens. In: Brunton L, Chabner B, Knollmann B, editors. Goodman
& Gilman's The Pharmacological Basis of Therapeutics. 12th ed. New York: McGrawHill; 2011. Available from:
http://www.accessmedicine.com/content.aspx?aID=16673856
This article was used to provide details of the testosterone biosynthetic pathway.
Through this article it was learned that testosterone, the primary androgen secreted in
men, is synthesized in the Leydig cells of the testes as well as the adrenal cortex. The
precursors to this sex hormone, androstenedione and dehydroepiandrosterone, are
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
converted here to testosterone by the enzyme 17 alpha-hydroxylase (CYP17). Also, this
article describes in detail how increased levels of bioavailable testosterone (BT) affect
libido in males by binding to the androgen-specific receptors NR3A present in the basal
epithelial cells throughout the body, and more specifically in glands. It was learned that
of the two forms of testosterone found in the blood, BT and bound testosterone, it is the
BT which is important in the onset of sexual urges. Given that BT is either free-floating
or loosely bound to blood proteins, it is available to bind on androgen receptors and
illicit a response. This article was credible because it was extracted from a textbook
published by a reputable source and was well-referenced allowing the information to be
corroborated.
5.
Attard G, Belldegrun AS, Bono JSD. Selective blockade of androgenic steroid
synthesis by novel lyase inhibitors as a therapeutic strategy for treating metastatic
prostate cancer. BJU International [serial on the Internet]. 2005; 96(9): Available from:
http://onlinelibrary.wiley.com/doi/10.1111/j.1464-410X.2005.05821.x/full. Doi
10.1111/j.1464-410X.2005.05821.x.
In most cases, testosterone levels are mediated in males by a negative feedback loop
involving the hypothalamus and the pituitary gland. When the hypothalamus detects
low testosterone levels, it releases gonadotropin-releasing hormone (GdRH) to the
anterior pituitary gland, resulting in release of luteinizing hormone (LH) and folliclestimulating hormone (FSH). LH travels to the testes via the bloodstream, stimulating
production of testosterone upon binding to receptors on Leydig cells. This loop then
continues to maintain testosterone levels by responding to the detected levels in the
blood. If this loop is disturbed, however, there are other mechanisms by which these
levels stabilize. For example, plasma testosterone can be converted into testosterone
from adrenal androgenic steroids by 17-ketoreductase. This synthesis can be targeted by
inhibiting enzymes involved in this process, though this approach has not been proven
to be fully effective. These results indicate that it is very difficult to properly inhibit
levels of testosterone in the body, giving Team Inanna support for discounting it as a
potential target molecule.
6.
Schmidt C. Abiraterone and MVD3100 take androgen deprivation to a new level.
J Natl Cancer Inst [serial on the Internet]. 2011; 103(3): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/21242338. Doi 10.1093/jnci/djr014.
This article focuses on the mechanisms of action of two drugs, abiraterone acetate (AA)
and MDV3100, to respectively reduce testosterone production in the body and decrease
its ability to bind to receptors to elicit a response. Through this article it was learned
that abiraterone acetate targets the conversion of pregnenolone into
dehydroepiandrosterone, via activity of the enzyme CYO450c17, in the testes as well as
adrenal glands. By acting on the aforementioned areas, this drug inhibits the body from
producing testosterone. Despite its ability to act on two different areas, AA has toxicity
issues and is not effective in the long term because the body compensates for a reduction
of testosterone by upregulating other hormones such as adrenocorticotropic hormones.
An increase in levels of these hormones causes hypertension. In order to mitigate this
side effect, this medication needs to be prescribed with steroids. This article was used to
gain a thorough understanding of the mechanisms used by the body to inhibit
testosterone production in the testes and other areas, as well as the effects of drugs to
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
disrupt natural testosterone synthesis pathways and its functioning. Also the
information extrapolated from this article was used to rule out testosterone as a target
molecule because a drug potentially acting as an analogue of AA, having already passed
phase III clinical trials, would require co-prescriptions and would still not be effective in
the long-term due to the compensatory mechanisms of the body.
7.
Cooper AJ. A placebo-controlled trial of the antiandrogen cyproterone acetate in
deviant hypersexuality. Compr Psychiatry [serial on the Internet]. 1981; 22(5): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/6230209.
This article describes the inefficiency of cyproterone acetate in treating sexual deviants.
It states that androgen levels are "transient" when using the drug and may
spontaneously return to pre-administration levels. This suggests that androgens may
not be the best locus of intervention for an anti-lust drug.
8.
Cooper AJ, Sandhu S, Losztyn S, Cernovsky Z. A double-blind placebo controlled
trial of medroxyprogesterone acetate and cyproterone acetate with seven pedophiles.
Can J Psychiatry [serial on the Internet]. 1992; 37(10): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/1473073.
In this study, 10 pedophiles underwent androgen therapy and responded positively to
the first phases of treatment. Before the experiment had finished though, specific sexual
behaviours returned, implying that androgen therapy was not effective in the long-term.
This article states that although FSH and LH levels decreased during drug therapy, they
ultimately returned to normal pre-antiandrogen administration levels. This suggests
that antiandrogens may not be completely effective at reducing testosterone in sexual
predators, weakening its viability as a potential locus of interference.
9.
Melmed S, Jameson J. Disorders of the Anterior Pituitary and Hypothalamus. In:
Longo D, Fauci A, Kasper D, Hauser S, Jameson J, Loscalzo J, editors. Harrison's
Principles of Internal Medicine. 18th ed. New York: McGraw-Hill; 2012. Available from:
http://www.accessmedicine.com/content.aspx?aID=9139876.
This passage explains the synthesis, secretion and action of prolactin. It also cites
possible effects of hyperprolactinaemia, including hirsutism and galactorrhea. PRL
consist of 198 amino acids and is secreted from lactotrophs, which constitute about 20%
of the anterior pituitary. Unlike other hormones in the pituitary, PLR‟s function is
inhibitory; more specifically, it reduces libido. In males, PLR levels range from 10–20
μg/L and 10–25 μg/L in females. When PLR concentrations elevate, PLR receptors on
dopaminergic neurons in the hypothalamus become bound. This increases the
production of the rate limiting enzyme in the dopamine synthesis pathway. The increase
in released dopamine acts on the lactotrophs and decreases the production of PLR. This
passage was used to explain and analyze the prolactin pathway in order to identify
possible points of drug intervention. It is credible due to its extensive use of peerreviewed citations and endorsement by a reputable publisher.
10.
Kruger T, Exton MS, Pawlak C, von zur Muhlen A, Hartmann U, Schedlowski M.
Neuroendocrine and cardiovascular response to sexual arousal and orgasm in men.
Psychoneuroendocrinology. [Research Support, Non-U.S. Gov't]. 1998 May;23(4):40111. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9695139.
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
In this study, tests were conducted on males before, during, and after the sexual
refractory period. Concentrations of various neurotransmitters were monitored during
these periods. It was indicated that a significant increase in prolactin levels occurred
directly preceding and during the sexual refractory period, lending support to the theory
that prolactin causes the onset of this refractory period.
11.
Correll CU. Monitoring and management of antipsychotic-related metabolic and
endocrine adverse events in pediatric patients. Int Rev Psychiatry. [Research Support,
N.I.H., Extramural Review]. 2008 Apr;20(2):195-201. Available from:
http://www.ncbi.nlm.nih.gov/pubmed/18386212.
This article analyzes the efficacies of several antipsychotics and provides details on side
effects. It states that elevated prolactin levels consistently reduce libido. The article also
notes that in trials up to 30 weeks, low prolactin levels have not been associated with
specific consequences. Thus, increasing prolactin levels could form the basis of an antilust drug but low prolactin levels would be ineffective.
12.
Fitzgerald P, Dinan TG. Prolactin and dopamine: what is the connection? A
review article. J Psychopharmacol [serial on the Internet]. 2008; 22(2 Suppl): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/18477617. Doi:
10.1177/0269216307087148.
This article explains the negative feedback loop and physiological effects of prolactin.
The study explains how high prolactin levels cause dopaminergic neurons to increase
production of a rate-limiting enzyme in dopamine synthesis, which converts L-tyrosine
to L-DOPA. The resulting increase in dopamine levels acts on receptors on
prolactinergic neurons. This reduces prolactin gene expression and exocytosis. After
reading this article, we were able to determine the link between dopamine and
prolactin; we were also able to discount prolactin as a potential locus, since it would be
more efficient to simply target dopamine directly. This source is credible due to its
acceptance in a reputable peer-reviewed journal and its proper employment of citations.
13.
Snyder P. Clinical manifestations and diagnosis of hyperprolactinemia. Wolters
Kluwer; 2011 [updated July 20; cited 2012 March 30]; Available from:
http://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-ofhyperprolactinemia?source=search_result&search=Hyperprolactinemia&selectedTitle=
2%7E127.
This article describes the clinical manifestations of hyperprolactinemia. It was used to
examine the consequences of raising prolactin levels in order to reduce libido. Males
with sustained high levels of prolactin often exhibit gynecomastia (significant breast
enlargement) and, in some cases, galactorrhea (the production and secretion of milk),
side effects which would likely affect drug adoption and compliance. The article is
credible due to UpToDate's reputation as a reliable source for medical information and
the articles extensive use of citations from peer-reviewed journals.
14.
Elsworth JD, Roth RH. Dopamine synthesis, uptake, metabolism, and receptors:
Relevance to gene therapy of Parkinson's disease. Exp Neurol [serial on the Internet].
1997; 144(1): Available from: http://www.ncbi.nlm.nih.gov/pubmed/9126143.
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
This article gives an overview of the 5 stages of the life cycle dopamine as a
neurotransmitter. It begins by giving a detailed description about dopamine synthesis,
storage, release and response in terms of receptor and autoreceptor interactions, and
reuptake. All DA receptors are 7-transmembrane G-coupled protein receptors that are
activated by heterotrimeric GTP-binding proteins and D1 and D2 receptors are the most
abundant in the CNS. Then, it focuses on the changes that occur in the nigrostriatal
system, a pathway that uses dopamine as its primary neurotransmitter to function in
individuals who suffer from Parkinson's disease. This article was used to gain a
thorough understanding of how DA was synthesized, stored, released, how it elicited a
particular response in the body and how this response is terminated. Through this
article it was learned that dopamine is synthesized from the dietary amino acid tyrosine.
After being integrated into the bloodstream, tyrosine is transported to the brain‟s
extracellular fluid by a low-affinity amino acid transport system. From this point,
tyrosine is transported by high and low-affinity amino acid transporters into
dopaminergic neurons in the brain where it is converted to dihydroxyphenylalanine (LDOPA) by tyrosine hydroxylase (TH). The latter is the rate-limiting step in the synthesis
of dopamine. L-DOPA is then converted to dopamine by aromatic amino acid
decarboxylase (AADC). AADC decarboxylates L-DOPA at a very fast rate and this causes
the levels of this amino acid to be low in the brain. In the dopaminergic neurons, the
neurotransmitter is transported from the cytoplasm into specialized storage vesicles,
from where it is released upon stimulation by an action potential. After producing its
effect, DA is terminated so that the neurotransmitter homeostasis is maintained. Highaffinity membrane carriers reuptake DA back into the nerve terminals by actively
pumping it from the synaptic cleft. Some evidence has shown that the glia and nondopaminergic neurons may take up and metabolize, to limited extent, extracellular
dopamine. Some enzymes responsible for the metabolism of DA are: MAO, COMT,
ALD-D, ALR and ADH. The fact that this article was published in a reputable scientific
database and was well referenced makes it a credible source.
15.
Melis MR, Argiolas A. Dopamine and sexual behavior. Neurosci Biobehav Rev.
[Research Support, Non-U.S. Gov't Review]. 1995 Spring;19(1):19-38: Available from:
http://www.ncbi.nlm.nih.gov/pubmed/7770195.
This review article outlined the effect of DA on both sexually consummatory (i.e. penile
tumescence) and appetitive behaviour. From this study, it was determined that
activation of the dopaminergic projections in the MCL system, which encompasses the
mPOA, and the NAcc is responsible for sexual motivation and the manifestation of
sexually appetitive behaviour. A commonly used test for sexually appetitive behaviour is
the bilevel chambers test designed by Mendelson and Gorzalka in which male rats chase
female rats from one level to another. Studies determined that upon direct injection of
D1 or D2 antagonists into the mPOA and NAcc, this behaviour was significantly reduced.
It was found that, except at relatively high doses of DA receptor antagonists, DA
antagonism typically affects the sexual motivation and appetitive behaviour rather than
copulatory ability. DA antagonism has also been shown to reduce sexual motivation
through the lever press test; DA receptor antagonist ct-flupenthixo resulted in a 70-80%
decrease in lever pressing. From this we were able to infer that overall decrease in CNS
levels of DA would result in reduced sexual motivation and appetitive behaviour while
having minimal locomotor side effects as long as the decrease is not extreme (i.e. it was
found that a substantial effect on rat locomotion was observed only after 0.25 mg/kg of
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
flupenthixo). This study proves that our method of intervention via PTB‟s decrease of
synaptic DA is valid and that DA activation in the MCL and NAcc is very significant in
the moderation of sexual motivation and appetitive behaviour. From this study, we can
infer that D1-D4 receptors within their respective regions are heavily involved in the
mediation of sexual desire and due to the diversity of DA receptors expressed in these
places, Team Inanna decided to lower intracerebral DA activity.
16.
Sanders-Bush E, Hazelwood L. 5-Hydroxytryptamine (Serotonin) and
Dopamine. In: Brunton L, Chabner B, Knollmann B, editors. Goodman & Gilman's The
Pharmacological Basis of Therapeutics. 12th Edition ed. New York: McGraw-Hill; 2011.
Available from: http://www.accessmedicine.com/content.aspx?aID=16662305
This chapter talks about the role of serotonin and dopamine, neurotransmitters present
in the central nervous system, in many peripheral functions. It explains that the highest
concentrations of dopamine are present in the brain, in the adrenal medulla and to
limited extent in the plexuses of the GI tract and the enteric nervous system. It also
explains that serotonin is found in high concentrations in enterochromaffin cells
throughout the GI tract, in storage granules that are present in the platelets and broadly
throughout the central nervous system. Along with aiding in the understanding of the
fourteen different receptor subtypes of serotonin and the five dopamine receptor
subtypes, this chapter focuses on the fact that the effects of serotonin in the brain are
modulated by serotonergic neurons that are extensively concentrated in the dorsal
raphe nucleus (DRN) of the midbrain. Its general overview of serotonin‟s action in the
GI tract and platelet cells gave us the basis for further research for taking the side-effects
of increased [5-HTP] in the body in to consideration. This chapter is credible because it
is well referenced to corroborate information and is published by reputable and credible
publishers, McGraw- Hill.
17.
Sanders-Bush E, Hazelwood L. 5-Hydroxytryptamine. In: Brunton L, Chabner B,
Knollmann B, editors. Goodman & Gilman's the Pharmacological Basis of Therapeutic.
12th Edition ed. New York: McGraw-Hill; 2011. Available from:
http://www.accessmedicine.com/content.aspx?aID=16662308
Basis of Therapeutic. 12th Edition ed. New York: McGraw-Hill; 2011.
This textbook outlined the serotonin pathway from synthesis all the way up to
termination. Its chapters also included a brief overview of serotonin receptors as well as
the mechanism of action for certain SSRIs. This textbook was a major source of
information and reference for us considering our work with serotonin. We learned about
serotonin synthesis and were able to finalize our point of intervention based on the
information this book presented to us. We also learned that tryptophan was not a viable
target to use in order to increase 5-HT levels in the brain. Tryptophan competes with
other branched-chain and aromatic amino acids for the carrier protein, LAT1, in order
to cross the blood brain barrier. Since it has to compete with other molecules for this
carrier protein, it is a critical step in the serotonin synthesis pathway. The exposure to
SSRIs was also helpful as it gave us a starting point from which we could look at the use
of serotonin in treatment. The information was used to solidify a foundation for our
drug pathway and allowed us to make our research more refined in terms of the role of
neurotransmitters and sexual desire. We accessed this textbook through the Health
- 15 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
Sciences Library online under the Access Medicine link. The textbook is credible as it
had an extensive bibliography and list of acknowledgements as the end.
18.
Maurer-Spurej E. Serotonin reuptake inhibitors and cardiovascular diseases: a
platelet connection. Cell Mol Life Sci [serial on the Internet]. 2005; 62(2): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/15666087. Doi 10.1007/s00018-0044262-1.
This article outlines the role of 5-HT in the peripheral nervous system, where it is stored
and carried by platelets, as well as synthesised in enterochromaffin cells of the
gastrointestinal tract before being released into the blood. 5-HT is also involved in
thromogenesis, acting as an aggregating agent via the activation of peripheral 5-HT2
receptors. It further provides detail of the neurotransmitter‟s synthesis pathway. In the
dorsal raphe nuclei, serotonergic neurons contain the rate-limiting enzyme TPH2,
whereas in peripheral tissues only its isoform TPH is present. [5-HT] is modulated by
the serotonin transporter which allows for the reuptake of 5-HT.
19.
Dumuis A, Sebben M, Bockaert J. Pharmacology of 5-Hydroxytryptamine-1a
Receptors Which Inhibit Camp Production in Hippocampal and Cortical-Neurons in
Primary Culture. Mol Pharmacol [serial on the Internet]. 1988; 33(2): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/2828913.
This journal outlines the secondary messenger-mediated signal transduction pathways
used upon activation of 5-HT1A. It is concluded that the 5-HT1A receptor is negatively
coupled to adenylyl cyclase, inducing an inhibitory cascade resulting in a reduction in
cAMP formation.
20.
Parrish JC, Nichols DE. Serotonin 5-HT2A receptor activation induces 2arachidonoylglycerol release through a phospholipase c-dependent mechanism. J
Neurochem [serial on the Internet]. 2006; 99(4): Available from:
http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2006.04173.x/full Doi
10.1111/J.1471-4159.2006.04173.X.
This article outlines the activation pathway of 5-HT2A receptors. 5-HT2A receptors are
coupled to heterotrimeric G-proteins and, when bound to by 5-HT, induce a
phosopholipase C-mediated cascade leading to the production of the secondary
messengers diacylglycerol (DAG) and inositol triphosphate (IP3).
21.
Rodriguez-Manzo G, Lopez-Rubalcava C, Hen R, Fernandez-Guasti A.
Participation of 5-HT(1B) receptors in the inhibitory actions of serotonin on masculine
sexual behaviour of mice: pharmacological analysis in 5-HT1B receptor knockout mice.
Br J Pharmacol [serial on the Internet]. 2002 1573454]; 136(8): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/12163345. 10.1038/sj.bjp.0704827.
This study was imperative in furthering our understanding of the effects when agonizing
the 5-HT1B receptor. By increasing general serotonin levels throughout the brain, we had
to take the fact that activity on other serotonin receptors would increase into
consideration. When researching which serotonin receptors were involved in sexual
arousal or desire, 5-HT1B came up as having involvement in sexual arousal. From this
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
paper, the exact response of serotonin on the 5-HT1B receptor was learned to be the
physical aspects of sexual arousal such as erection and ejaculation. This study
discovered the role of 5HT1B in the physical manifestations of sexual arousal by
introducing a 5HT1B agonist, TFMPP, into 2 separate groups of rats. At specific
concentrations of TFMPP, both groups of rats showed significant decreases in what they
considered the three main aspects of male sexual physical behavior: mounting,
intromission (deep pelvic thrusts/penile insertion), and ejaculation. Observing the clear
actions of specifically agonizing the 5-HT1B receptor resulting in disrupted physical
actions of copulation in males, provided strong evidence allowing us to make the
statement that a general increase in serotonin, resulting in activation of this receptor,
would be successful at inhibiting the physical manifestations of sexual arousal in males.
22.
Jorgensen H, Knigge U, Warberg J. Involvement of 5-HT1, 5-HT2, and 5-HT3
receptors in the mediation of the prolactin response to serotonin and 5hydroxytryptophan. Neuroendocrinology [serial on the Internet]. 1992; 55(3): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/1386914.
This article describes the regulation of PRL by 5-HT, mediated by 5-HT1A, 5-HT2A and 5HT3 receptors. The precursor 5-HTP was administered to male rats alongside the SSRI
fluoxetine. A dose-dependent increase in plasma PRL concentration was observed, an
effect which was inhibited to varying degrees upon administration of 5-HT1A, 5-HT2A
and 5-HT3 antagonists.
23.
Sershen H, Hashim A, Lajtha A. Serotonin-mediated striatal dopamine release
involves the dopamine uptake site and the serotonin receptor. Brain Res Bull [serial on
the Internet]. 2000; 53(3): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/11113592.
This article explains the mechanisms behind mesocortical DA release upon activation of
the 5-HT1A receptor by 5-HT. The binding of 5-HT from the raphe nuclei to 5-HT1A
autoreceptors in the PFC and VTA induces an increase in DA in the mesocorticolimbic
and the medial preoptic area (mPOA). Regulation of DA release upon activation of 5-HT
receptors is thought to be mediated by reversal of DAT which results in increased
synaptic DA concentrations, facilitating movement of DA from the cytoplasm to the
extracellular space. This implies that any decrease in sexually appetitive behaviour
through a simple increase in 5-HT will be compensated by a rise in levels of DA.
24.
Esposito E. Serotonin-dopamine interaction as a focus of novel antidepressant
drugs. Curr Drug Targets [serial on the Internet]. 2006; 7(2): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/16475959.
This article describes the functional connections between the dopaminergic and
serotonergic systems in the brain in order to explain the mechanisms of action of SSRI
antidepressants. Fibrous innervations from the dorsal and median raphe nuclei project
to dopaminergic neurons in the VTA and SN, as well as to the NAcc, PFC and striatum.
Electron microscopy has revealed direct synaptic transmission between dopaminergic
dendrites and 5-HT neurons, implying communication between 5-HT in the DRN and
dopaminergic regions involved in sexual behaviour.
- 17 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
25.
Berridge KC. The debate over dopamine's role in reward: the case for incentive
salience. Psychopharmacology. [Research Support, N.I.H., Extramural Research
Support, Non-U.S. Gov't Review]. 2007 Apr;191(3):391-431: Available from:
http://www.ncbi.nlm.nih.gov/pubmed/17072591
This article outlines the role of dopamine in the functioning of the mesocorticolimbic
system. There are three functions of this dopamine pathways that are identifiedwanting, pleasure and learning, and the first two roles are very significant for the
consideration of dopamine in our drug. These functions are important to address as they
are what motivate the sexual offender, our target audience, to commit the crime.
26.
Crouzel C, Guillaume M, Barre L, Lemaire C, Pike VW. Ligands and tracers for
PET studies of the 5-HT system--current status. Int J Rad Appl Instrum B [serial on the
Internet]. 1992; 19(8): Available from: http://www.ncbi.nlm.nih.gov/pubmed/1428914.
This article was used because it stated that 5-HT cannot pass the blood brain barrier,
meaning its chemical pre-cursor 5-HTP, which can cross the blood brain barrier, had to
be used instead.
27.
Birdsall TC. 5-Hydroxytryptophan: a clinically-effective serotonin precursor.
Altern Med Rev [serial on the Internet]. 1998; 3(4): Available from:
http://www.altmedrev.com/publications/3/4/271.pdf.
The purpose of this report was to help in the narrowing down of areas to intervene in the
serotonin synthesis pathway, specifically the idea to introduce tryptophan. This article
outlined the specific ways of how directly administering tryptophan would not be an
effective way to increase serotonin within the brain. This article states four potential
ways tryptophan can be used. Encouraged conversion of serum tryptophan to
kynurenine due to any of; high dosages of tryptophan, increased stress, heightened
cortisol levels, or deficiency of vitamin B6. These potential elevated levels in kynurenine
would then result in an inhibition of transport of the introduced tryptophan into the
CNS, which would ultimately result in unchanged levels of serotonin within the brain,
despite initially added tryptophan. In addition to elevated kynurenine inhibiting
transport across the blood-brain barrier, tryptophan naturally depends on a transport
molecule to get past the barrier and enter the brain. The molecule responsible for
carrying tryptophan is also responsible for transporting five other amino acids. Lastly,
simply adding more tryptophan would not ensure more tryptophan passing into the
brain. Lastly, tryptophan is not only used for serotonin production in the body, but also
for various metabolic and molecular processes, such as protein synthesis, as well as the
production of niacin. Seeing all the potential areas for failure in increasing levels of
serotonin in the brain by using tryptophan, this article was significant in narrowing
down our potential loci.
Another use of this article was just for clarification purposes. We understood that by
introducing more of the serotonin precursor, 5-hydroxytryptophan, that more
intracellular serotonin would be produced, but we were not certain that it would be
released and therefore increase synaptic serotonin levels. This article provided strong
evidence illustrating that introduced 5-HTP will increase synaptic levels of serotonin.
After this was clarified we were able to move on knowing that 5-HTP would increase
serotonin synthesis, which would in fact lead to an increase in synaptic serotonin.
- 18 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
28.
Retroviral Vectors and Gene Therapy. In: Kufe D, Pollock R, Weichselbaum R,
editors. Holland-Frei Cancer Medicine. 6th ed. Hamilton: BC Decker; 2003. Available
from: http://www.ncbi.nlm.nih.gov/books/NBK13295/
This book section comes from the 6th edition of the "Holland-Frei Cancer Medicine"
textbook. It outlined the method of gene therapy for increasing levels of endogenous
enzymes in the body. It details the specific viruses currently under use and investigation
for gene therapy. This source was used to confirm that gene therapy is required for
increasing enzymes (i.e. TPH2) in the body.
29.
Csermely P, Agoston V, Pongor S. The efficiency of multi-target drugs: the
network approach might help drug design. Trends Pharmacol Sci [serial on the
Internet]. 2005; 26(4): Available from:
http://www.sciencedirect.com/science/article/pii/S0165614705000556.
This article was used to understand the mechanism of action of multi-target drugs and
to gain a deeper understanding of how these drugs are designed. According to this
article multi-target drugs bind with lower affinity to their targets.
30.
Strobel A, Gutknecht L, Rothe C, Reif A, Mossner R, Zeng Y, et al. Allelic
variation in 5-HT1A receptor expression is associated with anxiety- and depressionrelated personality traits. J Neural Transm. 2003 Dec;110(12):1445-53: Available from:
http://www.ncbi.nlm.nih.gov/pubmed/14666415.
This study examines the effect of decreased ligand binding on 5-HT1A receptors in
relations to feelings of anxiety and depression, particularly in patients clinically
diagnosed with the disease. In a group of 284 healthy university students, the
prevalence of a particular gene present on these receptors were used to develop a
relationship between bound receptors and scores on a neuroticism test. Individuals with
increased concentration of bound receptors displayed higher scores on tests of
neuroticism. A significant correlation was determined, and later attributed to further
tests conducted on clinically diagnosed individuals. The publication of this study in a
peer-reviewed journal add to itís credibility.
31.
Khan ZU, Mrzljak L, Gutierrez A, de la Calle A, Goldman-Rakic PS. Prominence
of the dopamine D2 short isoform in dopaminergic pathways. Proc Natl Acad Sci U S A
[serial on the Internet]. 1998 22740]; 95(13): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/9636219.
This article presented a comprehensive view of the location, structure, function and
pharmacology of DA receptors with focus especially on the D2 receptors. The five
dopamine receptors D1, D2, D3, D4, and D5 are categorized into D1-like and D2-like based
on structural and signaling properties. All DA receptors are 7-transmembrane Gcoupled protein receptors that are activated by heterotrimeric GTP-binding proteins and
D1 and D2 receptors are the most abundant in the CNS. D2 receptors are further
differentiated into the D2-short (D2S) and D2-long (D2L) subtype based on the insertion
of 29 amino acids in the third cytoplasmic loop. Although these receptors are very
similar in structure, they differ greatly in function; D2L is postsynaptic receptor while
D2S‟s role as the sole dopaminergic autoreceptor is strongly suggested by studies which
- 19 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
have shown the loss of autoreceptor function in D2-deficient mice. Immunoprecipitation
studies using D2S and D2L selective antibodies show that the presence of D2S is
prominent in the hypothalamus and the midbrain. These areas project to the NAcc,
mPOa and PFC; all of which are areas heavily involved in the mediation of sexual desire.
These information led to the selection of D2S receptor agonism as our loci of
intervention.
32.
Higano CS. Side effects of androgen deprivation therapy: monitoring and
minimizing toxicity. Urology [serial on the Internet]. 2003; 61(2 Suppl 1): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/12667885.
Existing treatments targeting this pathway have failed to prevent relapse of abnormal
sexual behaviour, as well as normalize levels of testosterone in paraphiliacs . Side effects
of such a treatment method also include muscle loss, erectile dysfunction, hot flashes,
and gynecomastia. This review article provides information regarding the current antiandrogen treatments being imposed on sex offenders. Treatment reevaluations have
shown to be ineffective and sex offenders displayed recidivism. Side effects of nausea,
vomiting, hot flashes, dizziness, and breast tenderness were also observed. Although
this study focused on androgen treatment it did mention that a break in treatment, no
matter how long, can have disastrous effects. A break in treatment resulted in regression
and a return to predation. This outlined the serious need for a long term treatment
option. We adapted the information in this source to stress the importance of long-term
therapy. The article‟s credibility is observed in its reference to numerous other studies
and testing procedures.
33.
Hans M, Lowman A. Biodegradable nanoparticles for drug delivery and
targeting. Current opinion in solid state and materials science [serial on the Internet].
2002; 6(4): Available from:
http://www.sciencedirect.com/science/article/pii/S1359028602001171.
This review article explored the use of polymeric nanoparticles as an administration
technique in a wide variety of drug therapies. We used this review article to select the
polymers which would be used, as well as the size of the particles. This article showed us
the advantages of using poly(lactide-co-glycolide) polymers such as biocompatability
and resorbability. It also outlined some ideas on what the ideal size for a microparticle
should be.
34.
Saavedra JM. Distribution of serotonin and synthesizing enzymes in discrete
areas of the brain. Fed Proc [serial on the Internet]. 1977; 36(8): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/872947.
One of the initial worries we had when developing the idea of simply introducing 5-HTP
to increase serotonin, was that we did not know where it would be converted to
serotonin in the brain. This was important to find out because if all the 5-HTP was
converted in one area of the brain not related to any regions involved in lust, we were
uncertain as to whether our idea would work. We had previously learned that levels of
serotonin in the raphe nuclei were associated with the production of sexual thoughts,
before we learned that this region is most concentrated with serotonin. This article
taught us that the amount of serotonin synthesis production in a region of the brain, is
dependent on the concentration of the decarboxylation enzyme. This article also
- 20 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
provided us with the information that this enzyme is most concentrated within the
raphe nuclei, which cleared up our prior concerns and confusions.
35.
Alex KD, Pehek EA. Pharmacologic mechanisms of serotonergic regulation of
dopamine neurotransmission. Pharmacol Therapeut [serial on the Internet]. 2007;
113(2). Doi 10.1016/J.Pharmthera.2006.08.004.
This review article outlines the connection between the dopaminergic system and the
serotonergic systems in the brain. It identifies the location of twelve serotonin receptors
and their subsequent relationship with dopamine in the mesolimbic and mesocortical
pathways in the brain. This article was very important in helping us understand the
correlation of serotonin receptor activation and changes in the levels of dopamine. We
were able to map out the interconnectivity of the two pathways that had an involvement
with lust and were able to solidify the need for a multi target drug. The most important
information we used from this review was the location of the serotonin receptors that
correlate with lust and their role in the dopaminergic pathway. The review had all the
appropriate elements, including an acknowledgements section and two hundred ninety
six references.
36.
Blier P, Abbott FV. Putative mechanisms of action of antidepressant drugs in
affective and anxiety disorders and pain. J Psychiatry Neurosci [serial on the Internet].
2001 1408043]; 26(1): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/11212592.
This review article talked about the desensitization of the 5-HT1A autoreceptor due to
an increase of 5-HT. This was helpful information for our drug as we aim to increase 5HT levels. This alleviates our concerns about the 5-HT levels decreasing because of the
activation of the autoreceptor.
37.
Giulian D, McEwen BS, Pohorecky LA. Altered development of the rat brain
serotonergic system after disruptive neonatal experience. Proc Natl Acad Sci U S A. 1974
Oct;71(10):4106-10: Available from: http://www.pnas.org/content/71/10/4106.full.pdf.
The following study was conducted to determine the effect of disruption to the postnatal
serotonergic system in rat brains. The disruptive event was a cold treatment applied to
the rats after their first postnatal day. Serotonin levels of those treated with a cold
treatment were compared to a control group of rats. Results displayed the levels of 5-HT
released in the forebrains and midbrains of the species examined. These values were
used as a basis for the values of serotonin to be produced by our developed drug. This
article gave us a baseline average serotonin level of 625 ng/g of brain mass in rats, for
the average rat this is 1500 ng. Considering the serotonin range falls within 20% of the
average value, the concentration range should be 520-750 ng/g. Assuming the average
rat brain weighs 2.4 g this gives an actual range of 1248-1800 ng. This means that the
actual difference between the average and extreme value is 300 ng. To increase it this
much, 429 ng of 5-HTP has to be added per day because only 70% of 5-HTP is actually
turned into serotonin. Lastly, to administer this drug over 6 months 77.2 micrograms
has to be put in the capsule. This source is credible due to its extensive use of citations,
and publication through a prominent university institution.
- 21 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
38.
Bowyer JF, Tank AW, Newport GD, Slikker W, Jr., Ali SF, Holson RR. The
influence of environmental temperature on the transient effects of methamphetamine
on dopamine levels and dopamine release in rat striatum. The Journal of Pharmacology
and Experimental Therapeutics [serial on the Internet]. 1992; 260(2): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/1346646.
This article gave us a baseline average dopamine level of 9,400 ng/g of brain mass in
rats, for the average rat this is 22,500 ng. Considering the dopamine range falls within
20% of the average value, the concentration range should be 7,520-11,280 ng/g.
Assuming the average rat brain weighs 2.4 g this gives an actual range of 18,000-27,000
ng. This means the actual difference between the average and extreme values is 4,500
ng. The EC50 of Patrabandole is 7.58 x 10^(-9) mol/L. This has a 20% range of effect
above and below the EC50. Since Patrabandole decreases dopamine levels, to reach the
lower end of the range, the concentration of Patrabandole must be 20 % higher. This
means 9.10 x 10^(-9) mol/L of patrabandole will result in 18,000 mg of dopamine. To
get a mass this value was multiplied by its molar mass of 358 g/mol and multiplied by
the volume of blood in the rat (0.031 L). This gave a value of 10 ng/day. Lastly, the
capsule which has to last 6 months will contain 1800 ng.
39.
Vorvick L. Serum serotonin levels. U.S. National Library of Medicine; 2011
[updated January 2; cited 2012 April 3]; Available from:
http://www.nlm.nih.gov/medlineplus/ency/article/003562.htm.
This article was used to find the normal serum range for serotonin: 101-283 ng/mL.
Based on this information, we decided to target the higher end of this range; namely,
250-280 mg/mL. Even though this source is not from a peer reviewed journal, it is from
MedlinePlus, a well-known medical encyclopaedia. It was also written by the medical
director at the University of Washington School of Medicine.
40.
Hoare T. Microparticles and drug administration. In: Innana T, editor.
[Interview] ed. Hamilton; 2012.
Our interview with Dr. Todd Hoare allowed us to determine our finalized method of
administration (controlled-release microparticles). One thing which was learned in
meeting with Dr. Hoare, is that patrabandol, our roxindole analogue, would be very
hydrophobic given the four aromatic rings in its structure, and 5-HTP would likewise be
hydrophobic due to its two existing aromatic rings. Since both 5-HTP and patabrandole
are hydrophobic, a pellet depot injection (comprising of a water-based capsule) would
not be feasible. Additionally, it was noted that caution must be taken in choosing to
utilize a biodegradable microsphere due to the launch of a possible immune response
against the introduced capsule. With respect to our drug, the rate of release is based on
the ratio of glycolic acid to lactic acid. A pure glycolic acid polymer will take 3 weeks to
degrade, while an all lactic acid one will take 8 months. Bioaccumulation of lactic acid
can lead to undesirable side effects like muscle soreness, however, adjustment to the
rate of release can minimize this result. The release rate of the molecule can be changed
by changing the ratio of the acids. Thus a 6 month release would require a composition
of 70% lactic acid and 30% glycolic acid. Our interview with Dr. Hoare also allowed us to
develop the following test to determine if microparticles were an effective method of
administration.
- 22 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
Test: Efficiency of Controlled-Release Microparticles
Purpose: To determine the efficiency of Nadamo controlled-release microparticles
Procedure: 1 mg of each of 5-HTP microparticles and patabrandole microparticles were
placed in a dialysis bag containing 5% saline solution, which was then submerged in a
phosphate buffer bath at 37 degrees Celsius. Samples were taken from the buffer
solution in order to measure the concentrations of the two ligands. For the first 62
hours, these measurements were taken hourly. For the next 14 days following that,
measurements were taken daily. For the following 3 weeks, measurements were taken
weekly. Subsequent measurements were taken on a monthly basis.
Results: The results indicated that Nadamo had completely dissolved after
approximately 178 days.
41.
Banks WA. Characteristics of compounds that cross the blood-brain barrier. Bmc
Neurol [serial on the Internet]. 2009; 9: Available from:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697631/. Artn S3
Doi 10.1186/1471-2377-9-S1-S3.
Despite understanding that Nadamo would be hydrophobic, we needed to learn what
was required of a molecule to pass the blood brain barrier. This paper provides a very
clear breakdown of what exactly is needed for passing of the blood brain barrier. This
source provided the evidence needed to clarify that patrabandol and 5-HTP would pass
the blood brain barrier, since the mechanism of transmembrane diffusion, which would
facilitate the crossing, favors hydrophobic substances.
42.
Newman-Tancredi A, Cussac D, Audinot V, Millan MJ. Actions of roxindole at
recombinant human dopamine D2, D3 and D4 and serotonin 5-HT1A, 5-HT1B and 5HT1D receptors. Naunyn Schmiedebergs Arch Pharmacol [serial on the Internet]. 1999;
359(6): Available from: http://www.springerlink.com/content/p85um6hyuqwqu30g/.
10.1007/PL00005374.
This study examines the affinity of roxindole on 5-HT and dopamine receptors and the
dosages of roxindole needed to elicit an effect on 5-HT and dopamine receptors. pKi
values describe affinity, E(max) values describe the maximal obtainable effect when all
receptors are occupied, and EC50 values describes the value needed to achieve half of
the maximum dose response in the average person. In our studies, 100% Emax was set
to the body‟s endogenous serotonin and dopamine levels; this served as a comparative
value for the ideal binding affinity and efficacy. The D2, D3, and D4 receptors have pKi
values of 8.93, 8.55 and 8.23, respectively; E(max) values of 10.5, 30.0, and 35.1,
respectively (all in %); and pEC50 values of 7.88, 9.23, and 7.98, respectively. The 5HT(1A), 5-HT(1B), and 5-HT(1D) have pKi values 9,42, 6.00 and 7.05, respectively;
E(max) values of 59.6, 27.1, and 13.7, respectively (all in %); and pEC50 values of 8.28,
5.83, and 7.80, respectively. This article was used to establish the comparative effect of
roxindole on serotonin and dopamine. For the purposes of the research conducted by
our department, [35S]GTPS binding assays were used, along with Chinese hamster
ovary (CHO) cells programmed to express either human D1, D2L, D2S (autoreceptor),
D3, D4, D5, 5-HT1A, 5-HT1B or 5-HT2A receptors. Each CHO cell membrane
- 23 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
expressing a specific receptor were incubated for 20 min at 22°C in triplicate with 5-HT,
DA, and PTB in a buffer containing 20 mM HEPES, 3 PM GDP , 3 mM MgS04, and ~0.1
~nM [35S]GTPyS ~(1300 Ci/mmol, ~NEN) . Incubations were terminated by rapid
filtration. This process was repeated for each CHO cell expressing a 5-HT receptor.
Information gathered helped in not only identifying agonist efficacy, but also the
necessary modifications needed for roxindole in order to prevent serotonin syndrome
when roxindole and 5-HTP (a serotonin precursor) are administered in the same depot
capsule. This conclusion is based on the fact that the difference in pEC50 of 5-HT(1A)
and D2 separated by only 0.4 log units (2.0 log units is needed to avoid unwanted
effects). Our group determined that all serotonergic effects of roxindole needed to be
eliminated. This article is credible due to its acceptance in a reputable peer-reviewed
journal and proper use of citations.
Using the experimental methods of the study, the roxindole analog patrabandole,
developed by Team Inanna, was analyzed. It was determined that patrabandole had the
following pEC50 values: 8.12, 5.49, and 5.51 for D2, D3, and D4, respectively; and 5.98,
6.78, and 5.69 for HT(1A), HT(1B), and HT(1D), respectively.
43.
Extracts L. L-5-Hydroxytryptophan Griffonia Seed Extract. Easton; 2000
[updated January 17; cited 2012 April 2].
This review of a 5-HTP supplement was used to determine the rate of absorption and
excretion from 5-HTP pill. This was used as a basis for developing the percentages
pertaining to our own drug. According to this review, about 70% of 5-HTP is absorbed,
and 30% excreted. We chose an absorption rate of 73% for our own drug. Knowing the
higher ends of the normal ranges of intracerebral serotonin and dopamine in humans,
these combined values would have to account for the 73% absorbed. Therefore, 127% of
the drug dosage should be found within our capsule in order to release the required
levels. This would correspondingly account for 73% of our drug absorbed. To determine
the percent release of our drug per day, the dosage present in our capsule (127%) was
divided by the 180-day time span to obtain a value of 0.778% daily. This article is
credible due to its use of numerous citations.
44.
Ryckmans T, Edwards MP, Horne VA, Correia AM, Owen DR, Thompson LR, et
al. Rapid assessment of a novel series of selective CB(2) agonists using parallel synthesis
protocols: A Lipophilic Efficiency (LipE) analysis. Bioorg Med Chem Lett [serial on the
Internet]. 2009; 19(15): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/19500981. Doi 10.1016/j.bmcl.2009.05.062.
This article provides information on how to calculate lipophilic efficiency (LiPe) based
on logP and pEC50 values using the following formula: pEC50-logP. This formula was
then used to calculate LiPe for patrabandole in the following manner: pEC50-LogP =
8.12 - 3.038 = 5.082. The article is credible due to its use of proper citations and its
acceptance in a peer-reviewed journal.
45.
Jabeen I, Pleban K, Rinner U, Chiba P, Ecker GF. Structure-Activity
Relationships, Ligand Efficiency, and Lipophilic Efficiency Profiles of BenzophenoneType Inhibitors of the Multidrug Transporter P-Glycoprotein. J Med Chem [serial on the
- 24 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
Internet]. 2012: Available from: http://www.ncbi.nlm.nih.gov/pubmed/22452412. Doi
10.1021/jm201705f.
This article analyzes a several features of capabilities of benzophenones. It also provides
detailed information on the ideal logP and LiPe values. This article was used to help
determine and calculate logP and LiPe values for patrabandole. Most significantly, it
identified that the ideal logP values of an effective receptor based drug to be 2-3 and the
ideal LiPe value to be greater than 5. It is credible due to its acceptance in a credible
peer-reviewed journal and use of citations.
46.
White B. Calculate Approximate logP. University of Massachusetts Boston;
[cited 2012 March 30]; Available from: http://intro.bio.umb.edu/111112/OLLM/111F98/jlogp/test.html.
This site was was used to calculate logP. Based on its structure, patrabandole has a logP
value of 3.038. The site is credible as it was created and is maintained by the University
of Massachusetts Boston.
47.
Eyles DW, Rogers F, Buller K, McGrath JJ, Ko P, French K, et al. Developmental
vitamin D (DVD) deficiency in the rat alters adult behaviour independently of HPA
function. Psychoneuroendocrinology [serial on the Internet]. 2006; 31(8): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/16890375. Doi
10.1016/j.psyneuen.2006.05.006.
This website introduced us to the Sprague Dawley rat which was used for testing on
multiple occasions. Sprague Dawley rats were used in test one, test four and test five.
These rats are commonly used in testing areas including toxicology, pharmacology,
reproduction and behavioural testing. This article gave us information about testing in
rats such as age (7 to 8 months), life span (2.5-3.5 years), and average litter size (6-12
pups). It also referenced pre-testing conditions such as observing the rats for rashes on
the skin and parasite infections, and testing to determine the age of sexual maturity, by
testing each rat for first copulation by exposing them to a receptive female within 35
days of birth. In certain tests, such as test 4, this article referenced specific testing
criteria. In test 4, pregnant females were occasionally removed from the room and
replaced with non-pregnant, sexually active females. After this period of time, that
lasted 1 month, males were put into revolving drums (Slonaker type) and kept there for
5 weeks – one week for preliminary adjustment and 4 weeks for collecting data on
activity and vigor. Then, they were moved back to the laboratory cages with dimensions
of 45 cm. x 45 cm. x 20 cm in groups of 4 and were tested for their copulatory frequency.
48.
Hyttel J, Overo KF, Arnt J. Biochemical effects and drug levels in rats after longterm treatment with the specific 5-HT-uptake inhibitor, citalopram.
Psychopharmacology. 1984;83(1):20-7: Available from:
http://www.ncbi.nlm.nih.gov/pubmed/6429698.
The testing method outlined in this research study was applied to our own drug testing
procedures. A population of rats were decapitated, their brains rapidly removed, and
subsequently placed in a saline solution. Sections of the prefrontal cortex were
subsequently homogenized (i.e. diluted through crushing of the tissue sample till evenly
dispersed in solution). The samples were then measured for fluorescence to determine
- 25 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
the presence of each neurotransmitter and their concentrations. Our research methods
were adopted from this study to apply to both serotonin and dopamine release in tests
one and three. The measurement of 5-HT and DA content followed the procedures of
Yuwiler et. al. referenced in this paper. In test one of our studies brain levels of 5-HT
and DA were measured in rats administered increasing concentrations of 5-HTP and
PTB. Rats (n=100) were divided into groups of four with each group receiving a specific
concentration of 5-HTP and PTB via intramuscular injection. Resulting concentrations
of serotonin and dopamine were attributed to given dosages of 5-HTP, solidifying the
doses of the precursor and agonist required to attain the desired heightened and
lowered neurotransmitter levels respectively.
For the purposes of test three male rats (n=160) were tested in 32 groups of 5 at
specified time intervals. Each rat was administered the same concentration of Nadamo
(77.2 µg of 5-HTP and 1.8 µg of PTB). A group of rats were randomly selected for
decapitation daily for 14 days. CNS 5-HT and DA concentrations were measured
following homogenization of the raphe nuclei, mPOA, PFC, and VTA using
spectrofluorometry. After this 14 day period, another group of rats was decapitated
every week for 14 weeks. After this 14 week period, a newly selected group was
decapitated every 2 weeks for 8 weeks. The same methods in the first time interval were
applied to the following two. Standard reagent samples of 5-HT and DA were measured
for fluorescence, and a wavelength maxima of 545 nm and 447 nm respectively was
determined. Fluorescence of the samples were then determined and attributed to the
levels of 5-HT converted from its precursor 5-HTP and DA released. A gradual increase
of neurotransmitter levels was observed in the first 14 days followed by stabilization of
these levels after this period in time, validating our drugs ability to maintain levels of
our targeted molecules. This study serves as a credible source for its publication in a
well-established peer review journal.
49.
Curzon G, Green AR. Rapid method for the determination of 5hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain. Br J
Pharmacol [serial on the Internet]. 1970 1702616]; 39(3): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/5472211.
The testing method in this study also provides detail into the testing of 5-HT
concentrations in rat population‟s brains. Rats were decapitated, and whole brains
homogenized in n-butanol. 10 mL aliquots of these homogenized solutions were
extracted and tested using spectrofluorometry. Wavelengths of 360 nm and 470 nm
were determined. This article presented us with the method of fluorescence
measurement for the identification of the neurotransmitters in our test samples. The
wavelengths of the fluorescence emitted indicated to us what neurotransmitters were
existent, while the absorption measurement was used to determined the amount of the
neurotransmitter present. Standard reagent samples were then tested for fluorescence
and showed identical wavelength peaks to the rat population. The validity of this article
is displayed due to its numerous citations.
50.
Korf J, Venema K, Postema F. Decarboxylation of exogenous L-5hydroxytryptophan after destruction of the cerebral raphe system. J Neurochem.
[Comparative Study]. 1974 Jul;23(1):249-52: Available from:
http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.1974.tb06941.x/pdf.
- 26 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
This research article was used to determine the levels of 5-HT produced in response to
5-HTP both in a control group of male rats, as well as in two separate groups of rats
pretreated an enzyme inhibitor. Normal conversion levels of 5-HTP to 5-HT in the
control group was used as the basis for the data in our own drug testing methods. Values
of 5-HTP were extrapolated to fit our heightened range of 5-HT to be produced.
Additionally, data from this study displayed decreased conversion of 5-HTP to 5-HT
following pretreatment with an synthesis enzyme inhibitor (L-a-hydrazino-a-methyl
dopa). This source is credible due to its numerous use of citations.
51.
Seeley TT, Abramson PR, Perry LB, Rothblatt AB, Seeley DM. Thermographic
measurement of sexual arousal: a methodological note. Arch Sex Behav [serial on the
Internet]. 1980; 9(2): Available from: http://www.ncbi.nlm.nih.gov/pubmed/7396689.
This article outlines the method of Thermographic Measurement to test different stages
such as excitement phase, plateau phase, orgasm phase and resolution phase of sexual
arousal. The changes in levels of sexual arousal were measured by analyzing the created
thermograms at different stages. Through this article it was learned that thermograms
detect subtle elevations in temperature due to excessive cellular and metabolic activity
that are typical during changes in levels of sexual arousal. The method and test type
outlined in this article was used to test the levels of sexual arousal in male rats during
the activation trials for test # 4.
52.
Stone C, Tomilin M, Barker R. A comparative study of sexual drive in adult male
rats as measured by direct copulatory tests and by the Columbia obstruction apparatus.
Journal of Comparative Psychology [serial on the Internet]. 1935; 19(2): Available from:
http://psycnet.apa.org/index.cfm?fa=buy.optionToBuy&id=1935-04183-001.
This article describes two testing methods, direct copulation frequencies and the
Columbian obstruction apparatus, which can be used to measure sexual drive in male
rats. The information extrapolated from this article was used to design the test to
measure the effects of our drug, Nadamo, on sexual desire in rats. In the study a group
of albino rats, between the ages of 7 to 8 months with weight ranging between 278 ±3.94
grams were used. All the pre-test conditions, such as no rashes on the skin and no
parasite infestations, were kept constant for all the rats used throughout the experiment.
In order to determine the age of sexual maturity, indicated by the first copulation, each
rat was tested with a receptive female within 35 days after birth. After the copulatory
ability of each rat was established, each male rat was separated from the female and put
in a group of 5 or 6 rats in a separate cage until the age of 5 months. After the rats
reached the age of 5 months, they were all released in a room where about 24 female
rats were introduced so that the males would be exposed to sexual experiences and
promiscuity. From time to time, the pregnant females were removed from the room and
re-placed with non-pregnant sexually active females. After this period of time, that
lasted 1 month, males were put into revolving drums (Slonaker type) and kept there for
5 weeks – one week for preliminary adjustment and 4 weeks for collecting data on
activity and vigor. Then, they were moved back to the laboratory cages with dimensions
of 45 cm x 45 cm x 20 cm in groups of four and were tested for their copulatory
frequency. Five or ten minutes before the beginning of the testing an „activation trial‟
was introduced where each male was exposed to a highly receptive female and allowed
- 27 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
to copulate 2 to 3 times. The purpose of these „activation trials‟ was to arouse each male
to a high state of copulatory readiness. Following the above mentioned preparation, all
the rats were tested for their copulatory frequency. It was noticed that most rats showed
high levels of sexual desire, followed by productive sexual activity, and only 2 male rats
failed to copulate.
This article is credible because it is published by the American Psychological
Association, a reputable source that contains a collection of psychology journals.
53.
Harms LR, Eyles DW, McGrath JJ, Mackay-Sim A, Burne TH. Developmental
vitamin D deficiency alters adult behaviour in 129/SvJ and C57BL/6J mice. Behav Brain
Res. [Comparative Study]. 2008 Mar 5;187(2):343-50: Available from:
http://www.ncbi.nlm.nih.gov/pubmed/16890375.
This website introduced us to the Sprague Dawley rat which was used for testing on
multiple occasions. Sprague Dawley rats were used in test one, test four and test five.
These rats are commonly used in testing areas including toxicology, pharmacology,
reproduction and behavioural testing. This article gave us information about testing in
rats such as age (7 to 8 months), life span (2.5-3.5 years), and average litter size (6-12
pups). It also referenced pre-testing conditions such as observing the rats for rashes on
the skin and parasite infections, and testing to determine the age of sexual maturity, by
testing each rat for first copulation by exposing them to a receptive female within 35
days of birth. In certain tests, such as test # 4, this article referenced specific testing
criteria. In test 4 pregnant females were occasionally removed from the room and
replaced with non-pregnant, sexually active females. After this period of time, that
lasted 1 month, males were put into revolving drums (Slonaker type) and kept there for
5 weeks – one week for preliminary adjustment and 4 weeks for collecting data on
activity and vigor. Then, they were moved back to the laboratory cages with dimensions
of 45 cm x 45 cm x 20 cm in groups of four and were tested for their copulatory
frequency.
54.
Rozas G, Garcia JLL. Drug-free evaluation of rat models of parkinsonism and
nigral grafts using a new automated rotarod test. Brain Research [serial on the
Internet]. 1997; 749(2): Available from:
http://www.sciencedirect.com/science/article/pii/S0006899396011626.
This article was used to design a rotarod test for Nadamo. This test measured the
balance of rats walking on a rod rotating at varying speeds. Before the test began, the
rats were placed on the rotarod machine, where they were allowed to acclimate to the
new environment. Practice trials on the rotarod were also run (before the initial drug
administration). Rats received a mild electrical shock after falling on the pad; this was
intended as a deterrent against falling. The rotarod machine was placed at subsequently
increasing revolutions per minute (r.p.m.): 3, 8, 12, 15, 18, 21, 25, 29, 33, 37, 41, 45, 49,
and 54 r.p.m. Each revolution was maintained for 5 minutes. Rats were allowed 25-30
minutes to rest between each speed increase. The time and rotation at which the rats fell
off the rotomotor machine was recorded. The trial was stopped once the rat fell off the
machine. This test was repeated on three consecutive days each week.
Results: No significant difference was found between the experimental group and the
control group. Skill was found to increase with time. This is likely due to the rats
becoming accustomed to the test.
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
55.
Burnea T, McGrathb J, Eylesb D, Mackay-Sim A. Behavioural characterization
of Vitamin D receptor knockout mice. Archives of Biochemistry and Biophysics [serial
on the Internet]. 2005; 157(2): Available from:
http://www.sciencedirect.com/science/article/pii/S0166432804002827. Doi
10.1016/j.bbr.2004.07.008.
This article discusses behavioural tests run on vitamin D receptor knockout mice. It was
used to determine the stages of SHIRPA testing. Based on this information, SHIRPA
tests were conducted to determine Nadamo's effects on mood and behaviour. This
article also provided the basis for all of the mood tests conducted thus far and served as
an introduction to the various mood tests available, including SHIRPA, rotarod, gait
analysis, marble burying test, Y-maze, and holeboard tests. Rotarod and gait analysis
tests measured locomotion, while the latter three measured anxiety. This article was also
used to design a gait analysis test for Nadamo. In this test, rats were placed on a glass
runway lit by a dim red light in an otherwise dark room. A camera was also placed in the
room. The rats were observed for three minutes, during which time the rats walked
across the runway. Video frames were played afterwards, and gait was observed.
Results: A marked difference was seen in the experimental group‟s results when
compared to the control group‟s. This is not surprising, considering PTB's hypotonic
effects. The greatest difference in gait analysis was seen in the size of steps taken by the
rats. Rats in the experimental group showed a significantly shorter average step (x bar =
2.77 mm, P < 0.05) than the control group (x bar = 3.67 mm, P < 0.05).
56.
Njunge K, Handley SL. Evaluation of Marble-Burying Behavior as a Model of
Anxiety. Pharmacol Biochem Be [serial on the Internet]. 1991; 38(1): Available from:
http://www.sciencedirect.com/science/article/pii/009130579190590X.
This article outlined the role of anxiogenic agents, such as diazepam yohimbine and
zimeldine, in altering the locomotive behaviour and the burying reflexes associated with
feelings of anxiety. The article describes the marble-burying test, used to determine
Nadamo's effects on mood/anxiety. Twenty groups of female rats were prepared for
testing by being kept in conditions with twelve hours of light and twelve hours of dark a
day, along with a free access to food and water three days prior to testing. Each rat was
placed in a cage with 1-inch deep sawdust.12 marbles were semi-buried in various
locations around the cage. The marbles were planted by ungloved hands; this allowed
the rats to detect the presence of a foreign body and, consequently, become guarded and
territorial. This territoriality leads to burying reflexes. Rats remained in the cage for 30
minutes. At the end of this time, the number of buried marbles (at least 2/3 covered)
were counted.
This test was performed once a week for 18 weeks. Three trials were performed during
each test. Results: It was found that the number of marbles buried after 30 minutes in
both the experimental and control groups was roughly equal. These results suggest
Nadamo has no effect on anxiety. The article had all the appropriate formatting and
referencing, and included an acknowledgements section as well as a list of twenty-two
references.
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
57.
Yamazaki K, Yamaguchi M, Baranoski L, Bard J, Boyse EA, Thomas L.
Recognition among Mice - Evidence from the Use of a Y-Maze Differentially Scented by
Congenic Mice of Different Major Histocompatibility Types. J Exp Med [serial on the
Internet]. 1979; 150(4): Available from:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2185685/pdf/je1504755.pdf.
This article describes the Y-maze test, which analyses learning and memory (spatial
memory recognition) in rats exposed to a drug. In this case, Nadamo was used. The Ymaze is a structure specifically designed to test learning and memory. It is made of gray
plastic, and consists of one long arm, which eventually forks into two smaller arms.
Gates are present in all three arms. The floor of the maze was covered with sawdust
purified to remove any odours. Before the test begun, the mouse was exposed to a novel
substance.
58.
Dellu F, Mayo W, Cherkaoui J, Le Moal M, Simon H. A two-trial memory task
with automated recording: study in young and aged rats. Brain Research [serial on the
Internet]. 1992; 588(1): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/1393562.
This article gave us a basis for the Y-maze that was conducted on mood. In this
experiment, the novel substance was a small rock of unusual texture and pattern. The
rock was purified to remove all odours. In the beginning of the test, the mouse was
placed in compartment S. The gate was opened, and the mouse was allowed to roam
freely between the starting arm and the “other” arm (namely, the arm without the rock).
The gate leading to the rock was closed until 15 minutes of the experiment had passed.
The amount of time the rat spent in each of the two/three arms was recorded. It was
hypothesised that the rat should spend equal times in each of the two arms when only
they were open, and then the majority of the time in the rock arm once the gate was
opened, since rats have an innate attraction to novelty.
59.
Wright RL, Conrad CD. Chronic stress leaves novelty-seeking behavior intact
while impairing spatial recognition memory in the Y-maze. Stress [serial on the
Internet]. 2005; 8(2): Available from:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2185685/pdf/je1504755.pdf. Doi
10.1080/10253890500156663.
This article gave us a basis for the results of the Y-maze experiment. The results were as
follows. There was a 12% increase in the time taken to cross the Y-maze when the rats
were under the influence of Nadamo. This signifies a significant difference between the
results of the Y-maze in the experimental and control groups. Note: One rat from the
experimental group did not enter the novelty arm at all; thus, its data was excluded from
the compilation of results. An interesting pattern to note is that the rats seemed to
prefer (excluding the experimental weeks 1-9) spending time in the other arm to the
starting arm.
60.
Gil M, Marti J, Armario A. Inhibition of catecholamine synthesis depresses
behavior of rats in the holeboard and forced swim tests: influence of previous chronic
stress. Pharmacol Biochem Behav [serial on the Internet]. 1992; 43(2): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/1359582.
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Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
This article talks about the catecholamine pathways in the brain and how they are
increased during stress. This increase in pathway activity can have a very serious effect
on physiology and behaviour. If stressed enough this could even induce adaptive
changes that have an even longer lasting effect. This article was used to not only predict
the results of our behavioural tests but also to interpret what the results mean. The
effect Nadamo played on the behaviour could be indicative of depression-like or high
anxiety states.
61.
Sanz EJ, De-las-Cuevas C, Kiuru A, Bate A, Edwards R. Selective serotonin
reuptake inhibitors in pregnant women and neonatal withdrawal syndrome: a database
analysis. Lancet [serial on the Internet]. 2005; 365(9458): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/15705457. Doi 10.1016/S0140-6736(05)178659.
This article describes severe conditions associated with excess serotonin in the body.
These include dyskinesia and serotonin syndrome. Dyskinesia is a movement disorder
characterized by decreased voluntary, and increased involuntary movement. Serotonin
syndrome results from excess serotonin production, which in some cases can be life
threatening. Furthermore, serotonin withdrawal symptoms have been experienced
when serotonin-increasing drugs (such as SSRI's) were stopped. This information
helped us decide to create a constant, slow-release drug, rather than bombarding the
body with a high dose at one time.
62.
Jose PA, Raymond JR, Bates MD, Aperia A, Felder RA, Carey RM. The renal
dopamine receptors. J Am Soc Nephrol [serial on the Internet]. 1992; 2(8): Available
from: http://www.ncbi.nlm.nih.gov/pubmed/1627751.
This article records the presence of dopamine receptors in the renal system. This
presence explains the possible adverse renal effects of Nadamo, which include
vasoconstriction of renal tubular pathways involved in excretion processes. This article
also verifies the presence of the D2short and D2long receptor subtypes. D2short
receptors are autoreceptors that, when agonised, inhibit the release of dopamine,
leading to the aforementioned side effect.
63.
De Ponti F, Tonini M. Irritable bowel syndrome: new agents targeting serotonin
receptor subtypes. Drugs [serial on the Internet]. 2001; 61(3): Available from:
http://www.ncbi.nlm.nih.gov/pubmed/11293643.
This article explores different treatment methods for gastrointestinal problems. It was
used to gain a thorough understanding of the connection between increased serotonin
peripheral levels and high motility rates. Through this paper it was learned that
increased serotonin levels would potentially affect the motility of the GI tract because of
the presence of 5-HT3 and 5-HT4 receptors in this location. Specifically, binding of
serotonin to 5-HT4 receptors leads to diarrhea, while antagonism of 5-HT3 receptors
causes constipation. This knowledge helped determine some of Nadamo's potential
adverse effects. This source is credible because it was published in a reputable scientific
database and was well referenced allowing the information to be corroborated.
64.
Fozard JR, Palfreyman MG. Metoclopramide antagonism of 5hydroxytryptophan-induced 'wet-dog' shake behaviour in the rat [proceedings]. Br J
- 31 -
Group
Name:
Team Inanna
Student Priyank, Nathan, Alexa, Beatrice, Tul-Zahra, Alex, Amrit,
Names: Danusha, Tina
Pharmacol [serial on the Internet]. 1978 1668426]; 63(2): Available from:
http://www.springerlink.com/content/wr8676g1328rxl66/.
AADC inhibitor would be needed if severe side effects, most likely in the GI tract, are
exhibited due to a significant increase in peripheral serotonin. When taking this into
consideration we looked into how side effects as a result of increased peripheral
serotonin could be reduced. This paper was used in researching the effectiveness of
using an AADC inhibitor on produced serotonin levels. What was discovered while
reading this paper, is that when using an AADC inhibitor in lowering serotonin levels,
another side effect called wet dog shakes, is a result. What was most significant in this
report, is that they compared 5-HTP injections and AADC inhibitors on brain serotonin
and experienced wet dog shakes. This was important to look at in the report, since
increasing peripheral 5-HTP is what Nadamo is doing as well. What was discovered in
this report is that when excess 5-HTP was introduced in the periphery, minor tremors
were experienced in the rats, yet when an AADC inhibitor was introduced the frequency
of the wet dog shakes increased from 1 to 78 in 140 minutes. This significant increase in
shakes as a result of using an AADC inhibitor is an additional side effect which would
have to be taken into consideration if it were to be co-prescribed.
65.
Cohen MA. The monetary value of saving a high-risk youth. J Quant Criminol.
1998 Mar;14(1):5-33: Available from:
http://www.byep.org/cost%20of%20not%20saving%20youth.pdf.
This article describes the necessity of having youth-programs for high-risk youth in
Canada and elsewhere. Also, it outlines some of the benefits of having these youth
oriented programs. Through this article it was learned that the government faces heavy
losses in terms of resources due to sexual crimes and that the criminal career of highrisk adult sex offenders in Canada exceeds one million dollars. This source is credible
because it is well referenced, allowing the readers to corroborate the information in it.
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