A comparison of two formulations of intradermal capsaicin as models of neuropathic pain in healthy volunteers

A comparison of two formulations of intradermal capsaicin
as models of neuropathic pain in healthy volunteers.




Master Thesis in Pharmacy
The Pharmacy Programme





Johanna Åkesson










Supervisor: Prof. Paul Rolan
Discipline of Pharmacology, School of Medical Sciences,
University of Adelaide, Australia







Göteborg, Sweden 2007

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Abstract
Background: Intradermal capsaicin is a putative human pain model that produces reliable pain
perception, replicating the symptoms for neuropathic pain. It facilitates controlled testing of
analgesic efficacy via cross-over-design in healthy volunteers. A formulation with capsaicin, which
is a solution in lower concentrations and a colloidal suspension in higher concentrations, has been
used extensively but it is associated with several disadvantages. A new beneficial formulation, a
solution in all tried concentrations, with capsaicin dissolved in a (2-hydroxypropyl)-?-cyclodextrin-
vehicle, (HP-?-CD), has been investigated. In order to have confidence in the utility of the
HP-?-CD-formulation of capsaicin, a clinical validation-study comparing the two formulations was
required.
Methods: 1, 10, 30 and 100 µg doses of both formulations were given as 10 µL intradermal
injections in a blinded, randomized, cross-over manner to sixteen volunteers (8M/8F). Spontaneous
pain (rated on VAS) and hyperalgesia (standardized von Frey hair) were assessed at intervals up to
one hour post-injection.
Results: The two formulations produced pharmacodynamic comparable responses to each other, for
the three lowest doses for both outcomes. The fixed effects of formulation, dose and
formulation•dose were significant affecting each outcome. Gender, arm position and dominance
were also significant affecting hyperalgesia.
Conclusions: The formulations can be considered to be pharmacodynamic comparable for the three
lowest doses, which may be the doses most suitable for clinical use. Both formulations can be
considered to be safe and tolerable but the HP-?-CD-formulation exhibits pharmaceutical benefits.
These findings complemented and extended the knowledge about intradermal capsaicin as a model
for neuropathic pain.

Key Words: human pain model, capsaicin, allodynia, flare, hyperalgesia, pain, neuropathic pain

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ABSTRACT __________________________________________________________________________________ 2
DECLARATION ______________________________________________________________________________ 4
1. INTRODUCTION ___________________________________________________________________________ 5
1.1 BACKGROUND_____________________________________________________________________________ 5
1.1.2 Neuropathic pain ______________________________________________________________________ 5
1.1.3 Pain models __________________________________________________________________________ 5
1.1.4 Capsaicin and the TRPV1-receptor ________________________________________________________ 5
1.1.5 Capsaicin formulations _________________________________________________________________ 6
1.2 AIM FOR THE STUDY ________________________________________________________________________ 7
1.2.1 Hypothesis ___________________________________________________________________________ 7
2. METHODS AND MATERIALS ________________________________________________________________ 8
2.1 TRIAL DESIGN AND SUBJECTS _________________________________________________________________ 8
2.2 CAPSAICIN INJECTION _______________________________________________________________________ 9
2.3 HPLC ANALYSIS___________________________________________________________________________ 9
2.4 PAIN ASSESSMENTS________________________________________________________________________ 10
2.4.1 Determination of the spontaneous pain ____________________________________________________ 10
2.4.2 Determination of the average radius of hyperalgesia _________________________________________ 10
2.5 STATISTICAL ANALYSIS ____________________________________________________________________ 10
3. RESULTS _________________________________________________________________________________ 12
3.2 SPONTANEOUS PAIN (VAS) _________________________________________________________________ 13
3.3 HYPERALGESIA (VON FREY TEST) ____________________________________________________________ 17
3.4 HPLC ASSAY ____________________________________________________________________________ 20
4. DISCUSSION ______________________________________________________________________________ 21
4.1 SPONTANEOUS PAIN (VAS) _________________________________________________________________ 22
4.2 HYPERALGESIA (VON FREY TEST) ____________________________________________________________ 22
4.3 HPLC ASSAY ____________________________________________________________________________ 23
4.4 SOURCES OF VARIABILITY___________________________________________________________________ 24
5. CONCLUSIONS____________________________________________________________________________ 26
6. ACKNOWLEDGEMENTS ___________________________________________________________________ 27
7. REFERENCES _____________________________________________________________________________ 28
APPENDIX 1: PROTOCOL APPLICATION FOR SUBMISSION TO THE ROYAL ADELAIDE HOSPITAL
RESEARCH ETHICS COMMITTEE & INVESTIGATIONAL DRUG SUB-COMMITTEE
APPENDIX 2: PHARMACY MANUAL

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Declaration
This thesis is prepared as a part of our degree of Master of Science in Pharmacy at Göteborg
University.

The thesis is based on original data, obtained while we were exchange students at the Discipline of
Pharmacology at the University of Adelaide, South Australia in the period July - December 2007.

The clinical trial was conducted in a team environment and the trial was dependent on contributions
from:
Chai Li Lau, for the development of a HP-?-CD formulation of capsaicin and HPLC-
assay after the trial.
Pharmacist Sharon Yap, for assisting us in the preparation of the actual formulations
used in the trail.
Statistician Lisa Miller for creating a randomization list and conducting the statistical
analysis.
Prof. Paul Rolan for assisting us in the writing and submitting of the protocol to the
Research Ethics Committee.

However, we were the persons responsible for conducting the complete trial, which includes the
following:
• Co-ordinate the whole study
• Write the protocol
• Write answers to the Investigational Drug Subcommittee
• Prepare and perform test-batches of both formulations
• Write the pharmacy manual and batch sheets
• Perform the first production steps of the actual formulations used in the trial
• Perform a literature study on previous capsaicin studies with similar design
• Develop the methods and writing the SOPs
• Create case report forms
• Prepare material required in the trial and write the schedule for the trial
• Execute and plan all pharmacodynamic measurements
• Assess the four outcomes during the trial
• Work-up the data and create a data file with all results
• Interpret and analyse the data and write a own personal thesis







Adelaide, December 2007





Johanna
Åkesson
Helena
Gustafsson



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1. Introduction
1.1 Background
1.1.2 Neuropathic pain
The mechanisms responsible for different types of clinical pain are only partly understood.
Neuropathic pain is a type of chronic pain caused by the damage or malfunctioning of the peripheral
or central nervous system and it may be unrelated to ongoing tissue damage or inflammation [1]. It
often occurs in a number of clinical conditions, such as post-herpetic neuralgia, diabetic neuropathy,
phantom limb pain, post stroke pain and peripheral neuropathies. Unlike physiological pain, which
serve to warn and protect humans from injury, neuropathic pain serves no useful purpose. Current
treatments are only partly effective (e.g. opioids, gapabentin, pregabalin), are limited by side effects
(e.g. tricyclic antidepressants (amitryptylline) or carbamazepine) or are or are difficult to control
(NMDA receptor antagonists). Beside these factors, a high degree of variability exists clinically
between patients in their response to treatment. The prevalence of neuropathic pain is still unknown,
but estimates indicate that 1 % of individuals in UK experience some form of neuropathic pain.
This rate is probably an underestimate [1]. There are many potential new treatments in development
for neuropathic pain.
1.1.3 Pain models
The use of the clinical pain state has evident limitations in evaluating analgesic interactions. Pain
states are often multi-factorial (e.g. tissue- and nerve injury and inflammation) with treatment-
regimens involving multiple medications which convey that controlled crossover interactions
cannot typically be conducted on the same subject [2]. Evaluation of potential new agents treating
neuropathic pain may be helped by the use of pain models in early clinical development. Volunteer
models of pain permit a more well-controlled study design and are useful in the study of specific
pain mechanisms. Due to these reasons, volunteer models are used in clinical drug development to
demonstrate the analgesic potential of new compounds [2]. A pain model deployed in a limited
number of human subjects would be clearly advantageous in reducing early development time and
costs [3] as well as the number of volunteers needed. The selection of a model should be based on
the mechanism of pain targeted by the compound under investigation. One putative model of
neuropathic pain is the intradermal capsaicin model [3]. When capsaicin is injected into the skin it
causes a transient characteristic burning and sensitivity to light touch. Such symptoms replicate the
key symptoms of neuropathic pain and raise the possibility of using this tool to assess drug
response.
1.1.4 Capsaicin and the TRPV1-receptor
Capsaicin is the primary active component of the heat and pain-eliciting, lipid-soluble fraction of
the capsicum pepper. It is an agonist to the transient receptor potential vanilloid 1, TRPV1, a ligand
gated ion channel, expressed predominantly by nociceptive afferent neurons [4]. Binding to TRPV1
causes influx of Na+ and Ca2+, hence depolarisation and initiation of action potentials [5].

Capsaicin influences the pain perception when injected into the skin. The pain sensation of
capsaicin can be divided into four physiologically based categories:
1. Spontaneous pain, which is short-lived (10-30 minutes) and consist of a burning/aching
sensation experienced at the site of administration.
2. Allodynia, which is pain that is evoked by a previously non-painful stimuli. The allodynia is
usually short-lived (20 minutes) and appears as both primary and secondary allodynia.
3. Hyperalgesia, which is increased pain evoked by a previously painful stimulus, e.g. a pinprick.
This can last between 6 and 24 hours and occurs at the site of administration (primary
hyperalgesia) and in the surrounding skin area (secondary hyperalgesia).
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4. Flare or neurogenic inflammation, which is the area of redness extending beyond the site of
injection. The inflammation lasts between 30 and 90 minutes.

The afferent pain transmission is mediated by sensory nerves with varying anatomical dimensions.
The nociceptors, sensory receptors, detect different types of stimuli and are classified afterwards.
Large diameter sensory fibres have either low- (A?-mechanoreceptors) or high –threshold receptors
(A?-mechanoreceptors). These finely myelinated fibres are sensitive to various mechanical
stimulations. Low- threshold A?-mechanoreceptors do normally only convey non-painful
stimuli [2]. Smaller diameter sensory fibres contain receptors sensitive to the various kinds of pain;
these polymodal C-fibre receptors, are un-myelinated and hence more slowly conducting compared
to the A-fibres and as the name suggests, these response to mechanical deformation, to intense heat
or cold and to irritant chemicals. All three types of fibres synapse in the dorsal horn ganglion.

Capsaicin elicits burning pain and cutaneous neurogenic vasodilatation, after TRPV1-binding on the
nociceptor by causing release of substance P and calcitonin gene related peptide (CGRP) from
sensory C-fibres. The spontaneous burning and aching pain caused by administration of capsaicin is
mediated by the C-fibre polymodal receptors, the pinprick hyperalgesia is mediated via A?- and
C-fibres and the allodynia are mediated via A?-fibres. This has been suggested in several studies in
both man and animal [2].
The characteristics of this capsaicin irritation vary across the body because of physical properties
such as skin thickness and some differences in autonomic and sensory functions in different parts of
the body [6]. The volar part of the forearm is the body part that is generally used in this model.
[2,3,9,18].

Capsaicin can even be intended to function as a potential ‘biomarker’ of central sensitisation, since
both allodynia and hyperalgesia are believed to be mediated by central sensitization. Central
sensitisation is an altered central processing, which is thought to underlie many clinical pain states
such as post herpetic neuralgia. This altered central processing of the pain input in the spinal cord
explains why the normally non painful stimulation of A?-fibres in example allodynia is experienced
as painful [2].

The response properties of nociceptors and the peripheral neural mechanisms contributing to pain
and altered pain states are similar in monkeys and humans, however some species differences have
been found. The flare that surrounds a local cutaneous injury in human skin is believed to be
mediated by an axon reflex which not is present in monkey skin. So the conclusion is that if the
hyperalgesia is caused by the axon reflex it is best seen in humans [7]. In addition to anatomical
differences between animals and humans, animal pain models also have the disadvantage that the
interpretation of pain usually relies on the observation of animals behaviour and the lack of
possibility to measure allodynia and hyperalgesia and their underlying mechanism under
standardized experimental conditions [8].
1.1.5 Capsaicin formulations
Capsaicin is insoluble in water and this causes a problem for intradermal use. It is highly soluble in
ethanol but a formulation of 10 % ethanol causes significant pain on injection due to the vehicle,
which confounds the scientific integrity of the model. A formulation with capsaicin, which is a
solution in lower concentrations and a colloidal suspension in higher concentrations, has been used
extensively but it is associated with several disadvantages [9]. A colloidal suspension may reduce
the effective local concentration, result in unequal given dosages or act as a depot. The formulation
is also difficult to prepare and must be made freshly for use. However, such a formulation has been
used extensively [9]. An alternative formulation, which is a sterile solution within the whole
investigated dose range, is easier to prepare and does not need to be made freshly, would make the
use of this technique more practicable [2].
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Figure 1. Structure of capsaicin molecule [10].

Because of the poor water solubility, excipients such as solvents might be methods for increasing its
solubility. The use of ethanol, propylene glycol and surfactants are unsuitable because of pain and
tissue damage. Adjusting pH in this case will be unsuitable because the pKa of capsaicin is 9.76
(CAS 404-86-4), which means that high pH is needed to solubilise the compound. In addition, a
low pH will cause pain and tissue irritation on injection since TRPV1 is activated by acidic
conditions (pH < 5.9) [11] and by elevated temperatures (~ 43oC) [12,13].

Cyclodextrins (CD) are potentially useful agents for increasing the aqueous solubility of lipophilic
compounds like capsaicin. They have lipophilic inner cavities and lipophobic outer surfaces,
capable of interacting with a large variety of guest molecules to form non-covalent inclusion
complexes [14]. See Figure 2 for chemical structures of the three cyclodextrins. Cyclodextrins are
cyclic oligosaccharides, containing at least 6 D-(+) glucosapyranose units attached by
?-1,4 glucosidic bonds. The three natural CDs, ?-CD, ?-CD and ?-CD (with 6, 7, and 8 glucose
units respectively) differ in their ring size and solubility. The cavity size of ?-CD is insufficient for
many substances. The ?-CD has been widely used in the pharmaceutical applications because of its
ready availability and cavity size suitable for the widest range of substances [14]. In addition to
increased solubility, cyclodextrin can also improve the stability of substances against dehydration,
hydrolysis, oxidation, and photodecomposition and thus increasing the shelf life of the substances
[15]. Substitution of any of the hydrogen bonds forming hydroxyl groups, even by hydrophobic
moieties such as methoxy and ethoxy functions, will increase the solubility of the ?-CD further, due
to transformation of the crystalline cyclodextrin into more amorphous mixtures of isomeric
derivatives [15]. (2-Hydroxypropyl)-?-cyclodextrin, (HP-?-CD), has been shown to both increase
the solubility of capsaicin by 205 fold and increase the shelf life of capsaicin [16].



a b




Figure 2. (a) The chemical structure of different cyclodextrins [17] and (b) the cone shape of the ?-CD molecule [14].

1.2 Aim for the study
A study has been published in which the dose-response relationship of capsaicin in a HP-?-CD
formulation used in volunteers was investigated [2]. Howeve
udy
r, to our knowledge; no previous st
has compared the performance of such a formulation against the performance of a standard
formulation on which most of the clinical validation is based. In or
the
der to have confidence in
utility of a HP-?-CD formulation of capsaicin, a clinical validation study comparing the 2
formulations was required.
The objectives were to compare the dose-response and dose-duration curves for pain, flare,
hyperalgesia and allodynia of two formulations of intradermal capsa
s.
icin in healthy volunteer
1.2.1 Hypothesis
The hypothesis was that the 2 formulations would produce similar pharmacodynamic profiles.
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2. Methods and Materials
2.1 Trial design and subjects
Sixteen healthy Caucasian human volunteers, eight men and eight female, aged 19-58 (mean 25.7),
participated in this randomized, blinded, cross over study comparing two different formulations of
capsaicin, after given written informed consent. The study was approved by the Royal Adelaide
Hospital Research Ethics Committee and the Investigational Drug Subcommittee.
The objectives of the study were to clinically compare the performance of a HP-?-CD formulation,
against the performance of a standard formulation on which most of the clinical validation is based.
The study took place at the Pain and Anaesthesia Research Clinic (PARC) within the Royal
Adelaide Hospital. This study was carried out in accordance with Principles of International
Conference on Harmonisation (ICH) Good Clinical Practice (GCP), as adopted in Australia, which
build upon the ethical codes contained in the Declaration of Helsinki and The Australian National
Statement on Ethical Conduct in Research Involving Humans.
The subjects were recruited to the study through flyers.

Twenty-two subjects were screened. An initial test session performed during the screening
familiarized the subjects with the four assessments. To minimize withdrawals from the study and to
ensure recruitment of test drug ”responders”, the screening also contained a familiarization event
with the injection of the highest dose (100 µg) of the HP-?-CD formulation into the dominant
forearm [3]. The screening event also contained an alcohol swab response test to confirm that the
subjects did not respond with a localised flare due to swabbing of the skin with alcohol.
The screening took place no more than 7 days prior to the first scheduled dosing date. Each
potential participant had to meet the following inclusion and exclusion criteria in order to qualify
for admission into the study:
Inclusion criteria
A subject was eligible for inclusion in this study only if all of the following criteria applied:
1. Healthy subjects not suffering from any clinically significant painful condition
2. Agree to and be capable of signing an informed consent form.
3. Be of either gender and aged between 18 and 65.
4. Fair skin colour (so that the flare can be observed)
Exclusion criteria
A subject was excluded from the study if any of the following criteria applied:
1. Pregnant or breastfeeding
2. Allergy or intolerance to capsaicin
3. Scarring or tattoos on the forearms.
4. Regular use of analgesics

During the trial phase, the eight injections were separated into two (dosing) occasions. During each
occasion the subjects received a total of four injections, each injection was separated by one hour
and five minutes. The two dosing occasions were separated by a minimum of four days. Each of the
sixteen subjects was scheduled to receive all eight injections.

The injections were administered according to a randomized Latin Square Design, produced by the
Discipline of Public Health, University of Adelaide, assuring that injection site, formulation and
session were balanced. This design assured that a formulation did not by chance always appear
before the other formulation which could lead to the possibility of confounding a treatment effect
with a time or carryover effect.
On each trial day, each subject came in on either mornings or afternoons to decrease the influence
of time of day variability within a subject [3]. Subjects were placed in a bed and superficial skin
temperature was fixed at 34 – 36oC, using a 250 W heat lamp (Philips) positioned about 50 cm from
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the subject’s volar arm. The temperature was monitored by a thermocouple placed on the skin of the
subject. A fixed temperature has been shown to decrease the variability of the model [18]. The
subjects were blindfolded during the assessments. After completion of the study, the subjects were
paid $200.

2.2 Capsaicin injection
Spontaneous pain and hyperalgesia were induced by intradermal injection of capsaicin,
(8-methyl N-vanillyl 6-nonamid) in two different formulations. Preparation of test batches
preceded the preparations of trial batches. The two formulations were given in the strengths
100 µg, 30 µg, 10 µg or 1 µg in 10 µL each, doses known to be tolerable to subjects while
producing areas of allodynia, hyperalgesia, spontaneous pain and neurogenic inflammation of
sufficient size to be measured accurately [2].

The conventional formulation was prepared according to Simone et. al. [9]. The dose of capsaicin
used in this trial was 100, 30, 10 and 1 µg in 10 µL of vehicle (Tween 80, 7.5 % w/v in normal
saline). The formulation appeared as solution for the three lowest doses and a colloidal suspension
beyond this.

The new HP-?-CD formulation was prepared according to Hughes et. al. [3] with the exception of
the concentration of the HP-?-CD vehicle, which was 38 % in this study, a concentration known to
be equivalent to isotonic solution [16]. The HP-?-CD formulation was passed through a sterile
syringe filter (Sterivex 0.22 ?m) into a sterile vial before syringes were prepared. Information about
the substances is given in Table I.

Table I. List of chemicals used in preparation of the two different formulations.
Chemical
Batch number
Manufactured by
Purity
Capsaicin
21 748
Fluka, Switzerland
? 97 %
Tween 80
073K00643
SigmaUltra
UK
(2-Hydroxypropyl)- ?-cyclodextrin, (HP-?-CD)
56 332
Fluka, Switzerland
UK
Sodium Chloride Injection BP 0.9 %, (saline)
30 18 92
Astra Zeneca
UK

The formulations were produced by a licensed pharmacist or under the strictly supervision of a
licensed pharmacist by the Pharmacy School of the University of South Australia and Pharmacy
Department at the Royal Adelaide Hospital, according to standards appropriate for a product for
human administration.

Each injection was drawn into syringes for administration no more than a maximum of one week in
advance, since the adsorption of the formulation to syringes could be a problem for the HP-?-CD
formulation [16]. The both formulations were used within one month of preparations due to stability
data [16,18].
In each experiment, a volume a 10 µL was injected intradermally into the skin to the midline of
either the dominant or non-dominant arm and to either forearm or upperarm, avoiding any veins.
The syringes used were 0.3 mL sterile insulin syringes (BD Ultra-Fine II). All injection sites were
marked with a blue pen.

2.3 HPLC analysis
After the last occasion in the trial, HPLC analyses were implemented to determine the
concentrations of capsaicin in all given formulations and doses. The neat formulations were diluted
1:1 with mobile phase before 10 µL were injected. The isocratic mobile phase was circulated at a
flow rate of 1.0 mL/min at ambient temperature of 21°C. The composition of the mobile phase is
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showed in Table II and the mobile phase was degassed (500T degasser, Soniclean, SA, Australia)
30 minutes before use. The UV-detector was set to 280 nm. Under these given circumstances,
capsaicin appeared as a single peak after approximately eight minutes.

Table II. HPLC conditions including column and mobile phase for detection of capsaicin.
Substance
Running time
Column
Mobile phase
capsaicin
15 minutes
Luna C18 (2) RP-column methanol / water /acetic acid, 75:25:0.1 v/v/v
(5µm, 4.6 x 250 mm)

2.4 Pain assessments
Information about the assessments and testing schedule were repeated to the subject before every
occasion. The assessments were measured five minutely intervals up to 30 minutes post injection
and then every 10 minutes to one hour post injection. The assessments of pain were performed in
the following order: spontaneous pain, area of flare, allodynia and hyperalgesia. One staff member
performed all measurements of each assessment to decrease observer bias. A parallel master thesis
investigated allodynia and flare [19].
2.4.1 Determination of the spontaneous pain
Perception of spontaneous pain was assessed using a numeric visual analogue scale (VAS). The
scale was 100 mm in length and was calibrated from 0 to 100, where 0 = “no pain” and
100 = “worst pain imaginable”. Actual measurements were converted to a scale of 0 – 100 mm
to correspond with the VAS calibration and recorded in the CRF.
2.4.2 Determination of the average radius of hyperalgesia
The average radius of pin pricked-induced hyperalgesia was assessed by applying a standard
von Frey hair, number 5.46 [2,3] with microfilament bending threshold 26G, (TouchTest
800-821-9319, Semmes Weinstein, Stoelting, IL, USA). The subjects were told
when the
to report
hair caused a greater or changed pain sensation compared to the pinprick sensation felt in the area
of normal sensitivity.

The hair was applied in 8 compass point directions and assessments started in the area of normal
sensitivity. The point of commencement for the procedure was the highest point in line with the
glenohumeral joint (shoulder), 10 cm above the inje
.
ction site. This point was referred to as north
The hair was reapplied at approximately 1 cm intervals every second, moving towards the injection
site [3]. The procedure continued until the site of injection if, the subject did report any change in
pain state/sensation. The procedure was repeated using the following sequence; S, E, W, NE, SW,
SE and finally NW. The resulting points, demarcating transition from normal sensitivity to
hyperalgesia, were traced onto acetate, using a red water resistant pen [20]. The transition was
made directly after the measurement to decrease the bias. Each radius was then measured with a
ruler and recorded in the CRF before an average radius for each injection was calculated. Since not
all assessments resulted in eight points, an average radius was used in preference to a calculated
area of hyperalgesia.

2.5 Statistical analysis
The subject numb
ilar to th
er was sim
at in previous studies; hence an adequate statistical power
should be provided.

ll calculations were perfor
A
med using Microsoft Office Excel Professional edition 2003 (Redmond,
WA, USA) and SAS version 9.1 (Cary, NC, USA).

A mixed model was fit to all outcomes separately with subject as a random effect and gender, arm
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