Results of a Pilot Randomized Placebo-Controlled Trial in Primary and Secondary Raynaud's Phenomenon with St. John's Wort: Detecting Changes in Angiogenic Cytokines When RP Improves.

Abstract

Objectives.To perform a 6-week double-blind RCT in Raynaud's phenomenon (RP) comparing the plant extract St. John's Wort (SJW) to placebo. Methods. RP patients having at least 7 attacks per week were stratified by primary and secondary RP and within secondary by systemic sclerosis or other connective tissue disease. Subjects completed a daily standardized diary recording all RP attacks (frequency, duration and severity). Serum levels of 18 inflammatory and angiogenic cytokines were measured pre- and post-treatment. Results. Eighteen patients completed the study; 8 received SJW and 10 placebo. The decrease in mean number of attacks per day was 0.75 with SJW and 1.01 with placebo, P = 0.06. Attack duration and severity were not different between groups. Cytokine analyses demonstrated no between-groups differences. Combining treatment groups, those with >50% improvement in frequency of attacks yielded a significant increase in E-selectin (P = 0.049), MMP-9 (P = 0.011), G-CSF (P = 0.02), and VEGF (P = 0.012) pre- versus post-treatment. A ≥50% improvement in severity of attacks corresponded to a significant increase in levels of sVCAM-1 (P = 0.003), sICAM-1 (P = 0.007), and MCP-1 (P = 0.004). Conclusions. There were no clinical or biomarker benefit of SJW versus placebo in RP. However, combining all patients, there were changes in some cytokines that may be further investigated.

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International Scholarly Research Network
ISRN Rheumatology
Volume 2011, Article ID 580704, 6 pages
doi:10.5402/2011/580704
Research Article
Resultsof a Pilot RandomizedPlacebo-ControlledTrial in
Primary andSecondaryRaynaud’s Phenomenonwith
St.John’sWort:DetectingChangesinAngiogenicCytokinesWhen
RP Improves
D´ eanne Malenfant,1Kelly Summers,2,3Shannon Seney,2Donna McBain,4Lisa Petrlich,4
SharonWatson,4LouiseVanderhoek,4NooshinSamadi,1,4AshleyBonner,5andJanetPope1,4
1Department of Medicine, University of Western Ontario, London, ON, Canada N6A 5C1
2Screening Laboratory for Immune Disorders, Lawson Health Research Institute, London, ON, Canada N6A 4V2
3Department of Microbiology & Immunology, University of Western Ontario, London, ON, Canada N6A 5C1
4Rheumatology, St. Joseph’s Health Care London, London, ON, Canada N6A 4V2
5Department of Mathematics and Statistics, McMaster University, Hamilton, ON, Canada L8S 4K1
Correspondence should be addressed to Janet Pope, janet.pope@sjhc.london.on.ca
Received 6 May 2011; Accepted 27 June 2011
Academic Editors: M. G. Danieli, B. A. Eberhard, C. G. Mackworth-Young, and C.-H. Suh
Copyright © 2011 D´ eanne Malenfant et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Objectives.To perform a 6-week double-blind RCT in Raynaud’s phenomenon (RP) comparing the plant extract St. John’s Wort
(SJW) to placebo. Methods. RP patients having at least 7 attacks per week were stratified by primary and secondary RP and within
secondary by systemic sclerosis or other connective tissue disease. Subjects completed a daily standardized diary recording all RP
attacks (frequency, duration and severity). Serum levels of 18 inflammatory and angiogenic cytokines were measured pre- and
post-treatment. Results. Eighteen patients completed the study; 8 received SJW and 10 placebo. The decrease in mean number of
attacksperdaywas0.75withSJWand1.01withplacebo,P = 0.06.Attackdurationandseveritywerenotdifferentbetweengroups.
Cytokine analyses demonstrated no between-groups differences. Combining treatment groups, those with >50% improvement in
frequency of attacks yielded a significant increase in E-selectin (P = 0.049), MMP-9 (P = 0.011), G-CSF (P = 0.02), and VEGF
(P = 0.012) pre- versus post-treatment. A ≥50% improvement in severity of attacks corresponded to a significant increase in
levels of sVCAM-1 (P = 0.003), sICAM-1 (P = 0.007), and MCP-1 (P = 0.004). Conclusions. There were no clinical or biomarker
benefit of SJW versus placebo in RP. However, combining all patients, there were changes in some cytokines that may be further
investigated.
1.Introduction
Raynaud’s phenomenon (RP) affects 3–5% of the popu-
lation [1] and is characterized by episodic vasospasm of
digital arteries causing pallor, cyanosis, and/or rubor upon
rewarming. It is caused by poor blood flow to digits, often
occurring with cold exposure and stress. The severity of
symptoms ranges from mild to severe pain, and in secondary
RP there may be ischemic complications. RP can be primary
(idiopathic)orsecondary.SecondaryRPisassociatedwithan
underlying disease (often a connective tissue disease {CTD}
suchassystemicsclerosis {SSc, scleroderma},systemiclupus
erythematosus {SLE}, and Sjogren’s syndrome).
The exact mechanisms causing RP are unknown, but
inflammatory and angiogenic cytokines secreted by cir-
culating cells and endothelial cells (ECs) may stimulate
vascular abnormalities that ultimately reduce blood flow.
Underlyingendothelialdysfunctionlikelyinitiatesandmedi-
ates this pathological process [2]. ECs in RP promote
an inflammatory response through elevated production of
proinflammatory cytokines [2] and increased expression of

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adhesion molecules [3, 4] and secretion of vasoconstric-
tor and profibrotic mediators, such as endothelin-1 and
angiotensin, instead of vasodilators [5].
Primary RP is most often controlled with lifestyle
changes (keeping warm, smoking cessation), whereas sec-
ondary RP in patients with SSc frequently requires systemic
treatment. Depending on disease severity, the choice of
medication includes calcium channel blockers (CCBs), such
as nifedipine, and other vasodilators. CCBs in both primary
andsecondaryRaynaud’shavedemonstratedmodestbenefit;
however these medications frequently have side effects such
as hypotension, flushing, peripheral edema, and headaches
[6–8]. There is an unmet need for an effective and well-
tolerated treatment in moderate-to-severe RP.
One trial suggested that fluoxetine (an antidepressant)
was effective in the treatment of both primary and secondary
RP [9]. Fluoxetine is a selective serotonin reuptake inhibitor
(SSRI) which in RP may be improving vascular endothelium
[9]. Ketanserin, a selective antagonist of serotonin, has also
been studied in RP [10]. Serotonin receptors have been
shown to be important in vasospasm in patients with RP
[11].St.John’sWort(SJW,hypericumperforatum)isanatural
herbal product with some SSRI-like properties. In mild-to-
moderate depression, SJW appears to be equally effective,
with fewer side effects than prescription SSRI drugs [12–
17]. Thus, SJW may be an effective, safe, and well-tolerated
alternative treatment for RP via effects on serotonin. In this
study, the dose of SJW was selected to be 600mg a day (given
as 300mg BID). Depression trials have dosed SJW from 300
to as high as 900mg BID [12–17], where 300mg BID was
superior to placebo and well tolerated [16, 17].
We performed a trial with SJW in subjects with primary
and secondary RP. The hypothesis of this trial was that SJW
would be more effective than placebo in the treatment of RP,
as defined by decreased frequency, duration, and severity of
attacks.
2.Methods
2.1.
placebo-controlled trial was registered at clinicaltrials.gov.ca.
It was approved by Health Canada (in the Natural Health
Product division). Ethics approval was obtained from the
University of Western Ontario Ethics Committee.
Study Design. Thisrandomized, double-blinded,
2.2. Subjects. Subjects were recruited from a rheumatology
clinic over two winters, as Raynaud’s symptoms are more
severe at this time. All subjects had symptomatic RP and
were stratified by primary versus secondary Raynaud’s, and
within secondary RP between systemic sclerosis and other
connective tissue diseases. Inclusion criteria were primary
or secondary RP as diagnosed by a rheumatologist, clinical
need for treatment of RP with at least 7 attacks per week
over a two-week run in, age at least 16 years, able to
provide informed consent, and ability to complete the daily
standardizedRPsymptomdiarypriortorandomization.The
exclusion criteria consisted of a prior allergic reaction to
SJW, depression requiring treatment, use of SSRIs or other
antidepressants with the exception of low-dose amitriptyline
usedforreasonsotherthandepression,useofdrugsthatmay
interact with SSRIs such as cyclosporine, warfarin, digoxin,
and theophylline, clinically significant noncompliance, and
anticipated need for surgery (sympathectomy) within 3
months of the trial. Subjects that met these criteria were
randomized using computer-based randomization 1:1 to
receive either SJW dosed at 300mg twice daily (BID)
or a lactose placebo BID for 6 weeks. St. John’s Wort
(SJW) was provided by Webber Pharmaceuticals. SJW was
reencapsulated by our research pharmacists who also made
the encapsulated placebo. Subjects completed a daily journal
with standardized questions about each attack (severity and
duration).Usingthediary,thenumberofattacksperdaywas
calculated. They also recorded any potential adverse events
(AEs). Every 2 weeks subjects were seen by a study nurse and
asked about side effects and digital ulcers, and patient global
assessments were performed.
Blood was drawn into EDTA tubes at 0 and 6 weeks post-
treatment,immediatelyspun,andplasmafrozenat-80◦Cfor
later analysis of 18 inflammatory and angiogenic cytokines
using multiplexed immunoassays (Millipore Corp., MA) and
ELISA for TGF-β1 (BD Biosciences, NJ).
2.3. Efficacy Measures. Primary outcome measures were the
frequency of RP (number of attacks daily), severity of RP on
a 0 to 10 scale, duration of RP attacks (in minutes) (from
the RP diary), and changes in the levels of inflammatory
and angiogenic cytokines between baseline and 6 weeks
post-treatment. Secondary outcome measures assessed at the
beginning and end of the treatment period were the health
assessment questionnaire (HAQ) disability index, patient
global assessment of RP, and the presence and location of
digital ulcers.
2.4. Sample Size Calculation. Using a between-group differ-
ence in number of RP attacks at the end of the study of 2.4
attacks per day in placebo and a 45% reduction in attacks in
SJW, and assuming an equal SD of 0.75 with 80% power and
a two-sided P value of 0.05, we would need only 8 patients
per group to complete the study. This was an early phase
study (the first of SJW in RP), and the data were to direct
future potential RP trials with SJW.
2.5. Statistics. Between-group comparisons (SJW compared
to placebo) were done using 2-tailed, paired t-tests to
analyze for significant changes (fold changes) in cytokine
levels between baseline and 6 weeks post-treatment. Similar
analyses were then performed combining all completers
based on patient-reported improvement (≥ or <50%) in the
frequency and severity of attacks, irrespective of treatment
allocation, to find a correlation between cytokines and
symptoms. Within-group comparisons were done using
paired t-tests in completers to analyze for significant changes
in the frequency, duration, and severity of attacks per day.

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3.Results
3.1. Subjects. Thirty-three patients with symptomatic RP
were screened (30 females, 3 males) and 25 were randomized
(as five had <7 attacks per week over the two week run in
and three did not want to continue in the study). In those
randomized, 5 had primary RP and 20 had secondary RP,
of whom 11 had SSc and 2 were undifferentiated CTD, 2
SLE, 2 RA, and one each of mixed connective tissue disease,
Sjogren’s syndrome, and dermatomyositis. Eighteen subjects
completedthetrial(2hadprimaryRPand8withSSc),with8
on active SJW and 10 on placebo. Adverse events (AEs) were
common (57% SJW, 43% placebo), especially headaches.
While these AEs were generally mild, there was one serious
AE in a subject with secondary RP and scleroderma that
involved hospitalization due to atrial fibrillation. Seven who
were randomized dropped out, 4 due to AEs (3 on SJW
and one on placebo). There were no significant differences
between SJW and placebo using ITT and also comparing
only the completers.
3.2. Severity, Duration, and Frequency of Attacks. There
were no significant differences in the severity, duration or
frequency of attacks between the groups. However, there
were significant within-group differences observed from
beginning to end of the trial in both SJW and placebo groups
(Table 1 shows the completers only analysis). There were
no important differences for the secondary endpoints when
comparing SJW to placebo.
The within-group pre- and post-treatment frequency
and severity of attacks were not significant in the SJW group.
In contrast, within the placebo group, the frequency of
attacks per day decreased significantly (P = 0.001) as did
the severity of attacks (P = 0.004). The duration of daily
RP attacks decreased significantly with SJW (P = 0.002)
and also with placebo (P = 0.009). The absolute change
was numerically less with SJW than placebo. This difference
in duration of attacks was further analyzed within the SJW
group by the subtype of secondary RP (SSc and other CTD),
but not in primary RP as the number of participants was
too small. These analyses showed a significant improvement
(P = 0.033) in the duration of attacks in subjects with
secondary RP in the nonscleroderma group.
3.3. Cytokines. There were no significant changes in cytokine
levels from pre- to post-treatment between the SJW and
placebo. All patients who completed the study were collated
(combining SJW and placebo) based on patient-reported
scores into ≥ or <50% improvement in the frequency
and then severity of RP attacks to analyze the significance
between cytokines and symptoms, irrespective of treatment
allocation. Cytokines were analyzed as the percent change
relative to baseline and also as mean differences between the
start and end of treatment since both types of analyses may
have biological relevance.
3.4. Percent Change. We found that those with a >50%
improvement in patient-reported frequency of attacks was
significantly associated with a larger fold increase in levels
of MMP-9 (P = 0.027), MIP-1β (P = 0.045), and VEGF
(P = 0.002) between baseline and the end of treatment when
compared to a <50% improvement (data not shown). There
wasno significantcorrelationbetween severity ofattacksand
percent change in cytokine levels.
3.5. Mean Differences. Comparing subjects who improved
by at least 50% to those who did not with respect to the
frequency of RP attacks demonstrated significant changes
in E-selectin (P = 0.049), MMP-9 (P = 0.011), G-CSF
(P = 0.02), and VEGF (P = 0.012) (Table 2). Using the
same comparison for ≥ or <50% improvement in severity
of attacks, a significant increase in sVCAM-1 (P = 0.003),
sICAM-1 (P = 0.007), and MCP-1 (P = 0.004) was observed
(Table 3).
4.Discussion
SJW was not superior to placebo, and in fact, placebo was
numerically but not significantly better than SJW on some
outcomes. It is likely that shared properties between SSRIs
and SJW in the treatment of depression are not similar in
RP treatment at the dose studied [9]. SJW should not be
pursued for the treatment of RP in a larger trial as there
was no biomarker or clinical change that was superior to the
placebo group.
Thereweresignificantwithin-groupresultswhichunder-
scores that any open-label treatment trial needs to be
interpreted with caution in RP as there is likely a large
placebo effect or regression to the mean. Even proof-of-
concept trials testing new agents in RP should have a double-
blinded, randomized trial design.
Therearemultiplepotentialreasonswhythisisanegative
trial which could include inefficacy of SJW, underdosing,
heterogeneity of the patients, small sample size, inadequate
outcome measurements, or spurious results, but there was
not even a trend of improvement compared to placebo.
Manypublishedtrialshavecombinedprimaryandsecondary
RP stratifying by type of RP, which we did. We tried to
have a more homogenous group of patients to study by
using only patients with a high frequency of RP attacks.
Also, the majority of patients were secondary RP, so perhaps
they are more difficult to treat than primary RP, and in
this group the numbers are incredibly small to make any
treatment conclusion within this small subset of completers.
The SJW dose was at the lower end of what has been used
for depression trials but nevertheless the dose we chose had
demonstrated efficacy [16, 17]. The Raynaud’s Condition
Score (a daily self-assessment questionnaire of RP on an
ordinal scale) [18] was not collected within the diaries but at
each visit patient global RP assessment was done. Biomarker
data also did not demonstrate that SJW is effective in RP.
Some biomarkers improved in patients whose RP was better,
but they did not improve more in the SJW group compared
to placebo. The placebo changes for RP outcomes in this
study may have occurred as there is often regression to the
mean in RP trials. For instance, when patients enroll in
RP trials requiring a large number of attacks, they enter at
a high level of RP activity which may decrease over time,

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Table 1: Within-group characteristics for frequency, duration, and severity of RP in SJW compared to placebo in the completers. There were
no between-group differences.
Baseline SJW
(N = 8)
6-Week
Followup
SJW
Mean
difference
SJW
P-value
Within group
SJW
Baseline
Placebo
(N = 10)
6-Week
Followup
Placebo
Mean
difference
Placebo
P-value
Within group
Placebo
Frequency (mean
no. of attacks per
day)
Duration (mean
total of attacks in
minutes per day)
Severity (scale 0 to
10)
3.175 2.4238 .75130.1238 2.3661.3531.0130 0.001
28.27918.009 10.2703 0.001926.01013.51012.49920.009
4.7523.785.96680.07582.9541.947 1.0066 0.004
Table 2: Within-group comparisons of patient-reported improvements in the frequency of Raynaud’s attacks and the difference of cytokine
levels between pre- and post-treatment. P values were calculated using 2-tailed, paired t-tests. Data represents mean comparisons of changes
in cytokines in those who had more than 50% reduction in frequency (number) of RP attacks compared to those who did not.
Cytokine
Mean cytokine change
Difference Std. error difference
P value
Frequency of attacks
improved by ≥50%
(n = 7)
5843.8
134550.6
8861.6
68574.0
−188.4
−0.2
−2.6
−0.4
0.0
0.3
0.0
−4.6
1.0
15.0
−13.6
110.1
−12.4
−0.4
7.1
−133.4
−1.0
7.3
−2.4
Frequency of attacks
improved by <50%
(n = 6)
−3473.3
−14819.9
−13292.5
−104997.8
−10086.1
0.0
0.1
−1.1
0.0
0.1
−0.5
0.6
3.4
−12.1
0.6
78.9
−3.1
0.3
−3.5
−244.1
2.2
−11.5
−4.1
sE-Selectin
sVCAM-1
sICAM-1
MMP-9
tPAI-1
IL-4
IL-6
IL-8
IL-10
IL-12p70
IL-17
Eotaxin
Basic FGF
G-CSF
IFN-γ
IP-10
MCP-1
MIP-1α
MIP-1β
PDGF-BB
TNF- α
VEGF
TGF-β1
9317.1
149370.5
22154.1
173571.8
9897.7
−0.2
−2.7
0.7
4203.1
122138.5
17813.4
57260.1
7392.6
0.2
2.9
1.9
0.049
0.247
0.239
0.011
0.208
0.356
0.387
0.726
0.3
0.5
−5.2
−2.5
27.0
−14.1
31.2
−9.3
−0.7
10.6
110.7
−3.2
18.8
1.7
0.3
0.5
9.4
2.9
10.0
21.8
106.8
20.2
0.5
5.5
309.2
3.1
6.3
2.9
0.416
0.363
0.588
0.408
0.020
0.531
0.776
0.654
0.199
0.082
0.727
0.341
0.012
0.565
and attacks are often related to cold temperature, which
may vary over the study as a 6-week long trial started in
winter may finish for some patients in spring. However,
we cannot infer that SJW was worse than placebo as there
were no statistically significant between-group differences
and in both allocations there was improvement of RP. The
study is too small to comment on individual background
medications but patients were in a steady state if they were
using calcium channel blockers or losartan for RP. We did
notstopapartiallyeffectivedrugduetotheethicsofstopping
treatment during winter in patients with more than 7 attacks
per week on their current treatment if it was perceived to
have partial benefit. We did not find lack of tolerability with
otherRPmedicationswhenaddingSJW,butthenumbersare
too small. SJW does have many potential drug interactions
but the Health Canada-approved protocol excluded only

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Table 3: Within-group comparisons of patient-reported improvements in the severity of Raynaud’s attacks and the difference of cytokine
levels between pre- and post-treatment. P values were calculated using 2-tailed, paired t-tests.
Cytokine
Mean cytokine change
Severity of attacks
improved by ≥50%
(n = 2)
1429.1
443639.4
27421.2
31051.5
1203.7
0.0
0.0
1.4
0.0
−0.1
0.0
4.0
−1.0
8.8
9.0
188.5
27.8
0.0
3.1
3.0
−0.4
9.5
−3.5
Mean differenceStd. error difference
P value
Severity of attacks
improved by <50%
(n = 11)
1564.4
−3122.2
−6596.9
−19279.3
−5840.2
−0.2
−1.6
−1.2
0.0
0.2
−0.3
−3.3
2.7
1.3
−10.0
78.8
−14.7
0.0
2.1
−218.6
0.7
−3.4
−3.1
sE-Selectin
sVCAM-1
sICAM-1
MMP-9
tPAI-1
IL-4
IL-6
IL-8
IL-10
IL-12p70
IL-17
Eotaxin
Basic FGF
G-CSF
IFN-γ
IP-10
MCP-1
MIP-1α
MIP-1β
PDGF-BB
TNF-α
VEGF
TGF-β1
−135.3
446761.6
34018.1
50330.7
7043.9
0.2
1.6
2.6
6985.0
119191.6
10030.8
106102.6
10808.5
0.4
4.5
2.4
.985
.003
.007
.645
.528
.689
.724
.305
−0.3
0.3
7.3
−3.7
7.5
19.0
109.7
42.4
0.0
1.0
221.6
−1.1
12.9
−0.4
0.4
0.7
12.9
3.9
17.7
30.2
144.4
11.4
0.8
8.8
424.4
4.2
11.0
4.1
.447
.689
.582
.363
.680
.542
.463
.004
.956
.916
.612
.798
.265
.919
drug-SJW interactions which were thought to be of clinical
relevance.
AEs were common in this trial, and one SAE in a patient
using SJW occurred. Since our incidence of AEs was fairly
similar in SJW and placebo, many side effects were likely not
related to the study medication.
Patients that reported a >50% improvement in the
frequency of Raynaud’s attacks were associated with higher
levels of the proangiogenic cytokines, MMP-9, MIP-1β, sE-
selectin, G-CSF, and VEGF. Similarly, decreased severity of
these attacks corresponded to increases in plasma levels of
sVCAM-1, sICAM-1, and MCP-1. We did not correct for
multipletests,andsomeofthechangesmaybeduetowithin-
patient variability of the cytokine levels. However, testing
thesecytokinesmaybeusefulinotherproof-of-concepttrials
with different agents in RP to determine if any are associated
with clinically relevant RP outcomes.
Acknowledgments
St. John’s Wort was supplied by Webber Pharmaceuticals and
gel coated by St. Joseph’s pharmacy to make an identical
placebo. In no way did Webber influence the conduct or
results of the trial. D. Malenfant was a medical student
who was funded by the Schulich Research Training Program
(SRTP).
References
[1] L. G. Suter, J. M. Murabito, D. T. Felson, and L. Fraenkel, “The
incidence and natural history of Raynaud’s phenomenon in
the community,” Arthritis and Rheumatism, vol. 52, no. 4, pp.
1259–1263, 2005.
[2] R. Bakst, J. F. Merola, A. G. Franks, and M. Sanchez,
“Raynaud’s phenomenon: pathogenesis and management,”
Journal of the American Academy of Dermatology, vol. 59, no.
4, pp. 633–653, 2008.
[3] S. N. Lambova and U. M¨ uller-Ladner, “New lines in therapy
of Raynaud’s phenomenon,” Rheumatology International, vol.
29, no. 4, pp. 355–363, 2009.
[4] B. Kahaleh, O. Meyer, and R. Scorza, “Assessment of vascular
involvement,” Clinical and Experimental Rheumatology, vol.
21, no. 3, pp. S9–S14, 2003.
[5] S. Rajagopalan, D. Pfenninger, C. Kehrer et al., “Increased
asymmetric dimethylarginine and endothelin 1 levels in sec-
ondary Raynaud’s phenomenon,” Arthritis and Rheumatism,
vol. 48, no. 7, pp. 1992–2000, 2003.
[6] J. E. Pope, “The diagnosis and treatment of Raynaud’s
phenomenon: a practical approach,” Drugs, vol. 67, no. 4, pp.
517–525, 2007.