A Randomized, Double-Blind, Placebo-Controlled, Multicentre Trial of the Effects of a Shrimp Protein Hydrolysate on Blood Pressure

Abstract

In this randomized, double-blind, placebo-controlled, multicentre, parallel, 8-week study, the efficacy of a daily dose of 1200 mg of protein hydrolysate from Coldwater Shrimp ( Pandalus borealis ) on ambulatory and office blood pressure was investigated in 144 free-living adults with mild to moderate hypertension. The primary outcomes of the study were daytime ambulatory systolic blood pressure and office blood pressure. During the 8-week intervention period and in the intention-to-treat analysis (n=144), there were significant reductions in the group consuming the shrimp-derived protein hydrolysate relative to the placebo group in daytime ambulatory systolic blood pressure at 4 weeks (p=0.014) and at 8 weeks (p=0.002), and in office systolic blood pressure at 2 weeks (p=0.031) and 4 weeks (p=0.010), with a trend toward significance at 8 weeks (p=0.087). By 8 weeks, significant and favourable improvements in the group consuming the shrimp-derived protein hydrolysate relative to the placebo group were also observed for several secondary outcomes, including 24-hour ambulatory systolic and diastolic blood pressure, daytime ambulatory diastolic blood pressure, and daytime and 24-hour ambulatory mean arterial pressure. Also by Week 8, there was a statistically significant difference between groups in the distribution of subjects across National Institutes of Health-defined blood pressure categories (i.e., Normotensive, Prehypertensive, Stage 1 hypertension, and Stage 2 hypertension), with a more favourable distribution in the shrimp-derived protein hydrolysate group than in the placebo group (p=0.006). Based on exploratory analyses conducted only in participants in the shrimp-derived protein hydrolysate group, angiotensin II levels were significantly reduced relative to baseline. This study is registered at ClinicalTrials.gov NCT01974570 .

Full text

Clinical Study
A Randomized, Double-Blind, Placebo-Controlled,
Multicentre Trial of the Effects of a Shrimp Protein
Hydrolysate on Blood Pressure
Kathy Musa-Veloso,1Lina Paulionis ,1Tetyana Pelipyagina,2and Mal Evans 2
1Food & Nutrition Group, Intertek Scientific & Regulatory Consultancy, 2233 Argentia Road, Suite 201, Mississauga,
ON, Canada L5N 2X7
2KGKScienceInc.,255QueensAvenue,Suite1440,London,ON,CanadaN6A5R8
Correspondence should be addressed to Mal Evans; mevans@kgkscience.com
Received 21 January 2019; Accepted 18 June 2019; Published 5 August 2019
Guest Editor: Daniela Zanini
Copyright ©  Kathy Musa-Veloso et al. is 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.
In this randomized, double-blind, placebo-controlled, multicentre, parallel, -week study, the ecacy of a daily dose of  mg
of protein hydrolysate from Coldwater Shrimp (Pandalus borealis) on ambulatory and oce blood pressure was investigated in
 free-living adults with mild to moderate hypertension. e primary outcomes of the study were daytime ambulatory systolic
blood pressure and oce blood pressure. During the -week intervention period and in the intention-to-treat analysis (n=),
there were signicant reductions in the group consuming the shrimp-derived protein hydrolysate relative to the placebo group in
daytime ambulatory systolic blood pressure at  weeks (p=.) and at  weeks (p=.), and in oce systolic blood pressure at 
weeks (p=.) and  weeks (p=.), with a trend toward signicance at  weeks (p=.). By  weeks, signicant and favourable
improvements in the group consuming the shrimp-derived protein hydrolysate relative to the placebo group were also observed
for several secondary outcomes, including -hour ambulatory systolic and diastolic blood pressure, daytime ambulatory diastolic
blood pressure, and daytime and -hour ambulatory mean arterial pressure. Also by Week , there was a statistically signicant
dierence between groups in the distribution of subjects across National Institutes of Health-dened blood pressure categories
(i.e., Normotensive, Prehypertensive, Stage  hypertension, and Stage  hypertension), with a more favourable distribution in the
shrimp-derived protein hydrolysate group than in the placebo group (p=.). Based on exploratory analyses conducted only in
participants in the shrimp-derived protein hydrolysate group, angiotensin II levels were signicantly reduced relative to baseline.
is study is registered at ClinicalTrials.gov NCT.
1. Introduction
According to  data collected by the World Health Orga-
nization (WHO) on noncommunicable diseases, globally, the
overall prevalence of elevated blood pressure (BP) (dened
as a systolic blood pressure [SBP] ≥ mmHg or a diastolic
blood pressure [DBP] ≥ mmHg) in adults  years of age
and older is % [, ]. Elevated BP, if le untreated, can lead
to cardiovascular diseases (CVDs) (e.g., stroke, myocardial
infarction, cardiac failure, congestive heart failure, and atrial
brillation), renal disease and failure, cognitive decline (e.g.,
vascular dementia), and blindness [, ]. Worldwide, elevated
blood pressure is estimated to cause . million deaths, which
is about .% of all deaths []. us, as a global target to
be achieved by , the WHO has called for a % relative
reduction in the prevalence of raised BP or the contain-
ment of the prevalence of raised BP, according to national
circumstances []. In order to achieve this target, dietary
and lifestyle behaviours that eectively reduce BP levels
(e.g., achieving a healthy body weight, avoiding tobacco use,
drinking alcohol in moderation, increasing physical activity
levels, and following a low-sodium diet rich in fruits and
vegetables) must be adopted. With the worldwide surge in
the aging population, however, the prevalence of elevated BP
is steadily increasing, and additional interventions that could
assist with the maintenance of normal BP levels are needed.
Hindawi
International Journal of Hypertension
Volume 2019, Article ID 2345042, 13 pages
https://doi.org/10.1155/2019/2345042

International Journal of Hypertension
Marine-derived protein hydrolysates may be useful as
adjunctive treatments in the management of hypertension
or in the maintenance of normal BP levels. Using the
spontaneously hypertensive rat as a model of hypertension,
several dierent sh protein hydrolysates were demonstrated
to have antihypertensive eects. ese protein hydrolysates
were derived from the bowels of Skipjack tuna (Katsuwonus
pelamis), muscle of Bigeye tuna (unnus obesus), mus-
cle of sardine (Sardinops melanostictus), loach (Misgurnus
anguillicaudatus), head of cobia (Rachycentron canadum),
backbone of ribbonsh (Trichiurus haumela), and skin of
skate (Okamejei kenojei) [–]. In another study, however,
protein hydrolysates of wild Atlantic cod (Gadus morhua L.),
haddock (Melanogrammus aeglefinus L.), or farmed Atlantic
salmon (Salmo salar L.)werefoundtobeineectivein
reducing BP in spontaneously hypertensive rats []. While
the reasons for the apparent ineectiveness are not entirely
clear, it is likely that the source of the protein (sh species and
anatomical origin), the hydrolysing enzyme(s), the enzyme
to substrate ratio, and the protein hydrolysis conditions (i.e.,
temperature, time, and pH) all could impact the presence of
other potential bioactives, such as secretagogues, calciotropic
hormones, and growth factors, as well as the resulting amino
acid sequences [].
Studies of the potential antihypertensive eects of pro-
tein hydrolysates derived from crustaceans are limited.
In some studies, the in vitro inhibition of angiotensin-
converting enzyme (ACE) was greater with shrimp pro-
tein hydrolysates than with sh protein hydrolysates [,
]. Nii et al. [] previously demonstrated that the
oral administration of an izumi shrimp (Plesionika izu-
miae Omori) hydrolysate signicantly inhibited the age-
associated spontaneous increase in BP in stroke-prone spon-
taneously hypertensive rats. e group went on to iso-
late two ACE inhibitory peptides from the izumi shrimp
hydrolysate; their amino acid sequences were determined
to be valine-tryptophan-tyrosine-histidine-threonine and
valine-tryptophan []. e BP in stroke-prone sponta-
neously hypertensive rats was shown to decrease signicantly
aer just a single oral administration of synthetic versions of
the aforementioned two amino acid sequences []. Likewise,
Gildberg et al. [] reported high ACE inhibitory activity
of a desalted protein hydrolysate from Northern shrimp
(Pandalus borealis), with two novel ACE inhibitory tripep-
tides detected in the hydrolysate, namely, phenylalanine-
threonine-tyrosine and phenylalanine-serine-tyrosine. Fur-
ther, signicant improvements in BP were observed when
spontaneously hypertensive rats were administered  mg of
the shrimp protein hydrolysate per kg body weight per day
[].
Clinical studies of the ecacy of hydrolysates derived
from shrimp in reducing BP have not yet been conducted.
In this study, we report the results of a randomized,
double-blind, placebo-controlled, multicentre, parallel study
in which the primary objectives were to assess the ecacy of
desalted shrimp protein hydrolysate from Coldwater Shrimp
(Pandalus borealis)onthechangesfrombaselineindaytime
ambulatory SBP and oce SBP in individuals with mild to
moderate hypertension.
2. Methods
2.1. Study Objectives and Design. e primary objectives of
the study were to assess the eects of a shrimp-derived
protein hydrolysate [hereinaer also referred to as a Rened
Peptide Concentrate (RPC)] versus placebo on the changes
from baseline over  weeks in daytime ambulatory SBP and
oce SBP. Secondary objectives were to assess the eects of
theRPCversusplaceboon-hourandnight-timeambula-
tory SBP; -hour, daytime, night-time ambulatory and oce
DBP; and other endpoints (e.g., heart rate, fasting serum
glucose and serum lipids, serum C-reactive protein, dietary
variables from food records, urinary sodium). Exploratory
objectives were to assess the eects of the RPC on blood levels
of angiotensin I and II, aldosterone, renin, ACE activity, and
low-density lipoprotein (LDL) oxidation.
e study was multicentre ( centres/sites), random-
ized, double-blind (investigator, participants, and other site
personnel all blinded), placebo-controlled, and parallel (two
arms). e study was conducted from January  to
September . Initially, twelve sites in North America
(elevensitesinCanadaandonesiteintheUnitedStates),and
ninesitesinEurope(threesitesinGermanyandsixsitesin
theCzechRepublic)wereinvolvedinthestudy;however,two
sites in North America (both in Canada) were not successful
in enrolling patients. e study was performed in accordance
with the ethical principles that have their origins in the
Declaration of Helsinki and its subsequent amendments, and
in accordance with the International Council for Harmon-
isation of Technical Requirements for Registration of Phar-
maceuticals for Human Use [], and applicable regulatory
requirements. is study is registered at ClinicalTrials.gov
(ClinicalTrials.gov identier: NCT) [].
2.2. Study Population. Individuals were recruited via direct e-
mails, as well as via online and posted paper advertisements.
Individuals had to meet all of the inclusion criteria and none
of the exclusion criteria to qualify for the study.
e inclusion criteria for subject selection were as follows:
(1)male or female aged  to  years, inclusive (independent
and home-living); () if female, not of child-bearing potential
or having a negative urine pregnancy test result and agreeing
to use a medically approved method of birth control; ()
mild or moderate hypertension (SBP  to  mmHg and
DBP ≤ mmHg; mean of oce BP measurements from
three occasions used, i.e., the rst two study visits during
the run-in period and the baseline measurement); () body
weight ≥ kg; () stable body weight (self-reported weight
gain or loss <kginthepastmonths);()voluntary,
written, and informed consent to participate in the study;
and () agreement to comply with study procedures; to fast
(at least  hours) and abstain from alcohol ( days) prior
to blood sampling; to abstain from alcohol ( days), coee
( hours), and physical exercise ( hours) prior to blood
pressure measurement; and to abstain from donating blood
duringandfordaysaerthestudy.eexclusioncriteria
for subject selection were as follows: (1) females who were
pregnant, breastfeeding, or planning to become pregnant
during the course of the trial; () body mass index (BMI)

International Journal of Hypertension 
≥ kg/m2; () antihypertensive drug treatment, regular high-
dose nonsteroidal anti-inammatory drug treatment, or use
of cyclosporine or tacrolimusin; () Any history of CVD,
dementia/cognitive impairments, hypertensive retinopathy,
le ventricular dysfunction or peripheral artery disease,
secondary hypertension, diabetes (Types  and ), cancer
within the past  years (excluding basal cell carcinoma), or
any other disease or condition which, in the Investigator’s
opinion, could interfere with the results of the study or the
safety of the subject; () clinically signicant biochemistry,
haematology, and/or urinalysis, at the Investigator’s discre-
tion; () dietary restriction (sh and other seafood allergies,
citrus allergies, and multiple food allergies); () alcohol abuse
[dened as the consumption of more than  portions of
alcohol per week (one portion =  oz. spirits or  oz. wine
or oz. medium strength beer / cider)] and illicit drug use,
including smokers and tobacco/snu/nicotine users; () use
of natural health products intended for BP lowering within 
days before randomization; and () participation in another
clinical research trial within  days prior to randomization.
Each subject was allocated a randomization num-
ber according to a randomization scheme generated by
www.randomization.com. A sta member not involved in
any study procedure bottled and labelled the study product;
labels were applied according to the randomization list. e
investigator, study personnel (involved in product dispens-
ing, visit assessments, conduct of the study, monitoring, and
analysis), and the participants did not know what treat-
ment had been assigned. Sealed individual randomization
envelopes containing the randomization number and asso-
ciated treatment were prepared and kept at the coordinating
centre and as such, there was allocation concealment.
2.3. Description of Investigational Products. Study partici-
pants received either the shrimp-derived RPC or placebo
tablets. Study participants were instructed to consume two
 mg tablets of the shrimp-derived RPC or placebo,
once daily, with water, before noon (and between meals).
Each  mg tablet of active test product contained 
mg of protein hydrolysate from desalted Coldwater Shrimp
(Pandalus borealis); thus, those in the RPC group received
 mg of protein hydrolysate from desalted Coldwater
Shrimp daily.
e active and placebo products were identical in compo-
sition, except that the active product contained (per tablet)
 mg of protein hydrolysate from Coldwater Shrimp
(Pandalus borealis), while the placebo product contained
(per tablet)  mg of Rainbow trout sh oil (used to match
thetasteandsmelloftheinvestigationalproducts)and
a higher amount of microcrystalline cellulose. ere were
no dierences in the taste, smell, colour, size, texture, or
packaging between the active test product and placebo; thus,
both products were matched in taste, smell, and appearance.
Compliance with the intake of the tablets was assessed by
counting the returned product at each visit. Compliance was
calculated as:
#oftabletsconsumed
# of tablets expected to be consumedx100%()
2.4. Assessments
2.4.1. Office BP and Office Heart Rate. Oce SBP and DBP
and oce heart rate were measured at screening (- weeks),
run-in (- weeks), baseline (Week ), Week , Week 
(midpoint of study), and Week  (end of study). Oce BP was
measured according to Dieterle [] and Pickering et al. [].
Subjects were in a seated position with their legs uncrossed,
feet at on the oor, and backs comfortably ush against
the back of a chair for  minutes prior to and for the entire
period during the BP measurements. Using a random-zero
mercury or digital sphygmomanometer, trained personnel
measured BP. BP was initially measured in both arms and
the arm with the higher value was used for all subsequent
BP measurements; the arm was supported at heart level. At
each oce visit, three BP measurements were taken over -
minute intervals with the rst measurement discarded and
the latter two measurements averaged. Study eligibility was
determined by averaging the BP measurements taken over
three oce visits (screening [Week -], run-in [Week -], and
baseline [Week ]). e same recording method and the same
equipment were used for each subject throughout the study.
Oce heart rate was measured by radial arterial measure-
ment and counting of arterial pulses per minute. At each visit,
the number of arterial pulses per minute was measured three
times and the three measurements were averaged to represent
the participant’s heart rate during the visit.
2.4.2. Ambulatory BP and Ambulatory Heart Rate. Ambu-
l a t or y S B P, D B P, a n d h e a r t r at e w e r e m e a s u r e d a t b a s e l i n e ,
Week , and Week  using a -hour Ambulatory Blood
Pressure (ABP) monitor (Spacelabs Medical, model number
-Q), according to Dieterle [] and O’Brien et al. [].
Subjects were tted with an ambulatory BP monitor, with the
cu secured on the nondominant arm for at least  con-
secutive hours. All ambulatory BP monitors and cus were
coded, and each subject was tted with the same monitor and
cu for each wearing occasion. Ambulatory measurements
were programmed to occur at -minute intervals from :
AM to : PM inclusive and at -minute intervals from
: PM to : AM inclusive. Daytime ambulatory BP
and daytime ambulatory heart rate were dened as : AM
to : PM inclusive, and night-time ambulatory BP and
night-time ambulatory heart rate were dened as : AM
to : AM inclusive, similar to how “daytime” and “night-
time” were dened in other ambulatory BP studies [–].
e selection of these time ranges for “daytime” and “night-
time” resulted in the elimination, from the daytime and
night-time measures, values collected from : AM to :
AM and from : PM to : AM, during which subjects
would have been awakening or falling asleep, respectively,
and during which there would be considerable variations in
blood pressure.
2.4.3. Height, Weight, BMI. Height was measured at screen-
ing and weight was measured at screening, run-in, baseline,
and Weeks , , and . e Health O Meter Professional
Scale was used to measure height (reported in centimetres)
and weight (reported in kilograms). Measurement of height

International Journal of Hypertension
was performed with shoes removed, knees straight, and head
held upright. Measurement of weight was performed with
the subjects in light clothing, shoes removed, and bladder
empty. Subjects were weighed on the same scale at all visits.
At least two separate body weight measurements were taken
at each visit. If the two measurements were more than . kg
(. lbs) apart, a third measurement was taken, and the two
closest values were selected and averaged. BMI was calculated
at screening, run-in, baseline, and Weeks , , and .
2.4.4. Other. ree-day food records (two weekdays and
oneweekendday)werecompletedintheweekpriortothe
baseline,Week,andWeekvisits.Adverseevents(AEs)
wereassessedatWeeks,,and.
2.5. Laboratory Analysis. Collection of blood (subjects were
fasted ≥ hours) for the laboratory analysis of lipids
(total cholesterol, high-density lipoprotein- (HDL-) choles-
terol, LDL-cholesterol, triglycerides), C-reactive protein,
angiotensin I and II, aldosterone, renin, ACE activity and
LDL oxidation occurred at baseline and Week . A -hour
collectionofurine,forthemeasurementofurinesodium,
urine creatinine, and urine volume also occurred at baseline
and Week . Blood collection (subjects were fasted ≥ hours)
for safety endpoints (complete blood count, glucose, crea-
tinine, estimated glomerular ltration rate, sodium, potas-
sium,chloride,aspartateaminotransferase,alanineamino-
transferase, gamma glutamyltransferase, and bilirubin) was
conducted at screening and Week . It should be noted that
the assessment of angiotensin I and II, aldosterone, renin,
ACEactivity,andLDLoxidationwasconductedusingthe
baseline and Week  blood samples only for subjects who
were randomized to the RPC group, to explore the potential
mechanismofeect.
Subjects’ blood samples were stored at -∘Cor,ifthe
site did not have this capability, at -∘C, for a period not
exceeding  days aer blood collection. All the clinical
research sites within a particular country used the same
laboratory for the analysis of blood and urine (with the
exception of urine pregnancy tests, which were done at each
clinic). Quality assurance and clinical ranges used (for the
laboratory analysis) were in accordance with each respective
laboratory’s guidelines.
Just prior to analysis, blood samples intended for enzyme-
linked immunosorbent assay (ELISA) analysis were kept at
temperatures of between  and ∘C (aldosterone, ACE activ-
ity, renin) or at ∘C (angiotensin I, angiotensin II). ELISA
kits were used for their analysis (catalogue numbers: KGE,
DACE, DREN, ab, ab, respectively).
2.6. Statistical Analysis. Based on a previous unpublished
study, a standard deviation (SD) of  mmHg was used for
themeanchangeindaytimeambulatorySBPandaSDof
 mmHg was used for the mean change in oce SBP. With
an anticipated attrition rate of % and in order to detect a
dierence of  or  mmHg in the mean change in daytime
ambulatory SBP or oce SBP, respectively, aer an -week
supplementation period with a probability of % at alpha
level.,therequiredsamplesizewasestimatedasor
randomized subjects per group, respectively. erefore, the
sample size was determined to be  subjects ( subjects
per group), randomized in a : ratio to one of two groups.
Two populations were used for the ecacy analysis: (i)
the intention-to-treat (ITT) population which consisted of all
participants who received either product, and on whom any
post-randomization ecacy information was available; and
(ii) the per-protocol (PP) population, which consisted of all
participants who consumed at least % of either product,
did not have any major protocol violations and completed all
study visits and procedures connected with the measurement
of the primary variables. e safety analysis was conducted
on the safety population, which consisted of all participants
who received any amount of either product and on whom
any postrandomization safety information was available. All
missingvaluesintheITTanalysiswereimputedwiththe
most recent previously available value (“last-observation-
carried-forward” or LOCF imputation). No imputation was
performed for missing values of safety variables.
Numerical ecacy endpoints were tested for signicance
between groups by analysis of covariance (ANCOVA). e
dependent variable was the value at each visit, the factor was
thetreatmentgroup,andthevalueatbaseline(Week)was
the covariate. For parameters that required a transformation,
the transformed values were used in the ANCOVA model.
Numerical endpoints that were intractably nonnormal were
assessed by the Mann-Whitney U test. A within-group
analysis on numeric endpoints was done using the Student’s
paired t-test or, in the case of intractable nonnormality, the
Wilcoxon sign rank test.
For numeric safety endpoints, the data were presented
andanalysedusingthesamemethodsastheecacydata.
For AEs, a descriptive analysis was provided by body system
and treatment group; also, the nature, incidence, severity, and
causality were reported for each AE.
Probabilities ≤. were considered statistically signif-
icant. All statistical analyses were completed using the R
Statistical Soware Package Version .. for Microso Win-
dows [].
3. Results
As outlined in Figure , a total of  individuals were
screened for potential inclusion into the study with 
enrolled into the study ( in each group). Ecacy and safety
datawereavailableforallsubjects;thus,theITTand
safety analyses included data from all  subjects. A total
of  subjects completed the study ( in each group). Six
subjects (three in each group) dropped out from the study
for personal reasons. Of the  completers, eight were incor-
rectly enrolled into the study based on baseline BP and/or
thyroid stimulating hormone level, fasting glucose level, or
smoking status, and for an additional ve subjects, ambula-
tory BP data were not available. ese subjects were excluded
from the PP population which, as a result, consisted of 
subjects ( in the placebo group and  in the RPC group).
Subjects in both the RPC and placebo groups were gen-
erally well-matched, with no signicant dierences between

International Journal of Hypertension 
N=144
PASSED SCREENING
From: 10 North American sites
3 German sites
6 Czech Republic sites
N=269
POTENTIAL PARTICIPANTS SCREENED
From: 12 North American sites
3 German sites
6 Czech Republic sites
N=125
SCREENING FAILURES
From: 12 North American sites
3 German sites
6 Czech Republic sites
Baseline
N=1 Dropout
Week 2
N=0 Dropout N=1 Dropout N=0 Dropout N=0 Dropout N=0 Dropout
N=0 Dropout N=1 Dropout N=0 Dropout N=0 Dropout N=0 Dropout N=0 Dropout
Week 4
N=1 Dropout N=2 Dropout N=0 Dropout N=0 Dropout N=0 Dropout N=0 Dropout
Week 8
Number of Participants completing the study: N=138
Number of Participants Included in the ITT analysis: N=144
Number of Participants Included in the PP analysis: N=125
Czech Republic
(6 sites)
North America
(10 sites)
Germany
(3 sites)
N=144 PARTICIPANTS ENROLLED
N=24
Enrolled to
Placebo Group
N=23
Enrolled to
RPC Group
N=17
Enrolled to
Placebo Group
N=31
Enrolled to
Placebo Group
N=19
Enrolled to
RPC Group
N=30
Enrolled to
RPC Group
F : Flowchart of study participants.
groups in the majority of the demographic (Table ) and
baseline (Table ) variables assessed, except for SBP, which
was slightly but signicantly greater in the placebo group
compared to the RPC group. Average compliance with intake
of the tablets over the  weeks was high (≥.%) for both
groups, with no statistically signicant dierences observed
between groups in either population (data not shown).
e eects of RPC versus placebo on ambulatory BP
andoceBPintheITTpopulationaresummarizedin
Tables  and , respectively. Daytime ambulatory SBP was
signicantly reduced from baseline in the RPC group relative
to the placebo group, both at  weeks (p=.) and at 
weeks (p=.). Oce SBP was signicantly reduced in
theRPCgrouprelativetotheplacebogroupatweeks
(p=.) and  weeks (p=.), with a trend towards
signicance at  weeks (p=.). Similar to daytime ambu-
latory SBP, -hour ambulatory SBP was also signicantly
reduced from baseline in the RPC group relative to the
placebo group at both  weeks (p=.) and  weeks
(p=.), while night-time ambulatory SBP was signi-
cantly reduced in the RPC group relative to the placebo
group at  weeks (p=.) but not  weeks (p=.).
Although changes from baseline in night-time ambulatory
DBP were not signicantly dierent between groups at any

International Journal of Hypertension
T : Demographic and lifestyle characteristics of the placebo and RPC groups in the ITT population (n=).
Factor Placebo (n = ) RPC (n=) p-valuea
Age (years)
Mean ±SD . ±. . ±. 0.923b
Median (Min – Max)  ( – ) . ( – )
Location [n (%)]
North America  (%)  (%)
.
Czech Republic  (%)  (%)
Germany  (%)  (%)
Gender [n (%)]
Female  (%)  (%) .
Male  (%)  (%)
Alcohol Use [n (%)]
None  (%)  (%)
.
Occasionally  (%)  (%)
Weekly  (%)  (%)
Daily  (%)  (%)
Smoking Status [n (%)]
Current Smoker  (%)  (%)
.
Ex-Smoker  (%)  (%)
Non-Smoker  (%)  (%)
Race [n (%)]
Black or African American  (%)  (%)
.
Central American  (%)  (%)
EasternEuropeanWhite (%) (%)
Middle Eastern  (%)  (%)
North American Indian/Aboriginal  (%)  (%)
South American  (%)  (%)
South Asian  (%)  (%)
Western European White  (%)  (%)
Ethnicity [n ( %)]
Hispanic or Latino  (%)  (%) .
Not Hispanic or Latino  (%)  (%)
Status [n (%)]
Completed  (%)  (%) .
Dropout/Withdrew  (%)  (%)
ITT: intention-to-treat; Max: maximum; Min: minimum; n: number; RPC: Rened Peptide Concentrate; SD: standard deviation
aBetween-group comparisons were made using Fisher’s Exact Test, unless otherwise stated. p≤. was considered statistically signicant.
bBetween-group comparison was made using the Independent Student’s t-test.
of the time points assessed, daytime ambulatory DBP was
signicantly reduced in the RPC group versus the placebo
group, both at  weeks (p=.) and at  weeks (p=.),
while at  weeks (but not  weeks), -hour ambulatory
DBP was signicantly reduced in the RPC group relative
to the placebo group (p=.). ere were no signicant
dierences between the RPC and placebo groups in oce
DBP at any of the time points assessed. Daytime and -
hour ambulatory mean arterial pressure were signicantly
reduced from baseline in the RPC group relative to the
placebo group at  and  weeks; a between-group signicant
change in night-time mean arterial pressure, favouring RPC
over placebo, was observed only at  weeks (data not shown).
Taking into consideration the changes in the distribution
of subjects across National Institutes of Health- (NIH-)
dened blood pressure categories over the course of the study
(Figure ), in both groups, based on oce SBP and/or DBP
levels, there was a favourable increase in the proportion of
subjects classied as “Normal” or “Prehypertensive” from
baseline to  weeks. In the placebo group, the percentage
of subjects classied as “Normal” or “Prehypertensive” was
% (/) at baseline compared to % (/) at  weeks.
In the RPC group, the percentage of subjects classied as
“Normal” or “Prehypertensive” was % (/) at baseline
compared to % (/) at  weeks. Although there was a
placebo eect, which is a well-characterized phenomenon in

International Journal of Hypertension 
T : Baseline measurements for the placebo and RPC groups in the ITT population (n=).
Variable Placebo (n = ) RPC (n = ) p-valuea
SBP (mmHg)
Mean ±SD . ±. . ±. .
Median (Min – Max) 146.5 (116.7–182.3) 144.0 (125.0–160.0)
DBP (mmHg)
Mean ±SD . ±. . ±. .
Median (Min – Max) 89.7 (57.3–109.3) 86.0 (71.0–100.3)
Heart Rate (BPM)
Mean ±SD . ±. . ±. .
Median (Min – Max) 73.3 (53.0–87.3) 72.0 (57.7–97.7)
Height (cm)
Mean ±SD . ±. . ±. .
Median (Min – Max) 170.5 (149.2–195.0) 170.5 (150.0–188.0)
Weight (kg)
Mean ±SD . ±. . ±. .
Median (Min – Max) 79.7 (60.1–119.0) 81.1 (61.4–116.9)
BMI (kg/m2)
Mean ±SD . ±. . ±. .
Median (Min – Max) 26.8 (22.8–34.9) 27.4 (20.9–34.8)
BMI: body mass index; BPM: beats per minute; cm: centimeters; DBP: diastolic blood pressure; kg: kilograms; kg/m2:kilogrampersquaremeter;Max:
maximum; Min: minimum; mmHg: millimeter ofmercur y; n: number; RPC: Rened Peptide Concentrate; SBP: systolic blood pressure; SD: standard deviation
aBetween-group comparisons were made using the Independent Student t-test. p≤. was considered statistically signicant.
0248
0
20
40
60
80
100
Time (weeks)
0248
0
20
40
60
80
100
Time (weeks)
% of subjects (RPC)
Normal
Pre-hypertension
Stage 1 Hypertension
Stage 2 Hypertension
% of subjects (placebo)
∗∗∗ ∗∗∗
Normal
Pre-hypertension
Stage 1 Hypertension
Stage 2 Hypertension
F : Proportion of ITT subjects in NIH-dened blood pressure categories at weeks , , , and  of the study based on oce blood
pressure. ITT: intention-to-treat; NIH: National Institutes of Health; RPC: Rened Peptide Concentrate; ∗∗∗: p=., for the dierence
between groups in the distribution of subjects across NIH-dened blood pressure categories, favouring RPC over placebo.
hypertension studies, consumption of RPC versus placebo
resulted in the shiing of a greater proportion of subjects
from hypertensive categories (i.e., Stage  or Stage  hyper-
tension) into Normal/Prehypertensive categories. At Week ,
there was a statistically signicant dierence between groups
in the distribution of subjects across NIH-dened blood
pressure categories, favouring RPC over placebo (p=.).
By Week , the proportion of participants in the placebo and
RPC groups who were categorized as having Normal blood
pressure, Prehypertension, Stage  hypertension, and Stage 
hypertension was % versus %, % versus %, % versus
%, and % versus %, respectively.
Daytime, night-time, and -hour ambulatory heart rate,
oceheartrate,C-reactiveprotein,urinesodium,and
blood lipids (triglycerides, total cholesterol, HDL cholesterol,
LDL cholesterol, non-HDL cholesterol, total cholesterol:HDL
cholesterol), measured at Weeks , , and/or , remained
similar to baseline values, and there were no statistically
signicant dierences between groups in the changes in these
outcomes (data not shown). Results from -day food records

International Journal of Hypertension
T : Changes in ambulatory SBP and DBP for all subjects in the ITT population during the -week supplementation period.
Ambulatory Measure GroupaW W W
Change from
WtoW4bChange from
WtoW8b
Within-group
(p-value)
Between-group
p-value
Within-group
(p-value)
Between-group
p-value
SBP
(mmHg; mean ±SD)
24-hour Ambulatory
RPC . ±. . ±. . ±. -. ±.
.
-. ±.
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. +. ±.
(p=.) (p=.)
Daytime Ambulatory
RPC . ±. . ±. . ±. -. ±.
.
-. ±.
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. +. ±.
(p=.) (p=.)
Night-time Ambulator y
RPC . ±. . ±. . ±. -. ±.
.
-. ±.
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. -. ±.
(p=.) (p=.)
DBP
(mmHg; mean ±SD)
24-hour Ambulatory
RPC . ±. . ±. . ±.  +. ±.
.
-. ±.
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. +. ±.
(p=.) (p=.)
Daytime Ambulatory
RPC . ±. . ±. . ±. -. ±.
.
-. ±. 
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. +. ±.
(p=.) (p=.)
Night-time Ambulator y
RPC . ±. . ±. . ±. -. ±.
.
+. ±.
.
(p=.) (p=.)
Placebo . ±. . ±. . ±. +. ±. -. ±.
(p=.) (p=.)
ANCOVA: analysis of covariance; DBP: diastolic blood pressure; ITT: Intention-to-treat; mmHg: millimeter of mercury; RPC: Rened Peptide Concentrate; SBP: systolic blood pressure; SD: standard deviation;
W: we ek
aAt W , either  or  subjects per group had ambulatory blood pressure recording errors and thus data from  or  subjects per group were used to generate ambulatorySBPandDBPvaluesforW.Although
ambulatory SBP and DBP data were available for all  subjects per group at W  and W , due to the aforementioned reason, data from  or  subjects per group were used to calculate changes from W  to W 
andWtoW.
bWith in-group comparisons of the changes from W  to W  and W  to W  were made using the paired Student t-test. Between-group comparisons of the change from W  to WandWtoWweremade
using ANCOVA with the value at baseline used as the covariate. p≤. was considered statistically signicant.

International Journal of Hypertension 
T : Changes in oce blood pressure for all subjects in the ITT population during the -week supplementation period.
Group W  W  W  W 
Change from
WtoW2aChange from
WtoW4aChange from
WtoW8a
Within-
group
(p-value)
Between-
group
p-value
Within-
group
(p-value)
Between-
group
p-value
Within-
group
(p-value)
Between-
group
p-value
Office SBP (mmHg; mean ±SD)
RPC (n=) . ±. . ±. . ±. . ±. -. ±.
.
-. ±. 
.
-. ±.
.
(p<.) (p<.) (p<.)
Placebo (n=) . ±. . ±. . ±. . ±. -. ±. -. ±. -. ±.
(p=.) (p=.) (p<.)
Office DBP (mmHg; mean ±SD)
RPC (n=) . ±. . ±.   .  ±.    .  ±.  -. ±.
.
-. ±. 
.
-. ±.
.
(p=.) (p=.) (p=.)
Placebo (n=) . ±. . ±.   .  ±.   .  ±. -. ±. -. ±. -. ±. 
(p=.) (p=.) (p=.)
ANCOVA: analysis of covariance; DBP: diastolic blood pressure; ITT: intention-to-treat; mmHg: millimeter of mercury; RPC: Rened Peptide Concentrate; SBP: systolic blood pressure; SD: standard deviation; W:
week
aWith in-groupcomparisonsofthechangesfromWtoW,WtoW,andWtoWweremadeusingthepairedStudentt-test.Between-groupcomparisonsofthechangesfromWtoW,WtoW,
andWtoWweremadeusingANCOVAwiththevalueatbaselineusedasthecovariate.p≤. was considered statistically signicant.

 International Journal of Hypertension
T : Results from Exploratory Analyses for Participants in the RPC Group.
Oxidized
LDL (U/L)
ACE
(ng/mL)
Renin
(pg/mL)
Angiotensin
I (pg/mL)
Angiotensin
II (pg/mL)
Aldosterone
(pg/mL)
W
Mean ±SD . ±.  ±  ± , ± . ±.  ±
Median (Min – Max) . (. – .)  ( – )  ( – ,) , ( – ,) . (. – .)  ( – )
W
Mean ±SD . ±.  ±  ± , ±, . ±.  ±
Median (Min – Max)  (. – .)  ( – )  ( – ,) , ( – ,) . (. – .)  ( – )
ChangefromWtoW
Mean ±SD - ± - ± - ±  ± -. ±. - ±
Median (Min – Max) - (- – ) - (- – ) - (-, – ,) - (-, – ,) -. (-. – .) - (- – )
P-value p=.𝛼p=.∗p=.∗p=.∗p<.∗p=.¤
ACE: angiotensin converting enzyme; LDL: low-density lipoprotein; Max: maximum; Min: minimum; mL: milliliter; ng: nanogram; pg: picogram; SD: standard
deviation; U/L: International units per liter; W: week.
Within-group comparisons were made using the paired Student t-test.
∗Logarithmic transformation required to achieve normality.
𝛼Squared transformation required to achieve normality.
¤Square root transformation required to achieve normality.
Probability values P≤. are statistically signicant.
conducted in the week prior to the baseline, Week , and
Week  visits showed no statistically signicant dierences
between groups in the average daily intake of energy (calo-
ries) and the percent contribution of protein, carbohydrate,
and fat intake to total daily energy intake (data not shown).
In the RPC group, angiotensin II levels were signicantly
reduced from baseline to Week  (-. ±. pg/mL;
p<.); the remaining exploratory variables (oxidized LDL,
ACE activity, renin, angiotensin I, and aldosterone) did
not statistically signicantly change during the intervention
period (Table ).
For all variables and outcomes assessed, the PP popula-
tion analyses were generally similar (in direction and statisti-
cal signicance) to those of the ITT population analyses.
A total of  AEs ( in placebo group and  in
RPC group) were reported during the study ( mild, 
moderate, and one severe in intensity). One AE (in RPC
group) was determined as having the ‘most probable’ rela-
tionship to treatment, ten AEs (six in placebo group and
four in RPC group) were determined as having a ‘possible’
relationship to treatment,  AEs (ve in placebo group and
teninRPCgroup)weredeterminedtohavean‘unlikely’
relationship to treatment, and  AEs ( in placebo group
and  in RPC group) were determined to be ‘unrelated’
to treatment. e one AE that was rated as ‘severe’ in
intensity (accelerated hypertension) occurred in the placebo
group and was determined to be unrelated to treatment.
e one AE (nausea) that was determined as having the
‘most probable’ relationship to treatment occurred in the RPC
group and was rated as moderate in intensity. e ten AEs
determined to be ‘possibly’ related to treatment were RPC
group—euphoric mood (n=), fatigue (n=), upper abdomi-
nal pain (n=), headache (n=); placebo group—conjunctival
haemorrhage (n=), upper abdominal pain (n=), dermatitis
(n=), diarrhoea (n=); all ten AEs were rated as mild in
intensity. ere were no serious AEs reported in the study.
Changes from baseline to Week  in haematological and
clinical chemistry parameters, urine safety parameters (urine
creatinine concentration, urine volume, and urine creatinine
amount), and anthropometric variables such as body weight
and BMI were not signicantly dierent between groups.
4. Discussion
Elevated BP or hypertension is one of the key independent
risk factors for CVD, a global health issue that is estimated to
aect ∼% to % of the world’s adult population [, ].
Clinically, hypertension is characterized as having SBP above
 mmHg and/or DBP above  mmHg, and/or the current
use of any antihypertensive medication []. High BP is
strongly correlated with mortality, highlighting the need for
therapeutic approaches to reduce BP and reduce CVD risk.
ACE converts angiotensin I to angiotensin II (which is a
potent vasoconstrictor), inhibits the activity of the vasodila-
tor bradykinin, and increases aldosterone secretion from the
adrenal cortex, which has a tendency to cause elevated BP
by modulating renal sodium and water retention []. ACE
inhibition is therefore a target in the clinical management of
elevated BP.
It was suggested by Cheung et al. [] that the amino
acid residues at the COOH- and NH2-terminalsareimpor-
tant determinants of the ACE inhibitory potency of a sh
protein hydrolysate. Specically, when glycine was at the
NH2-terminal, the COOH- residues that most eectively
inhibited ACE were tryptophan, tyrosine, or proline. When
glycine was at the COOH-terminal, the NH2residues that
most eectively inhibited ACE were valine, isoleucine, and
arginine. In a sh protein hydrolysate derived from sardine
muscle, the valine-tyrosine hydrolysate is considered to have
the strongest ACE inhibitory eect, a nding aligned with

International Journal of Hypertension 
that of Cheung et al. [], who reported that valine-tyrosine
ranked in the top ten of a total of  dipeptides in terms
ofhavingthemostpotentinhibitionofACE.Inaprotein
hydrolysate of pink salmon (Oncorhynchus gorbuscha), it
was demonstrated, in vitro, that ten dipeptides and ten
tripeptides had ACE inhibitory activities, and that all 
peptides had aliphatic (i.e., glycine, alanine, valine, leucine,
and isoleucine) and aromatic (phenylalanine, tryptophan,
tyrosine, and histidine) amino acids in their sequence [].
e RPC administered in our study was derived from
Coldwater Shrimp (Pandalus borealis). e ACE inhibitory
tripeptides that have been detected in our hydrolysate from
Northern shrimp (Pandalus borealis) include phenylalanine-
threonine-tyrosine and phenylalanine-serine-tyrosine [].
e in vivo inhibition of ACE seems supported, given that
in our exploratory analysis reported herein, angiotensin II
levels were signicantly reduced at Week  relative to baseline
levels in the RPC group. As angiotensin I and II levels were
notassessedinparticipantsintheplacebogroup,future
studies are needed to determine whether RPC derived from
Coldwater Shrimp is, indeed, associated with a reduction
in angiotensin II levels. Also, whether the reduction in
angiotensinIIlevelsisaresultofACEinhibitionalso
requires further investigation. As reported herein,  weeks
aer supplementation with RPC, there was a reduction from
baseline in ACE activity; however, the reduction was not
statistically signicant.
In our study, treatment with RPC had a clinically relevant
impact on lowering BP compared to placebo in the study
participants, all of whom had mild to moderate hypertension
at study entry. Overall, the primary outcomes—oce SBP
and daytime ambulatory SBP—were favourably aected by
the consumption of RPC. With regard to oce SBP, both the
placebo and RPC groups experienced signicant reductions
from baseline at Weeks , , and , a nding that is not
surprising, given the known placebo eect in hypertension
studies []; however, between the two groups, the reductions
in oce SBP were signicantly greater in the RPC group
relative to the placebo group at Week  (by  mmHg; p=.)
and Week  (by . mmHg; p=.) and trended toward
a signicant reduction at Week  (by . mmHg; p=.).
Oce BP measurements can be subject to observer bias and
can be aected by temporary increases/decreases in BP due
to clinic surroundings or an observer’s presence, termed the
‘white-coat syndrome’; also, oce BP measurements can be
associated with blood pressure excursions that are situation-
dependent. Automated BP measurement techniques, such
as ABP monitors, overcome the limitations of oce BP
measurements []. With regard to ABP monitoring, par-
ticipants receiving RPC treatment experienced signicant
improvements over those receiving a placebo in daytime SBP
(at  and  weeks), night-time SBP (at  weeks), and -
hour SBP (at  and  weeks). For other hypertension param-
eters, including ambulatory DBP, mean arterial pressure, and
hypertension categorization, RPC outperformed placebo on
all assessments. Importantly, the participants enrolled in this
study were already in early progression towards developing
hypertension or had mild or moderate hypertension. us,
even modest reductions in BP are meaningful for this
population; in fact, by the end of the study, the proportion
of individuals who were categorized as being prehypertensive
was signicantly greater in the RPC group than in the placebo
group, while the proportion of individuals categorized as
having Stage  or  hypertension was signicantly lower in
theRPCgrouprelativetotheplacebogroup.Importantly,
treatment with RPC was safe and well-tolerated.
5. Conclusions
Reported herein are the results of the rst clinical study on
the ecacy of RPC (a shrimp-derived protein hydrolysate) in
reducing BP. Subjects recruited into this study had elevated
BP (mild or moderate hypertension). RPC versus placebo
signicantly reduced BP in these subjects. From baseline
to the end of the intervention period, RPC versus placebo
caused a greater proportion of subjects to be shied from
hypertensive categories (i.e., Stage  or Stage  hyperten-
sion) into Normal/Prehypertensive categories. Findings from
this study provide evidence that protein hydrolysates from
Coldwater Shrimp can safely reduce BP for subjects with
mild or moderate hypertension, possibly due to a reduction
in angiotensin II levels. Further research is recommended
to conrm the ndings from this study (both on BP and
mechanistic endpoints).
Data Availability
Requests for access to individual subject data may be made to
Marealis AS; please send an email to andreas@marealis.no.
Conflicts of Interest
Mal Evans is an employee of and Tetyana Pelipyagina is a
former employee of KGK Science Inc., which is the contract
research organization that was contracted by Marealis AS to
run the clinical study, conduct the statistical analyses, and
compile the clinical study report. Kathy Musa-Veloso is an
employee of and Lina Paulionis is a former employee of
Intertek Scientic & Regulatory Consultancy, which received,
from Marealis AS, the sponsor of the study, nancial com-
pensation for scientic, and regulatory consulting services,
including payment for the review of the clinical study report
and preparation of the manuscript.
Acknowledgments
We thank Judith Hill for her assistance in preparing the list
of numbered references and Emily Booth for her assistance
with the formatting of the manuscript. Marealis AS was
provided with nancial support from Innovation Norway
and SkatteFUNN/e Research Council of Norway for this
clinical trial and the research and documentation done
prior to the clinical trial. Marealis AS, the sponsor of the
study, provided nancial support for the preparation of the
manuscript.

 International Journal of Hypertension
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