Evaluation of the Efficacy of a Supplement With Spirulina for Amelioration of Benign Thyroid Nodules
Phase: NA
Conditions: Benign Thyroid Nodule
Sponsor: Fundatia Bio-Forum

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
 Protocol ID “TiroNod”,
version FB-F01
   Study ID number NCT03535974,  titled: 
“Efficacy of a Spirulina Supplement for Amelioration of benign thYroid nodules (ESSAY)”
The initial version of Protocol FB-F01 (in Romanian) written on February 12, 2018
first approved by the Ethics Committee of The Bio-Forum Foundation on March 03, 2018
English version (English translation) on February 24, 2020
Principal Investigator: Prof Dr Corin Badiu, Co-Principal  Investigator: Dr Felician Stancioiu

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
Protocol Cover Page
Protocol Title:
Efficacy  of  a  Spirulina  Supplement  for  Amelioration  of  benign
thYroid nodules (ESSAY)
Protocol Number:
Fundatia Bio-Forum 01
(FB-F01)
Protocol Date:
12 February 2018
Study Phase:
I-II
Principal Investigator:
Prof Corin Badiu, MD, Felician Stancioiu, MD
Research Team: 
Prof Corin Badiu, MD, Daniel Mihai, MD, Felician Stancioiu, MD,  
Protocol Author(s): 
Felician Stancioiu, MD
 
PI Prof Dr Corin Badiu, Co-PI Dr Felician Stancioiu
SPONSOR:    
Bio-Forum Foundation
 
Blv D Cantemir 2B, 
Bl P1, Sc 1 Apt 4
Bucharest, Romania
Principal Investigators:        
Prof Dr Corin Badiu
Felician Stancioiu, MD
National Institute of 
Endocrinology;
Bio-Forum Foundation
Bucharest, Romania

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
STUDY CONTACT LIST
For questions regarding the conduct of this study, please contact:
Project Leader:
Felician Stancioiu, MD
E-mail:
f elicians@bio-forum.net
 
 
Mobile:
0040727500402
Clinical investigator:
Daniel Mihai, MD
E-mail:
mihaimisudaniel@yahoo.com
 
For reporting a serious adverse event, please contact:
 Principal Investigator:
Prof Corin Badiu, MD
E-mail:
badicrin@yahoo.co.uk
 
For questions regarding data quality assurance, please contact:
Independent Study Monitor:
Ioana Scantei, RN
Mobile:
0763640163

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
PROTOCOL SYNOPSIS
TITLE
Efficacy of a Spirulina Supplement for Amelioration of benign
thYroid nodules (ESSAY)
Investigational 
site:
Outpatient Office, Prof Corin Badiu, National Institute of 
Endocrinology CI Parhon, Bucharest, Romania
Investigators:
Prof Corin Badiu, Dr Felician Stancioiu, Dr Daniel Mihai
Sponsor:
Bio-Forum Foundation, Bucharest, Romania
Representatives
:
Dr Felician Stancioiu
Study number:
FB-F01
Final Protocol:
February 2018
Clean File:
February 2018
Ethics 
Approval:
March 2018
Statistical 
analysis:
 
 
Clinical Phase:
 I-II
Study Report:
 
OBJECTIVES:
To establish the safety and efficacy of a supplement 
containing spirulina, curcuma and boswellia in decreasing 
the size of benign thyroid nodules
STUDY DESIGN:
 double-blind, placebo-controlled, crossover clinical trial. The
patient population will include approximately 30 consecutive
patients  initially randomized  to  placebo  (P)  or  active
ingredients (AI)
SUBJECTS:
Inclusion criteria
- adult patients over 18 years of age
- agreement to participate in the study by signing the 
Informed Consent Form
- euthyroid status (normal TSH and fT4 values), no active 
dysfunctional thyroid disease
- benign thyroid nodule confirmed by FNAB (Bethesda 2)
- no previous thyroid surgery, thyroid ablation treatment, 
substitution treatment with thyroid
hormones
- no steroid or beta-blocker treatment
- presence of one or more thyroid nodules documented by
ultrasound

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
Exclusion criteria

- pediatric patients (less than 18 years of age)

- diagnosis or suspicion of malignancy, including thyroid

- autoimmune disease, including Hashimoto

- Wilson disease

- hypo- or hyper-thyroidism

- contraception with intrauterine device

- acute infection

- known allergy to spirulina, curcuma or boswellia
TREATMENT TO
BE EVALUATED
Active Treatment AI:       Spirulina 400 mg, Curcumin 50 
mg, Boswellia 50 mg; 4 capsules/day
Placebo Treatment P:   an inert substance 
DURATION OF 
STUDY
6 months total, 3 months per patient
ENDPOINTS:
EFFICACY
1.    Reduction in the area of benign thyroid nodules
       2.    Examination by endocrinologist
SAFETY 
 
  
1. Adverse events, especially allergy, nausea
2. Modification of weight, digestion.
3. Modification of laboratory tests measured from 
venous blood: levels of TSH, fT4, Cu
  
 
SAMPLE SIZE:
approximately 30 patients  
STATISTICAL 
ANALYSIS:
Summary statistics will describe the variables.  Significance
testing will be done using T tests for two groups, by comparing
the means and standard deviation, also Pearson correlation,
and Wald Z test for proportions..  (α = 0,05)

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
 
Introduction
Benign thyroid nodules are among the most common endocrine disorder. Current progress in
imaging and pathology stratified the risk, but apart from surgery, current treatment options are
limited. Here we propose a first evaluation of an noninvasive treatment for benign thyroid nodules via 
an interventional, crossover, placebo controlled clinical study.
With a lifetime occurrence risk in general population estimated at 5-10% (1), thyroid nodules are a 
frequent pathology with a known gender bias, their prevalence being higher in women. Furthermore, 
along the lines of technological development with higher availability of high resolution ultrasound and 
other imaging modalities (ex. incidental findings on carotid or heart ultrasound, or routine scans), and 
also the increased focus on preventive medicine and improved access to healthcare resources, their 
prevalence seems to be higher than estimated, and a recent study revealed thyroid nodules in about 19-
68% of randomly selected individuals (1).
Considering the treatment, besides surgery, several noninvasive procedures developed, like 
radiofrequency ablation, alcohol injection, high intensity focused ultrasound, or laser ablation; a 
minimally invasive procedure was recently shown to be a safe and efficacious treatment for benign 
thyroid nodules: ablation with monopolar radiofrequency (2).
This last treatment is performed with a 16- or 18- Gauge internally cooled radiofrequency electrode 
after local anesthesia, can treat autonomously functional nodules (3), has better results for smaller and 
cystic nodules, and post-procedural complications in only 17% of patients (2). Other studies have 
shown various results with mean decreases of 40-75% at 6 months (4,5) and minimal complications in 
treating benign thyroid nodules with radiofrequency ablation (5,6), being noted that key success factors
are technical skill and patient selection (7).
Even though the vast majority (around 95%) of thyroid nodules are benign (1,8), and the 5% 
remnant thyroid malignancies are among the most successfully treated endocrine cancers (2,9), a 
medical noninvasive treatment which decreases the thyroid nodule dimensions would be greatly 
beneficial.
Patients
Patients and methods:
To evaluate the efficacy and safety of a supplement containing spirulina, curcuma and
boswellia in euthyroid patients with benign thyroid nodules, we will perform a 3 month, double-
blind, placebo-controlled study which aims to enroll approximately 30 patients. Patients with benign 
single thyroid nodules of less than 5 cm are evaluated in a prospective interventional placebo-
controlled cross-over trial, across 12 weeks (3 visits at six weeks intervals). At each visit, the target 
thyroid nodule was recorded in two dimensions. In addition, plasma levels of thyroid stimulating 
hormone (TSH), free thyroxine (fT4) and copper (Cu) were measured.
Participants in this clinical study are consecutive patients presenting for initial evaluation
or follow-up of thyroid nodules in the outpatient department of the National Institute of
Endocrinology in Bucharest, Romania during May-September 2018 and fulfill the inclusion
and exclusion criteria presented below. After being informed about the objective of the study,
including what is needed from each participant in terms of blood samples, ultrasound evaluations,
supplement administration with possible risks and benefits, the patients who agreed to the
terms signed the Informed Consent Form and were enrolled in the clinical study.

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
Inclusion criteria
- adult patients over 18 years of age
- agreement to participate in the study by signing the Informed Consent Form
- euthyroid status (normal TSH and fT4 values), no active dysfunctional thyroid disease
- benign thyroid nodule confirmed by FNAB (Bethesda 2)
- no previous thyroid surgery, thyroid ablation treatment, substitution treatment with thyroid
hormones
- no steroid or beta-blocker treatment
- presence of one or more thyroid nodules documented by ultrasound
Exclusion criteria
- pediatric patients (less than 18 years of age)
- diagnosis or suspicion of malignancy, including thyroid
- autoimmune disease, including Hashimoto
- Wilson disease
- hypo- or hyper-thyroidism
- contraception with intrauterine device
- acute infection
- known allergy to spirulina, curcuma or boswellia
Study design
For assessing the efficacy of the spirulina-based supplement in decreasing the size of
thyroid nodules we are performing a double-blind, placebo-controlled, crossover clinical trial. The
patient population will include aapproximately 30 consecutive cases randomized to placebo (P) or 
active ingredients (AI).
There are three visits for each patient, noted 
V1 – initial visit at enrollment; 
V2 – second (intermediary) visit, after six weeks of administration of placebo (P) or active ingredients 
(AI), and
V3 – final visit, after another 6 weeks of administration of P or AI. 
At each visit each patient is tested for plasma TSH, fT4 and Cu levels, by an independent 
laboratory and a thyroid ultrasound is performed by endocrinologist (DM) using the same ultrasound
machine at all study visits; the nodule images and dimensions are printed for comparison.
At V1, patients are randomly selected to receive each either AI or P, so that about half patients
receive P and the other half will receive AI, and after 6 weeks, at V2, the patients previously on P will 
receive AI, and the patients who first receive AI receive P. At the discretion of the study investigator a 
few more patients will initially receive AI than P.
Consequently, some patients receive the sequence P-AI, other patients receive sequence AI-P, 
and yet others will receive the sequence AI-AI.
The study Is registered on clinicaltrials.gov site with Protocol ID “TiroNod” and ID
number NCT03535974, and titled: Efficacy of a Spirulina Supplement for Amelioration of
benign thYroid nodules (ESSAY).
Outcomes
The primary outcome of the study is a change in size of the thyroid nodules and this is usually 
evaluated by comparing the biggest diameter of the nodules between measurements. However we have 

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
observed that there are patients in which the largest diameter decreases while the smallest diameter 
increases, and this variability may bring significant bias in comparisons; therefore for more consistent 
results and comparisons we will calculate the product of the largest (D) and smallest (d) diameters of 
the nodules in the same incident angle and compare the resulting area of the nodules (S = D x d) 
measured at visits V1, 2, 3.
As secondary outcome we will measure the serum values of TSH, fT4 and copper (Cu).
The first two are considered standard evaluations of the thyroid function, while the latter was chosen 
for multiple reasons: i. spirulina is a significant source of copper alongside with other minerals and 
vitamins; ii. most serum copper is bound and transported by ceruloplasmin (10), and iii. while there is 
an excellent correlation between serum copper and ceruloplasmin, changes of Cu plasma levels are 
faster than plasma ceruloplasmin levels, and so serum Cu would provide more accurate information.
To calculate statistical significance we use the NCSS software for T tests for two groups, by 
comparing the means and standard deviation, also Pearson correlation, and Wald Z test for proportions.
Quantitative data are expressed as mean ± standard deviation.
Treatment administered
The active treatment administered is a combination of spirulina, curcumin and boswellia extracts, 400-
50-50 mg per capsule, which were prepared alongside matching placebo capsules by DVR Pharm SRL 
and BioNovativ SRL. 
Spirulina is the common generic name for about 15 species from two genres of monocelular 
microalgae (Spirulina and Arthrospira), most common being Artrospira platensis (11), which grows 
naturally in freshwater and saltwater in all continents and most climates, optimally in an alkaline milieu
(pH 9.5-12). It was used as the only source of nutrition in some areas in Africa for more than 1 month 
at a time and its first documented use was in China around 2000 years ago also during periods of 
famine (12) . It has a very high protein content: 40-65% of dry mass depending on species and 
conditions of growth/cultivation (13) , it has all essential amino acids, minerals (Fe, K, Na, Ca, P, I, 
Mg, Zn, Cu, Se, Mn, Cr) and vitamins (carotenoids 5 mg/g vitamins B1, B2, B3, B6, B12, folate, 
biotin, pantothenic acid, vit K1, K2), gamma linolenic acid, chlorophyll and phycocyanin, thus being a 
complete food source.
Alongside this remarkable composition it has important quantities of antioxidants, such as superoxide 
dysmutase (360 UI/g), zeexantin (3 mg/g), vitamin E (14) .
Curcumin is natural extract from the roots of Curcuma longa, has also been in use for centuries 
in Asia for various ailments. It is a potent anti-inflammatory substance with pleiotropic actions, 
including antioxidant and metabolic modulation, and its effects are well studied, with multiple clinical 
trials showing benefits in arthritis, inflammatory bowel disease, prediabetes, and even in early stages of
neoplastic disease. A recent meta-analysis on 15 randomized clinical trials shows that curcumin down-
regulates inflammation and oxidation products by reducing levels of IL-6, hs-CRP, and MDA (15) .
Boswellia is also a potent anti-inflammatory molecule by blocking the NFkB pathway and
modulating the activity of regulatory and T-cell (16) . Together with Curcuma it was shown to
inhibit production of inflammatory cytokines Il-6, IL-8, TNF-α and reactive oxygen species (17) .
Patients are instructed to take two capsules twice daily, about 30 minutes before breakfast and lunch. 

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
Any and all adverse reactions are to be promptly reported to the study investigator.
 Benign thyroid nodules are among the most common endocrine disorders. Recent advances in 
diagnostic imaging and pathology have significantly contributed to better risk stratification of thyroid 
nodules. However, current treatment options, beyond surgical approaches are limited. The following 
placebo-controlled study presents, to the best of our knowledge, the first results of a  non-invasive 
therapy for benign thyroid nodules.  
The efficacy and safety of a supplement containing spirulina, curcumin and boswellia in euthyroid 
patients with benign thyroid nodules, is assessed by a 3-month, double-blind, placebo-controlled study. 
Patients with benign, single thyroid nodules with largest diameter of less than  5 cm are evaluated in a 
prospective placebo-controlled cross-over trial, across 12 weeks (3 visits with six-week intervals). 
At each visit, the target thyroid nodule was recorded in two dimensions. In addition, plasma levels of 
thyroid stimulating hormone (TSH), free thyroxine (fT4) and copper (Cu) are assessed.  
Study Summary
Design: double blind, placebo controlled, crossover 
Inclusion criteria: 
- adult F / M, euthyroid patients
- thyroid nodules documented ultrasound, without malignant suspicion (optimum 10-25 mm)
Exclusion criteria: 
- clinical suspicion of hyperthyroidism / Hashimoto
- ATPO increased
- TSH, T3, T4 in abnormal range
Three clinical and laboratory assessments (study visits V-V3) are made for each patient: 
- initially (upon enrollment in the study) - V1,
- after the first treatment (6 weeks after enrollment) – V2 
- final (12 weeks after enrollment) - V3
Thyroid ultrasound and TSH, fT3, fT4, serum copper analysis are performed at each of these study 
visits.
 
The treatment administered to each patient consists of 4 capsules / day, administered 2 + 2, with
½-1 hour before breakfast and lunch. Some capsules - supplement A  - contain spirulina (500 mg) + 
curcumin (50 mg) + boswellia (50 mg), -  and others are placebo - supplement B;
Each patient has 2 treatments of 6 weeks, each patient has both the active preparation and 
placebo, and the sequence is type and sequence of capsules is blinded to both the investigator and the 
patients.

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
The size of the nodules is evaluated (by measuring the largest diameters) after 6 weeks with the 
active substance, about 35 patients to make the 3 study visits.
Study Schedule
Enrollment/Initial
visit
6 weeks
between V1
and V2
Visit 2
6 weeks
between V2
and V3
Visit 3 - final
Thyroid Ultrasound
Thyroid Ultrasound
Thyroid Ultrasound
Laboratory TSH, 
fT3, fT4, seric Cu 
Laboratory TSH, fT3, 
fT4, seric Cu
Laboratory TSH, fT3, fT4, seric 
Cu
A / B supplement 
administration
A / B supplement 
administration
           Clinical Observations
There  are  three  visits  for  each  patient,  noted  as  V1  –  initial  visit  at  enrollment;  V2  –
intermediary visit, six weeks post-administration of placebo (P) or active ingredients (AI); and V3 –
final visit, after another 6 weeks of administration of P or AI. At each visit, each patient was tested for
plasma TSH, fT4 and Cu levels, by an independent laboratory and a thyroid ultrasound was performed
by an endocrinologist (DM) using the same ultrasound machine; the nodule images and dimensions
were printed for comparison.
At V1, patients were randomly selected to receive either AI or P, so that half of them (n=17)
received P and the other half (n=17) received AI. Subsequently at V2, the 17 patients previously on P
received AI, and 12 of the 17 patients previously on AI received P, while the remaining 5 randomly
chosen patients continued to receive AI. Consequently at V3, 17 patients received the sequence P-AI,
12 patients received AI-P, and 5 patients had AI-AI. The study was registered on clinicaltrials.gov site
with Protocol ID “TiroNod” and ID number NCT03535974, and titled: Efficacy of a Spirulina
Supplement for Amelioration of benign thYroid nodules (ESSAY).
 
Statistical analysis plan
NCSS 16 software  is used for data analysis. Quantitative data were expressed as mean ± standard
deviation (SD). Paired sample T test was used for comparisons of two groups (AI and P). Pearson’s
correlation was used to assess correlations between specific variables. Wald Z test for proportions was
used for  comparing differences in outcomes between two groups. One-way ANOVA test with the
Tukey and Bonferroni post-hoc can be used to compare values of TSH, fT4 and Cu.  α=0.05, and
P<0.05 indicated statistical significance. 
Total patients  enrolled, mean age, distribution by sex
Total completing the study and are evaluated for the primary outcome - the size of the thyroid nodules
evaluated by ultrasound at 1st (initial), 2nd (intermediate) and 3rd (final) patient visits. These patients will
also be tested for serum TSH, fT4 and Cu. 
The  overall  reduction  in  the  measured  surface  of  the  thyroid  nodules  for  all  the  study
participants, calculated as the mean percentage decrease in the surface of nodules (V3-V1),  

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
For all patients, mean initial nodule area at initial visit (V1 S) vs 2nd visit (V2 S)  and at 3nd visit (V3 S)
mean nodule area  
From the total patients with the 3 ultrasound evaluations, the percentage (%) who had decreases
in the areas of thyroid nodules of 5% or more at the end of the study,  and percentage (%)  with
decreases of 10%  or more of the thyroid nodule area at V3.
 
Percentage change from initial vs. final area of thyroid nodules, number of patients in groups
Initial vs. final area
decrease %
< 0
(increase)
~ 0
(unchanged)
5-9.9%
(decrease)
10-19.9%
(decrease)
20-49.9%
(decrease)
50-99%
(decrease)
Number of patients
 
A comparison of the effects of the active substance vs. placebo on decreasing the surface of
thyroid nodules  that after the initial administration of the placebo and active substances (V2-V1 for all
patients), the mean decrease with AI vs the mean decrease with placebo.
Comparing effects for the two groups (AI and P) is done with two-sided T-test for the two groups.
Additionally, we will use two statistical tests: the Wald Z test to compare the proportion of patients with
modifications in nodule areas, grouped in AI vs. P  and also tested both the equal variance and Aspin-
Welch variants for the difference of means and SD between the AI and P groups.
Will also calculate the difference in the proportion of patients with decreased thyroid nodule areas on
active substances  vs.. placebo groups. 
       For the overall effects of the placebo and the active substances on all administrations, we compare
the effects of the administration of the active substances by the mean decrease in nodule areas, versus
the mean decrease of nodules following placebo administration.
Testing for statistical significance between the two groups (active and placebo) at V3 versus V1 with T-
test for means and standard deviation, calculated with the equal variance T-test and the unequal
variance Aspin-Welch test.
Comparing the means, minimum and maximum values nodule areas in the AI (Act) and P (Plac) groups
after the initial 6 week administration of AI or P (V2-V1, noted “A”) and after 12 week of AI or P (V3-
V1, noted “A+B”, A being the initial 6 weeks, B the final 6 weeks of study). 
 
 
Beyond efficacy, we  are monitoring the influence of treatment on thyroid function and the
correlation with Cu levels. 
To determine whether the active substances or the placebo had a significant effect on thyroid
function, we compared the respective values of TSH and fT4 for all 3 visits (visit 1 vs.. 2, visit 1 vs.. 3
and 2 vs.. 3) with the one-way ANOVA test and the Tukey and Bonferroni post-hoc tests.

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
To verify the interdependence of the four variables measured in this study: nodule surface,
TSH, fT4 and Cu levels, we calculate the Pearson correlation indices between these variables.
 
The  link  between  thyroid  function  and  ceruloplasmin,  the  most  important  copper-based
enzyme, was observed a few decades ago, with a significant elevation of serum ceruloplasmin being
observed in patients on oral contraceptives together with an increase in thyroxine binding index, while
free thyroxine levels were unchanged  (20). Similarly, in male rats estradiol elevated ceruloplasmin
levels, while testosterone and thyroid hormones inhibited this action (21). Furthermore, it was shown
that  administration  of  L-thyroxine  suppresses  the  stimulation  of  ceruloplasmin  by  estrogen
administration  (22).  Opposite  modifications  of  ceruloplasmin  levels  in  hypo-  and  hyper-thyroid
patients, in tandem with thyroid function,   as well as oxidation status (malonyldialdehyde) and uric
acid levels were observed by a Romanian team (23). New direct links between ceruloplasmin, oxidative
stress and inflammation were recently shown with TNF-related apoptosis-inducing ligand (TRAIL)
down-regulating ceruloplasmin expression in inflammation  (24), and modulation of TNF-α-induced
neutrophil apoptosis by ceruloplasmin (25). Low levels of ceruloplasmin were shown in a hypothyroid
patient with Hashimoto thyroiditis (26).
Recent findings have shown that thyroid hormones increase serum copper levels by stimulating
the synthesis of ceruloplasmin in the liver as reflected in the mRNA levels, and this action is abolished
in cases of hyperthyroidism associated with receptor resistance to thyroid hormones (27). In addition,
the ratio of serum copper (Cu) and selenium (Se) was proposed as an indirect marker for receptor
resistance  to  thyroid  hormones.  Stimulation  of  hepatic  ceruloplasmin  mRNA by  thyroxine  and
dexamethasone was previously shown in rats  (28). It was also found that a copper-deficient diet
increases the levels of serum free triiodothyronine (fT3) but not free thyroxine (fT4) in rats  (29);
similarly a strong link between serum T3 and Cu was demonstrated in rat pups (30). Cu and Zn levels
in serum and tissue are modulated by thyroid hormones as shown by thyro-parathyroidectomy and T4
replacement in rat. Interestingly, it was seen that ceruloplasmin is present in follicular carcinoma and
absent in adenomas (31-33).  Further differences in the oxidative and inflammatory processes involved
in benign and malignant thyroid nodules versus normal thyroid tissue (hypoxia-inducible factor-1,
complement factor D, matrix metalloprotease 1, Von Willebrand Factor - were shown in different
studies (34, 35)  The interrelations between thyroid hormones, ceruloplasmin, adrenal hormones and
important metabolic processes are presented in Fig. 3.
Figure 3. Interactions between thyroid hormones, ceruloplasmin, adrenal hormones, and processes of 
intestinal absorbtion, oxidative metabolism and inflammation: T3, T4 – triiodothyronine, thyroxine, 
TRH/TSH – thyroid releasing/stimulating hormones, ACTH – adrenocorticotropic hormone, SOD – 
superoxide dysmutase, 

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
 
In summary, the combination of spirulina-curcumin-boswellia is effective in reducing the size
of benign thyroid nodules and can be safely administered in the doses used in the presented clinical
study. The mechanism by which the decrease of the nodule size is facilitated, is probably linked to the
anti-inflammatory effects, the improvement of the antioxidant status and the ceruloplasmin actions.
However, additional studies are needed in order to elucidate these associations, which may also provide
important insights into the pathogenesis of thyroid nodules. ot applicable.
Funding 
This  study  is  financed  by  a  grant  received  by  The  Bio-Forum  Foundation  from  DVR  Pharm SRL
and BioNovativ SRL which  intend  to  manufacture  and  bring  to  market  the  spirulina-curcumin-boswellia
combination under the name “TiroDren”.
Ethics approval and consent to participate

Bio-Forum Foundation
Clinical Study Protocol No: FB-F01  
 
             
 
                            
 
   Protocol Date:  
 
 February 12,
 
  2018
 
 
 All participants will provide written informed consent for the whole study. The study observes the ethical rules 
on clinical studies established by the Institute according to the World Health Organisation guidelines.
 
 
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  2018
 
 
  
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