APPENDIX 1 - The funding application (to be filled out in English)

B. The project leader

B1. Curriculum Vitae of the project leader1
LAST NAME AND FIRST NAME: LUPASCU FLORENTINA GEANINA
Email: lupascu.geanina@yahoo.com; florentina-geanina.l@umfiasi.ro
Date of birth: 07.04.1985
Professional experience:
-from october 2013- present - Assistant professor in department of Pharmaceutical Chemistry,
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa, Iasi;
Main activities and responsibilities
Know safety procedures and provide a safe environment in which students can work; prepare
for lab experiments or assignments; learn how to guide students' thinking and deal with questions
without giving away the answers, monitoring and recording the practice working results from students,
regularly giving feedback on the progress and behavior of students, -Participate at the research
activities of the Pharmaceutical Chemistry department.
- from october 2016–present - Lecturer in department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Medicine and Pharmacy Grigore T Popa, Iasi; developing course material and
laboratory practical work for educational activities with students, presentation of course materials to
students, research with didactic staff of discipline.
Education and formation
- october 2009 - octomber 2013 – PhD in medical sciences, Certificate in Medical Sciences;
Area of research: Pharmacy; Specialty: Pharmaceutical Chemistry, The PhD thesis “New xanthine
derivatives with biological potential”.
http://www.umfiasi.ro/ScoalaDoctorala/TezeDoctorat/Teze%20Doctorat/GHEA%C5%A2%C4%82%
20(LUPA%C5%9ECU)%20FLORENTINA%20GEANINA_rezumat.pdf
- october 2004 - july 2009 - Pharmacist licensed in Environmental chemistry and food”,
undergraduate degree No. B1 series. 0018534, 9.88 average at the graduation exam, the title of the
licence - furanocoumarins, bioactive compounds in foods
- september 2000-june 2004 - High School graduated, High school diploma series T no. 0462086,
marks average 9 to baccalaureate exam, High School „Vasile Alecsandri”, Iasi.

1 2
Sections B1 and B3 of the application will be published on UEFISCDI website. These will be uploaded in the online
platform for submission, both as two separate pdf. files as well as part of the funding application.
Internships abroad
- 19 august 2012 – 19 november 2012 - I was for three months at internship University of
Ghent, Faculty of Sciences, Polymer Materials Research Group, Ghent, Belgium Scholarship awarded
during doctoral studies within the project POSDRU „Doctoral scholarships for increasing
competitiveness in the medical and pharmaceutical fields” - project code 58965.
Research activity
- January the 1’st 2015 – June 30’th 2016 - Scientific researcher, I developed research activity
by inclusion in intern grant ”IMPROVING OF THE PHARMACOLOGICAL PROFILE OF ORAL
ANTIDIABETIC DRUGS USING POLYMER MATRICES” project Leader: Associate Professor Dr.
Stan Cristinel
-12 may 2014 – 7 october 2015 - Postdoctoral researcher. The research conducted was intitled
“Research regarding new therapies to treat type 2 diabetes” and done by the acceptance of my
membership in the target group of the project "Strategic partnership to improve the quality of
scientific research in medical universities through scholarships Doctoral and Postdoctoral - DocMed
.Net_2.0 "POSDRU 159 / 1.5 / S / 136 893”, co-funded by the European Commission
- 1 march 2010– 1 march 2013 - I benefited of doctoral scholarship by inclusion in the target
group of the project "Doctoral scholarships for increasing competitiveness in the medical and
pharmaceutical fields – Project code 58965, co-funded by the European Commission

Personal skills and competences, Acquired during career but not necessarily covered by a
certificate or a diploma:
- Course 1H-NMR, 13C-NMR, MRI organized by the Department, Faculty of Science, Ghent,
Belgium (October 2012).
- Course electrospinning technique to obtain nanofibers, organized by the Department of Polymer
Chemistry, Faculty of Science, Ghent, Belgium (September 2012).
Competence and aptitude in the field of research: - development of new structural entities by
synthesis, safer and more efficient, as potential antidiabetic therapeutic agents. Knowing methods of
spectral characterization and the interpretation thereof of spectral analyzes (UV, IR, NMR).
Scientific activities: - ISI works  Number 6 (author and co-author);
- BDI works  Number 9 (author and co-author);
- Number of papers in other journals, in proceedings of ISBN or ISSN
conferences: 22;
- Number of research projects - one (member of the project team)
B2. Significant and representative scientific achievements
The research activity began with my registration to doctoral studies from Faculty of Pharmacy,
University of Medicine and Pharmacy, Iasi. To increase scientific performance, I applied and received
financial support by my inclusion in the target group of the project "Doctoral scholarships for
increasing competitiveness in the medical and pharmaceutical fields – Project code 58965 in the
interval 1’st Mars 2010 / 28’th February, 2013 co-funded by the European Commission. The subject of
research had as main objective the development of new, safer and more efficient, potential antidiabetic
therapeutic agents. Through an original approach, 21 new heterocyclic compounds (derivatives of
xanthine-hydroxybutylidene-thiazolidine) have been obtained and characterized, having all the
theoretical premises to act as "multitarget" drug. These compounds may modulate the action of the
incretins, reduce the resistance of peripheral tissues to insulin and protect the pancreatic tissue from
harmful action of oxidative stress. This study has brought an important scientific contribution to
diabetes therapy and it represented the starting point in initiating other antidiabetic therapy research
studies, with impact on public health. These compounds were also biologically analyzed, the
compound 4-hydroxy benzylidene thiazolidine 4-one registering a remarkable antioxidant and
hypoglycemic action. The results of this research were valued by systematizing the obtained data and
by their inclusion in two articles published in the BDI Medical and Surgical Journal and one ISI article,
to which I am main author, published in Molecules journal.
The research area approached in doctoral studies, synthesis and characterization of new
structural entities with biological properties, was enlarged by knowing new directions in the field of
polymer chemistry: preparation and characterization of polymeric systems. This research experience
has been occasioned by the three months internship at the Faculty of Ghent, Department of Polymer
Chemistry and Biomaterials. The polymers constitute the class of functional and flexible materials, the
foundation of all modern industries not only in medicine and pharmacy but also in the chemical, textile,
etc.
Research direction of the doctoral studies constituted an important pillar in starting postdoctoral
studies that have been facilitated by the acceptance of my membership in the target group of the project
"Strategic partnership to improve the quality of scientific research in medical universities through
scholarships Doctoral and Postdoctoral - DocMed .Net_2.0 "POSDRU 159 / 1.5 / S / 136 893, co-
funded by the European Commission period: May 12th, 2014 - October 7th, 2015. In studies doctoral
research has been undertaken which covered analysis of in vivo biological xanthine derivative 4
hydroxy benzylidene thiazolidine 4-one. In order to improve the pharmacological and pharmacokinetic
profiles of the xanthine derivatives, it was developed new xanthine–chitosan formulation. After chronic
oral administration of this formulation on diabetic rats, an improvement of hypoglycemic effect was
observed, therefore the results obtained sustained the potential application of this formulation in the
treatment of the diabetes mellitus syndrome. The results of this research were valued by systematizing
the achieved data and by their inclusion in an one ISI article published in European Journal of
Pharmaceutical Science, a journal with 3.35 impact factor and in a second article, submitted to the
journal European Journal of Pharmacology with 2.73 impact factor; to both articles I am main author.
My inclusion in the research team of intern grant ”IMPROVING OF THE
PHARMACOLOGICAL PROFILE OF ORAL ANTIDIABETIC DRUGS USING POLYMER
MATRICES” Period January the 1’st 2015 – June 30’th 2016; project Leader: Associate Professor Dr.
Stan Cristinel constituted an occasion of communication and direct transfer of information and
experience among the team members. In this study, new binary polymeric systems based chitosan-
metformin-glybenclamide have been developed in order to improve the pharmacokinetic and
pharmacological profile of the used antidiabetic drugs. These polymeric systems showed improved
swelling degree, a good loading efficiency of antidiabetic drugs and a higher hypoglycaemic effect.
This means the new formulations could be a better therapeutic alternative for management of diabetes
mellitus treatment. The results of this research were used by systematizing data and by their inclusion
in two articles published in an ISI Journal, Farmacia. For one article, I am main author and co-author
for the second article.
The research activity conducted from 2010 to date has contributed to the accumulation of
experience on both teamwork and individual work. The chosen research themes have also contributed
to a better knowledge in this field which included:
-synthesis and physico-chemical and spectral characterization of new structural entities;
- in vitro and in vivo evaluation of biological properties of the compounds, regarding the
antioxidant and hypoglycemic action;
-getting new systems for controlled release of these compounds on the basis of chitosan in order
to improve their biological and pharmacokinetic profiles.
The research project has as main objective improving of pharmacokinetics and pharmacological
profile of glibenclamide, by developing polymer systems based on chitosan and embedding in the
polymer matrix the antidiabetic substance of glibenclamide with ferulic acid - substance with a
remarkable antioxidant action. These new polymeric systems with multi target action will constitute a
new approach to therapy for type II diabetes.
The whole research experience accumulated to present days and the research subjects, adjacent
to submitted project topics will contribute to the successful completion of its activities and objectives.
B3. Defining elements of the outstanding scientific achievements of the project leader 2
1. Articles
1. Florentina Geanina Lupascu, Mamoni Dash, Sangram Keshari Samal, Peter Dubruel,
Cătălina Elena Lupuşoru, Raoul Vasile Lupuşoru, Oana Dragostin, Lenuţa Profire, Development,
optimization and biological evaluation of chitosan scaffold formulations of new xanthine derivatives
for treatment of type-2 diabetes mellitus, European Journal of Pharmaceutical Science 2015, 77, 122-
134 (ISI Web of Science, IF 3,35).
New xanthine derivatives as potential antidiabetic drugs have been synthesized and their structure
was proved through spectral methods (IR, 1H NMR, 13C NMR). Based on the beneficial effects of
chitosan as drug delivery system as well as its demonstrated antidiabetic effect new xanthine–chitosan
formulation have been developed in order to improve the pharmacokinetic and pharmacological profile
of the developed xanthine derivatives. The optimized formulations were evaluated in terms of particle
size, morphology, swelling degree, X-ray diffraction and Fourier transform infrared analysis. The
results demonstrated that the loading efficiency of the xanthine derivatives in chitosan microparticles
was between 68.98% and 90.89% depending of the type of ormulation. The new developed formulation
showed also an increased cumulative release in the simulated biological fluids and a good
biodegradation rate. The xanthine derivatives with hydroxyl group, showed a good antiradical
scavenging effect, reduced toxicity level, and improved antidiabetic effect. Chronic oral administration
of xanthine derivatives with hydroxyl group on rats with streptozotocin induced diabetes mellitus was
associated to a lowered level of the blood glucose, while the glycosylated hemoglobin level was similar
to the one of pioglitazone. The antidiabetic effect of xanthine derivatives with hydroxyl group was
improved by loading in chitosan microparticles, the chitosan formulation, CS-6, showing an improved
hypoglycemic effect. All these results demonstrate the potential application of xanthine derivatives
with hydroxyl group and its chitosan formulation (CS-6) in the treatment of the diabetes mellitus
syndrome.
2. Florentina Geanina Lupascu, Oana Maria Dragostin, Liliana Foia, Dan Lupascu, and Lenuta
Profire, The Synthesis and the Biological Evaluation of New Thiazolidin-4-one Derivatives Containing
a Xanthine Moiety, Molecules, 2013, 18(8): 9684-9703 (ISI Web of Science, IF.2,67)
In this study new heterocyclic compounds that combine a xanthine structure with a thiazolidine
one have been synthesized. The structures of all new compounds were proven using spectral methods.
The compounds were evaluated for their antioxidant activity using in vitro assays: ferric reducing
power, total antioxidant activity, ABTS and DPPH radical scavenging ability. The all
benzylidenethiazolidin-4-one derivatives showed improved antioxidant effects in reference to the
corresponding thiazolidin-4-ones. The encouraging preliminary results support the antioxidant potential
of the synthesized compounds and their possible applications in several diseases mediated by reactive
oxygen species , including diabetes mellitus, and motivate our next research focused on their potential
antidiabetic effects.
3. Oana Maria Dragostin, Sangram Keshari Samal, Mamoni Dash, Florentina Lupascu,
Andreea Pânzariu, Cristina Tuchilus, Nicolae Ghetu, Mihai Danciu, Peter Dubruel, Dragos Pieptu,
Cornelia Vasile, Rodica Tatia, Lenuta Profire. New antimicrobial chitosan derivatives for wound
dressing applications. Carbohydrate Polymers 2016; 141:28-40. (ISI Web of Science, IF. 4.07)
New chitosan-sulfonamide derivatives were synthesized and characterized with regard to
structural, physico-chemical properties, swelling capacity, biodegradability, biocompatibility and tested
in respect with antimicrobial and antifungal activities. It has been found that all six chitosan-
sulfonamide derivatives exhibited better antimicrobial activity than the pristine chitosan, which
indicated that the antimicrobial ability of chitosan was strengthened by the introduction of sulfonamide
part to chitosan. The chitosan derivatives showed improved swelling and biodegradation rate and are
biocompatible and most of them are not cytotoxic. In vivo test proved that among the chitosan-
sulfonamide derivatives, the chitosan-sulfadiazine showed also improved healing effects. It can
conclude that these new chitosan derivatives could be useful in application as potential new dressing
materials for wound, especially for burn wounds.
4. Oana Dragostin, Sangram Samal, Florentina Lupascu, Andreea Pânzariu, Peter Dubruel, Dan
Lupascu, Cristina Tuchilus, Cornelia Vasile, Lenuta Profire. Development and Characterization of
Novel Films Based on Sulfonamide-Chitosan Derivatives for Potential Wound Dressing. European
Journal of Molecular Science 2015; 16(12): 29843-29855. (ISI Web of Science, IF. 2.86)
New films based on six chitosan-sulfonamide derivatives (CS-sulfametoxydiazine, CS-
sulfadiazine, CS-sulfadimethoxine, CS-sulfamethoxazol, CS-sulfamerazine, CS-sulfizoxazol) were
developed and characterized. The films showed a rough surface and a hydrophilic character compared
to chitosan film. For some of the chitosan-derivative films (CS-sulfamethoxydiazine, CS-
sulfamethoxazol, CS-sulfizoxazol) the swelling ratio was similar and even higher than that of chitosan
film. Also, based on the rough surface, all films of chitosan derivatives were biodegraded at a higher
ratio than chitosan, for which the biodegradation ratio was only 10%. The sulfonamide-chitosan
derivatives showed antioxidant effects compared with chitosan proved by in vitro assays (total
antioxidant capacity, ferric reducing power, DPPH radical scavenging ability). The antimicrobial
effects of chitosan-sulfonamide derivative films were also evaluated. The tested films, especially
chitosan-sulfadiazine (CS-S2) and chitosan-sulfamethoxazol (CS-S4), showed good antibacterial and
antifungal activity against all tested strains. According to the results, the most effective films for
biomedical applications such as wound-dressing materials are chitosan-sulfadiazine (CS-S2) and
chitosan-sulfamethoxazol (CS-S4), which showed good antimicrobial and antioxidant effects. In
addition, the chitosan-sulfamethoxazol also showed good mechanical properties, which are also
important for potential wound-dressing applications. However, even progress has been made in the
healing process, the control of infections, free radicals and inflammation remains a huge area of interest
for researchers.

5. Oana Maria Dragostin, Florentina Lupascu, Cornelia Vasile, Mihai Mares, Valentin Nastasa,
Ramona Florina Moraru, Dragos Pieptu, Lenuta Profire. Synthesis and Biological Evaluation of New 2-
Azetidinones with Sulfonamide Structures. Molecules 2013; 18, 4140-4157 (ISI Web of Science,
IF.2,67)
In this study new N-(arylidene)hydrazinoacetyl and new 2-azetidionone derivatives have been
designed and synthesized starting from sulfadiazine and sulfizoxazole. The structures of all new
compounds were proved using spectral methods. The compounds were evaluated for their antimicrobial
and antioxidant activity. Although their antimicrobial potential was reduced, they shown excellent
antioxidant properties; for some of them the potential is comparable with the antioxidant activity of
ascorbic acid. These results support the antioxidant potential of the synthesized compounds and their
applications in several disease mediated by reactive oxygen species (ROS) including the healing of the
wounds.

2. Books/ chapters (including monographs):

-
C. The mentor
C1. Curriculum Vitae of the mentor (maximum 2 pages);
LAST NAME AND FIRST NAME: LUPUȘORU CĂTĂLINA ELENA
EDUCATION AND TRAINING
1996 PhD in Medicine, specialty Pharmacology, “Grigore T. Popa” University of Medicine and
Pharmacy Iasi, confirmed by Order of the Education Minister no. 5200/4.10.1996. PhD thesis: New
synthetic immunomodulators. Research the possibility of selective immunomodulators identification
1986-1990 Specialist physician, Cantacuzino Institute – Iasi branch, specialty Clinical laboratory,
hematology and microbiology, Decision no. 4258/17.04.1986
1982-1986 Secondary physician, Cantacuzino Institute – Iasi branch, specialty Clinical laboratory,
hematology and microbiology, Order of Health Minister no. 478/1982
1979 Medical Doctor, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy
Iasi, MD diploma no. 6046/1979
PROFESSIONAL EXPERIENCE
2007-present PhD supervisor, Medical Sciences, specialty Pharmacology
11 PhD thesis confirmed, 6 PhD Students
2002-present Professor, Department of Pharmacology, Clinical Pharmacology, Algesiology, “Grigore
T. Popa” University of Medicine and Pharmacy Iasi
1999-2002 Associated professor, Department of Pharmacology-Toxicology-Algesiology, “Grigore
T. Popa” University of Medicine and Pharmacy Iasi
1996-present Main scientific researcher grade III
1990-1999 Lecturer, Department of Pharmacology and Toxicology, “Grigore T. Popa” University
of Medicine and Pharmacy Iasi
INVENTIONS AND PATENTS
• polibacterial immunostimulatory agent OROSTIM with oral administration, Inventor
Certificate no. 91006/22.10.1986 (co-author in team conducted by MD Hoişie Sylvia, Cantacuzino
Institute – Iasi branch); introduced in therapeutics since 1.01.1993;
• co-author at 2 products proposed to be patented: “Immunomodulatory drugs and synthesis
procedure” (OSIM 141281/14.VIII.1989); “Synthesis procedure of polibacterial immunomodulatory
agent (Imunostim) with external use (ocular, nasal and auricular)" (OSIM - nr. 14192/9.X.1989).
RESEARCH ACTIVITY
MD PhD Prof. Catalina Elena Lupusoru has experience in pharmacodynamics, toxicology, biomaterial
compatibility, experimental and clinical pharmacology. She participated at various activities such as:
protocol design and pharmacokinetic analysis of more than 10 bioequivalence studies (1999-2004);
fundamental studies and experimental researches for the development of immunopharmacology field,
an original screening that included the immunopharmacological testing of some active substances
(1990); development of some animals experimental methods used for testing new pharmacological
agents (new chemical derivatives, new polymers matrices) on laboratory animals: induced
inflammation (acute or chronic models), induced pain models (visceral or central pain), endotelin-1
inducing arterial hypertension, stress induced models (associated or not with arterial hypertension or
cardiac ischemia), induced experimental hepatic failure, ovalbumin induced experimental
bronchoconstriction, corticosteroids or cyclophosphamide induced experimental immunosuppression,
induced experimental diabetes II type.
RESEARCH ACTIVITY REPORT
• 20 national grants of which would be mentioned 2: “Pharmacological influence of nociception
through incorporated analgesics in original nanoparticulate systems” (“Grigore T. Popa” University of
Pharmacy and Medicine Iasi Grant no. 16407/2009, Project leader Univ. PhD Assist. Lecturer Tarţău
Liliana); “Researches concerning the development of new heterocyclic derivatives with improved anti-
inflammatory potential” (“Grigore T. Popa” University of Pharmacy and Medicine Iasi Grant no.
29244/2013, Project leader: PhD Assoc. Prof. CD Stan).
• 130 scientific publications on national and international journals, more than 300 scientific
publications on conference proceedings;
• 2 books as single author and 17 books as co-author.
Also, MD PhD Prof. has coordinated the master ʺPain and stress pharmacotherapyʺ (Subtopic
Pharmacological therapy of the stress) and the postgraduate course ʺImmunopharmacology – Actual
data and perspectivesʺ. Starting with 1990 there was around 200 diploma thesis of the Medicine
Faculty graduates. She was member in organizing committees of scientific manifestation (7), in exam
commissions: getting didactic titles (university assistant, lecturer, professor), public defense, specialist
physician in Clinical Pharmacology, organization residency exam, university admissions, thesis. CDI
Evaluator expert (UEFISCDI) in the project ʺProfessional counseling for medicine students and
practical integrative program on the general and dental medicine fieldsʺ, POSDRU/160/2.1/S/139881,
Project Manager MD PhD Norina Consuela Forna.

C2. The list with the most important scientific publications

1.Damian L, Ghiciuc CM, Dima-Cozma LC, Ungureanu MC, Cozma S, Patacchioli FR, Lupuşoru
CE. No definitive evidence for a connection between autoimmune thyroid diseases and stress in
women. Neuro Endocrinol Lett. 2016; 37(3): 155-162. [ISI Web of Science]
2.Popa G, Mititelu Tartau L, Stoleriu I, Lupusoru RV, Lupuşoru CE, Ochiuz L. The effect of
pregabalin - codeine combination on partial sciatic nerve ligation - induced peripheral
mononeuropathy in rats. J Physiol Pharmacol. 2016; 67(3): 465-469. [ISI Web of Science]
3.Ghiciuc CM, Dima-Cozma LC, Bercea RM, Lupuşoru Cătălina Elena, Mihaescu T, Cozma S,
Patacchioli FR. Imbalance in the diurnal salivary testosterone/cortisol ratio in men with severe
obstructive sleep apnea: an observational study. Braz J Otorhinolaryngol. 2016; 82(5): 529-535. [ISI
Web of Science – in process]
4.Ciubotariu D, Ghiciuc CM, Lupuşoru CE. Zinc involvement in opioid addiction and analgesia--
should zinc supplementation be recommended for opioid-treated persons? Subst Abuse Treat Prev
Policy. 2015; 10: 29. [ISI Web of Science]
5.Lupascu FG, Dash M, Samal SK, Dubruel P, Lupuşoru Cătălina Elena, Lupusoru RV, Dragostin O,
Profire L. Development, optimization and biological evaluation of chitosan scaffold formulations of
new xanthine derivatives for treatment of type-2 diabetes mellitus. Eur J Pharm Sci. 2015; 77: 122-
134. [ISI Web of Science]
6.Mititelu Tartau L, Popa EG, Lupusoru RV, Lupuşoru Cătălina Elena, Stoleriu I, Ochiuz L.
Synergic Effects of Pregabalin-Acetaminophen Combination in Somatic and Visceral Nociceptive
Reactivity. Pharmacology. 2014; 93(5-6):253-259. [ISI Web of Science]

D. Research project description

D1. Issues
Research subject chosen is modern and with a high importance in health area. Diabetes mellitus
type 2 (T2DM) is the most common form of diabetes, characterized by hyperglycemia resulting from
decreased insulin secretion caused by the β-cell dysfunction, insulin resistance in the liver, in the
adipose tissue and the skeletal muscles, or both. Diabetes reduces the quality of life, increases the risk
factors for mortality and morbidity worldwide and is considered one of the five main causes of death in
the world. The global prevalence of diabetes mellitus for all age groups was estimated to 2.8% in 2000
and is projected to rise to 4.4% in 2030 (Chandramohan et al., 2008). The chronic hyperglycemia of
diabetes is associated with micro and macrovascular complications leading to blindness, kidney failure,
heart disease, stroke, gangrene requiring leg amputation. Oxidative stress in diabetes coexists with a
reduction of the antioxidant status, an over production of free radicals, which produces an imbalance
between the levels of pro-oxidants and antioxidants, leading to cellular injury in biological systems
(Chowdhury et al., 2016). It is known the oxidative stress is acting adversely to the development and/or
progression of tissue insulin resistance. On the other hand, chronic exposure of endothelial cells to
oxidative stress leads to the initiation and progression of macro- and microvascular complications of
diabetes (Punithavathi et al., 2011).
Therefore, the goals of managing diabetes mellitus are the reduction of hyperglycemia and the
decline of oxidative stress through the neutralizing of free radicals. The decrease of oxidative
stress/toxicity may help the beta cells to proliferate and radiate more insulin in the pancreas. Increased
secretion of insulin causes a growth in the utilization of glucose from extra hepatic tissues that lessen
the blood glucose level.
 Different natural sources of antioxidants have been reported to exhibit their therapeutic
potential in various disease models, including diabetes. Like several other phenols, ferulic acid also
exhibits antioxidant activity, in response to free radicals, via donating one hydrogen atom from its
phenolic hydroxyl group. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is such a natural
phytochemical potent wich is extremely abundant in plants. Ferulic acid has an important antioxidant
effect and may be useful in the prevention and treatment of several diseases, for which oxidative stress
plays an important role, like: Alzheimer's disease, diabetes mellitus, cancer, hypertension and
atherosclerosis. Due to remarkable antioxidant action, ferulic acid may play a significant role in
treating diabetes by defending pancreatic beta cells from oxidative stress (Kimura et al., 2015).
Recent research showed that the ferulic acid scavenging effect is similar to that of superoxide
dismutase, a very efficient enzyme, naturally present in human body that can neutralize oxygen free
radicals with the protection of many types of cells from free radicals attack (Kumar and Pruthi , 2014).
In relation to its physicochemical and biological properties, FA has an aqueous solubility of
6.63 mg·dL−1 at pH 7.2, a half-life of 42 min when is administrate orally. In addition to antioxidant
potential, ferulic acid exhibits a wide range of biological activities such as anti-inflammatory,
antimicrobial, hepatoprotective, anticarcinogenic, antiviral, vasodilator actions, etc. (Kumar and Pruthi,
2014). In spite of this biological potential, its medical use is restricted due to some physicochemical
properties such as low aqueous solubility. FA has a hydrophobic nature, it can oxidize, and it is a
photosensitive compound. In that sense, its bioavailability is usually decreased, leading to a minimal in
vivo effect through oral administration (Nadal et al., 2016).
The pharmacological agents currently used for the treatment of type 2 diabetes include:
sulfonylureas, glinides, biguanides, thiazolidinediones and alpha-glucosidase inhibitors. Although
considerable progress has been made, these drugs have limited use because several undesirable sides
effects and fail to significantly alter the course of diabetic complications. A reference anti-diabetic
substance is Glibenclamide, a third generation sulfonylurea with high efficacy and long duration of
action but with low bioavailability (45%) due to the hydrophobicity. This substance may cause weight
gain, gastrointestinal disorders and severe and prolonged hypoglycemic episodes.
The research project aims to improve the pharmacokinetics and pharmacology profile of
glibenclamide, the physicochemical properties of ferulic acid by developing polymeric drug delivery
systems as binary active substance microparticles based on chitosan. Polymer matrices based on
chitosan, polyisobutylene, polyisoprene, Carbopol 934P are usually used to improve the bioavailability
of the drugs and also to obtain a sustained release of the drug. The desirable properties of an oral
delivery system for prolonged release are high drug loading capacity, good mucoadhesion and high
tolerance. Thus, microparticles are considered a promising approach for controlled and site specific
drug delivery, which minimize dosage frequency and improve patient compliance (Nidhi et al., 2016).
Chitosan is a biopolymer with broad practical applications in the pharmaceutical and
biomedical field because of its outstanding properties: biocompatibility, nontoxic, good
biodegradability, hypoglycemic, cholesterol-lowering, anti-inflammatory, antioxidant, etc. Chitosan
has found pronounced application in multiparticulate drug delivery and it enhances the dissolution of
drugs having poor solubility. CS possess mucoadhesive properties due to strong hydrogen bonding
groups like – OH, and –COOH, high molecular weight, sufficient chain flexibility, and surface energy
properties favoring spreading into mucus, reducing the clearance rate of drug from the body with
increasing the bioavailability of drugs incorporated in it. Various therapeutic agents such as anticancer,
anti-inflammatory, antibiotics, antithrombic, steroids, proteins, amino acids, antidiabetic and diuretics
have been incorporated in Chitosan micro spheres to achieve controlled release as well to enhance
bioavailability or for drug targeting to specific areas of the body.
Various techniques have been developed to prepare chitosan micro/nanoparticles, such as ionic
gelation, emulsion droplet, spray drying, coacervation and self-assembly chemical modification.
Among these methods, the ionic gelation method (also known as ionotropic gelation) with the non-
toxic multivalent poly anion tripolyphosphate (TPP) is the most widely used approach to physical
cross-linking. Chitosan, a cationic polysaccharide is particularly useful for constructing biopolymer
particles based on electrostatic interactions between positively charged amino groups of chitosan and
negatively charged counterion of TPP. This method is advantageous whereas the reaction is simple, the
conditions are relatively mild and do not require the use of organic solvents or high temperatures that
could contribute to degradation of the incorporated substances.
This new approach for the preparation of a binary polymer system based on chitosan have the
following strengths: increasing the glibenclamide bioavailability with reducing the frequency and
intensity of hypoglycemic episodes, enhancing its solubility, and improving the physicochemical
properties of ferulic acid as stability, bioavailability, and/or its biological effect.

D2. Objectives
The main objective of this research project is to improve the pharmacokinetics and
pharmacology profile of glibenclamide, the physicochemical properties of ferulic acid by developing
polymeric drug delivery systems as binary active substance microparticles based on chitosan.
The principal objective of the project will be based on the following specific objectives:
 1. Preparation and characterization of chitosan based microparticles as polymer matrices for
Glibenclamid and Ferulic acid controlled released. The first step in preparing these new
pharmaceutical formulations consists in optimization of the method used for the preparation of chitosan
based microparticles. In order to obtain stable microparticles, the experimental condition will vary:
type of chitosan, the concentration of the chitosan solution, the concentration of the cross-linking agent,
the ratio between chitosan and cross-linking agent and the reaction time. Considering the fact that
microparticles size greatly influences physico-chemical characteristics of the polymer matrix as:
microparticles stability, encapsulation characteristics (loading efficiency, retention, and release),
behavior within the gastrointestinal tract (transport, degradation, interactions, and penetration) we
propose to analyze several formulations of chitosan microparticles in terms of microparticles stability,
then to operate the extraction of three possible formulations to be proposed for drug delivery systems.

2. Development and characterization of new binary polymeric drug delivery systems Chitosan-
Glibenclamide-Ferulic acid. After establishing the necessary conditions for obtaining stable
microparticles, that provide a growth of ferulic acid and glibenclamide bioavailability, we intend to
realize the characterization of this three new binary polymeric drug delivery systems Chitosan-
Glybenclamide-Ferulic acid. The following aspects will be studied: the size, the swelling behavior, the
structural and morphological analysis of microparticles (SEM), physico-chemical and spectral (IR),
determination of loading efficiency (the amount of drug loaded and entrapped efficiency of the active
substance- Glybenclamide and ferulic acid into the chitosan microparticles), and evaluation of in vitro
biodegradation under the action of lysozyme. Another determining which brings valuable information
regarding in vivo substance release is the swelling test performed on chitosan microparticles in distilled
water and in simulated gastric environment, too, because our aim is to get oral administration of such
microparticles. We also want to analyze the in vitro kinetic studies on drug release in conditions that
simulate the body fluids: gastric fluid (pH 1.6), intestinal fluid and colonic fluid (pH 6.5). After all
characterizations and determinations, we will choose the best formulation to ensure an increased
glibenclamide and ferulic acid bioavailability.
This binary active substance microparticles based on chitosan was developed in order to improve
stability, increase bioavailability and/or enhance biological effect of FA and for increasing the
pharmacokinetic and safety profile of glybenclamide. In addition, this drug release system based on
chitosan will create a synergy between chitosan and glybenclamide, with an improvement of
hypoglycemic action.
 Through an original and interdisciplinary approach, it will be developed new binary polymeric
drug delivery systems Chitosan-Glibenclamide-Ferulic acid, having all the theoretical premises to
ameliorate glycemic control to people with diabetes, on the one hand, by improving the glibenclamide
bioavailability, reducing the frequency and intensity of hypoglycemic episodes and, on the other hand,
by protection of the pancreatic tissue from the harmful oxidative stress.

3. Biological evaluation of the new binary polymeric drug delivery systems type
microparticles in reference to active substances-glybenclamide and ferulic acid,
The best formulation that can ensure an increased glibenclamide and ferulic acid bioavailability
will be biological evaluate. These polymeric systems can achieve a good glycemic control through the
synergism of hypoglycemic action of glibenclamide and chitosan and, secondly, by reducing oxidative
stress due to the remarkable antioxidant action of ferulic acid.
The in vivo evaluation will consist in determining the next biological activities:
- hypoglycemic potential (fasting blood sugar, HbA1c) and antioxidant properties (superoxide
dismutase – SOD, glutathione peroxide - GPX and catalase - CAT). In type II diabetes, insulin
secretion decreases due to “β-cell glucose toxicity” and it represents a result of beta cells mass
affection, diminishing in the same time their proliferation. It is known that there is a statistically
significant relationship between appropriate long-term blood glucose control and reduction of diabetic
complications, therefore the maintenance of strict glycosylated hemoglobin (HbA1c) in normal
parameters is necessary, but it requires the preservation of β-cell function. An important role in the
proliferation of pancreatic beta cells it is played by the reduction of oxidative stress/toxicity which will
lead to increased secretion of insulin. Increased secretion of insulin causes a growth in the utilization
of glucose from extra hepatic tissues, that lessens the blood glucose level.
- evaluation of the renal toxicity degree (urea, creatinine, uric acid). Also we will monitorise and
analyze the clinical parameters-weight, intake of water and food of diabetic rats treated with analyzed
substances. too. Diabetic nephropathy, which appears in the early stage of diabetes, is associated with
reduced glomerular filtration rate and cause elevated serum creatinine, urea, uric acid (Kodera et al.,
2014). Prevention of diabetic nephropathy represents a challenge for public health systems because of
the exorbitant costs of renal replacement therapy and moreover, because it became the most common
cause of end-stage renal disease (Balakumar et al., 2014). Early diabetic nephropathy is characterized
by hypertrophy of the glomeruli and tubular epithelial cells, thickening of basement membranes and
enhanced renal blood flow.
- evaluation of lipids profile (total cholesterol, triglycerides, low-density lipoprotein - LDL, high-
density lipoproteins - HDL). Abnormal lipid metabolism plays an indispensable role in the
pathogenesis of diabetic nephropathy. Hyperlipidemia in diabetes occurs as a result of metabolic
disturbances such as regulatory processes, especially insulin deficiency, thereby rendering the diabetic
patient in hypercholesterolemia and hypertriglyceridemia (Kumar et al., 2013). In addition, the patients
with diabetes and profound alteration of lipoprotein profile have an increased risk of coronary heart
disease, peripheral vascular disease and cerebrovascular disease. The liver plays a key role in glucose
and lipid homeostasis and it is severely affected during diabetes. Hepatic steatosis associated with
diabetes mellitus occurs as a result of fatty acids accumulation by the liver, esterification with glycerol
phosphate and storage as triglycerides.
- evaluation hepatic profile (total bilirubin and direct, aspartate aminotransferase-AST, alanine
aminotransferase - ALT, lactate dehydrogenase - LDH). It is known that hyperglycemia plays a crucial
role in the development of diabetic complications, including hepatotoxicity.
- Performance histopathological study on kidney and liver tissue fragments. Because diabetes is
associated with failure of various organs, the goal of this study is the evaluation of the protective
effects of microparticles loaded with Glybenclamide – ferulic acid on hepatic and renal function, the
histopathological changes on kidney and liver tissue fragments on streptozotocin-diabetic rats.

D3. Impact
Through an original and interdisciplinary approach, it will be developed new binary polymeric
drug delivery systems Chitosan-Glibenclamide-Ferulic acid, having all the theoretical premises to
improve glycemic control to people with diabetes on the one hand, by amelioration of the
glibenclamide bioavailability, by reducing the frequency and intensity of hypoglycemic episodes and,
on the other hand, by protection of the pancreatic tissue from the harmful oxidative stress. Numerous
studies have shown that these polymeric systems would represent a first step to approve a potential
therapeutic system used in the treatment of diabetes mellitus.
This study will bring an important scientific contribution to new therapeutic approaches in
diabetes referring to the hypoglycemic management. If the results will be encouraging, it will also have
a public health impact.
The project results will make important contributions on:
(1) improving the health of people with diabetes by making polymeric systems with multi target
action: Chitosan-Glibenclamide-ferulic acid. These polymeric systems will achieve good glycemic
control through the synergism of hypoglycemic action of glibenclamide and chitosan and secondly by
reducing oxidative stress due to the remarkable antioxidant ferulic acid action.
(2) increasing the visibility of national/international University by harnessing and by inclusion
of the results in ISI scientific articles, and through presentation of the works at international scientific
events.
The economic impact refers to the attraction on laboratories/medicine factory interested in the
obtained results and also to develop a technology which can transfer the biopolymeric matrices
production to a pilot and even industrial scale.
This project opens new themes of research concerning the optimization of hypoglycemic effect
of current oral antidiabetics’ drugs, on the one hand, using a synergic effect with chitosan polymer
matrix and, on the other hand, by associating antidiabetic drug with one antioxidant substance and
inclusion in the polymer matrix for protection of the pancreatic tissue from the harmful oxidative
stress.

D4. Methodology
The methodology of the research follows certain key intermediate goals which will be spread in time as
following:
1. Preparation and characterization of chitosan microparticles (8 months)
These microparticles are designed as polymer matrices for controlled released of glybenclamide
and ferulic acid. In the last years, the chitosan based microparticles obtained using a cross-linking
agent like tripoliphosphate, is very useful for development of new delivery systems with improving
pharmacokinetic profile. Chitosan microparticles will be obtained by the ionic gelation of the
polycationic chitosan solution with anionic tripolyphosphate (TPP) according to the method described
in the literature (Hejjajia, Smith, Morris, 2017). At this stage, we will vary the experimental condition:
type of chitosan (chitosan low molecular weight and chitosan medium molecular weight), the
concentration of the chitosan solution (1-1.5-2%), the concentration of the cross-linking agent (1-2-
3%), the ratio between chitosan and cross-linking agent (3:20, 4:20, 5:20) and the reaction time for
cross linking process (12-24 h), and we will analyze these formulations regarding microparticles
stability, the size, the swelling behavior, the structural and morphological analysis of microparticles
(Lupascu et al., 2015). The particle size will be determined, using scanning electron microscopy
images with specific software. The composition, the structure and the morphology of the polymer
systems will be analyzed by Fourier transform Infrared Spectrometer and scanning electron microscopy
respectively, too. After we analyze these microparticles obtained through the mentioned methods, it
will be extracted three possible formulations to be proposed for drug delivery systems.

2. Development and characterization of new binary polymer drug delivery systems type
microparticles (10 months)
The microparticles type polymer-drug systems will be prepared by ionotropic gelation method
through a similar method with that of chitosan based microparticles preparation (Hejjajia, Smith,
Morris, 2017). So, after establishing the three formulations to prove a growth of ferulic acid and
glybenclamide bioavailability, we will proceed to encapsulation of Glybenclamide and ferulic acid in
polymer matrix. These three new binary polymeric drug delivery systems Chitosan-Glybenclamide-
Ferulic acid will be characterized in terms of:
- particles size determined using scanning electron microscopy;
- the swelling behavior performed in distilled water and simulated gastric fluid;
- shape, surface, composition analyzed by Fourier Transform Infrared Spectrometer and
Scanning Electron Microscopy respectively;
- the amount of drug loaded and entrapped efficiency estimated spectrofotometric at 230 nm
(Glybenclamide) and 718 nm (ferulic acid) (Gonçalves et al., 2016; Jadhav et al., 2012);
- evaluation of in vitro biodegradation under the action of lysozyme.
- in vitro drug release studies performed in conditions which simulate the body fluids: gastric
fluid (pH 1.6), intestinal fluid and colonic fluid (pH 6.5) (Lupascu et al., 2015).
After all these characterizations and determinations, we will choose the best formulation to ensure
an increase glibenclamide and ferulic acid bioavailability.
3. Biological evaluation of the new binary polymeric drug delivery systems type microparticles in
reference to active substances-glybenclamide and ferulic acid (6 months)
Determination of the antidiabetic and antioxidant effects will be achieved by recording the
active substance induced modulation upon glucose level (fasting blood sugar, glycated hemoglobin –
HbA1c) and antioxidant activity (superoxide dismutase – SOD, glutathione peroxide - GPX and
catalase - CAT) on diabetic (streptozotocin induced diabetes mellitus model) and nondiabeic rats,
knowing that metabolic activity during diabetes is strongly mediated by oxygen dependent systems.
The Fasting blood sugar will be measured using glucometer FORA G71a, Switzerland with FORA test
strips, while glycated hemoglobin will be evaluate by automatic analyzer through high performance
liquid chromatography (HPLC).
 Itt will also be evaluate the lipid profile (total cholesterol, triglycerides, low-density lipoprotein -
LDL, high-density lipoproteins - HDL) and hepatic toxicity, by analyzing bilirubine, the liver cytolysis
indicators (aspartate aminotransferase-AST, alanine aminotransferase - ALT, lactate dehydrogenase -
LDH) of diabetic rats treated with analyzed substances, of healthy control and diabetic control. Serum
HDL, triglycerides and total cholesterol will be measured by the enzymatic method using commercial
kits, spectrophotometric method and kinetic methods. In vivo renal toxicity by evaluation of
biochemical parameters as urea, creatinine, and uric acid will be also studied through the
spectrophotometric methods. Because diabetes is associated with failure of various organs, as
nephropathy hepatic steatosis, we will evaluate the histopathological changes on kidney and liver tissue
fragments on streptozotocin-diabetic rats using Nikon Eclipse 5i microscope (Lupascu et al., 2015).
We will also monitories and analyze the clinical parameters-weight, intake of water and food of
diabetic rats treated with analyzed substances (Kodera et al., 2014).
The research subject, through the proposed activities and realistic goals is an issue that can be
finalized without major risks. I believe that the scientific potential risks on the preparation and
characterization of microparticles based on chitosan and in vivo biological evaluation of these new
binary polymeric drug delivery systems can be solved given the knowledge and skills acquired during
doctoral and postdoctoral research. The potential risks for the research can be solved thanks to the great
research experience of my mentor.
The potential risks may appear due to insufficient research infrastructure which goes beyond the
laboratory of Pharmaceutical Chemistry, where the majority of biopolymeric matrices preparation is
going to be made and beyond CEMEX Centre where the in vivo study will be conducted. One
approach in order to solve this difficulty will be the utilization of the necessary infrastructure from
scientific partners (scanning electron microscope) available at the Macromolecular Chemistry Institute
"P. Poni" Iasi with which the Pharmaceutical Chemistry department has also collaborated in different
projects.

D5. Ethical aspects (if appropriate)

The experimental studies involve the use of animal (rats). The animal will be purchased through
biobase of University of Medicine and Pharmacy "Grigore T. Popa", Iasi, from Cantacuzino Institute,
Bucharest. The animals and the experiment will take place at the Cemex,
http://www.cemex.umfiasi.ro/. The protocol for animal experiment has advised by Ethics Research
Committee, University of Medicine and Pharmacy "Grigore T. Popa and will perform in compliance
with Law no. 206/27 2004 and EU / 2010/63 - CE86 / 609 / EEC directive. The experiment is
necessary to check the effects of the new binary polymeric systems on diabetic rats. The animal
experiment will use the minimum number of animals necessary for a statistical meaning (6 animals per
lot), only microparticles with the best efficiency in encapsulation will be used - reducing the numbers
of animals used for the experiment, no duplication of the experiment will do and the pain and distress
of animals will be minimized.

D6. Resources and budget

For the project implementation we have the majority of the research infrastructure necessary to
accomplish the proposed goals. For the preparation of the biopolymeric matrices it will be needed the
Freeze Dryer and for the spectral characterization we will use the Fourier transform spectrometer,
ABB-MB 3000 FT-IR MIRacleTM Single Bounce ATR-crystal ZnSe found in the Pharmaceutical
Chemistry laboratory. Link: http://erris.gov.ro/Research-Unit-on-Drug-Food-a
For the morphological characterization we will use the existent infrastructure of the
Macromolecular Chemistry Institute (Brucker-300 NMR spectrometer, scanning electron microscope)
and for the biological evaluation using in vivo study, the research will be conducted in the CEMEX
laboratory. Link: http://www.cemex.umfiasi.ro/
Budget Breakdown (in euro):
Budget chapter 2017 2018 2019 Total budget
(expenses) (euro) (euro) (euro) 2017-2019
(euro)
Personnel 6296 12592 6296 25184
Logistics 854.6 17777.7 4444.44 23076.88
Travel 0 1111.11 1111.11 2222.22
Indirect 1072.6 2222.13 1777.71 5072.44
Total 8223.20 33702.94 13629.26 55555.54

Budget Breakdown (in lei):

Budget chapter 2017 2018 2019 Total budget
(expenses) (lei) (lei) (lei) 2017-2019
(lei)
Personnel 28332 56664 28332 113328
Logistics 3846 80,000 20,000 103,846
Travel 0 5,000 5,000 10,000
Indirect 4826,7 9999,6 7999,7 22826
Total 37004,7 151663,6 61331,7 250,000
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680-684.
7. Kimura T., Kanet H., Shimoda M., Hirukawa H., Okauchi S., Kohara K., Hamamoto S.,
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