Aprotinin revisited: formulation, characterization, biodistribution and therapeutic potential of new...

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J Drug Target, Early Online: 1–13! 2015 Informa UK Ltd. DOI: 10.3109/1061186X.2015.1015537

ORIGINAL ARTICLE

Aprotinin revisited: formulation, characterization, biodistributionand therapeutic potential of new aprotinin microemulsionin acute pancreatitis

H. Yesim Karasulu1, Nevin Oruc2, Neslihan Ustundag-Okur1, Derya _Ilem Ozdemir3, Zeynep Ay Senyigit1,Funda Barbet Yılmaz4, Makbule Asıkoglu2, Hayal Ozkılıc5, Eren Akcicek2, Tamer Guneri1, and Omer Ozutemiz2

1Department of Pharmaceutical Technology, Faculty of Pharmacy, 2Department of Gastroenterology, Faculty of Medicine, 3Department of

Radiopharmacy, Faculty of Pharmacy, 4Department of Pathology, Faculty of Medicine, and 5Department of Nuclear Medicine,

Faculty of Medicine, Ege University, Izmir, Turkey

Abstract

The aim of this study was to develop aprotinin-loaded microemulsion (MA) for intravenousadministration and evaluate the biodistribution and therapeutic potential of developedformulation in acute pancreatitis models in rats. Phase diagrams were constructed to identifymicroemulsion region and the optimal microemulsion was evaluated for physicochemicalproperties and treatment effect in rats, and comparisons made with the solution of aprotinin(SA). To evaluate the biodistribution of the drug by gamma scintigraphy aprotinin wasradiolabeled with 99mTc radionuclide. Mild and severe acute pancreatitis was induced in rats bysubcutaneous injections of cerulein and introductal infusion of 3% sodium taurocholate intothe bile-pancreatic duct, respectively. In addition, serum amylase and pancreatic tissuemyeloperoxidase activities were measured to evaluate the pancreatic damage. According togamma scintigraphy and biodistribution studies, accumulation times and distribution of99mTc-MA and SA were different. While MA was highly uptake by reticuloendothelial system,SA was mostly excreted by kidneys and bladder. Compared with the mild acute pancreatitisgroup, treatment with MA significantly decreased the serum amylase activity and pancreasmyeloperoxidase activity. Furthermore, the protease inhibitor molecule aprotinin hastherapeutic potential in acute pancreatitis. Finally, MA may be suggested as a promisingalternative for treatment of acute pancreatitis.

Keywords

Aprotinin, microemulsion, 99mTc-aprotinin,mild acute pancreatitis, severe acutepancreatitis

History

Received 21 November 2014Revised 16 January 2015Accepted 2 February 2015Published online 4 March 2015

Introduction

Therapeutic peptides and proteins are usually administered

via parenteral route since their poor absorption after oral

administration [1]. Parenteral route is very important for drug

development and delivery research. Parenteral microemul-

sions are valuable drug delivery system, due to their excellent

thermodynamic stability, low viscosity, ease of preparation,

ability to withstand sterilization techniques and potential to

provide sustained release [2,3]. Small particle size may results

in higher blood circulation time which would be useful in

certain cases [2]. In addition, microemulsions are suitable

dosage forms for delivering peptide and protein drugs [1,4,5].

Aprotinin is a monomeric globular polypeptide which

derived from bovine lung tissue. It has a molecular weight of

6512 Dalton and consists of 16 different amino acids arranged

in a chain of 58 amino acid residues. Since aprotinin is a

Kunitz protease inhibitor and has action particularly against

trypsin, chymotrypsin and kallikrein, it is theoretically

attractive molecule in ameliorating the effects of acute

pancreatitis [6,7]. Acute pancreatitis is autodigestion of the

pancreas by activated pancreatic enzymes especially trypsin

within the pancreatic acinar cells [8]. This autodigestion

process leads to profound inflammatory changes both locally

and systemically [9]. Although 80% of the acute pancreatitis

cases have mild course 20% have severe necrotising pancrea-

titis with high mortality rates [10,11]. There is no effective

treatment of acute pancreatitis and current management

options are usually supportive [10]. Experimental studies

are generally focused on anti-inflammatory drugs or mol-

ecules while trials focused on stopping the mechanistic

pathways in acute pancreatitis are quite few [12].

Aprotinin has been used for over three decades as an

intensive care drug for acute pancreatitis and hyperfibrinoly-

tic hemorrhage due to shock of traumatic or septic origin [13].

Although aprotinin therapeutic potential on pancreatitis is

proven experimentally, its clinical therapeutic success is

limited due to low targeting to pancreas [7,14]. Therefore,

in this study, we aimed to formulate aprotinin microemulsion

formulation to improve aprotinin bioavailability in the

pancreas.

Address for correspondence: Hatice Yesim Karasulu, Faculty ofPharmacy, Department of Pharmaceutical Technology, Ege University,35100 Bornova, Izmir, Turkey. Tel: +90 232 3111010/3138. Fax: +90232 3885258. E-mail: [email protected]

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The purpose of this study was to formulate a novel o/w

microemulsion system of aprotinin (MA) for intravenous (i.v.)

administration and to investigate its feasibility for future

practice. For this aim, first, the physicochemical characteris-

tics and stability of microemulsion system were investigated.

Second, aprotinin was radiolabeled with 99mTc and quality

control and stability studies of 99mTc-aprotinin were per-

formed. Then, gamma scintigraphy and biodistribution studies

were performed to evaluate the biodistribution of aprotinin

solution (SA) and MA in rats with acute pancreatitis. Finally,

therapeutic potential of SA and MA for mild and severe forms

of experimental acute pancreatitis were evaluated.

Materials and methods

Materials

Aprotinin, Cremophor EL, cerulein, sodium taurocholate,

stannous chloride (SnCl2�2H2O) and Carregeenan were

purchased from Sigma (Steinheim, Germany) and oleic acid,

isopropanol, sodium hydroxide and sodium dihydrogen phos-

phate were purchased from Merck (Steinheim, Germany).

Glycine was purchased from Eczacibasi (Istanbul, Turkey).

Saline was purchased from Adeka (Istanbul, Turkey) and water

for injection was purchased from Sopharma (Istanbul, Turkey).99mTc was obtained from the Department of Nuclear Medicine

(Ege University, Istanbul, Turkey). All chemicals were used as

analytical grade.

Microemulsion formulation development

Phase diagram construction

Safe and biocompatible non-ionic surfactant (Cremophor EL)

and a co-surfactant (isopropanol) were chosen to form

microemulsions. Pseudoternary phase diagrams of oil, sur-

factant/cosurfactant and saline (0.9% NaCl) were developed

to formulate microemulsion systems for oleic acid (oil phase).

The weight ratio between Cremophor EL and isopropanol was

1:2. The mixtures of oil, surfactant/co-surfactant ranging at a

constant ratio from 5:95 to 95:5 (w/w), were titrated with

saline under constant magnetic stirring at 25 ± 2 �C. The

mixture was observed for transparency. The points from clear

to fuzzy and fuzzy to clear were defined as emulsion and

microemulsion, respectively. Based on the diagram, suitable

concentration of materials was selected. All the experiments

were repeated at least three times. The stability of

microemulsion was evaluated by measuring the turbidity

using a turbidimeter (Lovibond� Water Testing Tintometer�

Group, Dortmund, Germany). Centrifugal methods have been

also employed to subject the system to assess accelerated

stability [15,16]; The microemulsion was subjected to centri-

fugation at 5175� g for 30 min and observed for any phase

separation.

Preparation of aprotinin-loaded microemulsion (MA)

Aprotinin was slowly incorporated into the aqueous phase

under stirring and mixed the other composition of micro-

emulsion. After aprotinin was entirely dissolved in the

microemulsion, the clear microemulsion was obtained. The

final concentration of aprotinin in microemulsion system was

0.2 mg/mL [5000 kallikrein inactivator unit (KIU)]. MA was

filtered by 0.22 mm membrane sterile filter (Minisart,

Sartorius) into a sterile vial under laminar airflow Class II

cabinet (EsCo Class 2 ACIIG34M, Selangor, Malaysia). In

order to verify sterilization, 100 mL of MA was inoculated on

Eosin Methylene Blue and Blood agar mediums and

incubated at 37 �C for 48 h. The results were assessed by

bacteria colony counting. All the measurements were carried

out in triplicate.

Preparation of aprotinin solution (SA)

Two milligrams of aprotinin was dissolved in saline

(0.2 mg/mL). SA was filtered by 0.22 mm membrane sterile

filter (Sartorius) into a sterile vial under laminar airflow Class

II cabinet (EsCo Class 2, Selangor, Malaysia).

Characterization of microemulsions

The microemulsion was analyzed for various physicochemical

attributes. The average droplet size and polydispersity index of

microemulsions with or without aprotinin were measured by

photon correlation spectroscopy (Nano ZS, Malvern

Instruments, Worcestershire, UK). The viscosity values of

microemulsions were measured at 25 ± 2 �C using a viscosim-

eter (ULA spindle, Brookfield, WI). The pHs of the

microemulsions were detected at 25 ± 2 �C using a digital

pH-meter (HI 221, Ontario, Canada). The refractive indexes of

microemulsions were evaluated using a refractometer (Atago

RX-7000, Tokyo, Japan). Conductivity of the microemulsions

was studied at 25 ± 2 �C using a conductometer (Jenway 4071 –

Staffordshire, UK). The phase inversion temperature (PIT) of

the microemulsions with or without aprotinin was measured

using a conductometer [17,18] Experiments were performed in

triplicate for each sample. The osmolality measurements of

water phase with/without aprotinin were based on the freezing-

point method. After calibration of the osmometer (Knauer

Semi, Micro Osmometer, Berlin, Germany) with reference

standards (water and 400 miliosmol NaCl solution), the

osmolality was measured with 0.15 mL of sample [19]. All

the experiments were replicated at least three times. The

protein content of MA was determined using Lowry’s method

[20].

Morphology

The form and superficies morphology of microemulsions

with/without aprotinin were examined by transmission elec-

tron microscopy (TEM, FEI Tecnai G2, Netherlands). A drop

of microemulsion with or without aprotinin was placed on 300

mesh copper grids. Then, they allowed standing for 10 min.

The sample was quickly frozen at liquid nitrogen, followed by

freeze-dried at �55 �C [21]. One drop of 1% osmium

tetrachloride was applied for fixation and allowed to dry for

5 min. All the measurements were carried out in triplicate.

Preparation of 99mTc-aprotinin

Aprotinin was labeled by 99mTc with small modification on

previously described method [22]. Glycine phosphate alkaline

buffer (GPB) was prepared by mixing 1 mL 3.5% sodium

dihydrogen phosphate in 1 mL 1 M NaOH solution, 6 mL

1.5% glycine, 6 mL saline and 9 mL water for injection. Two

2 H. Y. Karasulu et al. J Drug Target, Early Online: 1–13

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milligrams aprotinin was dissolved in 0.6 mL GPB and 400 mL

freshly prepared stannous chloride solution (2 mg

SnCl2 � 2H2O/1 mL of 0.005 M HCl) was added under nitro-

gen atmosphere. The mixture was mixed well and filtered

from a 0.22 mm membrane filter into a vial. Four millicurie

(mCi) 99mTc was used for radiolabeling. After nitrogen

bubbling was observed the solution was allowed to stand for

20 min at room temperature prior to radiochemical analysis.

Quality control and the stability of 99mTc-aprotinin

Radiochemical purity of 99mTc-aprotinin was analyzed by

paper and thin layer chromatography (TLC) studies. Albumin-

impregnated instant thin layer chromatography silica gel

strips (ITLC-SG) were prepared by soaking in 5 mg/mL

bovine serum albumin (BSA) solution for 30 min, then clean

with water, air dried and stored at 4 �C until required [22].

Whatman No:3 papers and albumin-impregnated ITLC-SG

strips were used as stationary phases while acetone and

ammonia/ethanol/water (A/E/W) (1/2/5) were mobile phases.

For radiochemical purity studies, strips were placed into

two developing chambers containing acetone and A/E/W

(1/2/5). After the solvent front reached the end point (0.5 cm

from the bottom), the strips were removed and allowed to

dry. Radioactivity on the strips was detected by an AR-2000

Radio-TLC Scanner (Bioscan Inc., Washington, DC). The

radioactive contaminants were identified as reduced/

hydrolyzed (R/H) 99mTc and free 99mTc-pertechnetate. Free99mTc was determined using Whatman No:3 papers as

stationary phase and acetone as the mobile phase. Reduced/

hydrolyzed (R/H) 99mTc was determined by albumin-impreg-

nated ITLC-SG strips which developed in A/E/W solvent

system. Percentage the radiochemical purity (RP%) of 99mTc-

aprotinin was calculated from the following equation by

subtracting from 100 the sum of measured impurities

percentages.

RP% ¼ 100� Free 99mTc%þ R=H 99mTc%� �

Stability of 99mTc-aprotinin was determined using TLC

studies at room temperature up to 6 h.

Preparation of 99mTc-aprotinin-loaded microemulsion

and solution

Two milligrams of aprotinin was labeled with 99mTc accord-

ing to above studies and loaded into 10 mL of microemulsion

or saline (0.2 mg/mL).

In vivo studies

The experimental protocol was approved by the Local Animal

Ethical Committee of Ege University (Approval No. E.U.

1344-249) according to the Declaration of Helsinki. Female

Wistar albino rats (3–6 months age) weighing 250 ± 20 g were

purchased from the Experimental Animal Center of Ege

University (Turkey). Rats were housed in a room maintained

at 22 ± 1 �C with an alternating 12 h light-dark cycle. Animals

had free access to pellet diet and water ad libitum.

Hemolysis test

The effect of microemulsions on the integrity of erythrocyte

membranes was investigated by in vitro hemolysis assay [23].

The release of hemoglobin from the erythrocytes (RBC) was

used as a measure of toxicity of microemulsions. Briefly, rat

RBC were separated from 20 mL fresh rat blood by centri-

fugation at 2000� g for 15 min and then washed thrice with

20 mL of saline. The purified RBC was resuspended in saline

to obtain 2% (v/v) of RBC suspension. Immediately there-

after, 2.5 mL of the RBC suspension was incubated with

2.5 mL of tested microemulsions (with or without aprotinin)

at 37 �C for 0.5, 1, 2 and 3 h in an incubator shaker and then

centrifuged at 3000� g for 10 min. The percentage of

hemolysis was measured by UV–Vis analysis of the super-

natant at 545 nm absorbance (ABS). Saline was used as the

negative control with 0% hemolysis, and distilled water was

used as the positive control with 100% hemolysis. All the

hemolysis data points were presented as the percentage of the

complete hemolysis. The percent hemolysis caused by the test

sample was calculated according to the following equation

(n¼ 3):

Hemolysis percent %ð Þ ¼ ABSsample � ABSsaline

ABSdistilled water � ABSsaline

� �� 100

Experimental model of pancreatitis with cerulein (mild acute

pancreatitis)

Rats were randomly divided into six experimental groups and

all the groups consisted of seven rats (n¼ 7). An experimental

model of mild acute pancreatitis was created on first three

groups. Mild acute pancreatitis was induced in rats by four

subcutaneous injections of 20 mg/kg cerulein every hour up to

4 h [12]. On the other hand, to create control group, last three

groups was injected with saline instead of cerulein. Mild

acute pancreatitis induced rats (AC1–C3) were treated with MA

or SA (AC1MA, AC2SA) or saline (AC3S) by i.v. injection 1 h

before the first cerulein injection. 5000 KIU aprotinin was

selected as therapeutic dose of pancreatitis according to the

literature [24]. Last three control groups (AS4–S6) were

applied with MA or SA (AS4MA, AS5SA) or saline (AS6S),

respectively, by i.v. injection 1 h before the first saline

injection.

AC1MA: Mild pancreatitis group, rats were treated with MA.

AC2SA: Mild pancreatitis group, rats were treated with SA.

AC3S: Mild pancreatitis group, saline was injected into rats as

control.

AS4MA: Control group, rats were treated with MA.

AS5SA: Control group, rats were treated with SA.

AS6S: Control group, saline was injected into rats as

control.

Experimental model of pancreatitis with sodium taurocholate

(Severe acute pancreatitis)

Rats were randomly divided into six experimental groups and

all the groups consisted of seven rats survived (n¼ 7). Severe

acute pancreatitis was induced by intraductal (ID) infusion of

3% taurocholate in BT1, BT2 and BT3 experimental groups.

Under sterile conditions, a midline laparotomy was performed

and the common biliopancreatic duct was cannulated with

27-gauge needle. The seromuscular layer of duodenum was

punctured 2–3 mm away from the entrance of biliary duct.

The needle was introduced into the duct. The distal

DOI: 10.3109/1061186X.2015.1015537 Aprotinin microemulsion in acute pancreatitis 3

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biliopancreatic duct ligated to avoid reflux of taurocholate

into the duodenum. To prevent leakage of bile salt into the

liver, the bile duct was clamped temporarily near the hepatic

hilum. Sterile 3% sodium taurocholate 0.1 mL/100 g of body

weight was infused slowly. After the infusion completed, the

needle and the ligature were removed, the hepatic hilum was

decamped and the abdominal wound was closed in two layers

[12]. On the other hand, for creating control groups, saline

was applied instead of sodium taurocholate (BS4, BS5 and

BS6). Severe acute pancreatitis induced rats (BT1–T3) were

treated with MA or SA (BT1MA, BT2SA) or saline (BT3S) by

injection into femoral vein. Furthermore, last three control

groups (BS4–S6) were treated with MA, SA (BS4MA, BS5SA) or

saline (BSP6S), respectively, by injecting into the femoral vein.

Experimental groups were as follows:

BT1MA: Severe pancreatitis group, rats were treated with MA.

BT2SA: Severe pancreatitis group, rats were treated with SA.

BT3S: Severe pancreatitis group, saline was injected into rats

as control.

BS4MA: Control group, rats were treated with MA.

BS5SA: Control group, rats were treated with SA.

BS6S: Control group, saline was injected into rats as control.

Air pouch formation

Rats were randomly divided into two experimental groups and

all the groups were consisted of seven rats (n¼ 7). The air

pouch was formed by initial subcutaneous (SC) injection of

20 mL air into back of male Wistar albino rats and injection of

10 mL air every 3 days to sustain its patency (Figure 1) [25].

Six days after the initial injection of air, 2 mL of 1%

carrageenan solved in 0.9% saline was injected into the cavity.

After that, air pouch induced rats (C1–2) were treated with i.v.

injection of MA or SA (C1MA, C2SA). Six hours after

carrageenan injection, the animals were anesthetized with

ether and 1 mL of heparinized saline was given to wash out

the cavity. The pouch cavity was opened and the exudate was

harvested 6 h after injecting carrageenan. Experimental

groups were as follows:

C1MA: Rats were treated with MA.

C2SA: Rats were treated with SA.

Gamma scintigraphy and biodistribution experiments

For gamma scintigraphy and biodistribution experiments

radiolabeled aprotinin-loaded microemulsion and saline

were administered into the tail vein of anesthetized rats

with mild acute pancreatitis (ACMA, ACSA), severe acute

pancreatitis (BTMA, BTSA), air pouch models (CMA, CSA) or

untreated rats. The scintigraphic images were obtained with

a dual head gamma camera (Infinia General Electric, GE

Healthcare, Waukesha, WI) equipped with a low-energy high-

resolution collimator viewing the whole body of rats. After

administration of formulations, serial anterior static images

were taken in a 256� 256 matrix at different time intervals

up to 90 min.

For quantitative evaluation, regions of interest were drawn

around the interested organs and soft tissue of rats. The99mTc-aprotinin uptake was calculated by dividing the average

counts per pixel within the region of target to the average

counts per pixel within the region of background.

After scintigraphy studies, the rats were sacrificed and

biodistribution was determined. Furthermore, results of the

biodistribution studies were compared with healthy (no drug

treated) rats. Samples of interested organs including pancreas,

kidney, spleen, liver and soft tissue were weighed and the

radioactivity was measured using a gamma counter (Sesa

Uniscaller). The results were expressed as the counts per gram

of tissue. The ratio of blood radioactivity to relevant tissue

radioactivity was calculated for each rat.

Assessment of the efficacy of treatment

Rats were isolated to minimize stress and they were

anesthetized with ketamin hydrochloride (50 mg/kg IM) and

xylazine hydrochloride (10 mg/kg IM). They were sacrificed

12 h after cerulein-induced acute edematous pancreatitis

model and 8 h after severe acute pancreatitis model were

created. Blood samples were taken and centrifuged at

3000 rpm for 10 min. Serum samples were separated and

stored in special tubes at �20 �C. Serum amylase activity was

determined by the enzymatic photometric method. The

abdomen of the rats was opened and the pancreatic tissue

was removed. Pancreatic samples were divided into two parts.

One pancreatic tissue was used for the myeloperoxidase

(MPO) activity assays. MPO activity was determined as

previously described [26]. To carry out the assays, pancreas

tissue samples were homogenized in 1 M PBS (pH 7.4) and

centrifuged at 12 000� g for10 min at 4 �C. The supernatant

was assayed for MPO activity with standard ELISA kits

(Hycult HK105). Another part of pancreas tissue was used for

pathological examination.

Histopathological examination

The abdomen of the rats was opened and the pancreatic tissue

was excised. Pancreatic tissues separated for pathological

analysis was immediately inserted into 10% buffered forma-

lin. The tissue samples were subsequently processed with

conventional techniques. Hematoxylin–eosin stained slides

were reviewed under standard light microscope.

Histopathological alterations were assessed by an experi-

enced pathologist blinded to the experimental groups accord-

ing to the Schoenberg grading system in mild acute

pancreatitis groups [27]. Oedema was graded as: 0, no

oedema; 1, interlobular oedema; 2, moderate interlobular and

intra-acinar oedema; and 3, severe interlobular and intra-

acinar oedema. Neutrophil infiltration was graded as: 0, noFigure 1. Air pouch formed rats.


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infiltration; 1, minimal infiltration, intravascular margination

of granulocytes in capillaries and post-capillary venules; 2,

moderate infiltration, granulocytes present in the perivascular

tissue; and 3, diffuse infiltration of entire pancreatic gland.

Fatty necrosis was graded between 0 and 3 according to the

extent of necrosis. Total possible maximum score was 9.

Severity of necrotizing pancreatitis was determined by a

scoring system described by Spormann et al. [28] This score

includes the graded assessment of pancreatic edema (0–3),

inflammatory infiltration (0–3), pancreatic necrosis (0, 3, 5

and 7), pancreatic hemorrhage (0, 3, 5 and 7) and extra

pancreatic fatty necrosis (0, 3, 5 and 7) for a possible

maximum score of [27,28].

Statistical data analysis

Statistical analysis was performed using a one-way analysis of

variance (ANOVA) Kruskal–Wallis analysis and Chi-square

to test the difference between the means of MA and SA in vivo

studies. Results were given as mean or median as appropriate.

Data were considered statistically significant at p50.05.

Results and discussion

Preparation of aprotinin-loaded microemulsions

The formulation of parenteral dosage form of lipophilic and

hydrophilic drugs has confirmed to be challenging [29].

Microemulsions are suitable dosage forms for delivering

peptide and protein drugs [4]. O/w microemulsions are

beneficial in the parenteral delivery of drugs. In this study,

o/w microemulsion was developed by titration method using

pseudo-ternary phase diagrams.

The construction of pseudo-ternary phase diagrams was

used to define the composition of the aqueous phase, oil

phase, surfactant and co-surfactant from which the trans-

parent, one-phase microemulsions were formed. Most of the

time, cosurfactant would be beneficial to the formation of

microemulsions by penetrating surfactant monolayer [30].

The pseudo-ternary phase diagrams with various weight

ratios of oleic acid, Cremophor EL, isopropanol and saline

are shown in Figure 2. The HLB value of the formulation

was calculated as 8.5. The transparent microemulsion

region is presented in the shady side of phase diagrams

and the other areas signify for the non-microemulsion

region with an opaque appearance. Once the microemulsion

region was identified, the microemulsion at preferred

component ratios was prepared from gravity center of the

phase diagram. According to these diagrams, it was

considered that the optimum microemulsion contained

9.34% oleic acid, 18% Cremophor EL, 36% isopropanol

and 36.66% saline.

In this study, i.v. injection was suggested as administration

route for MA. Therefore, all the ingredients of the micro-

emulsions were chosen according to biocompatible, non-

irritating, injectable, sterilizable, non-hemolytic characteris-

tics [2]. The choice of the surfactant is not only critical for

the safety of injections but also for the formulation of

microemulsion. Thus, the aim in the formulation of o/w

microemulsion was to have lowest amount of surfactant with a

suitable composition for microemulsion system. Based on

these criteria, Cremophor EL was selected as a surfactant and

as an emulsifier with good in vivo tolerance after parenteral

administration. Previously, it was stated that, in some i.v.

formulations, Cremophor EL was used at high concentrations

such as 40% [31,32]. On the other hand, in this formulation,

Cremophor EL was used as 18% (w/w) concentration.

Similarly, oleic acid has been used safely in the oil phase of

i.v. microemulsions [33].

Figure 2. The pseudo-ternary phase diagramsof oleic acid/Cremophor EL/propanol/saline.The area of the shady side represents thesingle-phase microemulsion region. Point (o)represents ratios of 9.34:54:36.66 for micro-emulsion formulation (oil surfactant/co-surfactant:water).


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According to the turbidity stability studies, microemulsion

did not show any evidence of instability about turbidity.

Furthermore, after 30 min of the centrifugation at 3000 rpm,

the developed microemulsion did not show any phase

separation.

Microemulsion containing 0.2 mg (5000 KIU) of aprotinin

per milliliter of formulation was prepared by dissolving the

appropriate amount of drug in water phase and mixing the

other composition of formulation. After dissolving process,

the clear microemulsion-based formulation was obtained. The

formulations were stored at 25 and 37 �C during 24 h and no

difference was observed.

Sterility is one of the pre-requisites for the parenteral

preparations. Microemulsions are considered as suitable

formulations for sterile filtration. Fort this reason, the

developed microemulsions were easily filtered through

0.2mm sterile filters under sterile conditions. The sterile

microemulsions were kept in sealed sterile vials until the

experiment. The sterility of the developed formulation was

evaluated by bacteria colony counting technique. According

to the sterility studies no growth was observed on the agar

mediums and the formulations were detected to be sterile.

Characterization of microemulsions

The physicochemical parameters of the microemulsions were

listed in Table 1. The average droplet size of microemulsion

without aprotinin was 67.3 ± 0.387 nm. However, in aprotinin-

loaded microemulsion, the average droplet size was

64.55 ± 3.217 nm. The incorporation of aprotinin into micro-

emulsion resulted in a 2.75 nm decrease in the droplet size.

This finding supports a recent study that found the mean

droplet size was decreased after the drug loading [34]. In a

previous study, Washington and Davis [35] described that i.v.

emulsions should have a droplet diameter below 0.5 mm, with

most commercial products being in the range 0.25–0.4 mm. It

can be concluded that aprotinin-loaded microemulsion may

be suitable for i.v. administration since the droplet size is in

these limitations.

The polydispersity value shows the homogeneity of the

droplet size. In all of the formulations, polydispersity values

were smaller than 0.3 and this indicate that the droplet size

had high homogeneity.

The viscosity values of microemulsions with or without

aprotinin were found to be 17.000 ± 0.017 and 14.806 ± 0.032

cP, respectively (Table 1). The results showed that incorpor-

ation of aprotinin into microemulsion significantly increased

the viscosity (p50.05). The relatively low viscosity values

indicated that the microemulsions formulated were composed

of individual spherical droplets or bicontinuous structures and

non-isometric aggregates were present [36]. The low viscosity

of the developed microemulsion ensures ease of syringeability

as well as ease of mixing with i.v. fluids with minimum

mechanical agitation [15].

The acceptable range is pH 2–12 for i.v. and intramuscular

formulations [15]. The average pH of all the microemulsions

ranged from 4.907 ± 0.015 to 5.500 ± 0.010 and the aver-

age refractive indexes of microemulsions ranged from

1.447 ± 0.014 to 1.394 ± 0.005 (Table 1). The incorporation

of aprotinin into microemulsion significantly increased the

pH and the refractive index values (p50.05). The pH of the

microemulsions was appropriate for parenteral delivery of

aprotinin.

The phase systems (o/w or w/o) of the microemulsions

were detected by evaluating the conductivity. The investigated

microemulsion could be divided into w/o and o/w [37,38].

Therefore, electrical conductivity of microemulsion was

determined. The results presented that it has the conductivity

value of 1.155 ± 0.035 mS/cm and identified as o/w system

(Table 1).

Figure 3(a) and (b) show that rising the temperature

induced an increase in the electrical conductivity of M and

MA up to PIT. As it can be seen, first an increase in

conductivity of the microemulsions with temperature was

observed; however; after reaching PIT, abrupt decrease were

determined (Table 1). The results showed that electrical

conductivity and PIT slightly decreased in MA. Similarly,

Figure 3. (a) Change of conductivity of microemulsion with various temperatures and (b) change of conductivity of MA with various temperatures.

Table 1. Characterization of microemulsions in with or without ofaprotinin at 25 ± 2 �C and values are means of three experiments ± SD.

Parameters M MA

pH 4.907 ± 0.015 5.5 ± 0.01Viscosity (cP) 14.806 ± 0.032 17.00 ± 0.017Droplet size (nm) 67.300 ± 0.387 64.55 ± 3.217PDI 0.290 ± 0.005 0.252 ± 0.012Electrical conductivity (mS) 1.155 ± 0.021 0.43 ± 0.02Refractive index 1.447 ± 0.014 1.394 ± 0.005PIT (�C) 50.666 ± 0.577 46.5 ± 0.707


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Warisnoicharoen et al. [39] showed that the presence of

electrolytes as aprotinin could lower PIT, which shows the

importance of defining microemulsion phase behavior in the

presence of drug.

The water phase of the parenteral microemulsions should

be formulated by the incorporation of ionic or osmotic agents

as emulsified oil exerts no osmotic effect. Therefore, the

osmolality values of water phase (saline) and 0.2 mg/mL

aprotinin in saline were measured. The osmolality of the

water phase was found to be 307.5 ± 10.606 milliosmol/kg.

The water phase of the microemulsion was found iso-osmotic.

Cellular damage does not occur when endothelial cells

contact an iso-osmotic solution [40].

The Lowry method is a sensitive technique to determine

low concentrations of proteins. Most of the techniques use

universally Bovine Serum Albumin (BSA) as a standard

protein, due to its low cost, high purity and ready availability.

The method is sensitive enough for low protein concentrations

(10mg/mL) and is probably the most widely used protein

assay despite its being only a relative method. Aprotinin

content of the microemulsion was determined according to

the method of Lowry et al. using bovine serum albumin

standard [20]. According to the results, aprotinin concentra-

tion in the microemulsion was found to be between 0.2 and

0.205 mg/mL.

Morphology

TEM analysis presented the circular form and uniform droplet

size of microemulsions (Figure 4a and b). This uniformity

reflects the manufacturing procedures and results in droplets

with a narrow range of sizes. TEM image of microemulsion

and MA showed that the diameters were below 100 nm with

narrow scattering in most of the particles. Moreover, the

particle size which was determined by dynamic light scatter-

ing method was definite by TEM. It was also detected that the

droplet size of MA was smaller than that of microemulsion.

It could be announced that the presence of aprotinin in

microemulsion affected the droplet size. The diameters of the

microemulsion particles observed by TEM were in good

agreement with the particle sizes determined as described

in Table 1.

In vivo evaluation

Hemolysis test of formulation

The ability of any nanoproduct to cause hemolysis after

parenteral delivery is one of the most restrictive properties in

all the pharmaceutical applications. Furthermore, surfactants

are known to cause hemolysis of red blood cells [22].

Therefore, the disruption of erythrocytes is a major barrier

for in vivo application. The hemolytic activity has been

recommended as an indicator for toxicity display in vitro and it

is a simple measurement for estimating the membrane damage

caused by formulations with surfactants or solvents. In these

experiments, the complete hemolysis was clearly observed

with distilled water for positive control at 15 min presenting as

the red clear-diaphanous and no erythrocyte survived at the

bottom of the tube. While the erythrocytes precipitated at the

bottom of the tube for negative control with saline and

different concentration of microemulsion or MA, they redis-

persed after shaking within the 3 h observation. This indicates

that increasing the incubation time led to a negligible change

in the membrane damage induced by formulations. The

hemolysis percent was below 1% and altered insignificantly

with the incubation time (p40.05). Considering that micro-

emulsion would be injected intravenously and would be

diluted by blood, it could be assumed that microemulsions

may be relatively safe carriers for aprotinin in the circulation.

Radiolabeling, quality control and stability of 99mTc-aprotinin

The role of gamma scintigraphy techniques is well established

for designing new drugs, determining the biodistribution of

promising pharmaceuticals [41,42]. 99mTc is one of the most

popular radionuclide for gamma scintigraphy studies with its

versatile chemistry, good photon energy (140 keV) and

suitable half-life (6 h) [43,44]. In this study, a modified

formulation was prepared for radiolabeling of aprotinin with99mTc radionuclide. The radiochemical purity of the modified99mTc-aprotinin formulation described here was determined

Figure 4. Transmission electron microphotographs of (a) microemulsion (the scales bar for image represents 200 nm and 160 000�) and (b) aprotinin-loaded microemulsion (MA; the scale bar for image represents 200 nm and 135 000�).


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by Whatman No: 3 papers, albumin-impregnated ITLC-SG

strips in acetone and A/E/W solvent systems.

Aprotinin was radiolabeled with 99mTc previously by

Smyth and Tsopelas and Schaadt et al. [22,45]. Although the

authors used different radiolabeling and quality control

procedures both the formulations were found to have good

labeling yield and stability.

Smyth and Tsopelas [22] prepared aprotinin ready to use

cold kits which contained aprotinin, stannous chloride, tetra

sodium pyrophosphate (PYP), glycine and sodium di-hydro-

gen phosphate. The authors concluded the presence of excess

PYP ions played an important role in stabilizing stannous and

reduced technetium in the alkaline environment of the cold kit

minimizing the formation of insoluble hydroxides. According

to quality control studies, 99mTc-aprotinin labeling yield was

found to be 98.1 ± 0.2% and 97.6 ± 0.5% at 0.4 and 4 h post-

labeling.

In this study, 99mTc-aprotinin was prepared with small

modification on previously described method in the absence

of PYP. According to quality control studies, 99mTc-aprotinin

labeling yield was found slightly poor than Smyth and

Tsopelas’s [22] method for later hours. These results can be

explained by the positive effect of PYP on stability of the

complex.

Researchers improved some quality control methods

previously to assess the impurities in 99mTc-aprotinin using

different stationary and mobile phases such as Whatman

No:1/Methylethylketone (MEK), ITLC-SG/MEK etc

[22,45,46].

In this study, radiochemical purity and stability of the

product were evaluated successfully by chromatographic

studies as described previously [22]. The chromatographic

studies indicated that 99mTc-aprotinin is stable up to 6 h

at room temperature (Figure 5). Radiolabeling yield was

found as 95.430 ± 0.946, 96.160 ± 1.235, 93.853 ± 0.745,

95.150 ± 0.870, 91.617 ± 1.602, 93.913 ± 1.879 and

90.180 ± 2.320% at 0, 1, 2, 3, 4, 5 and 6 h post-labeling,

respectively.

Experimental model of mild and severe acute pancreatitis

Gamma scintigraphy and biodistribution studies. In prelimin-

ary experiments, MA and SA biodistribution were evaluated by

gamma scintigraphy at 5, 15, 30, 60 and 120 min in healthy

rats (data not shown). Effective dosage and timing of

biodistrubution were determined from preliminary studies.

Then, in vivo behavior of 99mTc-aprotinin was assessed in the

different pancreatitis models induced in rats.

Mild pancreatitis. According to the gamma scintigraphy

studies, 99mTc-aprotinin-loaded microemulsion (AC1MA) and

saline (AC2SA) formulations show different in vivo behavior in

the body. Figure 6 shows the gamma scintigrams of mild

acute pancreatitis induced rats 90 min after administration of

AC1MA and AC2SA. As expected, while 99mTc-aprotinin-

loaded saline (AC2SA) has higher uptake in the kidney, 99mTc-

aprotinin-loaded microemulsion (AC1MA) has higher uptake

in the spleen and liver (Table 2). In mild pancreatitis, 99mTc-

aprotinin-loaded microemulsion has slower distribution speed

compared to 99mTc-aprotinin-loaded saline since it reaches

lower radioactivity in tissues at 90 min. Besides, its excretion

by kidneys is less, more stable blood and tissue levels might

be expected and in vivo maintenance time would be longer.

According to the biodistrubution studies for this pancrea-

titis group, 99mTc-aprotinin-loaded microemulsion uptake

Figure 6. Scintigrams of 99mTc-aprotinin-loaded microemulsion andsaline for mild acute pancreatitis, severe acute pancreatitis and air pouchmodels at 90th min (L: Liver, S: Spleen, B: Bladder, K: Kidney).Figure 5. Stability of the 99mTc-aprotinin.


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ratios were found 1.201, 6.023 and 0.373 for liver, kidney and

pancreas, respectively. On the other hand, the highest uptake

ratio of 99mTc-aprotinin-loaded saline administered rats was

found in kidneys (53.314). The differences for the uptake

ratios were found to be statistically different (p50.05). In this

group, the uptake of 99mTc-aprotinin-loaded saline (AC2SA) in

spleen, liver and pancreas was much lower.

Severe pancreatitis. Scintigrams and uptake ratios of 99mTc-

aprotinin-loaded microemulsion (BT1MA) and saline (BT2SA)

administered rats with severe acute pancreatitis model are

shown in Figure 6 and Table 2, respectively. Gamma

scintigraphy studies indicate that 99mTc-aprotinin-loaded

saline show two times higher uptake than 99mTc-aprotinin-

loaded microemulsion in kidneys. The radioactivity rates for

both the experimental groups at 90 min were lower in organs

compared to mild pancreatitis model. Taurocholate-induced

pancreatitis model is severe form of pancreatitis and usually

accompanied by circulatory failure and fluid loss. This might

be explanation for slower biodistribution of both the formu-

lations in that model. According to the biodistribution studies

after the i.v. administration of 99mTc-aprotinin-loaded micro-

emulsion; the uptake ratios of 99mTc-aprotinin in liver, kidney

and pancreas were found 1.493, 0.601 and 1.436 at 90th min,

respectively. On the other hand, the highest uptake ratio of99mTc-aprotinin-loaded saline administered rats was found to

be in kidneys (26.600). The differences for the uptake ratios

were found to be statistically different (p50.05). For

achievement of passive targeting, colloidal drug delivery

systems (such as micelles, microemulsions, nanoemulsions,

etc.) have to circulate in blood for longer periods and their

size determines their biological fate. Polymeric micelles

smaller than 5 nm and 5–10 nm were easily eliminated

through the renal glomeruli, whereas the larger micelles

with the size range of 50–100 nm were removed by the liver

and spleen [47]. As it was expected, the results of the

scintigraphy and biodistribution studies showed that, i.v.

administration of 99mTc-aprotinin-loaded microemulsion,

with droplet size of 64.550 ± 3.217 nm, has high uptake in

liver and spleen while 99mTc-aprotinin-loaded saline distrib-

uted mostly in kidneys and bladder.

Natural protease inhibitors are present in the circulation

and play an important role in limiting tissue damage [48].

However, they remain insufficient when the protease activity

increases as in acute pancreatitis. Decreased levels of

circulating protease inhibitors and the delayed clearance of

serum protease-inhibitor complexes by the mononuclear

phagocyte system (MPS) have been associated with the

more severe forms of pancreatitis [48,49]. The increased

uptake of 99mTc-aprotinin in liver and spleen might show

increased clearance of aprotinin and trypsin complexes by

MPS. On the other hand, decreased clearance of MA by

kidney guaranties high levels of aprotinin in the pancreas as

well as circulation and decreased aprotinin nephrotoxicity.

Therefore, new developed microemulsion of aprotinin might

supply a protease inhibitor activity with high bioavailability.

In conclusion, the results of scintigraphic and biodistribu-

tion studies showed that, in vivo behavior of 99mTc-aprotinin-

loaded microemulsion and saline are different. While99mTc-aprotinin-loaded saline has higher uptake in the

kidney, 99mTc-aprotinin-loaded microemulsion has higher

uptake in spleen and liver.

The assessment of the efficacy of treatment of mild and

severe acute pancreatitis

To confirm the successful induction of pancreatitis, pancre-

atic histopathology scores and serum amylase levels and

tissue MPO levels were measured in all the experimental

models (Tables 3 and 4). Compared with the untreated group,

serum amylase activity, pancreatic MPO activity and

histopathologic scores in the mild and severe acute pancrea-

titis significantly increased after the induction of pancreatitis

in groups AC3S and BT3S (Tables 3 and 4).

Table 2. 99mTc-Aprotinin uptake ratio in related organs for mild acute pancreatitis, severe acute pancreatitis and air pouch models at 90th min.

Formulations Heart Lung Spleen Liver Kidney

AC1MA 11.663 ± 2.5 8.356 ± 1.8 14.223 ± 4.5 13.155 ± 5.2 6.721 ± 0.32AC2SA 22.307 ± 16.9 10.808 ± 11.9 19.784 ± 7.9 30.268 ± 16.4 47.052 ± 0BT1MA 3.639 ± 2.7 3.792 ± 3.5 6.652 ± 5.4 5.943 ± 5.0 6.812 ± 3.2BT2SA 5.086 ± 2.1 4.191 ± 2.1 6.198 ± 2.4 6.123 ± 0 12.052 ± 3.4C1MA 28.942 ± 8.928 9.653 ± 8.874 44.392 ± 6.888 41.118 ± 5.191 23.963 ± 17.027C2SA 10.780 ± 4.682 6.727 ± 2.150 8.504 ± 2.261 15.176 ± 5.499 47.379 ± 10.365

Table 3. The results of the mild acute pancreatitis treatment efficacy ofaprotinin in rat groups (n¼ 7).

Groups Amylase (U/L) MPO (ng/dL)Histopathological score

(median, range)

AC1MA 3542.1 ± 699.8 973.6 ± 876.9 4 (2–6)AC2SA 4037 ± 2017.8 1094.3 ± 840.5 4 (0–6)AC3S 8313.8 ± 3800.1 1256.5 ± 666.9 6 (3–7)AS4MA 1620.9 ± 325.1 70.3 ± 20.4 0 (0–3)AS5SA 1447.4 ± 285.8 44.1 ± 24.9 0 (0–0)AS6S 1613.1 ± 355.6 52.2 ± 63.9 0 (0–0)

Table 4. The results of the severe acute pancreatitis treatment efficacy ofaprotinin in rat groups (n¼ 7).

Groups Amylase (U/L) MPO (ng/dL)Histopathological score

(median, range)

BT1MA 7063.6 ± 4860.4 1000.3 ± 755.5 3 (0–15)BT2SA 8214.4 ± 4712 1373.7 ± 842.1 8 (2–9)BT3S 9221.0 ± 4909.2 1494.1 ± 980.8 6 (3–17)BS4MA 2758.6 ± 452.7 406.8 ± 755 0 (0–1)BS5SA 2845.3 ± 457.4 240.4 ± 291 1 (0–6)BS6S 3707 ± 1895.3 306.4 ± 260.5 2 (0–2)


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Mild pancreatitis. In cerulein-induced mild pancreatitis

model, MA significantly decreased serum amylase levels.

Serum amylase activity was 3542.1 ± 699.8 U/L in AC1MA

group whereas amylase activity in AC3S untreated group

was 8313.8 ± 3800.1 U/L (p50.05; Table 4). Similarly, SA

decreased serum amylase activity in cerulein-induced acute

pancreatitis to less extend 4037 ± 2017.7 (p50.05). MA

suppressed pancreatic MPO activity from 1256.5 ± 666.9 to

973.6 ± 876.9 ng/mL, however the differences between

AC1MA and AC2SA pancreatic MPO activity were not

significant.

Severe pancreatitis. In taurocholate-induced severe acute

pancreatitis model, serum amylase levels of BT1MA, BT2SA

and BT3S were significantly higher compared to control

groups (p50.05; Table 4). Both MA and SA treatment

decreased serum amylase activity but the difference was not

significant in that model of pancreatitis (p40.05). In group

BT1MA, formulation suppressed the MPO in severe form

of the pancreatitis compared to BT3 but the differences were

not significant.

In addition to pancreatic proteases, activated leukocytes

play an important role in the pathogenesis of acute pancrea-

titis [50]. MPO activity was evaluated in both cerulein and

bile pancreatic-duct obstruction with sodium taurocholate-

induced models. MPO activity, which is accepted as an

indicator of neutrophil infiltration, was decreased in the

pancreatic tissue of the pancreatitis group treated with MA

than that of the non-treated groups (AC3S, BT3S; Tables 3

and 4).

According to the obtained results, aprotinin was able to

ameliorate inflammation of pancreatitis in both models.

Air pouch models

Gamma scintigraphy and biodistribution studies of air pouch

formation. The results of biodistribution studies in air pouch

inflammation model showed that i.v. administration of 99mTc-

aprotinin-loaded microemulsion had high uptake in liver and

spleen while 99mTc-aprotinin-loaded saline distributed mostly

in kidneys and bladder. The scintigrams of rats 90 min after

application of 99mTc-aprotinin-loaded microemulsion and

saline are shown in Figure 6. According to the gamma

scintigraphy studies, 99mTc-aprotinin uptake ratios are shown

in Table 2.

After the i.v. administration of 99mTc-aprotinin-loaded

microemulsion in air pouch formed rats, the uptake results in

liver, kidney, pancreas and exudate were 1.901, 11.639, 0.472

and 0.027 ± 0.021, respectively. On the other hand, the uptake

results in liver, kidney, pancreas and exudate in air pouch

formed rats administered 99mTc-aprotinin-loaded saline

were 0.818, 75.847, 0.335 and 0.007 ± 0.006, respectively at

90 min.

Previously, acute carrageenan-induced air pouch inflam-

mation was successfully used as sham inflammation model in

rats [25]. In this study, air pouch model was created to

investigate whether 99mTc-aprotinin-loaded microemulsion

was uptaken by sham inflammation or pancreas. The results

showed that the uptake of 99mTc-aprotinin-loaded microemul-

sion in an exudate was five times lower in this sham

inflammation group than pancreas. Moreover, the uptake in

pancreas was higher in 99mTc-aprotinin-loaded microemul-

sion than 99mTc-aprotinin-loaded saline in rats with air pouch

model. Therefore, it can be concluded that aprotinin-loaded

microemulsions may be suggested as promising formulations

for targeting to pancreas but not the inflammation. Here,

carrageen-induced inflammation was served as inflammatory

focus poor in protease activity compared to pancreatic

inflammation rich in protease activity. This new MA has

affinity to tissues rich in proteases.

Histopathological evaluation of pancreas

Histological examination of pancreas sections from mild and

severe acute pancreatitis group revealed tissue damage

characterized by edema, fat necrosis, hemorrhage, neutro-

philic infiltration and pancreatic tissue necrosis. The results of

histopathological evaluation of the pancreases were shown in

Figures 7–9.

In cerulein-induced mild acute pancreatitis model, histo-

pathological scores were not significant different between

AC1MA, AC2SA and AC3S groups. Cerulein-induced model of

acute pancreatitis represents mild form of acute pancreatitis

and usually ameliorates spontaneously by supportive treat-

ment [50]. The histopathological scores were similar between

MA and SA treated or untreated cerulein-induced pancreatitis

groups. This form of mild pancreatitis may not need treatment

with further drugs even aprotinin although both forms of

aprotinin has treatment effect.

In group BT1MA, formulation suppressed the histopatho-

logical median score in severe form of the pancreatitis

compared to BT3S but the differences were not significant. In

previous trials, intravenously applied commercial formulation

of aprotinin did not show a significant effect on acute

pancreatitis. However, higher i.v. dosages reduced mortality

related to acute pancreatitis in elderly [51,52]. In mild

pancreatitis groups, no mortality was observed. On the other

hand, in taurocholate-induced severe pancreatitis mortality

rates of BT1MA, BT2SA and BT3S were 6, 12 and 19%,

respectively.

The hypothesis that there is local microcirculatory failure

in pancreatitis led to the examination of intra-arterial

administration to increase local tissue levels by targeted

administration intra-arterial infusion of aprotinin decreased

serum amylase levels and improved clinical symptoms.

Relevant to that aprotinin-loaded microemulsion decreased

serum amylase levels and improved mortality in groups. The

histopathological damage was also improved to some extent

[53]. Moreover, MA has favorable bioavailability as demon-

strated by biodistrubution studies.

In addition, aprotinin-loaded microemulsion (MA) was

applied to control groups (AS5SA and BS4MA) and the

median histopathological score was determined 0 and 1,

respectively. It is known that some drugs or formulations are

listed in the etiological factors of acute pancreatitis [54] For

this reason, the effects of MA and SA in rats without

pancreatitis were determined and compared. The results

showed that the aprotinin-loaded microemulsion does not

lead any pancreatic damage itself and it may be considered

as safe.


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Figure 7. Histopathological evaluation of rat groups for mild acute pancreatitis model.

Figure 8. Histopathological evaluation of rat groups for severe acute pancreatitis model.


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Conclusions

The parenteral aprotinin-loaded microemulsion (o/w) was

successfully developed with droplet size less than 70 nm.

According to the obtained results, physicochemical charac-

terizations showed that microemulsion may be appropriate

vehicle for parenteral delivery of aprotinin.

This comparative study indicates that, both MA and SA

have beneficial effects to decrease amylase activity in mild

and severe acute pancreatitis. Moreover, in vivo behaviors of

aprotinin-loaded microemulsion and saline have totally

different biodistribution in rats with pancreatitis. It can be

concluded that, with this newly developed formulation, more

effective treatment against pancreatitis would be provided and

side effects would be reduced. Although the tissue levels of

aprotinin in pancreas, liver and spleen was not measured,

biodistrubution studies clearly showed that new formulation

has different affinity to MPS. For a new modality and

revisited to an old drug, a new formulation of aprotinin loaded

to microemulsion may be suggested to safely use in medicine

in the future.

Acknowledgements

The authors would like to thank to Ege University, Faculty of

Pharmacy, Department of Microbiology and Department of

Toxicology. The authors would like to thank Assoc. Prof. Dr.

Timur Kose for statistical advice and Caglar Us, _Ismail

Zonguldak, Bulent Ata for assistance at in vivo experiments.

Declaration of interest

The authors declare no conflict of interest. The authors alone

are responsible for the content and writing of the paper. This

study was supported by The Scientific and Technological

Research Council of Turkey (Tubitak 108S083). The authors

also would like to thank to the T.R. Prime Ministry State

Planning Organization Foundation (Project Number:

09DPT001).

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Aprotinin revisited: formulation, characterization, biodistribution and therapeutic potential of new...

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