Chapter Eight RADIOLABELLING, BIODISTRIBUTION ...shodhganga.inflibnet.ac.in/bitstream/10603/43134/12/12_chapter 8.p… · 8.1.4. Biodistribution and Pharmacokinetics Studies M ice

Chapter Eight

RADIOLABELLING, BIODISTRIBUTION,

PHARMACOKINETICS & PHARMACODYNAMIC

STUDIES

In-vivo biodistribution & pharmacokinetics studies were performed by using

readiolabelling based methods, where drugs were labeled with technetium and

administered.

Pharmacodynamic studies of the developed formulations were performed against

chemical and electrical models of seizure.

8.1. MATERIALS & METHODS

Diethylene triamine penta acetic acid (DTPA) and stannous chloride dihydrate

(SnCl2.2H2O) were purchased from Sigma Chemical Co. (St.Louis, MO). Sodium

pertechnetate, separated from molybdenum-99 (99m) using a solvent extraction

method, was provided by Regional Center for Radiopharmaceutical Division

(Northern Region), Board of Radiation and Isotope Technology (BRIT, Delhi, India)

to Institute of Nuclear Medicine & Allied Sciences (INMAS) were the radiolabelling

studies carried. All other chemicals and solvents were of analytical reagent grade and

were used without further purification.

8.1.1. Radiolabelling of Drug solution and Nanoemulsions

The labelling of drug solution (DS: Amiloride solution = AS), Nanoemulsion (NE:

Amiloride nanoemulsion = ANE) and mucoadhesive nanoemulsion (MNE: Amiloride

mucoadhesive nanoemulsion = AMNE) was performed by direct labelling using

technetium as per the reported method with some modifications (Richardson et al.,

1977; Babbar et al., 1991). Briefly, 0.75 mL of drug solutions, DS, NE and MNE

were mixed with sufficient stannous chloride solution, prepared in ethanol (5 mg/mL)

to get highest labelling. The pH was adjusted with 0.5 M sodium bicarbonate solution.

Further, the preparation was incubated with 99mTc pertechnetate solution (111-131.3

MBq) for required time at room temperature. The final volume was made up to 1.0

mL using sterile sodium chloride solution.

8.1.2. Labelling efficiency (% LE)

The labelling efficiency of 99mTc-DS, 99mTc-NE and 99mTc-MNE was determined

using ascending instant thin layer chromatography (ITLC) using silica gel (SG)-

coated fibre glass sheets (Gelman Sciences Inc, Ann Arbor, MI). The ITLC was

performed using acetone as the mobile phase. Approximately 2 to 3 ^L of the radio

labelled complex was applied at a point 1 cm from one end of an ITLC-SG strip. The

Chapter 8: Radiolabelling, Biodistribution, Pharmacokinetics & Pharmacodynamic Studies

160

s t r i p w a s e l u t e d i n a c e t o n e a n d s o l v e n t f r o n t w a s a l l o w e d t o r e a c h 7 - 8 c m f r o m t h e

p o i n t o f a p p l i c a t i o n . T h e s t r i p w a s c u t h o r i z o n t a l l y i n t o t w o h a l v e s , a n d t h e

r a d i o a c t i v i t y i n e a c h h a l f w a s d e t e r m i n e d i n a g a m m a r a y c o u n t e r ( G a m m a r a y

s p e c t r o m e t e r , C a p t e c - R , C a p i n t e c , U S A ) . T h e f r e e 99mT c - p e r t e c h n e t a t e t h a t m o v e d

w i t h t h e s o l v e n t ( R f = 0 . 9 ) w a s d e t e r m i n e d . T h e r a d i o c o l l o i d s ( r e d u c e d / h y d r o l y z e d )

t e c h n e t i u m a l o n g w i t h t h e l a b e l l e d c o m p l e x r e m a i n e d a t t h e p o i n t o f a p p l i c a t i o n .

T h e a m o u n t o f r a d i o c o l l o i d s w a s d e t e r m i n e d u s i n g I T L C w i t h p y r i d i n e : a c e t i c a c i d :

w a t e r ( 3 : 5 : 1 . 5 v / v ) a s m o b i l e p h a s e (Saha, 1993; Saha, 2005). T h e r a d i o c o l l o i d s

r e m a i n e d a t t h e p o i n t o f a p p l i c a t i o n , w h i l e b o t h t h e f r e e p e r t e c h n e t a t e a n d t h e l a b e l l e d

c o m p l e x m o v e d a w a y w i t h t h e s o l v e n t f r o n t . T h e a c t i v i t y m i g r a t e d u s i n g p y r i d i n e :

a c e t i c a c i d : w a t e r a s a m i x t u r e w a s s u b t r a c t e d f r o m t h a t w i t h t h e s o l v e n t f r o n t u s i n g

a c e t o n e , t h e n e t a m o u n t o f 99mT c - D S ( D r u g s o l u t i o n ) , 99mT c - N E ( N a n o e m u l s i o n ) o r

M N E ( M u c o a d h e s i v e n a n o e m u l s i o n ) w a s c a l c u l a t e d .

T h e r a d i o l a b e l l i n g w a s o p t i m i z e d f o r i n c u b a t i o n t i m e a n d t h e c o n c e n t r a t i o n o f

S n C l 2 .2 H 2 O . T h e p H o f t h e s o l u t i o n a n d t h e f o r m u l a t i o n s w a s m a i n t a i n e d a t a r o u n d 6

7 . T h e i n f l u e n c e o f t h e i n c u b a t i o n t i m e o n l a b e l l i n g e f f i c i e n c y o f A M B l o a d e d N E s

a n d M N E w e r e r e c o r d e d . T h e i n f l u e n c e o f c o n c e n t r a t i o n o f S n C l 2 . 2 H 2O o n l a b e l l i n g

e f f i c i e n c y o f A M B l o a d e d N E s a n d M N E w e r e r e c o r d e d .

8.1.3. In-vitro Stability of labelled complex

T h e s t a b i l i t y s t u d y o f r a d i o l a b e l l e d f o r m u l a t i o n s w a s d e t e r m i n e d in vitro u s i n g m i c e

s e r u m b y a s c e n d i n g t h i n l a y e r c h r o m a t o g r a p h y (Garron et al., 1991). T h e c o m p l e x

( 0 .1 m L ) w a s m i x e d w i t h 1 .9 m L o f m i c e s e r u m a n d i n c u b a t e d a t 3 7 ° C . T h e s a m p l e s

a t d i f f e r e n t t i m e p o i n t u p t o 4 8 h w e r e s u b j e c t e d t o I T L C u s i n g a c e t o n e s o l v e n t

s y s t e m s . T h e % l a b e l l i n g e f f i c i e n c y w a s d e t e r m i n e d . T h e r e s u l t s f o r s t a b i l i t y i n m i c e

s e r u m f o r A M B l o a d e d N E s a n d M N E w e r e r e c o r d e d . S u m m a r y o f r a d i o l a b e l l i n g

s t u d y o f A M B l o a d e d N E s a n d M N E w e r e a l s o r e c o r d e d .

8.1.4. Biodistribution and Pharmacokinetics Studies

M i c e ( n = 3 ) w e r e u s e d a t e a c h t i m e p o i n t f o r e a c h f o r m u l a t i o n . T h e m i c e w e r e d i v i d e d

i n t o t h r e e g r o u p s . G r o u p I , g r o u p I I a n d g r o u p I I I w e r e a d m i n i s t e r e d 99mT c - A S , 99mT c -

A N E a n d 99mT c - A M N E r e s p e c t i v e l y . A S w a s u s e d f o r c o m p a r a t i v e e v a l u a t i o n . A l l

g r o u p s r e c e i v e d 4 4 . 4 - 5 3 . 2 8 M B q / k g o f r a d i o a c t i v i t y i n c o r p o r a t e d i n 1 0 m L o f 99mT c -


161

AS, 99mTc-ANE, and 99mTc-AMNE, administered via intranasal route. Before nasal

administration of the formulations, the mice were partially anesthetized with diethyl

ether. 4/5 mL of formulation was administered in the each nostril using micropipette

(10 ^L). The mice were held from the back in slanted position during nasal

administration (Jogani et al., 2008).

The mice were sacrificed at different time intervals of 0.5, 1, 2, 4, 6 and 8 h and blood

was collected via cardiac puncture. Brain was dissected and washed twice with

normal saline, made free from any adhering tissues, dried between adsorbent paper-

folds, placed in pre-weighed plastic tubes, and weighed. The radioactivity present in

each tissue/blood sample was determined using shielded well-type gamma

scintillation counter along with 3 samples of standard solution representing 100% of

the administered dose. The radioactivity in each organ/blood sample was determined

as fraction of administered dose per gram of the tissue (%A/g). The radioactivities

determined included the delivery system in the vascular space as well as in the tissue

parenchyma. Hence a correction was made for the radioactivity in the vascular space

using the following formula as reported (Hatakeyama et al., 2004).

Xtissue = Xorgan — V0 C(t)

Where V0 denotes the total volume of the vascular space and interstitial fluid, as

determined by the radioactivities in the whole organ samples divided by the blood

concentration 10 min after i.v. injection.

To evaluate the brain targeting efficiency, 2 indices [Drug targeting efficiency (DTE)

(%) and direct nose-to-brain transport (DTP) (%)] were adopted as mentioned below

(Jung et al., 2000; Zhang et al., 2004).

Brain targeting efficiency was calculated using two equations mentioned below. Drug

targeting efficiency (DTE %) represents time average partitioning ratio.

A U C b r a i t iD T E = --------------- X 1 0 0

A U C b n n l d

Where, AUC indicates area under the curve.

Brain drug-direct-transport percentage [DTP%] was calculated using equations:

Sx BiD T P = ---------------- X 1 0 0 R = — X Ap a: p rm

Where,


162

Bx = Brain AUC fraction contributed by systemic circulation through the blood-

brain-barrier (BBB) following intranasal administration.

Bi v = AUCo® 24 (brain) following intravenous administration.

P i v = AUCo® 24 (blood) following intravenous administration.

Bin = AUCo®24 (brain) following intranasal administration.

PIN = AUC0® 24 (blood) following intranasal administration.

AUC = Area under the curve.

The mice were administered with 100^Ci 99mTc-AMB and the radioactivity was

measured in percent per gram of tissue of the administered dose. Each value was the

mean ± S.E.M. of three estimations. PK Parameters were calculated by using Kinetica

Software Version 5.0 (ThermoScientific, USA). The results of radioactivity measured

for Amiloride formulations administered by intranasal route at various time points in99m 99m 99m

different organs were recorded for Tc-AS, Tc-ANE and Tc-AMNE

respectively. The blood concentrations of AMB formulations vs. time (h) were

plotted.

8.1.5. Nasal residence time studies

Gamma scintigraphy studies were carried out in New Zealand rabbits of either sex

weighing (1.5-3 kg) (n=3). Rabbits were housed under standard conditions and had

free access to water and were fed standard laboratory foods. The rabbits were

anaesthetized by injecting ketamine hydrochloride (50 mg/kg) and xylanine

hydrochloride 10 mg/kg i.m. injection to prevent sneezing after instillation of the

formulation or drug solution. For administration a 1mL tuberculin syringe was

attached to a scalp vein low density polyethylene tube (LDPE). 50 ^L of solution

administered at a depth of 4 cm. The animals were positioned posteriorly on the board

and the fate of distribution was studied by performing static/dynamic imaging at

different time intervals using gamma camera.

Total radioactivity administered was calculated by determining radioactivity (cps) of

syringe before and after dosing (pre & post syringe). Dynamic images were acquired

for 30 min. To assess in-vivo uptake or retention pattern, regions of interest (roi) were


163

drawn in the static image and radioactivity in the same was determined under MPS

acquisition system. A graph was also plotted between decay in radioactivity over the

time period from the head region.

8.1.6. Pharmacodynamic Studies

Pharmacodynamic studies of the developed formulation were performed as per the

protocol materials/methods discussed in chapter 3.

Antiepileptic pharmacodynamic studies performed on the developed formulations

were as follows:

1. Chemical induced Seizure : Pentylene tetrazole model (PTZ),

2. Electrical induced seizure: Increased current electroshock model (ICES) ,

8.1.6.1. Study design

Pharmacodynamic studies were designed as per the below mentioned design:

Table 32: Study design and drug treatment plan for PTZ/ICES studies for Amiloride (AMB) loaded formulations


Group TestCompound

Dose* Route of administration

Frequency No. of mice in

each group

Group I Normal Saline 10 mL/kg i.n. Single 6Group II AS 10 mL/kg i.n. Single 6

Group III ANE 0.16 mg/kg i.n. Single 6

Group IV AMNE 0.16 mg/kg i.n. Single 6

i.n. : intranasal *Dose of Amiloride selected was based upon experimental studies performed in chapter 3.

8.1.7. Statistical Analysis

All data were reported as mean ± SD (standard deviation). Pharmacokinetic

parameters were calculated using Kinetica (version 4.40, Innaphase, Philadelphia, PA,

USA) applying non compartmental kinetics. Data were analyzed using a one-way

analysis of variance (ANOVA) followed by Dunnett’s t-test at the 95% confidence by

using GraphPad Prism Version 5 Software.

164

8.2. RESULTS AND DISCUSSION

8.2.1. Radiolabelling studies

Drug Solution, Nanoemulsion (NE) & Mucoadhesive Nanoemulsion

Table 33: Influence of incubation time on the labelling efficiency


Incubation % Labelling Efficiency

time (min) 99mTc-AS

99mTc-ANE

99mTc-AMNE

5 95.57 ± 0.82 89.68 ± 0.93 90.26 ± 0.91

10 98.06 ± 1.18 92.26 ±1.26 93.78 ± 1.32

15 97.67 ± 1.46 94.43 ± 1.43 95.54 ± 1.14

30 97.03 ± 2.14 96.17 ± 1.25 96.33 ± 1.56

60 96.75 ± 1.31 95.86 ± 1.32 96.06 ± 2.07

Value are represented as mean ± SD, n=3.

AS: Amiloride solution; ANE: Amiloride Nanoemulsion;

AMNE: Amiloride Mucoadhesive Nanoemulsion

Table 34: Influence of the Amount of Stannous Chloride on the Labelling Efficiency

SnCl2. 2 H2O (^g)^ 50 100 150 200 300

AS

% Labelling (mean ± SD) 92.27 ± 1.12 98.18 ± 1.87 95.93 ± 1.32 93.76 ± 1.34 ppt

Colloids (mean ± SD) 3.41 ± 0.10 0.60 ± 0.24 2.89±0.13 5.23 ± 0.09 ppt

% Free (mean ± SD) 4.32 ± 0.44 1.22 ±0.12 1.18 ± 0.21 1.01 ± 0.10 ppt

ANE


% colloids (mean ± SD) 4.83 ± 0.11 1.08 ± 0.12 3.72 ± 0.28 5.13 ± 0.25 ppt

% Free (mean ± SD) 31.25 ± 0.62 19.1 ± 0.22 6.63 ± 0.32 0.55 ± 0.15 ppt

AMNE


% Colloids (mean ± SD) 4.59 ± 0.18 0.94 ± 0.09 3.68 ± 0.26 4.15 ± 0.16 ppt

% Free (mean ± SD) 31.71 ± 0.43 3.72 ± 0.27 3.18 ± 0.17 1.97 ± 0.12 ppt

165

Table 35: In-vitro stability of 99mTc -labelled complex of drug solution and formulations


Time (h)

0.5

4

24

48

“55mTc-AS

% Labelling Efficiency=55m--------------- p5m

98.18 ± 1.87

98.04 ± 0.82

97.84 ± 0.79

97.54 ± 1.21

96.92 ± 1.34

96.00 ± 1.12

95.44 ± 2.01

92.05 ± 1.43

Tc-ANE

99.82 ± 1.42

99.13 ± 1.84

96.75 ± 1.64

96.56 ± 0.95

96.06 ± 1.13

95.53 ± 1.65

94.75 ± 1.06

91.47 ± 1.32

Tc-AMNE

95.34 ± 1.46

96.23 ± 1.26

96.02 ± 1.57

95.85 ± 0.86

95.39 ± 1.35

95.02 ± 1.17

93.86 ± 2.11

90.83 ± 1.43

Values are represented as mean ± SD, n=3.

Table 36: Radiolabelling summary of drug solution and formulations

AS ANE AMNE

Method Direct Labelling Direct Labelling Direct Labelling

Amt. of SnCl2 (^g) 100 100 100

pH 6.5 6.5 6.5

Incubation duration (min) 15 30 30

Labelling efficiency (%) 98.18 99.82 95.34

Activity added (^Ci) 111-131.3 138.75-166.5 111-131.3

The optimum quantity of stannous chloride for high labelling efficiency and low free

and reduced/hydrolyzed 99mTc, was found to be 100 ^g for NE, MNE formulations

and drug solution respectively. The incubation time was optimized at 30min. for NE

and MNE formulations. AS require incubation of 15 min. The pH of all the

formulations was kept at around 6. The labelling efficiency for AS, ANE and AMNE

was found to be 98.18%, 99.82% and 95.34 % respectively. The radiolabeled complex

showed high stability in mice serum with radiolabelling efficiencies measured, greater

than 90%.

166

0

1

2

6

8.2.2. Biodistribution and Pharmacokinetics studies

The radiolabeled complexes of AS, ANE and AMNE were evaluated for

biodistribution between blood and brain in healthy Swiss albino mice for 8 h after

intranasal administration. ANE were administered by intravenous route.


Fig. 51: Amiloride concentration in mice blood at different time intervals following AS (i.n.), ANE

(i.n.), ANE (i.v.), and AMNE (i.n.) administrations

Fig. 52: Amiloride concentration in mice brain at different time intervals following AS (i.n.), ANE

(i.n.), ANE (i.v.), AMNE (i.n.) administrations.

167


Table 37: Pharmacokinetic parameters of blood and brain AS (i.n.), ANE (i.n.), ANE (i.v.), and AMNE (i.n.)

Formulation and route of

administration

Organ/

Tissue

Cmax(%/g)

Tmax

(h)

AUC 0- 480min

(h* %/g)

AUC 0-^

(h* %/g)

Clearance(g/h)

T1/2 (h) MRT (h)

ANE (i.v.)

Blood 2.52 ± 0.43 0.50 ± 0.10 5.21± 0.94 5.38± 0.63 18.59± 6.53 1.18± 0.41 1.69± 0.55

Brain 0.53 ± 0.22 1.00 ± 0.10 1.15± 0.17 1.16± 0.11 86.18± 6.32 0.71± 0.06 2.26± 0.73

AS (i.n.)

Blood 1.08± 0.07 1.00± 0.15 1.94± 0.63 2.26± 0.33 44.23± 6.85 1.94± 0.02 2.93± 0.53

Brain 0.39± 0.14 0.50 ± 0.10 1.20± 0.44 1.45± 0.29 68.49± 8.83 2.11± 0.06 3.48± 0.86

ANE (i.n.)Blood 0.89± 0.44 1.00± 0.10 2.16± 1.38 2.39± 1.92 41.77± 5.71 1.63± 0.38 2.65± 0.63

Brain 0.51± 0.07 1.00 ± 0.15 1.65± 0.95 1.95± 0.71 51.26± 7.22 2.04± 0.71 3.29± 0.58

AMNE (i.n.)

Blood 0.86± 0.31 0.50± 0.15 3.31± 0.43 6.34± 2.01 15.75± 2.04 5.26 ± 0.74 7.94± 0.57

Brain 0.71 ± 0.12 0.50 ± 0.10 2.65± 0.96 3.12± 0.52 32.03± 5.33 1.92± 0.94 3.33± 0.58

Each value is the mean±SEM of three estimations.

168


Fig. 53: Brain/blood ratio after intranasal administration of AS (i.n.), ANE (i.n.), AMNE and intravenous administration of ANE at 0.5 h post administrations in Swiss albino mice. Each value was the mean±S.E.M. of three estimations. * statistically significant different from A Sin, and ANEiv, P<0.05.

Table 38: Brain targeting efficiency and direct nose to brain transport percentage

following administration of 99mTc labelled AS (i.n.), ANE (i.n.), ANE (i.v.), and

AMNE (i.n.)

Brain Plasma%DTE = (Brain/

Plasma*100)Biv/Piv Bx=Biv/Pi

v*Pin

% DTP= (Bin-Bx)/ Bin*100

ANE IV 1.150 5.210 22.073 0.221

AS IN 1.200 1.940 61.856 0.221 0.428 64.32

ANE IN 1.650 2.160 76.389 0.221 0.477 71.10

AMNE IN 2.650 3.310 80.060 0.221 0.731 72.43*

* statistically sig;nificant different from ASin, , P<0.05.

169

8.2.3. In-Vivo Gamma Scintigraphy Dynamic (for Nasal retention time) and Static

Studies of ANE:


Fig. 54: Illustration showing Dynamic and static images taken between 0-30 min post ANE i.n.

administrations by using Gamma Camera. Black colour circled area is head zone of the animal.

170

8.2.4. In-Vivo Gamma Scintigraphy Dynamic (for Nasal retention time) and Static

Studies of AMNE:


INKUS .DiplatNUCLEAn

t n l C l4 l / 1? ; 1V : 13.Q /ro lq/i '4lQ Cw c l

(I 2 l| 6 8 IB 1Z U 1& 1ft 2D Z? 24 26 2ft 3B

i f f %hjhdjb/!,hjdjb»0»/LAit>: R E M L

Fig. 55: Illustration showing Dynamic and static images taken between 0-30 min. post AMNE i.n.

administrations by using Gamma Camera. Black colour circled area is head zone of the animal.

171

Biodistribution studies of 99mTc-AMB formulations following intravenous

administration (ANE) and intranasal administrations (AS, ANE and AMNE) on Swiss

albino mice were performed and the radioactivity was estimated at different intervals

up to 8 h and plotted in Fig. 51 & 52. The brain-blood ratio of the drug at all

sampling time points for different formulations was also calculated and is recorded in

and presented in Fig. 53. The amiloride concentration in brain following the intranasal

(i.n.) of AMNE (Fig. 52) were found to be significantly higher at all the time points

compared to both ANE (i.n.) and ANE (i.v.). While the brain concentration of

amiloride after i.n. administration of ANE was comparable to that of i.v.

administration of ANE at all the time points. The brain/blood ratios of 0.62, 0.76,

0.80, and 0.22 of AS (i.n), ANE (i.n), AMNE (i.n) and ANE (i.v), respectively, at 0.5

h are indicative of direct nose to brain transport bypassing the blood-brain barrier,

hence prove the superiority of nose to brain delivery of amiloride by nanosized

colloidal dispersion like nanoemulsion (Qizhi et al., 2004). Table 37 shows the

calculated pharmacokinetic parameters for the AMB formulations. The lower Tmax

values for brain (0.5 h) when compared to blood (1 h) may also be attributed to

preferential nose to brain transport following i.n. administration. When the Cmax and

AUC of brain concentration of AS (i.n.), ANE (i.n.) and AMNE (i.n.) were compared,

the Cmax (0.71%/g) and AUC0-t (2.65 h %/g) of AMNE were found to be significantly

higher because the addition of mucoadhesive agent decreased the mucociliary

clearance, which under normal circumstances rapidly clears the instilled formulation.

Reports in the literature (Illum, 2000, 2003; Vyas et al., 2005; Mathison et al., 1998;

Chow et al., 1999; Kumar et al., 2008) reveal that the drug uptake into the brain

from the nasal mucosa mainly occurs via two different pathways. One is the systemic

pathway by which some of the drug is absorbed into the systemic circulation and

subsequently reaches the brain by crossing the BBB. The other is the olfactory

pathway by which the drug partly travels from the nasal cavity to CSF and/or brain

tissue. It can be concluded, that the amount of drug in the brain tissue after nasal

administration was attributed to these two pathways. The DTP% and DTE% represent

the percentage of drug directly transported to the brain via the olfactory pathway

mechanism for the same is illustrated in Fig. 53. DTP% and DTE% were calculated

using tissue/organ distribution data following intranasal and intravenous

administration and are recorded in Table 37. AMNE showed the highest DTE% (80)

Chapter 8: Radiolabelling, Biodistribution, Pharmacokinetics &Pharmacodynamic Studies

172

and DTP% (72.4) amongst the three tested formulations, followed by the ANE i.n.

and then by AS i.n.. Statistically significant (*p<0.05) difference in DTP% of AMNE

as compared to AS and ANE showed the benefit of mucoadhesive nanoemulsion

formulation. The higher DTE% and DTP% suggested that AMNE had better brain

targeting efficiency and the findings are in consequence with reports of Qizhi et al.

(2004) that mucoadhesive nanoemulsion increase nose to brain uptake of drugs. In

order to visualize brain uptake following intranasal and intravenous administrations of

99mTc AMB formulations, gamma scintigraphy were performed and the scintigrams of

mice 0.5 h post i.v. administration of ANE and i.n. administration of AMNE. The

scintigrams (Fig. 54 and 55) clearly demonstrate the accumulation of formulations in

brain administered via respective routes. Major radioactivity accumulation was seen

in brain following intranasal administration of AMNE as compared to intravenous

administration of ANE. Additionally, a part of activity was also noticed in oesophagus

and in the abdominal region, which was in conformity with the results of

biodistribution studies. For the reported results we have postulated the pictogram

showing the movement of nanoemulsions across olfactory to reach CSF as per the

Fig. 56.


Fig. 56: Proposed Mechanism of Amiloride loaded nanoemulsion transport to brain via olfactory route

after intranasal administration showing neuronal / transneuronal ways of movement.

173

8.2.5. Pharmacodynamic Studies

Amiloride formulations against PTZ Seizure Model


■ao o

■ | o.E ^ o)pd ^ 5 2 .

S S S 1 ^>. cu ■— O c ® c s CO® ore

250

200

150

100

5 0

0

I f 'Jo n r ;!

S a l i ne

{i.n.ll A S [ i . n . )

lANE{i.n.

lAMN^[i.n.l

T'ea t men t Type

Fig. 57: Effect of AMB formulations on PTZ-induced latency to generalized seizures in mice

1 6 0

1 4 0

120

100

SO

6 0

4 0

20

0T e e a tm e nt T ^f pe

I N o r m a l

Sa l i n e

(Ln.) l A S { i . n . )

l A N E { i , n .

l A M M E

(i.n.)

Fig. 58: Effect of AMB formulations on PTZ-induced latency to myoclonic jerks in mice

174


Fig. 55: Percentage protection of AMB Formulations against mortality due to PTZ Induced seizures

Amiloride Formulations against ICES Seizure Model

35 -|

30 -LU

25 -~l~

O 20 -

TSO 15 -

toa> 10 -r—i *

CO 5 -LU<J> 0 -

I Normal Saline{i.n.

lAS{i.n.)

IANE(i.n.)

lAMNE{i.n.)

TreatmentType

Fig. 60: Effect of AMB formulations against ICES induced threshold for HLTE in mice

175


<_>oCO

oo

o>o<_>o

COo

70

60

50

40

30

20

10

0T r e a t m e n t T y p e

I rjorna Sa l i ne ( i . n . l

I AS( i . n . l

I A N E { . n . ' i

Fig. 61: Effect o f AMB Formulations against ICES induced Post HLTE recovery time in mice

Fig. 62: Percentage Protective Effect o f AMB Formulations against ICES induced mortality in mice

176

We found an improved anticonvulsant action with mucoadhesive nanoemulsion

formulation administered through intranasal route as compared to other routes

(mentioned in chapter 3) in Chemical induced seizure model (Pentylenetetrazole

model: PTZ) and in Electrical induced seizure (Increased current electroshock model:

ICES). While pre-treatment with AMH alone at high dose (0.65 mg/kg) through oral

route significantly affected seizure threshold in the ICES test or latencies to

myoclonic jerks and clonic generalized seizures in the PTZ test, but that could be

achieved with a significant lower dose of amiloride (0.16 mg/kg) loaded formulations

(ANE/AMNE) when administered through intranasal route.

From these observations, it can be hypothesized that nanometric formulations of

Amiloride when administered through intranasal route may enter the brain to a greater

extent than free Amiloride when administered through other routes i.e., per oral or

intraperitoneal. This is in line with the earlier reports that suggested enhanced

anticonvulsant action of drugs like Clonazepam, Lamotrigine, Midazolam and

Diazepam when administered through intranasal route as

nanoemulsion/mucoadhesive nanoemulsion formulations (Vyas et al., 2006; Shende

et al., 2007; Botner & Sintov, 2011). The augmented action of nanoemulsion based

formulation may be attributed to greater ability of the nanometric carrier to cross the

BBB. Further mucoadhesive nanoemulsion could be better adhere and retained in

nasal cavity as compared to non-mucoadhesive formulations and it may lead to better

patient compliance.


177


8.2.6. CONCLUSIONS

Direct radiolabelling using technetium was found to be useful tool to study

biodistribution of selected formulations and drug solution. Radiolabelling of

nanoemulsion, mucoadhesive nanoemulsion and solution preparations of amiloride

were successfully performed and the results indicated good stability and bonding

strength of the radiolabeled complex. Hence, these formulations were found stable

and suitable to study biodistribution and to study gamma scintigraphy imaging of

these formulations on animals. Significant quantity of amiloride was quickly and

effectively delivered to the brain by intranasal administration of formulated

mucoadhesive nanoemulsion of amiloride. The study conducted in mice clearly

demonstrated effectiveness of intranasal delivery of amiloride as an antiepileptic drug;

however clinical data is needed to evaluate the risk vs. benefit ratio.

In conclusion, our findings indicate that nanoformulations exhibited improved

anticonvulsant action at lower dose (1/4th) when administered through intranasal route

as compared to when given by other route (p.o.) however the pharmacokinetic finding

supported that mucoadhesive formulation clearly showed improved brain uptake as

compared to drug solution, but the same increased pharmacokinetic levels did not

correlated to pharmacodynamic studies, it could be postulated that the minimum

levels of drug could be reached by intranasal route even administered at intranasal

route either as solution or with nanoformulation. These findings add to the

cumulative evidence suggesting nanotechnology based products like nanoemulsion

enhance drug delivery to brain.

178

Chapter Nine

SAFETY ASSESSMENTSTUDIES

Chapter 9: Safety Assessment Studies

9. SAFETY ASSESSMENT OF DEVELOPED NANOFORMULATIONS

In order to confirm the safety of the optimised and selected nanoemulsion

formulations, In vivo toxicity evaluation of ANE and AMNE was carried out in

Wistar rats.

Following studies carried out for safety assessment of the developed formulations:

1. In-vivo toxicity studies:

a. Mortality Count

b. Nasal mucosal histology

c. Brain Histology

2. Nasal cavity (mucosa) temperature measurement using NIR camera

3. Neurotoxicity studies by Rotarod method

4. In-vitro safety assessment

Methods:

9.1. In vivo toxicity

In vivo toxicity evaluation of ANE/AMNE was carried out to assess the mortality (if

any) followed by nasal and brain histology studies (after 14 days treatment) at an

equivalent dose respective to amiloride 0.16 mg/kg. The toxicity study was carried out

on optimised and selected formulations using rats as animal models as per the

reference taken from national toxicology programme

(http://ntp.niehs.nih.gov/go/9987).

Animals used:

Rats (n=6 per group) were used as experimental animals because they are most

suitable, easily available and widely used for research especially in vivo tissue toxicity

studies. Specific-pathogen-free, healthy, adult Wistar rats of either sex (3 months old;

200-250 g) were used in the tissue toxicity study. Tap water and rats food pellets were

available ad libitum throughout the study. They were maintained in a room that was

kept at 25° ± 2°C with relative humidity of 50 ± 5%.

179

http://ntp.niehs.nih.gov/go/9987


Sample Preparation:

The samples were prepared and used as such in case of ANE/AMNE and

administered the dosages of amiloride for rats except control group.

Methodology:

The rats were dosed once daily in morning (between 9.00 am to 10.00 am) with 50

of prepared ANE/ANME equivalent to 0.16 mg/kg of amiloride, by intranasal route

for 14 days. Prior to treatment, every day the animals were examined for any

abnormal behaviour, mortality and morbidity. For each formulation six rats (n=6)

were used and divided in the following groups;

Table 39: In-Vivo toxicity study protocol for Amiloride Nanoformulations

S.No. Group Treatment Number of Animals Dose

1 Group I Normal Saline 6 50 ^L

2 Group II ANE 6 0.16 mg/kg

3 Group III AMNE 6 0.16 mg/kg

4 Group IV Placebo 6 50 ^L

Amount administered 50 (20-25 in each nostril by keeping rat in supine position)

After the completion of study period of 14 days, the rats were sacrificed by keeping

them in desiccators containing diethyl ether for inhalation anaesthesia. Brain and

Nasal mucosa were dissected out, fixed in 10% neutral buffered formalin solution.

This prevented the post-mortem changes such as putrefaction and autolysis and

preserved the cell-constituents in as life-like manner as possible. Transverse sections

(T.S.) of the tissues were stained with hematoxylin and eosin and were examined

microscopically for the severity of mucosal irritancy or brain tissue damage loss and

atrophy. To evaluate any potential toxic effects of excipients used in the formulation

on the nasal mucosa, the nasal mucosa of was dissected and microscopically

evaluated for the toxic effects.

9.2. NASAL CAVITY (MUCOSAL) TEMPERATURE MEASUREMENT

USING IR CAMERA

Infrared (IR) imaging was shown to be a useful method to diagnose the signs of

certain diseases by measuring the local skin temperature (Herman et al., 2011;

180


Sumbera, et al., 2007). Inflammation in human and animals is marked by several

parameters such as pain swelling, immobility, and a rise in temperature of the affected

part. Since human skin, irrespective of its pigmentation, is an almost perfect radiator

of infrared radiation, there is a direct relationship between the temperature and

emissivity of this organ (Collins & Ring, 1972).

By using the above mentioned hypothesis that skin/mucosal irritation or sensitization

leads to the inflammation and this inflammation leads to localized temperature

change, hence forth nasal cavity (mucosal) temperature measurements were obtained

using an infrared camera (IR Camera (Extech i5 Infrared camera)) placed at an angle

of approximately 90° from the surface, 25 cm from the plantar region of the nose of

the animal and 75 cm above the floor post treatment every day (0-8h) for 7 days. This

camera has a thermal sensitivity of approximately 0.08°C, with an error of 2°C. The

images were digitally recorded at 30-second intervals. The thermographic procedures

were conducted in a room at a constant temperature of 23°C (±1°C) and humidity

between 30 and 50% (Thermo/hydro/clock, MT-230, Minipa, Brazil). Surface

temperature was measured from 9:00 to 12:00 a.m.

9.3. NEUROTOXICITY STUDIES BY ROTAROD METHOD

The rotarod test according to Lima et al. (1993) was used to determine the effect of

developed formulation on motor coordination. The integrity of the motor system was

evaluated with the rotarod test. Briefly, the rotarod apparatus consists of a rod 30-cm

long and 3 cm in diameter that is subdivided into three compartments by discs 24 cm

in diameter. The rod rotates at a constant speed of 10 rpm. The trained animals were

then evaluated for motor coordination at 7 and 14th days after i.n. administration of

0.16 mg/kg ANE/AMNE everyday for 14 days schedule. The fall off time of each

animal was recorded. Grouping for neurotoxicity studies would be as per above

mentioned Table 39.

9.4. IN-VITRO SAFETY ASSESSMENT

In-vitro safety of optimized formulation was assessed by observing their effect on

histology of goat nasal mucosa which was used for permeation study. Out of three

nasal mucosa pieces, one mucosa was used as control (0.6 mL water), the other was

181


t r e a t e d w i t h 0 . 6 m L o f o p t i m i z e d f o r m u l a t i o n a n d t h e l a s t o n e w a s t r e a t e d w i t h K C l

s o l u t i o n .

9.5. RESULTS AND DISCUSSION

1. In Vivo toxicity Studies: a. Mortality Count

Table 40: I n - V i v o t o x i c i t y s t u d i e s f o r A m i l o r i d e N a n o f o r m u l a t i o n s : M o r t a l i t y C o u n t s

S.

No.

Group Treatment Number

of

Animals

Dose Upon completion of

studies (14 Days) No.

of Animals survived

1 G r o u p I N o r m a l S a l i n e 6 5 0 ^ L 6

2 G r o u p I I A N E 6 0 . 1 6 m g / k g 6

3 G r o u p I I I A M N E 6 0 . 1 6 m g / k g 6

4 G r o u p I V P l a c e b o 6 5 0 ^ L 6

Amount administered 50 ^L (20-25 ^L in each nostril by keeping rat in supine position

b. Nasal mucosa histology

. '* * '/V ■ . * * - •

■ ' . > , ” ■ * r * "

Control Group Placebo

! : . r _ ? i 5 - A J g M g K a f t s

; > ■ ■ ■ ' : '

ANE AMNE

Fig. 63: Photomicrographs showing the T.S. of rats’ nasal mucosa for vehicle control (control group),

Placebo, ANE, AMNE treated groups after 14 days

182


c. Brain Histology

Fig. 64: Photomicrographs showing the T.S. of rats’ brain for vehicle control (control group), Placebo,

ANE, AMNE treated groups after 14 days

183


2. Nasal cavity (mucosa) temperature measurement using IR camera

Table 41: N a s a l C a v i t y T e m p e r a t u r e ( ° C ) M e a s u r e m e n t I R C a m e r a f o r A m i l o r i d e m u c o a d h e s i v e n a n o e m u l s i o n :

Time

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Control AMNE Con

trol AMNE Control AMNE Con

trol AMNE Control AMNE Con

trol AMNE Control AMNE

0 h32.2

±1.6332.5

± 2.9432.9 ±

1.4431.9 ±

1.9633.2 ±

1.3130.4±2.91

33.2 ± 2.14

31.7 ± 1.65

31.3 ± 1.83

34.7 ± 2.42

31.9 ± 1.33

32.3 ± 3.81

33.4 ± 2.13

32.4 ± 2.19

0.5 h31.5

±2.8333.4

± 1.8333.2 ± 2.14

32.4 ± 1.34

32.6 ± 2.16

32.6±1.87

33.6 ± 3.03

33.4 ± 2.14

32.8 ± 2.44

33.4 ± 1.53

33.1 ± 4.02

33.9 ± 2.52

34.1 ± 1.82

33.3 ± 1.99

2 h32.4

±1.9633.1

± 2.0232.4 ±

1.9634.0 ±

4.8231.7 ±

1.9833.7±3.71

30.4 ± 5.33

32.8 ± 2.63

33.4 ± 1.95

32.3 ± 2.02

32.6 ± 3.13

30.7 ± 3.02

32.8 ± 2.94

33.7 ± 2.06

4 h33.7

±2.0432.6

± 1.8433.1 ±

1.6233.1 ±

2.6230.5 ± 2.06

32.4±2.12

32.1 ± 2.62

33.1 ± 2.02

31.3 ± 2.56

31.1 ± 0.93

32.6 ± 1.44

32.4 ± 2.82

31.9 ± 3.01

33.5 ± 2.61

6 h32.3

±1.9333.1

± 2.0232.9 ± 2.00

30.8 ± 3.26

32.5 ± 2.72

30.2±3.51

30.9 ± 0.96

31.6 ± 4.71

31.6 ± 1.04

32.8 ± 1.73

31.9 ± 3.00

33.1 ± 1.32

32.4 ± 2.13

31.8 ± 1.92

8 h31.96±4.02

33.4 ± 2.94

32.9 ± 2.63

32.4 ± 1.95

31.8 ± 2.44

32.5±2.10

33.4 ± 1.15

32.4 ± 2.11

32.1 ± 2.94

34.0 ± 3.89

32.1 ± 2.72

32.6 ± 2.25

30.4 ± 5.13

32.5 ± 2.73

181


3. Neurotoxicity studies by Rotarod method

Fig. 65: Effect of i.n. administration of AMNE on rotarod test endurance time in seconds at different

time intervals 7 & 14 days (experiment performed post 1 h of treatment).

4. In-vitro Safety Assessment

Fig. 66: Histology of goat nasal mucosa; treated with (a) distilled water, (b) optimized formulation and

(c) KCl solution

T h e r e w e r e n o m o r t a l i t i e s o f r a t s o b s e r v e d i n a n y o f t h e g r o u p s d u r i n g t h e 1 4 -

d a y t r e a t m e n t p e r i o d w i t h i n t r a n a s a l a d m i n i s t r a t i o n o f d e v e l o p e d f o r m u l a t i o n

( A N E / A M N E ) . C l i n i c a l e x a m i n a t i o n o f t h e r a t s ’ b r a i n t i s s u e s p r i o r t o a n d a f t e r

a d m i n i s t r a t i o n o f e a c h ( A N E / A M N E ) f o r m u l a t i o n f o r 1 4 d a y s r e v e a l e d n o s i g n s o f

i r r i t a t i o n o r t i s s u e d a m a g e f o r a l l t h e r a t s a s c o m p a r e d t o t h e v e h i c l e c o n t r o l g r o u p s .

185


M a c r o s c o p i c e x a m i n a t i o n o f t h e b r a i n t i s s u e s e x p o s e d t o t h e p o l y m e r i c

( A N E / A M N E ) f o r m u l a t i o n , v e h i c l e a l s o d i d n o t s h o w a n y c h a n g e i n t h e m o r p h o l o g y

o r t i s s u e m i c r o s t r u c t u r e . A s c o m p a r e d t o v e h i c l e c o n t r o l , t h e ( A N E / A M N E )

f o r m u l a t i o n t r e a t e d g r o u p s s h o w e d n o v i s i b l e s i g n o f i n f l a m m a t i o n o r n e c r o s i s

d e m o n s t r a t i n g t h e s a f e t y o f ( A N E / A M N E ) f o r m u l a t i o n (Fig. 64).

T h e Fig. 63 s h o w s t h e d i s s e c t e d n a s a l m u c o s a t r e a t e d w i t h v a r i o u s t r e a t m e n t s , s h o w e d

n o n a s o c i l i a r y d a m a g e a n d t h e n a s a l m e m b r a n e r e m a i n e d i n t a c t . I n p l a c e b o g r o u p , n o

d a m a g e t o n a s a l m u c o s a i n t h e f o r m o f i n t a c t c i l i a t e d p s e u d o s t r a t i f i e d n a s a l

e p i t h e l i u m ( n o c i l i a e r o s i o n ) c o u l d b e o b s e r v e d , t h u s s u b s t a n t i a t i n g t h e s a f e t y o f t h e

e x c i p i e n t s u s e d i n t h e f o r m u l a t i o n s . T h e m u c o s a l h i s t o l o g y i m a g e s f o r f o r m u l a t i o n s

t r e a t e d w i t h c h i t o s a n c o n t a i n i n g n a n o e m u l s i o n ( A M N E ) s h o w e d p r e s e n c e o f

u n a l t e r e d t i g h t j u n c t i o n s w h i c h i s s i m i l a r t o n o n c h i t o s a n b a s e d f o r m u l a t i o n t r e a t e d

n a s a l m u c o s a ( A N E ) s u p p o r t i n g t h a t c h i t o s a n r e v e r s i b l y a l t e r t h e m u c o s a l

p e r m e a b i l i t y b y o p e n i n g t h e t i g h t c e l l u l a r j u n c t i o n f o r i n c r e a s e d r u g p e r m e a b i l i t y b u t

n o t a l t e r i n g c e l l u l a r s t r u c t u r e p e r m a n e n t l y . T h e s e f i n d i n g s c o r r o b o r a t e o b s e r v a t i o n s

r e p o r t e d b y G a v i n i a n d c o - w o r k e r s t h a t o n e x p o s u r e o f n a s a l m u c o s a t o f o r m u l a t i o n

c o n t a i n i n g m u c o a d h e s i v e a g e n t s h o w e d o p e n e d t i g h t j u n c t i o n s (Gavini et al., 2005).

F r o m t h e n e u r o t o x i c i t y s t u d i e s i t c a n b e c o n c l u d e d t h a t a m i l o r i d e m u c o a d h e s i v e

n a n o f o r m u l a t i o n s d o e s n o t c a u s e a n y n e u r o t o x i c i t y o r m o t o r c o o r d i n a t i o n i m p a i r m e n t .

T h e h i s t o l o g y o f g o a t n a s a l m u c o s a i n c o n t r o l , t r e a t e d w i t h o p t i m i z e d f o r m u l a t i o n a n d

t r e a t e d w i t h K C l s o l u t i o n i s s h o w n i n Fig. 66. T h e m i c r o s c o p i c o b s e r v a t i o n s i n d i c a t e

t h a t w i t h t h e o p t i m i z e d f o r m u l a t i o n , s u r f a c e e p i t h e l i u m l i n i n g a n d t h e g r a n u l a r

c e l l u l a r s t r u c t u r e o f t h e n a s a l m u c o s a w e r e t o t a l l y i n t a c t , w h e r e a s K C l c a u s e s m a j o r

c h a n g e s i n t h e u l t r a s t r u c t u r e o f m u c o s a . T h i s i n d i c a t e s a m i l o r i d e l o a d e d

m u c o a d h e s i v e n a n o e m u l s i o n f o r m u l a t i o n s a r e n o n t o x i c o n g o a t n a s a l m u c o s a a s w e l l

a n d c a n b e g i v e n b y i n t r a n a s a l r o u t e f o r e f f e c t i v e t r e a t m e n t o f e p i l e p s y .

9.6. CONCLUSION

T h e s h o r t - t e r m ( 1 4 d a y s ) t o x i c i t y s t u d i e s , r e p e a t e d i n t r a n a s a l a d m i n i s t r a t i o n o f t h e

a m i l o r i d e l o a d e d n a n o f o r m u l a t i o n s t o r a t s c a u s e d n o s i g n i f i c a n t i n f l a m m a t i o n , o r

t i s s u e t o x i c i t y . T h e s e p r e - c l i n i c a l s t u d i e s p r o v e d t h e s a f e t y o f d e v e l o p e d b r a i n -

t a r g e t e d a m i l o r i d e n a n o f o r m u l a t i o n s i n r a t s ; h o w e v e r c l i n i c a l d a t a i s n e e d e d t o

e v a l u a t e t h e r i s k v s . b e n e f i t r a t i o .

186

Documents

Chapter Eight RADIOLABELLING, BIODISTRIBUTION ...shodhganga.inflibnet.ac.in/bitstream/10603/43134/12/12_chapter 8.p… · 8.1.4. Biodistribution and Pharmacokinetics Studies M ice