Chapter 6

Evaluation of pharmacokinetic,

biodistribution, pharmacodynamic

and toxicity profile of juglone as

free and sterically stabilized

liposomal formulation

Journal of Pharmaceutical Sciences (Under Review)

Chapter 6

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ABSTRACT

The aim of this study was to comparatively investigate the in vivo behavior of

juglone (as free and sterically stabilized liposomal forms) in terms of its

pharmacokinetics, biodistribution, pharmacodynamic as well as toxicity profiles. The

pharmacokinetics and biodistribution profile of juglone following single intravenous

injection was carried out using tritium-labeled juglone. For all other studies,

unlabeled juglone was used.

The pharmacokinetic studies revealed that free juglone had a short plasma

half life of about 2 h and was rapidly eliminated from the systemic circulation. In

contrast, formulation of juglone as sterically stabilized liposomal form significantly

improved the pharmacokinetics with a 12-fold increase in the plasma half-life, 4.5-

fold increase in the AUC0-∞, 10-fold reduction in the renal clearance rates and a 3-

fold increase in the mean residence time of juglone. Further, from the biodistribution

studies of free juglone, large accumulation of juglone induced radioactivity in the

kidneys was observed indicating that the juglone may be rapidly eliminated through

the renal route. Also, accumulation in other organs was observed in the following

order kidney > liver > heart > spleen ≥ tumor. However, formulation of juglone as

sterically stabilized liposomes reduced the accumulation in the kidneys and heart

significantly with significantly higher accumulation in the liver, spleen as well as the

tumor tissue. This increased tumor accumulation of juglone was further confirmed

from the pharmacodynamic studies where liposomal juglone exhibited significantly

better anticancer as well as radiosensitizing potential in comparison to free juglone.

The toxicity studies were also carried out where significant levels of nephrotoxicity

was observed in the free juglone treated group which significantly reduced in the

mice treated with liposomal juglone, consistent with the biodistribution studies.

However, in spite of higher accumulation of liposomal juglone in organs like the liver

and spleen, no significant toxicities were seen from the histological studies, which

may be due to the slow release of juglone from the liposomes.

Based on these studies, liposomes encapsulation may be a promising

approach for the intravenous delivery of hydrophobic compounds like juglone.

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6.1. INTRODUCTION

Entering the 21st century, the pharmaceutical industry is challenged to further

transform its research and development operations to meet an ever-growing demand

for more and more affordable drugs brought to a highly competitive market in a

shorter time period. Demand for innovative and highly efficacious medications will

increase due to higher lifestyle expectations and changing demographic profiles. The

link between genomics and disease and the fallout of the Human Genome Projec t are

expected to provide numerous new molecular targets (Debouck and Metcalf, 2000;

Emilien et al., 2000). High throughput screening and combinatorial chemistry will

identify an exploding number of potential therapeutic agents for these targets that

need to be evaluated (Boulnois, 2000; Ohlstein et al., 2000; White, 2000).

Keeping that in the perspective, understanding of the behavior of drugs in

biological system has constantly been the subject of primary importance in treatment

of various diseases. This comprehension has immensely contributed to optimizing

dosage regimens, potentiating the therapeutic efficacy and tailoring of drug delivery

systems to meet specific needs, and to reduce side effects. In this regard, development

of animal models, interspecies scaling and physiologic based pharmacokinetic

modeling concepts have brought about pragmatic changes in establishing

pharmacokinetic/pharmacodynamic relationships (Andes and Craig, 2002). Presently,

pharmaceutical industry is faced with the challenge of introducing drug delivery

systems with reliable performance but also with a greater emphasis on patient

compliance (Meibohm and Derendorf, 2002; Scaglione, 2002). Following discovery

of potent molecules, these challenges of drug development go through successive

stages, such as, design of suitable delivery systems and in vivo evaluation. In this

context, preclinical pharmacokinetic, tissue distribution and metabolism studies

provide valuable information on safety profile since, usually good correlation exists

between pharmacokinetic and toxicological profile (Duncan et al., 1998; Hopewel et

al., 2001; Mahmood, 2001).

Therapeutic concepts in the treatment of diseases have undergone thorough

refinement with emerging novel drug delivery systems, which are marked with an

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ability to alter pharmacokinetics of drugs (Harris et al., 2001; Lavasanifar et al.,

2002). The utility of these novel systems has always demanded establishment of proof

of concepts on performance since, technology is also associated with inherent

complexities (Ciordia et al., 2000). For example, the efficacy of presently available

promising anticancer agents like doxorubicin, paclitaxel and camptothecin is limited

by toxic side effects due to non-specific distribution, especially to the rapidly

proliferating cells in body. Also, limitations result from the fact that, these agents per

se fail to selectively localize in tumors, which emphasizes the need for novel drug

delivery systems. To suppress toxic effects and to improve efficacy (drug burden on

malignant cells), numerous delivery systems based on specific carrier properties are

being extensively studied and evaluated in the field of cancer chemotherapeutics

(Howell, 2001; Maeda et al., 2001; Goldenberg, 2002; Park, 2002). It is the alteration

in pharmacokinetics brought about by these novel strategies, which reduces access to

normal cells but at the same time offer opportunities for tumor targeting (passive or

active).

Juglone has been studied for its potential as anticancer compound (Segura-

Aguilar et al., 1992; Kamei et al., 1998; Sugie et al., 1998; Cenas et al., 2006; Ji et

al., 2009; Ji et al., 2011). From the previous studies (described in chapter 3), the

anticancer potential of juglone against melanoma cells was established in vitro and

was attributed to ROS mediated genotoxic effect and cell membrane damage thereby

leading to cell death by a combination of apoptosis as well as necrosis. In the

subsequent studies (described in chapter 4), the in vivo anticancer as well as

radiosensitizing potential of juglone against B16F1 melanoma cells growing as solid

tumor on C57/BL6J mice was evaluated where juglone was also found to cause

significant amount of normal tissue toxicity against nucleated blood cells in mice.

Also it is reported in the literature that quinones generally have short plasma half life

which may diminish the anticancer efficacy of these compounds (Schellens et al.,

1994). Therefore, in the previous study (described in chapter 5), the formulation,

characterization and optimization (in terms of size, zeta potential, polydispersity

index, entrapment efficiency and in vitro release profiles) of sterically stabilized

liposomes of juglone was undertaken. Further, the free- and sterically stabilized

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liposomes of juglone were also comparatively evaluated for their in vitro cytotoxic

potential against melanoma cells in vitro where sterically stabilized liposomes of

juglone was found to be more cytotoxic in comparison to free juglone. In the present

study, an attempt was made to evaluate the in vivo behavior of the optimized sterically

stabilized liposomes of juglone (in comparison to free juglone) for its

pharmacokinetic, biodistribution, pharmacodynamic as well as toxicity profiles.

6.2. MATERIALS AND METHODS

6.2.1. Chemicals and reagents

Tritium (3H) labeled juglone (with a specific activity of 3.9 Ci/mmol) was

procured from Board of Radiation and Isotope Technology (BRIT, Navi Mumbai,

India). Unlabelled juglone, Minimum essential medium (MEM), L-glutamine,

gentamycin sulfate were obtained from Sigma Chemicals Co., (St. Louis, MO, USA).

Fetal bovine serum (FBS) was purchased from Genetix Biotech Asia, India.

6.2.2. Cancer cell lines

B16F1 cells were used in this study (as mentioned in Chapt 3, sect. 3.2.2).

6.2.3. Animals, tumor model and Irradiation procedures

The details regarding animal, tumor model and irradiation procedures are

same as described in the earlier section (Chapter 4, section 4.2.4 and section 4.2.5).

Sterilization of SSL juglone: The optimized liposomal formulation was passed

through 0.22 µ syringe filter, and the filtrate was analyzed using HPLC method for

entrapment efficiency and drug content. Based on the concentration of juglone in the

liposomal suspension, it was diluted with PBS (under sterile conditions) and injected

intravenously into the lateral tail vein of C57BL/6J mice.

6.2.4. Pharmacokinetic and biodistribution studies of free and SSL juglone

In the present study, the in vivo behavior of free juglone was compared with

the optimized sterically stabilized liposomal formulation (from the previous study).

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Tumors were induced in the mice by injecting 5 × 105 viable cells intradermally on

the dorsal side. Once the tumor size reached 100 ± 10 mm3, animals were divided into

following groups of 4 each:

Group 1: Tumor bearing animals in this group received single dose of 100 µl vehicle

(vehicle treated control group), Group 2: Animals in this group were injected

intravenously with single dose of free 3H-labelled juglone (0.02 mg/kg b. wt., 10

µCi/animal), Group 3: Animals in this group were injected intravenously with single

dose of sterically stabilized liposomes encapsulated with 3H-labelled juglone (equal

dose).

At various pre-set intervals of time, blood was collected into heparinized tubes

by retro-orbital plexus and plasma was collected and stored at -20 °C until further

analysis. These animals were also then euthanized and the organs (kidneys, liver,

spleen and heart) were dissected out, weighed gravimetrically and stored at -20 °C

until analysis.

Sample preparation and analysis

Amount of tritiated juglone in the blood (systemic circulation) either as free or

SSL encapsulated forms was assessed by directly measuring the radioactivity of the

plasma as a function of time. The amount of radioactivity in all other tissues was

studied according to the previously described methods (Mahin and Lofberg, 1966;

Sun et al., 1988). The pre-weighed tissue was first oven-dried, finely cut and

transferred into individual glass scintillation vials. In order to ensure accurate

measurement of the low energy beta radiation emitted by tritium, the prepared tissues

were completely dissolved using a solubilizing mixture of 0.2 ml perchloric acid (70

%) and 0.4 ml hydrogen peroxide (30 %). The sample digestion was carried out at 60

°C for about an hour (or until the sample turned almost colorless) and cooled to room

temperature.

The amount of tritium in the tissue samples was then determined by liquid

scintillation counting using 10 ml Ultima Gold™ (Perkin Elmer Life & Analytical

Sciences, Boston, USA) per sample. The counts per minute obtained per sample were

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corrected for efficiency of counting and used to derive the number of disintegrations

per minute and thus radioactivity (micro curies) per sample. The radioactivity per

milligram (µCi/mg) recovered from tissues was normalized to the amount of tritium

injected per respective mouse (i.e., the injected dose) and the results were then

expressed as a percentage of the injected dose per gram of tissue (% ID/g) versus

time.

6.2.5. Pharmacodynamic evaluation of the SSL of juglone

In the present study, the optimum dose of juglone 1 mg/kg b. wt. (for both

anticancer and radiosensitizing activity) was selected based on the previous studies

(described in chapter 4).

6.2.5.1. Anticancer activity

Tumors were induced by injecting 5 × 105 viable cells intradermally on the

dorsal side. Once the tumor size reached 100 ± 10 mm3, animals were divided into

following groups of 8 each:

Group 1: Tumor bearing animals in this group received 100 µl of vehicle (vehicle

treated control group); Group 2: Animals in this group were injected intravenously

with optimum dose (1 mg/kg b. wt.) of free juglone consecutively on day 1, 3 and 5;

Group 3: animals in this group received repeated injections of the optimum dose of

sterically stabilized liposomal formulation of juglone on day 1, 3 and 5 consecutively.

6.2.5.2. Radiosensitizing activity

Group 1: Animals in this group received 100 µl of vehicle, Group 2: Animals in this

group received 10 Gy of local tumor irradiation, Group 3: Animals in this group

received optimum dose of free juglone on day 1, 3 and 5 followed by local tumor

irradiation with 10 Gy. Group 4: Animals in this group received optimum dose of

sterically stabilized liposomal juglone on day 1, 3 and 5 followed by local tumor

irradiation with 10 Gy.

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At the end of various treatments, the important pharmacodynamic parameters

like volume doubling time (VDT) and growth delay (GD) were assessed as described

earlier (Chapter 4, section 4.2.6.3). The animals were also monitored for survival and

the median survival times were determined and reported using the Kaplan Meier

analysis of survival (Matthews and Farewell, 1996) as mentioned previously (Chapter

4, section 4.2.6.3).

6.2.6. Toxicity evaluation

The toxicity profile of free and SSL juglone was assessed in C57BL/6J mice

following repeated intravenous injections as mentioned below.

Group 1: Animals in this group received 100 µl of vehicle, Group 2: Animals in this

group were injected intravenously with optimum dose of free juglone (1 mg/kg b.

wt.), on 7 consecutive days Group 3: Animals in this group were injected

intravenously with optimum dose of sterically stabilized liposomal formulations of

juglone (1 mg/kg b. wt.), on 7 consecutive days.

6.2.6.1. Alkaline comet assay

Twenty four hours after the last dose, blood samples were collected and the

toxicity of juglone (as free and SSL encapsulated forms) on the blood lymphocytes

was then studied using the alkaline version of single cell gel electrophoresis (comet)

assay according to the standard procedure (Singh et al., 1988) as described earlier

(Chapter 4, section 4.2.6.4). Olive tail moment (OTM) and the tail DNA (%) were

then used to assess DNA damage levels (Chapter 4, section 4.2.6.4).

6.2.6.2. Histology

The same sets of animals were also used for the histological evaluation.

Briefly, twenty four hours after the last treatment dose, the animals were euthanized

and the organs (kidneys, liver, spleen and heart) were excised, washed with 0.9% w/v

NaCl solution and fixed in 10% v/v buffered formalin. All tissues were embedded in

paraffin blocks and sectioned into 5 μm thickness using a rotary microtome (Leica

RM2125 RT, Germany). After hematoxylin-eosin (H & E) staining, the slides were

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observed and the photos were taken using an optical microscope and analyzed for

gross structural alterations.

6.2.7. Statistical analysis

The level of statistical significance after various experiments was analyzed

using either student’s t-test (in cases where only two groups were to be compared) or

one way ANOVA followed by Bonferroni’s post-hoc test (where more than two

groups were involved). Survival studies were performed by Kaplan–Meier survival

analysis and the median survival time was reported. A P value of < 0.05 was

considered as statistically significant.

6.3. RESULTS

6.3.1. Pharmacokinetic studies

The time course of reduction in the percentage of injected radioactivity of

juglone (free and SSL encapsulated form) in plasma of tumor-bearing mice was

studied (Figure 6.1). From the figure 6.1, the SSL juglone showed a clear

improvement in the pharmacokinetic aspects of juglone.

Figure 6.1. Pharmacokinetic studies in tumor bearing mice injected with single

intravenous dose of 3H-juglone as free and sterically stabilized liposomal forms (0.02

mg/kg b. wt., 10 µCi/animal). The data represents mean ± SD of four animals.

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The non-compartmental analysis of the pharmacokinetic parameters using the

WinNonlin (Table 6.1) revealed that free juglone had a plasma half life of around

120.83 ± 15.66 min, AUC0-∞ of 87.91 ± 7.05 nmol.min/ml, Clearance of 0.66 ± 0.06

ml/min and a mean residence time (MRT) of 674.13 ± 33.18 min. In contrast, SSL

juglone exhibited significantly improved pharmacokinetic properties as shown in

table 6.1.

Table 6.1. Pharmacokinetic parameters of juglone, free and SSL encapsulated, after a

single intravenous bolus administration (0.02 mg/kg b. wt., 10 µCi/animal) in tumor

bearing mice. Each value represents mean ± SD of four animals.

Pharmacokinetic parameter Unit Free juglone SSL juglone

Plasma half life (t1/2) Min 120.83 ± 15.66 1424.70 ± 106.43*

Area under curve (AUC0-∞) nmol.min/ml 87.91 ± 7.05 380.30 ± 18.63*

Renal Clearance (CL) ml/min 0.66 ± 0.06 0.06 ± 0.01*

Mean residence time (MRT) Min 674.13 ± 33.18 2025.30 ± 165.26*

Significance levels - * - P<0.001 in comparison to free juglone treated group

6.3.2. Biodistribution studies

Table 6.2 shows the biodistribution patterns of the free and SSL encapsulated

juglone after single intravenous bolus dose administration in tumor-bearing mice. A

large accumulation of free juglone in the kidneys (about 35.1 ± 1.91 % of injected

dose) was seen within the first 15 min following the administration which however

declined with time to reach a value of 1.43 ± 0.07 % at 24 h interval. In the other

organs, the accumulation of free juglone was significantly lower in the following

order; kidney > liver > heart > spleen ≥ tumor. The distribution patterns of SSL

encapsulated juglone was markedly different from free juglone where a significantly

lower accumulation in tissues like the kidney and heart was observed, 15 min

following intravenous administration. On the other hand, in tissues like liver, spleen

as well as the tumor, at 15 min post administration, almost similar accumulation as

that of the free juglone was observed. However, the accumulation in these tissues

gradually increased with time to reach a maximum at 24 h interval 22.27 ± 2.26 %,

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18.94 ± 1.79 % and 15.02 ± 1.22 % was observed in liver, spleen and tumor tissue

respectively.

Table 6.2. Biodistribution patterns of free and SSL juglone in tumor bearing mice

following a single intravenous injection of 3H-juglone (0.02 mg/kg b. wt., 10

µCi/animal). Each value represents mean ± SD of the percent injected dose remaining

in various organs (n=4).

Organ 15 min 60 min 120 min 360 min 1440 min

Heart

Free drug

SSL

10.86 ± 1.42

6.16 ± 0.65

2.09 ± 0.27

2.61 ± 0.55

1.65 ± 0.22

1.88 ± 0.09

1.57 ± 0.59

1.64 ± 0.09

0.90 ± 0.02

0.97 ± 0.13

Kidney

Free drug

SSL

35.10 ± 1.91

16.34 ± 0.71

14.05 ± 1.70

14.62 ± 0.89

11.26 ± 1.01

10.61 ± 0.53

5.91 ± 0.11

9.53 ± 0.76

1.43 ± 0.07

6.29 ± 0.22

Liver

Free drug

SSL

12.74 ± 0.65

11.17 ± 0.49

8.34 ± 0.27

14.36 ± 1.18

4.93 ± 0.75

16.32 ± 0.68

4.73 ± 0.90

19.13 ± 1.21

3.41 ± 0.30

22.27 ± 2.26

Spleen

Free drug

SSL

5.81 ± 2.03

5.19 ± 0.23

4.82 ± 1.04

7.40 ± 0.08

3.74 ± 0.43

9.07 ± 0.13

2.20 ± 0.19

12.86 ± 1.90

1.57 ± 0.33

18.94 ± 1.79

Tumor

Free drug

SSL

5.80 ± 0.53

4.62 ± 0.56

3.29 ± 0.10

6.53 ± 0.41

1.96 ± 0.05

9.78 ± 0.30

1.56 ± 0.14

12.95 ± 0.63

1.34 ± 0.19

15.02 ± 1.22

6.3.3. In vivo anticancer potential of SSL juglone

Tumor bearing animals were injected with repeated doses of 1 mg/kg b. wt.

juglone (on days 1, 3 and 5) either as free or liposome encapsulated form and the

tumor growth kinetics were monitored and recorded as shown in figure 6.2 and table

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6.3. The tumors in the vehicle treated group showed a rapid and steep increase in size

and reached an average volume of approximately 4500 mm3 (45-fold compared to day

1) within 12 days. On the other hand, treatment with repeated doses of free juglone

caused the tumors to grow in a more gradual fashion and reached a mean tumor

volume of 1800 mm3 after 12 days. This delay tumor growth kinetics was also evident

from the significantly higher volume doubling time as well as the 5X values

compared to controls (table 6.3). Besides, the treatment of tumor bearing mice with

SSL juglone showed a further delay in the tumor growth parameters compared to free

juglone treated group (table 6.3) with a mean tumor size of only around 1000 mm3

after 12 days.

Figure 6.2. Effect of free and SSL juglone on tumor growth in mice inoculated with

B16F1 melanoma cells. Once the tumor volume reached 100 ± 10 mm3, animals were

injected with repeated doses of 1 mg/kg b. wt. juglone on day 1, 3 and 5 as free or

liposome encapsulated form and tumor growth kinetics was monitored as a function

of time (n=8). Significant levels - * - P < 0.01 in comparison to vehicle treated control

group and a – P < 0.05 in comparison to free juglone treated group

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Table 6.3. Tumor growth kinetics for free and SSL juglone. VDT – Volume doubling

time (Time required for tumors to reach double the initial volume); 5X – Time

required for tumors to reach 5 times the initial volume; GD – Growth delay

(difference in time between treated and untreated tumors to reach 5X).

Treatment VDT ± SD

(days)

5 X ± SD

(days)

GD ± SD

(days)

Vehicle control 1.57 ± 0.54 3.55 ± 0.53 -

Free juglone (on days 1, 3 & 5) 2.86 ± 0.68a 5.84 ± 0.56b 2.55 ± 0.51

SSL juglone (on days 1, 3 & 5) 3.61 ± 0.70a* 8.73 ± 1.01b** 5.18 ± 1.40†

Significance levels a – P< 0.001 compared to vehicle treated controls; * - P<0.05

compared to free juglone; b – P<0.001 compared to vehicle treated controls; ** -

P<0.001 compared to free juglone; † - P<0.001 compared to free juglone

Survival analysis

The data regarding the animal survival was also analyzed using the Kaplan-

Meier curves (Figure 6.3).

Figure 6.3. Kaplan-Meier analysis of animal survival after various treatments. Once

the tumor volume reached 100 ± 10 mm3, animals were injected with repeated doses

of 1 mg/kg b. wt. juglone on day 1, 3 and 5 as free or liposome encapsulated form and

monitored for animal mortality.

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A median survival of 19, 28 and 32 days was observed for the vehicle treated,

free juglone treated and liposomal juglone treated groups, respectively (Figure 6.3).

From these results, the beneficial effect of reduction in tumor burden of animals

treated with juglone (either as free or liposome encapsulated form) on the survival

rates of animals was clearly evident.

6.3.4. In vivo radiosensitization studies of SSL juglone

The optimum dose of juglone (1 mg/kg b. wt., from the earlier study) either as

free or SSL encapsulated form was then combined with radiation (10 Gy) to evaluate

its radiosensitizing potential in vivo.

Figure 6.4. A) Radiosensitizing potential of free and SSL juglone against B16F1

melanoma growing as solid tumor on C57BL/6J mice. Once the tumor volume

reached 100 ± 10 mm3, animals were injected with repeated doses of 1 mg/kg b. wt.

juglone on day 1, 3 and 5 as free or liposome encapsulated form. On the last day,

tumors were locally irradiated with 10 Gy radiation (n=8). Significant levels - a – P <

0.001 in comparison to vehicle treated group and * - P < 0.001 in comparison to free

juglone plus radiation group. B) Kaplan Meier analysis of animal survival after

various treatments.

Tumor response was measured in terms of volume doubling time and growth

delay as mentioned in the materials and methods section. The results of this study are

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depicted in figure 6.4 and table 6.4. There was a significant increase in the volume

doubling time as well as tumor growth delay with combination therapy (juglone plus

radiation) in comparison to radiation alone group at 10 Gy (P<0.001) radiation dose.

The volume doubling time as well as growth further improved when radiation (10 Gy)

was combined with SSL juglone which was statistically significant in comparison to

free juglone plus radiation group. Analysis of the survival patterns (Figure 6.4B)

revealed that the treatment of tumor bearing mice with radiation alone (10 Gy), free

juglone plus radiation and SSL juglone plus radiation resulted in median survival

values of 31, 36 and 42 days respectively.

Table 6.4. Tumor growth kinetics for radiosensitizing potential of free and SSL

juglone.

Treatment VDT ± SD

(days)

5 X ± SD

(days)

GD ± SD

(days)

Vehicle control 1.43 ± 0.46 3.64 ± 0.51 -

Radiation alone (10 Gy) 2.01 ± 0.62 6.36 ± 0.60* 2.82 ± 0.60

Free juglone + Radiation (10 Gy) 5.46 ± 0.58* 9.87 ± 0.49* 6.74 ± 0.77†

SSL juglone + Radiation (10 Gy) 9.22 ± 1.07*a 13.38 ± 1.33*a 9.83 ± 1.33†a

Significance levels * - P < 0.001 in comparison to vehicle treated control; † - P <

0.001 in comparison to radiation alone group; a – P < 0.001 in comparison to free

juglone + radiation group

6.3.4. Toxicity evaluation

6.3.4.1. Comet assay using blood

The genotoxic potential of juglone as free and liposome encapsulated form

was evaluated by performing comet assay using the whole blood from mice treated

with repeated doses (1 mg/kg b. wt.) for 7 consecutive days. As can be seen from

figure 6.5, the treatment of mice with repeated doses of 1 mg/kg b.wt juglone

intravenously resulted in significant (P < 0.01) elevation in the Olive tail moment and

the tail DNA values compared to vehicle treated controls. In contrast, the DNA

damage levels (Olive tail moment & tail DNA) in the mice treated with liposomal

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juglone was drastically reduced in comparison to free juglone treated group.

However, these levels were still marginally higher than the control levels.

Figure 6.5. Effect of free of SSL encapsulated juglone (1 mg/kg b. wt.) on the DNA

damage levels in blood cells of mice treated with optimum dose of juglone (on 7

consecutive days). Significant levels - * - P<0.01 in comparison to vehicle treated

control cells. Each data represents mean ± SD of four animals

6.3.4.2. Histological studies

The histological studies were also performed using various tissues (heart,

kidney, liver and spleen) isolated from the same sets of animals and the results are

depicted in figure 6.6.

From the figure, the heart sections of the untreated mice showed cardiac

muscles with peripheral nuclei as well as normal branching and striations of cells. The

presence of necrotic tissue if any would show loss of nuclei and striations, with only

cell outline being maintained, which was not seen in the present study. The heart

sections of the animal treated with either the free or SSL juglone did not show any

observable changes in the morphology of the heart and resembled that of the control

tissues.

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Figure 6.6. Toxicity evaluation of free and SSL juglone in C57BL/6J mice assessed

using H & E staining of various tissues. Black arrows showing necrosis of the renal

convoluted tubules and glomeruli in animals treated with free juglone. Red arrows

showing reduction in the juglone induced nephrotoxicity.

In the case of renal cortical sections of control mice, distinct glomeruli and

adjacent tubules were seen with normal histological architecture (evidenced by

distinct glomerular basement membrane as well as normal tubular interstitium). On

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the other hand, the renal cortical sections of the animals treated with free juglone

resulted in clear distortion of the glomeruli as well as the tubular architecture (as

evidenced by the coagulative necrosis of the proximal convoluted tubules and the

glomeruli, with loss of cellular morphology, nuclei in both tubules and glomeruli, and

loss of brush border also in proximal convoluted tubules) in comparison to the control

animals, indicating the potential of juglone to induce nephrotoxicity. However, unlike

in the case of free juglone, mice treated with SSL juglone resulted in the restoration of

the normal cortical architecture.

The hepatic section of the control mice showed distinct central vein, sinusoids

as well as hepatocytes with occasional apoptotic bodies. Similarly, the hepatic

sections of the animals treated with either free or SSL juglone did not show any

significant changes in the morphology.

From the spleen sections of the control animals, normal splenic corpuscles

were seen. Similarly, the spleen sections of free as well as SSL juglone did not alter

significantly and no evidence of any toxic changes could be seen.

6.4. DISCUSSION

Even though the pharmaceutical industry has been successful in discovering

many new cytotoxic drugs that are potential candidates for the treatment of cancer,

this life-threatening disease still causes more than 6 million deaths every year

worldwide and the number is growing. The clinical use of most conventional

chemotherapeutics is often limited due to inadequate delivery of therapeutic drug

concentrations to the tumor target tissue or due to severe and harmful toxic effects on

normal organs. Current efforts to meet this challenge are focused on developing

targeted therapeutics specific to the cancer cell. Another important approach being

actively investigated is to develop novel microcarrier technologies that can be used to

selectively deliver cytotoxic agents to the tumor site (Cheong et al., 2007). Way back

in 1974, liposomes were suggested as potential drug carriers in cancer chemotherapy

(Gregoriadis et al., 1974). Since then, the interest in liposomes has increased and

liposome systems are now being extensively studied as drug carriers (Andresen et al.,

2005).

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Initially, the research in liposome drug delivery systems suffered from the

very fast blood clearance by the reticuloendothelial system (RES). It was recognized

that particle size, surface charge and liposome composition had a strong influence on

the clearance profile (e.g., incorporation of phosphatidylinositols or

monosialogangliosides prolongs liposome circulation in the blood) (Allen and Chonn,

1987). However, liposomes were only fully recognized as successful drug delivery

candidates when it was discovered that liposomes coated with the synthetic polymer

polyethyleneglycol (PEG) had significantly increased half- life in the blood (Klibanov

et al., 1990; Senior et al., 1991).

Since then, the use of nanotechnology in medicine and more specifically drug

delivery has spread rapidly (De Jong and Borm, 2008). It is now generally accepted

that the specific tumor targeting leads to better profiles of pharmacokinetics and

pharmacodynamics, controlled and sustained release of drugs, an improved

specificity, an increased internalization and intracellular delivery and, more

importantly, a lower systemic toxicity. The tumor targeting consists in “passive

targeting” and “active targeting”; however, the active targeting process cannot be

separated from the passive because it occurs only after passive accumulation in

tumors (Danhier et al., 2010). Based on these developments, several plant based

compounds with anticancer properties including plumbagin (Tiwari et al., 2002),

lapachone (Blanco et al., 2010), curcumin (Li et al., 2005; Wang et al., 2008; Chen et

al., 2009) etc., have been formulated using liposomal platforms with improved

anticancer efficacies and better toxicity profiles.

In the previous study, formulation, characterization and optimization of the

sterically stabilized liposomal formulation of juglone was undertaken. In the present

study, an attempt was made to evaluate the pharmacokinetic, biodistribution,

pharmacodynamic and toxicity profile of sterically stabilized liposomal formulation

of juglone in comparison to free juglone.

The in vivo pharamacokinetic and biodistribution studies were carried out after

injecting tumor bearing mouse with single intravenous bolus dose of tritium labeled

juglone either as free of SSL encapsulated forms. As can be seen from this study, free

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juglone had a short plasma half life of about 2 h. Plumbagin, another structurally

related naphthoquinone with potent anticancer and radiosensitizing properties, is also

reported to possess a similar short plasma half life (Chandrasekaran and Nagarajan,

1981). In contrast, formulation of juglone as SSL encapsulated form resulted in a 12-

fold increase in the plasma half life of juglone. In addition, the other pharmacokinetic

parameters were also significantly improved as evidenced by the 4.32-fold increase in

the AUC0-∞ values, a 10-fold reduction in the renal clearance rate with a 3-fold

increase in the mean residence time of juglone. This improvement in the

pharmacokinetic parameters could potentially lead to enhanced tumor accumulation

of juglone and thereby improve the therapeutic efficacy of encapsulated juglone. To

test this hypothesis, the biodistribution patterns of free and SSL juglone was

evaluated, where free juglone was found to localize in the kidneys in large quantities

(approximately 35 % of the injected dose) indicating that kidney could one of the key

organ responsible for the elimination of juglone from the body. It also means that

kidney could be highly susceptible to the toxic effects of juglone. Also, there was

moderate levels of accumulation in the liver as well as the heart tissue and lower

levels of localization in spleen and tumor tissue at initial time-point which however

reduced with time. On the other hand, the distribution patterns of SSL juglone was

more in agreement with the hypothesis where significantly higher localization in the

tumor tissue was observed. However, the accumulation in the spleen as well as the

liver also increased which may be attributed to the uptake of the liposomes by the

mononuclear phagocyte system (MPS). Also, it is a known fact that particles in the

size of > 10 nm may not be filtered through the kidney (Danhier et al., 2010) which

may have contributed to the observed reduction in juglone-derived radioactivity in the

kidneys following SSL encapsulation.

Recently, Chen and co-workers (2005) studied the metabolism and disposition

of 14C-labelled juglone in male F344 rats following oral, intravenous and dermal

administration. They reported high levels of juglone-derived radioactivity in kidney

for all three dosing routes which is similar to what was observed in the present study.

They concluded that the accumulation in kidney could be attributed to covalent

binding of juglone and/or metabolites to cytosolic protein. From their study, liver

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microsomal incubations of juglone in the presence of NAD(P)H and UDP-glucuronic

acid gave rise to two 1,4,5-trihydroxynaphthalene mono-glucuronides indicating the

ability of juglone to undergo metabolism in the liver.

To investigate the therapeutic advantage of the improved pharmacokinetic and

biodistribution profile of juglone when administered as sterically stabilized liposomal

juglone, the comparative evaluation of the anticancer activity of SSL juglone and free

juglone against B16F1 melanoma cells grown as solid tumor on the dorsal side of the

C57/BL6J mouse was performed. A significantly higher anticancer potential of SSL

juglone in comparison to free juglone was observed (as evidenced by the tumor

growth kinetic parameters) with corresponding increase in the animal survival.

Further, a significant improvement in radiosensitizing potential of juglone was

observed when formulated as sterically stabilized liposomal form. It is well

documented that the in vivo behavior of liposomes depends on some liposome-related

factors viz., vesicle size, lipid composition, cholesterol, charge and surface

hydrophilicity (Senior, 1987; Allen et al., 1995). Significant advancement in the area

of membrane biophysics has provided a new approach to fabricate liposomes that

offer steric hindrance (through attachment of hydrophilic polymers to the liposome

surface) and prevent the process of opsonization leading to delayed recognition from

the MPS. Consequently, the liposomes evade destruction by MPS cells and stay in

circulation for longer time periods (Torchilin and Papisov, 1994; Storm et al., 1995;

Torchilin, 1996) resulting in enhanced circulation half life of the encapsulated drug.

From the present study, the observed improvement in the anticancer and

radiosensitizing efficacy of SSL juglone may be attributed to its higher tumor

localization in vivo through the enhanced permeability and retention (EPR) effect

(Fang et al., 2010), in agreement with the earlier reports where a direct correlation

between prolonged circulation time and increased liposome localization in tumors has

been demonstrated (Gabizon and Papahadjopoulos, 1988; Wu et al., 1993; Gabizon,

1995; Gabizon et al., 1997).

Although several earlier studies have demonstrated the anticancer potential of

juglone against in vitro and in vivo tumor models, its perceived toxicity against

normal tissues undermines its therapeutic potential. It is reported that juglone causes

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contact irritant dermatitis when applied topically (Neri et al., 2006). From the

previous study (presented in chapter 4), juglone was found to induce some damage to

the nucleated blood cells as well. However, not much has been studied in terms of its

toxic effects. In the present study, repeated intravenous administration of free juglone

(1 mg/kg b. wt) resulted in significant damage to the renal convoluted tubule as well

as glomeruli as evidenced by the observed coagulative necrosis in these cells. This

corroborates the earlier finding where large localization of juglone-derived

radioactivity in the kidneys was observed. To our knowledge, this is the first report

where juglone is shown to cause nephrotoxicity using mouse model. Also, no

observable toxicity to other tissues was seen which is in agreement with the

biodistribution studies (significantly lower accumulation was seen in these tissues).

On the other hand, in the animals treated with repeated doses of SSL juglone, the

damage to the kidney was drastically reduced, which again as in agreement with the

biodistribution studies (much lower accumulation was seen), indicating the potential

of sterically stabilized liposomes to reduced the juglone induced toxicity. In contrast,

although higher levels of accumulation of SSL juglone was seen in other tissues like

liver and spleen, no significant change in the morphology was observed which may be

due to the slow release of the drug from the liposome (lower than the toxic dose for

the particular tissue). Further, the toxicity to the blood lymphocytes in the SSL

juglone treated group was also significantly reduced in comparison to free juglone

treated group as evidenced by the 1.36-fold reduction in the OTM as well as % tail

DNA values. Results from these studies, clearly demonstrates the ability of liposomes

to reduce the toxicity associated with anticancer agent like juglone.

In conclusion, SSL significantly improved the pharmacokinetic and

biodistribution attributes of juglone resulting in enhanced plasma half life and higher

tumor accumulation in vivo. Also, the formulation of juglone as sterically stabilized

liposome resulted in significantly better anticancer efficacy against in vivo melanoma

model with reduced toxicity profiles. Based on the findings of this study, liposomes

encapsulation may be a promising approach for the intravenous delivery of

hydrophobic compounds like juglone.

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