Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α -tocopherol or γ -tocopherol in beagle dogs

2013

http://informahealthcare.com/txmISSN: 1537-6516 (print), 1537-6524 (electronic)

Toxicol Mech Methods, Early Online: 1–13! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.774079

ORIGINAL ARTICLE

Safety and pharmacokinetic studies of liposomal antioxidantformulations containing N-acetylcysteine, a-tocopherol orc-tocopherol in beagle dogs

Misagh Alipour1, Panagiotis Mitsopoulos2, Milton G. Smith3, Gordon Bolger4, Kresimir Pucaj4,and Zacharias E. Suntres1,2

1Medical Sciences Division, Northern Ontario School of Medicine and 2Department of Biology, Lakehead University, Thunder Bay, ON, Canada,3Amaox Ltd, Melbourne, FL, USA, and 4Nucro-Technics, Scarborough, ON, Canada

Abstract

The safety and pharmacokinetic profile of liposomal formulations containing combinations ofthe antioxidants a-tocopherol, g-tocopherol or N-acetylcysteine in beagle dogs was examined.Each group consisted of beagle dogs of both genders with a control group receiving emptydipalmitoylphosphatidylcholine (DPPC) liposomes (330 mg/kg DPPC, EL), and test groupsreceiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60 mg/kg NAC[L-NAC]); (ii) NAC and a-tocopherol (aT) (60 mg/kg NAC and 25 mg/kg a-tocopherol [L-aT-NAC])and (iii) NAC and g-tocopherol (60 mg/kg NAC and 25 mg/kg g-tocopherol (gT) [L-gT-NAC]). Thedogs in the control group (EL) and three test groups exhibited no signs of toxicity during thedosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathologyfindings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogsand in all groups. Results from the pharmacokinetic study revealed that the inclusion oftocopherols in the liposomal formulation significantly increased the area under the curve (AUC)and b-half life for NAC; the tocopherols had greater impact on the clearance of NAC, wherereductions of central compartment clearance (CL) ranged from 56% to 60% and reductions oftissue clearance (CL2) ranged from 73% to 77%. In conclusion, there was no treatment-relatedtoxicity in dogs at the maximum feasible dose level by a single bolus intravenousadministration while the addition of tocopherols to the liposomal formulation prolonged thecirculation of NAC in plasma largely due to a decreased clearance of NAC.

Keywords

a-Tocopherol, g-tocopherol, antioxidants,liposomes, N-acetylcysteine, toxicity

History

Received 7 October 2012Revised 4 February 2013Accepted 4 February 2013Published online 25 April 2013

Introduction

When reactive oxygen species (ROS) production exceeds the

cellular antioxidant capacity, oxidative damage to cellular

components such as proteins, lipids and DNA occurs

(McCord, 2000; Suntres, 2011; Ward, 2010; Ziech et al.,

2010). A potential pharmacological strategy in preventing or

treating oxidant-induced cellular and tissue damage involves

the use of antioxidants. Antioxidants are substances which are

able to prevent, delay or remove oxidative damage to a

molecule (Benzie, 2000; Evans & Halliwell, 2001; Sies, 1997;

Suntres, 2002). Yet, their efficacy is hindered with challenges

such as poor solubility, inability to cross cell membrane

barriers, extensive first pass metabolism and rapid clearance

of antioxidants from cells (Ratnam et al., 2006; Steinhubl,

2008). To improve the pharmacological and pharmacokinetic

properties of antioxidants, diverse systems such as antioxidant

chemical modifications, coupling to affinity carriers, micelles

and liposomes are being developed (Beg et al., 2010;

Carnemolla et al., 2010; Muzykantov, 2001a,b; Ratnam

et al., 2006; Stone & Smith, 2004; Suntres, 2002).

Liposomes can facilitate intracellular delivery of several

therapeutic agents via fusion with the plasma membrane

lipids, receptor-mediated endocytosis and phagocytosis

(Allen, 1998; Gregoriadis, 1991; Torchilin, 2006).

Liposomes have been used for the transport of water-soluble

and lipid-soluble antioxidants as well as antioxidant enzymes

to different organs and tissues for the treatment of oxidative

stress-induced damage. The lipophilic antioxidant

a-tocopherol (Minko et al., 2002; Mukherjee et al., 2009;

Suntres & Shek, 1997, 1998), the hydrophilic antioxidants

glutathione, N-acetylcysteine (Alipour et al., 2007;

McClintock et al., 2006; Mitsopoulos et al., 2008) and the

antioxidant enzymes superoxide dismutase and catalase

(Freeman et al., 1985; Turrens et al., 1984) have been

shown to confer additional protection against oxidant-induced

injuries when delivered as liposomal formulations. To date,

toxicological profile of liposomal antioxidants have been

Address for correspondence: Zacharias E. Suntres, PhD, MedicalSciences Division, Northern Ontario School of Medicine, LakeheadUniversity, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada. Tel:807-766-7395. E-mail: [email protected]

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reported only in the rat (Alipour et al., 2012). In order for a

liposomal antioxidant formulation to be further investigated

for its clinical applications, regulatory agencies such as the

Federal Drug Administration (FDA) also requires acute

toxicity testing in at least one non-rodent species such as

the dog and monkey.

This study was designed to evaluate the safety and

pharmacokinetics of a liposomal formulation containing

combinations of the antioxidants a-tocopherol, g-tocopherol

and N-acetylcysteine in beagle dogs.

Materials and methods

Chemicals

Dipalmitoylphosphatidylcholine (DPPC) was obtained from

Northern Lipids Inc. (Burnaby, Canada). N-acetylcysteine,

alpha (a)-tocopherol and gamma (g)-tocopherol as well as

all other chemicals were purchased from Sigma-Aldrich Co.

(Oakville, Canada).

Animals

Male and female beagle dogs (approximate body weight

6–9 kg, n¼ 2 animals per sex per group) were purchased from

Ridglan Farms Inc. (Mt. Horeb, WI) individually. The animal

room environment was controlled (targeted ranges: tempera-

ture 18–26 �C, relative humidity 30–70%) and monitored

daily. The photo-cycle was 12 h light and 12 h dark. The cage-

cleaning schedule, air filtration and recirculation, health

checks and facility maintenance was carried out in accordance

with the applicable institutional Standard Operating

Procedures, and such activities were recorded in the animal

room records. Each animal was assigned a unique ear tattoo

number by the supplier. The individual animal number and

the institution’s study number comprised a unique identifica-

tion for each animal. The animal cage was identified by the

study number, animal number, group number and sex. Upon

arrival all animals were submitted to a general physical

examination and only those found healthy were admitted.

Animals used in this study were treated and cared for in

accordance with the guidelines recommended by the

Canadian Council on Animal Care (CCAC) and Association

for Assessment and Accreditation of Laboratory Animal Care

(AAALAC).

This study was carried out at Nucro-Technics, Inc.

(Scarborough, Canada) in accordance with the Good

Laboratory Practices of the United States Food and Drug

Administration (21 CFR Part 58) and OECD Principles of

Good Laboratory Practice.

Acclimatization period

The animals were acclimatized for 2 weeks. During the

acclimation and study periods, Teklad Certified 25% Lab Dog

Diet (#8727C) was offered once daily (approximately 1 h

post-injection), and was available to the dogs for consumption

for a period of approximately 4–5 h. Water was offered

ad libitum throughout the study period. During this period the

animals were observed for any clinical signs of disease. At the

end of the acclimatization period, all animals were found to

be healthy and were admitted to the pool of animals for

randomization for the study.

Preparation of liposomal antioxidants

All liposomal formulations were prepared by a modified

dehydration–rehydration method (Alipour et al., 2012). The

lipophilic tocopherols and/or hydrophilic NAC were mixed

with DPPC lipids when required, for the preparation of the

specific formulations at steps indicated below. DPPC lipids

(4:1 molar ratio with the a- or g-T) were mixed and dissolved

in a 100 ml round-bottomed Erlenmeyer flask with chloro-

form and dried at 45 �C on a rotary evaporator (Buchi

Rotavapor R 205, Brinkmann, Toronto, ON, Canada).

Thereafter, the lipid film (with or without the tocopherols)

was further dried with N2 gas (to eliminate traces of organic

solvents) and then hydrated with sterile phosphate-buffered

saline (PBS; pH 7.4) or with sterile PBS containing NAC

(40 mg/mL). The solution was briefly warmed at 45 �C and

vortexed to promote the formation of multilamellar vesicles.

The solution was subsequently sonicated (Model 500

Dismembrator, Fisher Scientific, Ottawa, Canada) to produce

smaller multilamellar vesicles for 15 min (cycles of 40 s on

and 20 s off). The liposomal formulation was then freeze-

dried for 3 d and then stored at �20 �C. The freeze-drying

step ensured that the formulations were kept stable until use.

Reconstitution of lyophilized liposomal antioxidantformulations

Upon rehydration of liposomes, unentrapped antioxidants

were separated by centrifugation at 24 400 g at 4 �C, for

30 min, a step that was performed twice. Liposomal vesicle

size for all formulations was determined with Submicron

Particle Sizer (Nicomp Model 270; Santa Barbara, CA) after

rehydration and was found to have an approximate mean

diameter of 200 nm. The content of antioxidants in the

liposomal formulations before treatment and post-treatment

were determined by ultra performance liquid chromatography

(UPLC) using Waters Acquity system equipped with a binary

solvent manager, an automated sample manager and a

photodiode array detector (Waters, Milford, MA). Samples

were lysed by sonication (20 s, 100% amplitude; Sonic

Dismembrator Model 500), centrifuged, then passed through

a 0.2 mm filter prior to injection onto an Acquity UPLC HSS

T3 analytical column (2.1 mm I.D.� 150 mm length, 1.8 mm

particles) with a Vanguard 2.1 mm I.D.� 5 mm length guard

column, at 30 �C. The mobile phase consisted of 23 mM

ammonium formate (pH 3) at a flow rate of 0.250 ml/min.

NAC, a-T and g-T were measured at wavelengths of 200.2,

205.2 and 206.4 nm, respectively. The amount entrapped was

measured using respective antioxidant standard curves.

Stability of liposomal antioxidant formulations

To determine the stability of the liposomal antioxidant

formulations, the entrapment efficiency of NAC in the

different liposomal antioxidant formulations incubated in

PBS buffer or rat plasma was examined. Liposomes contain-

ing NAC with or without tocopherols were incubated with

equal volumes of rat plasma or PBS buffer, at 4 or 37 �C, for

2 M. Alipour et al. Toxicol Mech Methods, Early Online: 1–13

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different incubation times. Following the completion of each

incubation period, a sample aliquot was removed and

centrifuged at 24 400 g at 4 �C, for 30 min, a step that was

performed twice to separate the unentrapped antioxidants.

The resultant pellet, containing the stable liposomes contain-

ing NAC, were lysed with 0.1% Triton X-100 followed by

the addition of 0.004% 5,50-dithiobis(2-nitrobenzoic acid)

(DTNB) (dissolved in 10% sodium citrate) and read using a

spectrophotometer (OD412). The percentage entrapped was

measured using a known NAC standard curve.

Dose administration

The individual doses of the test articles were calculated for

each animal based on the body weight of the animal. Test and

control article suspensions were administered via the right

cephalic vein (Yang et al., 2006).

Dose-range finding study

Results from our previous study demonstrated that the

maximum feasible dose (MFD) levels, based on the physical

properties/viscosity of the test articles that could be safely

administered to dogs by an intravenous injection, were as

follows: (i) Empty liposomes (600 mg/kg DPPC); (ii) L-NAC

(600 mg/kg DPPC, 200 mg/kg NAC); (iii) L-aT/NAC

(600 mg/kg DPPC, 200 mg/kg NAC, 83.3 mg/kg a-T)

and (iv) L-gT/NAC (600 mg/kg DPPC, 200 mg/kg NAC,

71.4 mg/kg g-T) (Alipour et al., 2012). However, some dogs

experienced emesis and hypotension, most likely due to the

high rate of a large volume/viscosity of the liposomal

formulations; therefore, it was decided to reduce the volume

of the liposomal formulations administered to dogs by

almost 70%.

Acute toxicity study

For the acute toxicity study, the individual doses of the test

articles were calculated for each animal based on the body

weight of the animal (Table 1). Typically, in acute toxicity

studies, animals are given a single dose of the test article in a

24 h period and then observed for 14 days. In the present

study, the dose levels used were lower than those determined

from the dose-range finding study because some dogs

experienced emesis and hypotension during the intravenous

administration, an effect from which animals recovered

completely after 1 h post-administration.

Mortality

Mortality checks were performed twice per day throughout

the study.

Clinical signs

Animals were inspected twice daily during the course of the

study. In addition, they were monitored closely for 60 min

after each dosing. Elements of observation included reaction

to treatment such as changes in skin, fur, eyes and mucous

membranes. Respiratory, circulatory, autonomic, central ner-

vous system, somatomotor activity and behavior patterns were

also monitored along with any other signs of ill-health.

Clinical signs were recorded once a day during the morning

observation period. If the afternoon observations differed,

they were also recorded.

Blood pressure and heart rate monitoring

Blood pressure and heart rate monitoring in animals were

measured with the use of the Surgivet V60046 Non-Invasive

Blood Pressure monitor (SurgiVet, Inc, Waukesha, WI) as per

the manufacturer’s instructions.

Body weights and feed consumption

The unfasted body weight of each animal was recorded once

(for randomization) before its assignment to the relevant

group. Each animal was weighted again before dosing on

day 1, and on days 7 and 14. Finally, following an overnight

period (approximately 12–18 h) of food deprivation, each

surviving dog was weighed terminally prior to necropsy on

day 15. Feed consumption was recorded daily from days 0–14.

Clinical pathology

Clinical pathology investigations (e.g. hematology, coagula-

tion, clinical chemistry and urinalysis) were performed 24 h

post dosing and at necropsy for all animals. Blood (3 mL) was

collected from the left cephalic vein. Urine was collected by

placing dogs in metabolism cages overnight, prior to

Table 1. Dosing protocol.

Dosea Dose volumea Number of animalsFrequency/route

Group (mg/kg) (mL/kg) Male Female of administration

Control(DPPC)

330 10 2 2 SingleI.V. Bolus

Test 1(L-NAC)

330/60 10 2 2 SingleI.V. Bolus

Test 2(L-aT-NAC)

330/25/60 10 2 2 SingleI.V. Bolus

Test 3(L-gT-NAC)

330/25/60 10 2 2 SingleI.V. Bolus

EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine.

aMaximum feasible dose as determined from the range finding study based on the physical properties/viscosity of the testarticles that could be safely administered to dogs by an intravenous injection (Alipour et al., 2012). Dosing was followed by a14-day observation period.

DOI: 10.3109/15376516.2013.774079 Safety studies for liposomal antioxidant formulations 3

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necropsy. Baseline data for clinical pathology parameters

used in this study were taken from Nucro-Technics’ historical

database for the sex and age of animals.

Gross pathology

On day 15 after an overnight period of food deprivation, all

animals were euthanized with an overdose of sodium

pentobarbital (100 mg/kg), administered intravenously, fol-

lowed by exsanguination. For each animal, the necropsy

consisted of an external examination, as well as a detailed

internal examination. All necropsies were performed under

the supervision of a veterinarian.

Tissue preservation and histopathology

During necropsy, the following tissues and organs were

retained and fixed in neutral 10% formalin: abnormal tissues,

adrenals, aorta (thoracic), brain, cecum, colon, duodenum,

esophagus, femur and marrow, heart (sections of left and right

ventricles and atria, septum with papillary muscle), ileum,

jejunum, kidneys, liver (central and left lobes), lungs (left and

right diaphragmatic lobes), lymph node (mandibular and

mesenteric), ovaries, pancreas, pituitary, prostate, salivary

glands (mandibular), seminal vesicles, sciatic nerve, skeletal

muscle (quadriceps), skin (inguinal), spinal cord, stomach,

thymus, thyroid, parathyroids, tongue, trachea, urinary blad-

der, uterus (horns, cervix) and vagina. Epididymis, eyes, optic

nerves and testes were fixed in acid alcohol. With the

exception of the animal identification, the tissues above from

all animals were prepared for microscopic examination by

embedding in paraffin wax, sectioning and staining with

hematoxylin and eosin.

Pharmacokinetic profile of liposomal antioxidantformulations

For the pharmacokinetic analysis (PK), liposomal antioxidant

formulations were administered to animals (2 maleþ 2 female

dogs per group [n¼ 4]) via the right cephalic vein (Yang

et al., 2006): (A) L-NAC (330 mg DPPC/60 mg NAC/kg); (B)

L-aT/NAC (330 mg DPPC/25 mg a-T/60 mg NAC/kg) and

(C) L-gT/NAC (330 mg DPPC/25 mg g-T/60 mg NAC/kg).

Blood for pharmacokinetic analysis was collected as indicated

in Table 2. At each designated time point, 3 mL of blood was

collected from the left cephalic vein into K2EDTA tubes.

Blood samples were placed in a refrigerated centrifuge for

20 min at 2000 rpm in order to separate the plasma. Plasma

was transferred to duplicate cryovials that were stored frozen

(at �80 �C) pending analysis. Analysis was concluded within

1 month from collection.

Pharmacokinetic model selection

Attempts were made to fit the individual dog and mean

plasma concentration data to one, two and three compartment

models. None of the plasma concentration data could be fit to

a three compartment model; however, the plasma concentra-

tion data could best fit to either a one- or two-compartment

model according to Akaike Information Critera (AIC) as

follows: for the liposomal formulations containing NAC and

NAC in the presence of aT data was better fit to a two

compartment model; for NAC in the presence of gT, only two

individual dogs and the mean PK data could be better fit to a

two compartment model (Table 15).

Pharmacokinetic analysis

PK parameters were determined by initially fitting the i.v.

data to one and two compartment models with no lag time and

first-order elimination employing WinNonlin Professional

Software version 5.2.1. and determining the best fit model as

described above. For the one-compartment model the plasma

concentration data was fit to the following equation:

C Tð Þ ¼ Dose=Vss � e�K10xt

where C(T) is the plasma concentration in mg/mL,

Dose¼ dose in mg/kg, Vss¼ volume of distribution at steady

state in mL/kg, K10¼ terminal elimination rate constant per

hour and t¼ time in hours.

In addition to the primary PK parameters indicated above,

the calculated secondary PK parameters were: AUC (mg h/

mL) – area under the curve; AUMC (mg h2/mL) – area under

the first moment curve; K10 half-life (hours) – terminal

elimination half-life from the central compartment; Cmax

(mg/mL) – maximum plasma concentration (extrapolated) at

the instant of dosing; CL (L/kg/h) – total systemic clearance;

MRT (h) – mean residence time.

For the two-compartment model comprised of the central

and tissue compartments, the plasma concentration data was

fit to the following equation:

C Tð Þ ¼ A� e��t þ B� e��t

where C(T) is the plasma concentration in mg/mL, A¼ is the

extrapolated Cmax of the distribution phase in mg/mL, B¼ is

the extrapolated Cmax of the terminal elimination phase in

mg/mL, � and � (in h�1) are derived from the forward and

reverse micro rate constant, K12 and K21 in units of h�1 for

compound movement from and to the central compartment

(volume of distribution V1 in L/kg) with respect to the tissue

compartment respectively and the elimination rate constant

K10 (in h�1).

Table 2. Pharmacokinetic blood sampling schedule.

Bleeding time following I.V. administration

Dogs per group Pre-dose 5 min 15 min 30 min 1 h 2 h 4 h 6 h 8 h 12 h 24 h

2 Males ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ2 Females

At each designated pharmacokinetic sampling time point, 3 mL of blood was collected from the vena cephalica antebrachiisinistra into K2EDTA tubes. Plasma was separated by centrifugation (20 min, 4 �C, and 2000 rpm). Plasma was transferred toduplicate cryovials and was stored at �80 �C until analysis.

4 M. Alipour et al. Toxicol Mech Methods, Early Online: 1–13

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In addition to the primary PK parameters indicated above,

thecalculated secondary PKparameterswere:AUC(mg h/mL)–

area under the curve; AUMC (mg h2/mL – area under the first

moment curve; � half-life (h) – half-life of the central

compartment; � half-life (h) – half-life of the tissue compart-

ment; K10 half-life (h) – terminal elimination half-life; Cmax (mg/

mL) – maximum plasma concentration (extrapolated) at the

instant of dosing; CL (L/kg/h) – clearance from the central

compartment; MRT (h) – mean residence time; Vss (L/kg) –

volume of distribution at steady-state in the central compart-

ment; V2 (L/kg) – volume of distribution in the tissue compart-

ment; CLD2 (L/kg/h) – clearance from the tissue compartment.

Ultra-performance liquid chromatography analysis ofplasma antioxidant levels

The analysis of NAC by ultra-performance liquid chromatog-

raphy (UPLC) has been described previously (Mitsopoulos

et al., 2008). The mobile phase consisted of a mixture of

methanol and ammonium formate (20:80 v/v) with a C18

reverse phase column as the stationary phase. The method

was found to yield a quantitative recovery of NAC of more

than 96%, to be sensitive (20 pmole/10ml injection) and rapid

(less than 3 min).

Data collection and statistics

In-life data was collected using the Lab-Cat� In-Life module

(Version 6.1; July 5/00, Innovative Programming Associates

Inc., Lawrenceville, NJ). Necropsy data were collected using

the Organ Weights/Necropsy module (V3.28; Apr. 23/99 –

Innovative Programming Associates, Lawrenceville, NJ).

Data for the liposomal stability studies were analyzed by

the paired Student’s t-test, and a p-value of 0.05 or less was

considered significant. Pharmacokinetic analysis was calcu-

lated by using the WinNonLin Professional Software 5.2.1

(Pharsight, Montreal, Canada).

Numerical data collected during the course of the study

was subjected to the calculation of group means and standard

deviations (ANOVA with Newman–Keuls post hoc analysis).

Results

Entrapment efficiency of NAC in liposomes

The liposomal formulations used in the present acute toxicity

study were from the same batch that were used in the rodent

acute toxicity study (Alipour et al., 2012). Briefly, the

encapsulation efficiency of NAC in L-NAC, L-aT-NAC and

L-gT-NAC was measured as 25%, 23% and 22%, respectively.

The encapsulation efficiency of a-T in L-aT-NAC was

measured as 85% while that for g-T in L-gT-NAC was

measured as 79%. The particle sizes of L-NAC, L-aT-NAC

and L-gT-NAC were 192 nm, 207 nm and 174 nm, respect-

ively (Alipour et al., 2012).

Stability of liposomal antioxidant formulations in PBSbuffer and plasma

In order for the liposomal antioxidant formulations to be

effective in the treatment of oxidant-induced tissue injuries,

liposomes have to overcome structure destabilization as a

result of interaction with certain plasma components present

in blood (following intravenous injection). Thus, the stability

of liposomal NAC formulation in plasma as well as PBS

buffer as a control was examined and the results are shown in

Figure 1. Liposomal NAC with a-T or g-T showed better

stability in PBS buffer than plasma, but the differences were

not significant. Inclusion of a-T or g-T in liposomes improved

the retention of NAC under all conditions examined.

The maximum feasible dose from the dose-rangefinding study, blood pressure and heart rate

Results from the dose-range-finding study showed that the

maximum feasible dose (MFD) levels, based on the physical

properties/viscosity of the test articles that could be safely

administered to dogs by an intravenous injection, were as

follows: (i) Empty liposomes (600 mg/kg DPPC); (ii) L-NAC

(600 mg/kg DPPC, 200 mg/kg NAC); (iii) L-aT/NAC

(600 mg/kg DPPC, 200 mg/kg NAC, 83.3 mg/kg a-T) and

(iv) L-gT/NAC (600 mg/kg DPPC, 200 mg/kg NAC, 71.4 mg/

kg g-T). During these studies, it was decided to reduce the

volume of the liposomal formulations administered to dogs by

almost 70% since some dogs experienced emesis and

hypotension, most likely due to the high rate of a large

volume/viscosity of the liposomal formulations.

The pooled average systolic/diastolic blood pressure and

heart rate in male animals pre-dose were of 145.3� 5.98/

87.29� 3.90 mmHg and 120.4� 5.69 beats/min while

approximately 15 min post-dose the blood pressure and

heart rate had significantly decreased to 88.43� 3.39/

52.57� 9.53 mmHg and 109.1� 5.43 beats/min, respectively;

both parameters returned to normal levels 1 h post-dose.

Similarly, the average systolic/diastolic blood pressure and

heart rate for female animals pre-dose were 134.2� 5.48/

83.00� 3.44 mmHg and 117.00� 4.56 beats/min while

approximately 15 min post-dose the blood pressure and

heart rate had significantly decreased to 79.83� 9.7/

50.83� 8.65 mmHg and 107.80� 8.99 beats/min, respect-

ively, returning to normal levels 1 h post-dose. Thus, the

doses for all subsequent experiments used in the acute toxicity

Figure 1. The stability of the liposomal antioxidant formulations.Liposomes containing NAC with or without a- or g-T were incubatedwith equal volumes of rat plasma or PBS buffer, at 37 �C, for differentincubation times. The concentration of NAC (Mean� SD, n¼ 3 separateexperiments) in the liposomal formulations was measured spectrophoto-metrically (OD412) following its reaction with DTNB as described in theSection ‘‘Materials and methods’’. The percentage entrapped NAC wasmeasured using a known NAC standard curve.

DOI: 10.3109/15376516.2013.774079 Safety studies for liposomal antioxidant formulations 5

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study were as follows: (i) Empty liposomes (330 mg/kg

DPPC); (ii) L-NAC (330 mg DPPC/60 mg NAC/kg); (iii)

L-aT/NAC (330 mg/25 mg a-T/60 mg NAC/kg) and (iv) L-gT/

NAC (330 mg/25 mg g-T/60 mg NAC/kg).

Mortality

All animals from all groups received the specified treatment

(Table 1) and survived to the scheduled euthanasia and

necropsy dates.

Body weights, feed consumption and clinicalobservations

There were no appreciable differences in body weight

(Table 3) and feed consumption (Table 4) between the control

(empty liposomes) and corresponding test animals, both

genders. There was also no statistical difference for these

three parameters (ANOVA; p50.05). There were no adverse

reactions to the treatments during the injections or immedi-

ately after the dosing. Daily clinical observations (e.g. cage

side monitoring and detailed weekly physical examination

showed no drug-related toxicity in either the control group

(EL) or in any of the three test groups (i.e. L-NAC, L-aT-

NAC, L-gT-NAC groups).

Hematological, biochemical and urine parameters

The effects of empty liposomes and liposomal antioxidant

formulations on the hematological parameters of male and

female dogs 24 h and 15 d following treatment are shown in

Tables 5, 6 and 7, 8, respectively. RBC counts, reticulocytes,

hemoglobin, hematocrit and RBC indices (MCV, MCH and

MCHC) as well as platelet counts were all within the normal

physiological ranges for all groups and both genders. WBC

counts and differential were within the normal physiological

ranges for the control group (EL) and treatment groups.

Activated partial thromboplastin time (APTT) and prothom-

bin time (PT) were within the normal ranges in male and

female rats during the acute study (Tables 9 and 10).

The effects of empty liposomes and liposomal antioxidant

formulations on the serum biochemical parameters in male

and female dogs 24 h and 15 d following treatment are shown

in Tables 11, 12 and 13, 14, respectively. There was no

statistically significant difference between the control group

and test groups of animals (ANOVA; p40.05), for any of the

serum biochemical parameters in all groups.

The effects of empty liposomes and liposomal antioxidant

formulations on the urine parameters on Day 15 post-

treatment indicated that the specific gravity, pH and all

other substances (glucose, bilirubin, ketone, urobilinogen and

nitrite) remained within physiological ranges (data not

shown). There was no blood or leukocytes detected. Urine

appearance was clear and light yellow (data not shown).

Histopathology

No abnormalities were seen in the microscopic examination

of the internal organs. The cellular appearances were

unremarkable in all groups and between sexes. No histo-

logical findings were considered to be toxicologically

significant. Various known background or incidental condi-

tions were noted in the test and control groups (data not

shown).

Model fit of plasma concentration data

The results of fitting the plasma concentration data to one,

two and three compartment PK models is summarized in

Table 15. For the plasma NAC concentration data for L-NAC

and L-aT-NAC groups, there was highly likely fit to the two

compartment model as noted by an a difference in the Akaike

Information Criteria (AIC) values of 410. For the plasma

NAC concentration of the L-gT-NAC group, the two

compartment model was a likely preference (difference in

AIC values ranging from 5.63 to 8.97) for two individual and

the mean plasma concentration data in dogs; for two dogs (#2

and #3), the plasma concentration data could not be fit to a

two compartment model, but rather was fit to a one

compartment model, suggesting a lower preference for the

two compartment model.

Pharmacokinetic profile of liposomal antioxidantformulations

The plasma-concentration profiles and estimated PK param-

eters for liposomal NAC administered alone or in combin-

ation with either a- or g-tocopherol are presented in Table 16

Table 3. Body weights.

Mean body weights (g)� SD (n¼ 2)

Groups Day 1 Day 7 Day 14

MalesEL 8.9� 0.4 9.1� 0.4 9.5� 0.3L-NAC 8.8� 0.1 9.1� 0.1 9.3� 0.2L-aT-NAC 8.7� 0.1 8.5� 0.2 8.9� 0.3L-gT-NAC 8.8� 0.7 9.0� 0.4 9.3� 0.6

FemalesEL 6.2� 0.7 6.4� 0.8 6.5� 0.6L-NAC 6.6� 0.8 7.2� 1.2 7.2� 1.2L-aT-NAC 6.1� 0.1 6.4� 0.1 6.8� 0.2L-gT-NAC 6.5� 0.2 6.8� 0.1 6.8� 0.1

The NAC dose intravenously administered to each animal was 60 mg/kg.EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrappeda-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine.

Table 4. Feed consumption.

Feed consumption (g) (Mean� SD; n¼ 2)

Groups Day 1–8 Day 8–15

MalesEL 2619.5� 415.8 2883.0� 229.1L-NAC 2088.3� 196.9 2548.5� 101.1L-aT-NAC 1822.0� 197.3 2385.5� 197.3L-gT-NAC 2205.0� 384.7 3007.0� 592.6

FemalesEL 1848.3� 544.8 2149.0� 749.5L-NAC 2139.5� 227.7 2180.0� 527.5L-aT-NAC 2026.5� 45.3 2112.5� 92.6L-gT-NAC 1853.0� 340.1 2239.5� 303.3

The NAC dose intravenously administered to each animal was 60 mg/kg.EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrappeda-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine.

6 M. Alipour et al. Toxicol Mech Methods, Early Online: 1–13

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Table 5. Hematology group summaries of males – 24 h post-dose.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

RBC �1012/L 5.85� 0.24 5.84� 0.47 6.60� 1.12 6.50� 1.22 5.24–7.63Hb g/L 133� 7 134� 8 151� 23 148� 25 116–175Hct % 39.5� 2.7 38.9� 2.9 44.2� 6.2 43.9� 7.6 34.3–51.0MCV fL 67.5� 1.7 66.6� 0.5 67.2� 1.9 67.6� 0.8 61.7–71.1MCH Pg 22.7� 0.3 23.0� 0.4 23.0� 0.5 22.8� 0.4 21.0–24.3MCHC g/L 337� 4 345� 3 342� 3 336� 1 322–362Platelets �109/L 153� 62 215� 7 181� 10 156� 16 187–626WBC �109/L 16.99� 2.99 15.62� 0.20 15.88� 5.34 10.29� 0.96 5.47–17.95Neutrophils �109/L 12.24� 1.97 10.29� 0.45 13.28� 4.88 8.07� 1.47 2.48–11.15Lymphocytes �109/L 3.65� 0.96 4.11� 0.59 2.01� 0.35 1.52� 0.40 1.54–6.09Monocytes �109/L 0.53� 0.06 0.82� 0.04 0.27� 0.11 0.34� 0.01 0.16–1.23Eosinophils �109/L 0.40� 0.04 0.25� 0.02 0.20� 0.09 0.20� 0.04 0–0.62Basophils �109/L 0.12� 0.01 0.09� 0.01 0.08� 0.04 0.06� 0.05 0–0.13LUC �109/L 0.05� 0.00 0.07� 0.00 0.05� 0.05 0.11� 0.11 0–0.10Reticulocytes �109/L 238.0� 34.2 262.1� 63.9 327.8� 147.6 346.8� 122.3 10–128.4

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

Table 6. Hematology group summaries of males – day 15.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

RBC �1012/L 6.11� 0.59 6.65� 0.18 6.21� 0.44 6.43� 0.36 5.24–7.63Hb g/L 133� 14 145� 6 137� 7 141� 6 116–175Hct % 42.0� 4.2 45.5� 2.1 42.0� 1.4 43.5� 2.1 34.3–51.0MCV fL 68.6� 0.9 68.0� 1.4 67.4� 2.0 68.3� 0.4 61.7–71.1MCH Pg 21.8� 0.1 21.7� 0.3 22.1� 0.5 22.0� 0.4 21.0–24.3MCHC g/L 318� 1 319� 1 327� 3 322� 3 322–362Platelets �109/L 422� 97 520� 41 426� 51 427� 156 187–626WBC �109/L 12.01� 1.05 21.38� 0.83 16.61� 0.27 17.68� 3.86 5.47–17.95Neutrophils �109/L 5.91� 0.21 12.82� 0.52 10.74� 1.66 10.26� 2.28 2.48–11.15Lymphocytes �109/L 4.78� 0.73 6.39� 0.74 4.28� 1.52 5.58� 1.23 1.54–6.09Monocytes �109/L 0.74� 0.19 1.58� 0.26 0.98� 0.01 1.25� 0.02 0.16–1.23Eosinophils �109/L 0.45� 0.27 0.37� 0.32 0.47� 0.34 0.40� 0.17 0–0.62Basophils �109/L 0.08� 0.05 0.11� 0.01 0.08� 0.02 0.08� 0.08 0–0.13LUC �109/L 0.07� 0.03 0.13� 0.01 0.09� 0.05 0.12� 0.06 0–0.10Reticulocytes �109/L 47.5� 3.5 76.5� 3.5 47.5� 0.7 67.5� 19.1 10–128.4

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

Table 7. Hematology group summaries of females – 24 h post-dose.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

RBC �1012/L 6.25� 0.49 5.9� 0.66 6.88� 0.13 6.55� 0.04 5.34–7.54Hb g/L 141� 5 136� 16 157� 6 146� 4 119–174Hct % 41.2� 2.0 40.4� 4.1 45.7� 2.0 43.0� 0.4 35.1–51.0MCV fL 66.0� 2.1 68.6� 0.6 66.4� 1.7 65.6� 1.0 61.7–71.9MCH Pg 22.5� 1.0 23.1� 0.1 22.8� 0.3 22.4� 0.5 21.1–24.4MCHC g/L 341� 4 336� 4 344� 4 341� 13 323–358Platelets �109/L 197� 66 244� 82 145� 81 185� 49 223–545WBC �109/L 11.63� 0.76 15.52� 4.24 8.45� 1.64 11.85� 0.02 6.39–17.09Neutrophils �109/L 7.87� 1.12 9.23� 0.97 5.17� 0.34 8.65� 0.50 3.01–10.44Lymphocytes �109/L 3.01� 0.23 4.89� 3.34 2.64� 1.17 2.31� 0.58 1.63–6.38Monocytes �109/L 0.41� 0.02 0.68� 0.15 0.29� 0.09 0.32� 0.04 0.23–1.16Eosinophils �109/L 0.23� 0.13 0.53� 0.28 0.20� 0.10 0.41� 0.00 0–0.51Basophils �109/L 0.08� 0.02 0.12� 0.01 0.09� 0.02 0.10� 0.02 0–0.13LUC �109/L 0.05� 0.01 0.09� 0.04 0.07� 0.04 0.06� 0.01 0–0.10Reticulocytes �109/L 289.4� 59.1 268.7� 93.2 359.9� 8.8 327.5� 11.1 10–123.2

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

DOI: 10.3109/15376516.2013.774079 Safety studies for liposomal antioxidant formulations 7

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and Figure 2, respectively. No tendency toward gender

differences was noted for the PK of NAC and as such the

estimated PK parameters from the average plasma data and

for individual dogs are presented (Table 16). The majority of

the plasma concentration data could be fit to a two-

compartment model. The major impact of the inclusion of

tocopherols in the liposomal formulations on the PK of NAC

was a significant 2.2-fold and 2.6-fold increase of AUC for

NAC for a- and g-tocopherol, respectively. The tocopherols

reduced the K12 and K21 values for NAC, however the

significance of this observation could not be assessed. The

elimination constant K10 of NAC was significantly reduced

4.5-to-5.5-fold by adding tocopherols in the liposomes. The

tocopherols also increased the b-half life and elimination

half-life of NAC. The volume of distribution of NAC was

only affected by a-tocopherol where a reduction in the volume

of distribution in tissue, V2, was observed. However, the

tocopherols had greater impact on the clearance of NAC,

where reductions of central compartment clearance (CL)

ranged from 56% to 60% and reductions of tissue clearance

(CL2) ranged from 73% to 77%. Thus, the increased plasma

AUC for NAC in the presence of the tocopherols is largely

due to a decreased clearance of NAC.

Discussion

This study was undertaken to examine the acute toxicity of a

liposomal formulation containing combinations of the lipo-

philic antioxidants a-tocopherol, g-tocopherol and/or the

hydrophilic antioxidant NAC in beagle dogs. Liposomes are

spherical bilayered nanocarriers that have the capacity for

entrapping a diverse number of compounds within their inner

aqueous cores (e.g. hydrophilic compounds such as NAC), or

for incorporating them into their lipid bilayers (e.g. hydro-

phobic compounds such a-tocopherol or g-tocopherol).

The manipulation and design of liposomes endowed with

the ability for targeting specific cell sites (or alternately, the

temporary avoidance of these sites), results in long circula-

tion, increased biodistribution and favourable

pharmacodynamics.

Table 8. Hematology group summaries of females – day 15.

Group means� SD (n¼ 2)

Parameters Unitz EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

RBC �1012/L 6.87� 0.40 6.66� 0.64 6.51� 0.77 6.42� 0.74 5.34–7.54Hb g/L 148� 3 149� 14 143� 17 141� 14 119–174Hct % 46.5� 0.7 46.5� 4.9 43.0� 4.2 41.5� 6.4 35.1–51.0MCV fL 67.7� 2.1 69.9� 0.2 66.0� 0.6 65.2� 2.5 61.7–71.9MCH Pg 21.6� 0.9 22.4� 0.1 22.0� 0.0 22.1� 0.4 21.1–24.4MCHC g/L 319� 4 320� 0 334� 2 339� 18 323–358Platelets �109/L 515� 86 489� 62 492� 32 431� 11 223–545WBC �109/L 12.92� 2.81 14.12� 3.83 11.29� 0.41 12.46� 0.78 6.39–17.09Neutrophils �109/L 7.33� 2.09 7.65� 1.99 6.36� 0.08 6.82� 0.31 3.01–10.44Lymphocytes �109/L 4.18� 0.69 5.09� 2.22 3.71� 0.30 4.50� 0.42 1.63–6.38Monocytes �109/L 1.10� 0.08 0.89� 0.18 0.88� 0.14 0.69� 0.05 0.23–1.16Eosinophils �109/L 0.15� 0.01 0.29� 0.18 0.17� 0.04 0.31� 0.00 0–0.51Basophils �109/L 0.08� 0.06 0.10� 0.00 0.08� 0.04 0.08� 0.00 0–0.13LUC �109/L 0.10� 0.06 0.10� 0.01 0.10� 0.05 0.06� 0.01 0–0.10Reticulocytes �109/L 42.0� 0.0 96.5� 78.5 20.0� 5.7 47.0� 4.2 10–123.2

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

Table 9. Coagulation group summaries – 24 h post-dose.

Parameter – Mean� SD (n¼ 2)

Groups Prothrombin (s) APTT (s) Fibrinogen (g/L)

MalesEL 7.9� 0.0 11.6� 0.8 2.73� 0.08L-NAC 8.2� 0.1 11.3� 0.7 2.01� 0.14L-aT-NAC 7.9� 0.1 11.3� 1.1 3.17� 0.17L-gT-NAC 8.0� 0.4 11.4� 0.6 5.40� 1.20

FemalesEL 8.0� 0.0 11.6� 0.2 2.34� 0.00L-NAC 8.2� 0.0 10.4� 0.8 1.83� 0.00L-aT-NAC 8.1� 0.4 10.0� 1.2 2.69� 1.49L-gT-NAC 8.1� 0.4 10.2� 2.1 2.72� 0.49

Normal ranges 6.5–9.3 8.9–14.8 0.60–3.80

The NAC dose intravenously administered to each animal was 60 mg/kg.EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrappeda-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine.

Table 10. Coagulation group summaries – day 15.

Parameter – Mean� SD (n¼ 2)

Groups Prothrombin (s) APTT (s) Fibrinogen (g/L)

MalesEL 8.2� 0.1 11.2� 1.0 1.90� 0.13L-NAC 8.1� 0.4 11.9� 0.1 2.51� 1.35L-aT-NAC 8.0� 0.1 11.9� 0.4 2.17� 0.37L-gT-NAC 8.1� 0.0 11.2� 0.1 2.28� 0.21

FemalesEL 8.2� 0.0 11.9� 0.1 1.59� 0.02L-NAC 8.3� 0.1 11.7� 1.1 1.64� 0.16L-aT-NAC 8.1� 0.3 12.0� 0.6 1.72� 0.49L-gT-NAC 8.3� 0.1 10.5� 0.1 1.71� 0.06

Normal ranges 6.5–9.3 8.9–14.8 0.60–3.80

The NAC dose intravenously administered to each animal was 60 mg/kg.EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrappeda-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine.

8 M. Alipour et al. Toxicol Mech Methods, Early Online: 1–13

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In our study, DPPC, a neutral lipid which is also present in

the lungs where it serves as a natural surfactant was used.

Results from previous studies have shown that DPPC is well

tolerated in animal and human studies. It has been reported

that the toxicity of intravenously-administered liposomes

composed of DPPC is so low that accurate assessment of an

LD50 value is difficult, and has been estimated to be of the

order of 10 g/kg in mice (Storm et al., 1991). Intravenous

administration of DPPC (5–50 mg of lipid) did not induce

immediate or delayed toxicity in mice and did not produce

any changes in body weight and weight of major organs 2

weeks after administration. Results from our recent study

demonstrated that intravenous administration of liposomes

prepared from DPPC at 300 mg/kg b.wt with or without the

presence of NAC or tocopherols did not cause any adverse

effects (Alipour et al., 2012). Results from the in vitro study

examining the NAC stability showed that LNAC with a- or

g-tocopherols had better stability in PBS buffer than plasma,

Table 11. Serum chemistry group summaries of males – 24 h post-dose.

Group means� SD (n¼ 2)

Parameters Unit EL (0 mg/kg) L-NAC (126 mg/kg) L-aT-NAC (117 mg/kg) L-gT-NAC (149 mg/kg) Normal ranges

A/G – 1.1� 0.1 1.1� 0.1 1.1� 0.1 0.9� 0.0 0.9–1.4ALB g/L 26� 0 26� 0 28� 1 26� 3 23–34GLOB g/L 25� 0 24� 1 26� 2 29� 4 21–29ALP m/L 194� 7 136� 27 132� 1 184� 58 69–176Bil(T) mM 1.9� 0.3 51.7� 0.0 2.4� 0.9 2.7� 0.4 0–5.5BUN mM 3.7� 0.1 4.3� 1.0 3.3� 0.5 2.4� 0.0 2.5–7.0Ca mM 2.68� 0.08 2.70� 0.06 2.56� 0.04 2.67� 0.23 2.54–2.92Cl mM 115� 1 113� 2 113� 2 117� 1 108–122Creatinine mM 53� 5 60� 5 50� 1 48� 6 44–73Glucose mM 5.0� 0.1 5.2� 0.1 5.2� 0.5 5.2� 0.6 4.6–6.5LDH m/L 490� 421 215� 60 590� 560 482� 194 30–749P mM 2.28� 0.02 2.15� 0.18 2.14� 0.15 2.04� 0.25 1.33–3.10K mM 4.2� 0.1 4.2� 0.1 4.6� 0.8 4.3� 0.2 4.0–5.5Protein (T) g/L 51� 0 50� 1 54� 1 55� 6 47–60AST m/L 83� 13 50� 3 54� 1 54� 17 21–57ALT m/L 105� 57 35� 4 60� 31 35� 8 22–63Na mM 140� 0 138� 0 140� 0 140� 2 139–152Triglycerides mM 0.34� 0.01 0.23� 0.01 0.26� 0.08 0.24� 0.06 0.10–0.55CK m/L 299� 83 580� 245 220� 178 198� 63 100–519Cholesterol mM 5.13� 0.37 5.39� 0.30 4.41� 0.62 6.04� 0.96 2.62–5.19GGT m/L 9� 3 7� 1 8� 2 7� 1 4–15

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

Table 12. Serum chemistry group summaries of males – day 15.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

A/G – 1.1� 0.1 1.0� 0.1 1.0� 0.2 1.1� 0.1 0.9–1.4ALB g/L 28� 0 27� 1 25� 2 26� 4 23–34GLOB g/L 26� 2 28� 2 28� 4 25� 1 21–29ALP m/L 177� 1 144� 18 123� 9 136� 23 69–176Bil(T) mM 51.7� 0.0 51.7� 0.0 51.7� 0.0 51.7� 0.0 0–5.5BUN mM 4.1� 1.2 4.7� 0.6 3.6� 0.1 3.7� 0.8 2.5–7.0Ca mM 2.67� 0.04 2.73� 0.01 2.75� 0.01 2.75� 0.12 2.54–2.92Cl mM 113� 1 113� 0 114� 0 116� 1 108–122Creatinine mM 52� 1 62� 2 55� 1 57� 2 44–73Glucose mM 4.8� 0.0 4.7� 0.1 4.8� 0.3 4.9� 0.1 4.6–6.5LDH m/L 304� 54 526� 79 265� 22 355� 284 30–749P mM 2.22� 0.08 2.26� 0.23 2.23� 0.01 2.30� 0.13 1.33–3.10K mM 4.6� 0.0 5.1� 0.1 4.8� 0.1 4.6� 0.1 4.0–5.5Protein (T) g/L 53� 1 54� 1 52� 1 51� 5 47–60AST m/L 50� 4 49� 6 40� 4 42� 1 21–57ALT m/L 36� 1 24� 2 24� 14 27� 4 22–63Na mM 137� 1 138� 1 140� 0 139� 1 139–152Triglycerides mM 0.22� 0.13 0.13� 0.01 0.14� 0.04 0.18� 0.08 0.10–0.55CK m/L 340� 125 336� 204 191� 40 260� 35 100–519Cholesterol mM 3.89� 0.10 4.35� 0.00 3.51� 0.35 4.25� 0.26 2.62–5.19

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

DOI: 10.3109/15376516.2013.774079 Safety studies for liposomal antioxidant formulations 9

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but the differences were not significant. Inclusion of a- or

g-tocopherol in liposomes improved the retention of NAC

under all conditions examined to the same extent (Figure 1).

Similarly, data from the pharmacokinetic study in dogs

confirmed that the levels of NAC measured in plasma were

higher in the groups of animals treated with liposomes

containing a-tocopherol or g-tocopherol (Figure 2).

a-Tocopherol alters the membrane characteristics of lipo-

somes by making them more stable and less permeable to

aqueous solutes and highly resistant to protein-induced

disruption (Urano et al., 1990). It has been reported that the

suppression of protein-induced disruption is more pronounced

with a-tocopherol than with cholesterol (used in liposomal

preparation to increase the physical stability of liposomes

particularly in the presence of biological fluids such as

plasma), even at lower molar ratios. Also, a-tocopherol in

liposomes can undergo spontaneous intermembrane transfer

to an acceptor membrane without the fusion of the with

Table 13. Serum chemistry group summaries of females – 24 h post-dose.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

A/G – 1.1� 0.0 1.1� 0.1 1.1� 0.1 1.2� 0.0 0.7–1.5ALB g/L 26� 1 26� 4 26� 0 29� 3 21–33GLOB g/L 24� 0 24� 0 25� 4 25� 2 19–32ALP m/L 151� 50 125� 28 130� 21 157� 13 20–248Bil(T) mM 2.2� 0.3 51.7� 0.0 51.7� 0.0 4.0� 1.7 0–6.8BUN mM 4.0� 0.3 4.2� 0.5 4.4� 0.2 3.5� 0.2 2.1–7.7Ca mM 2.65� 0.05 2.68� 0.01 2.65� 0.01 2.67� 0.08 2.54–2.92Cl mM 115� 1 118� 1 114� 1 112� 0 108–121Creatinine mM 57� 3 54� 2 58� 0 49� 3 42–76Glucose mM 5.2� 0.1 5.3� 0.5 5.1� 0.2 5.3� 0.4 4.3–6.7LDH m/L 360� 69 250� 4 325� 90 254� 125 30–675P mM 2.06� 0.04 2.10� 0.04 2.00� 0.06 1.90� 0.12 1.37–3.08K mM 4.0� 0.1 4.2� 0.4 4.2� 0.1 4.3� 0.1 3.9–5.4Protein (T) g/L 50� 0 50� 4 51� 3 54� 5 29–72AST m/L 77� 5 54� 8 46� 1 74� 44 23–55ALT m/L 52� 6 45� 8 28� 1 225� 274 25–56Na mM 141� 1 140� 2 142� 2 140� 2 140–150Triglycerides mM 0.33� 0.04 0.22� 0.02 0.34� 0.11 0.17� 0.00 0.04–0.56CK m/L 413� 120 399� 179 344� 45 200� 91 100–489Cholesterol mM 3.90� 0.58 4.45� 0.51 3.87� 0.42 4.60� 0.33 2.39–5.19GGT m/L 7� 0 5� 0 6� 1 10� 3 3–9

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

Table 14. Serum chemistry group summaries of females – day 15.

Group means� SD (n¼ 2)

Parameters Unit EL L-NAC L-aT-NAC L-gT-NAC Normal ranges

A/G – 1.2� 0.1 1.3� 0.0 1.1� 0.1 1.1� 0.0 0.7–1.5ALB g/L 29� 1 29� 3 25� 2 27� 3 21–33GLOB g/L 25� 1 23� 2 23� 0 25� 1 19–32ALP m/L 153� 42 122� 33 95� 3 123� 6 20–248Bil(T) mM 2.2� 0.6 51.7� 0.0 51.7� 0.0 51.7� 0.0 0–6.8BUN mM 4.5� 0.7 4.9� 0.5 4.8� 1.4 4.2� 0.6 2.1–7.7Ca mM 2.68� 0.02 2.62� 0.02 2.61� 0.18 2.71� 0.06 2.54–2.92Cl mM 115� 2 116� 1 117� 4 115� 3 108–121Creatinine mM 57� 2 49� 6 53� 1 54� 1 42–76Glucose mM 4.7� 0.1 4.7� 1.2 4.8� 0.1 5.2� 0.6 4.3–6.7LDH m/L 642� 60 240� 175 184� 47 155� 25 30–675P mM 2.18� 0.15 2.27� 0.00 1.96� 0.01 2.04� 0.01 1.37–3.08K mM 4.8� 0.1 5.1� 0.6 4.3� 0.1 4.5� 0.1 3.9–5.4Protein (T) g/L 53� 0 51� 6 48� 1 53� 5 29–72AST m/L 60� 13 42� 3 40� 1 44� 1 23–55ALT m/L 36� 7 20� 1 26� 6 35� 12 25–56Na mM 137� 0 138� 1 140� 1 140� 2 140–150Triglycerides mM 0.15� 0.04 0.16� 0.04 0.12� 0.03 0.12� 0.01 0.04–0.56CK m/L 518� 285 226� 1 274� 139 188� 46 100–489Cholesterol mM 2.90� 0.52 3.59� 0.15 2.78� 0.26 3.27� 0.23 2.39–5.19

The NAC dose intravenously administered to each animal was 60 mg/kg. EL¼ empty DPPC liposomes; L-NAC¼DPPC liposome-entrappedN-acetylcysteine; L-aT-NAC¼DPPC liposome-entrapped a-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrappedg-tocopherolþN-acetylcysteine.

10 M. Alipour et al. Toxicol Mech Methods, Early Online: 1–13

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a-tocopherol liposomes (Verdon & Blumberg, 1988). Thus,

liposomes containing a-tocopherol or g-tocopherol may be

useful for delivering physiological quantities of this vitamin

component or other drugs to cells in culture or to tissues

in vivo.

The results of the range finding study showed that the dose

levels of liposomal antioxidants considered to be the maximum

feasible dose (MFD) levels, based on the physical properties/

viscosity of the test articles that could be safely administered to

dogs by an intravenous injection to be (i) Empty liposomes

(600 mg/kg DPPC); (ii) L-NAC (600 mg/kg DPPC, 200 mg/kg

NAC); (iii) L-aT/NAC (600 mg/kg DPPC, 200 mg/kg NAC,

83.3 mg/kg a-T) and (iv) L-gT/NAC (600 mg/kg DPPC,

200 mg/kg NAC, 71.4 mg/kg g-T). However, the volume was

reduced since some dogs experienced emesis and hypotension.

A decrease in the injectable volume and rate of administration

alleviated the side effect but it resulted in a decrease in the

amount of antioxidants delivered to the animals. It is not

unusual for intravenously administered liposomes or liposomal

formulations to cause infusion-related adverse reactions such

as shortness of breath, hypotension and vomiting, perhaps due

to complement activation (Chanan-Khan et al., 2003; Szebeni

et al., 1999, 2007). For example, some patients treated with

DOXIL� (doxorubicin HCl liposome injection, Janssen

Biotech Inc., Horsham, PA) experience infusion-related

adverse reactions that resolve over the course of several

hours to a day once the infusion is terminated. In some patients,

the reaction resolves with slowing of the infusion rate or

reducing the dose (Alberts & Garcia, 1997; Chanan-Khan

et al., 2003; Lyass et al., 2000; Riccardi et al., 2006; Udhrain

et al., 2007).

Reductions of doses to 70% of MFD (i.e. 60, 25 or

25 mg/kg body weight NAC, a-T, or g-T, respectively) levels

were well tolerated by the animals and were without any gross

and histopathological findings. Also, the unaffected hemato-

logical, biochemical and urinalysis parameters by the treat-

ments are evidence to indicate that the liposomal antioxidant

Figure 2. Plasma concentration-time profiles of NAC in beagle dogs. Adose of (A) L-NAC alone (60 mg/kg of NAC), (B) L-aT-NAC (60 mg/kgNAC and 25 mg/kg aT and (C) L-gT-NAC (60 mg/kg NAC and 25 mg/kggT) was intravenously administered to male (n¼ 2) and female (n¼ 2)dogs. The data is presented as the mean� SD of four dogs.

Table 16. Estimated pharmacokinetic parameters.

PK parameter Units L-NAC L-aT-NAC L-gT-NAC

AUC mg h/mL 25.1� 6.3 55.9� 10.2* 64.1� 7.2*AUMC mg h2/mL 41.6� 16.7 221.3� 103.6* 239.8� 76.2*Cmax mg/mL 26.6� 2.1 22.8� 1.8 21.0� 2.0A mg/mL 13.7� 0.3 9.7� 2.5 8.1� 0.8B mg/mL 11.7� 3.8 13.8� 2.7 14.6� 0.6K12 h�1 2.62� 1.55 0.84� 0.26 0.70� 0.08K21 h�1 3.36� 1.90 1.69� 0.65 1.68� 0.0K10 h�1 1.15� 0.36 0.42� 0.08* 0.33� 0.03*� half-life h 0.07� 0.04 0.27� 0.08 0.28� 0.01� half-life h 1.46� 0.73 4.10� 1.98* 3.12� 0.26K10-half life h 0.38� 0.34 1.72� 0.41* 2.11� 0.19*MRT h 1.64� 0.54 3.81� 1.07* 3.68� 0.76*V1 L/kg 1.5� 1.3 2.7� 0.2 2.7� 0.1V2 L/kg 2.6� 0.5 1.4� 0.5* 1.1� 0.1Vss L/kg 4.1� 1.4 4.0� 0.5 3.46� 0.4CL L/h/kg 2.5� 0.7 1.1� 0.1* 1.0� 0.1CL2 L/h/kg 8.2� 4.3 2.2� 0.6* 1.9� 0.2

Estimated pharmacokinetic parameters for NAC based on the averageplasma concentrations from animals intravenously administeredL-NAC at a dose of 60 mg/kg NAC alone or in the presence of25 mg/kg a- or g-tocopherols (n¼ 4). L-NAC¼DPPC liposome-entrapped N-acetylcysteine; L-aT-NAC¼DPPC liposome-entrappeda-tocopherolþN-acetylcysteine; L-gT-NAC¼DPPC liposome-entrapped g-tocopherolþN-acetylcysteine. PK parameters wereobtained by fitting the plasma concentration data to a two compartmentmodel.

*Significantly different from L-NAC, p50.05, ANOVA with theStudent–Newman–Keuls post hoc analysis

Table 15. AIC for fitting plasma concentration time data to one (1) andtwo (2) compartment pharmacokinetic models.

Dog

Compartment 1 2 3 4Mean plasma concentration

time data

L-NAC1 27.73 21.34 25.87 21.08 23.192 15.46 7.28 12.86 6.22 8.96L-aT-NAC1 27.49 31.02 19.27 23.85 25.872 17.79 17.10 4.9 14.26 7.49L-cT-NAC1 25.48 21.53 20.50 25.81 21.632 19.24 c.n.d. c.n.d. 16.84 15.00

c.n.d. – could not be determined.

DOI: 10.3109/15376516.2013.774079 Safety studies for liposomal antioxidant formulations 11

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formulations did not cause any treatment-related toxicity.

Although the liposomal antioxidant doses that are well

tolerated and hence can be used clinically safely are

significantly lower than the MFD levels, they still are

significantly higher than those reported in studies examining

the antioxidant effects of liposomal antioxidant formulations.

For example, the dose of NAC delivered as a liposomal

formulation against the ricin toxin A-, lipopolysaccharide

(LPS)-, shock- or 2-chloroethylethyl sulfide (CEES)-induced

injuries did not exceed the level of 25 mg/kg body weight

(Alipour et al., 2007; Buonocore et al., 2011; Fan et al., 2000;

McClintock et al., 2006; Mitsopoulos et al., 2008; Mukherjee

et al., 2009). Similarly, liposomal tocopherol doses used to

ameliorate the tissue injuries induced following ischemia/

reperfusion or the administration of LPS, acetaminophen,

phorbol myristate acetate were not higher than 8 mg/kg body

weight (Sinha et al., 2001; Suntres & Shek, 1995, 1996;

Werner & Wendel, 1990).

Inclusion of tocopherols in the liposomal formulation

significantly increased the amount of NAC in the blood as

demonstrated by the 2.2-fold and 2.6-fold increase of AUC for

NAC for liposomes containing a- and g-tocopherol, respect-

ively. The tocopherols also increased the b-half life of NAC

with a greater impact on the clearance of NAC, where

reductions of central compartment clearance (CL) ranged

from 56% to 60% and reductions of tissue clearance (CL2)

ranged from 73% to 77%. Thus, the increased plasma AUC for

NAC in the presence of the tocopherols is largely due to a

decreased clearance of NAC. This is consistent with data

reported in previous studies where inclusion of tocopherols in

liposomes increases the stability of liposomes and decreases

solute leakage and may enhance the duration of NAC’s

antioxidant effect (Suntres & Shek, 1994).

In conclusion, analysis of all generated data, including

clinical observations, clinical pathology, gross pathology and

histopathology revealed no drug/treatment related toxicity in

dogs at the maximum feasible dose level, by a single bolus

intravenous administration. Caution should be used on the

rate of administration.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

This work was supported by a grant (W81XWH-06-2-

0044) from the Department of Defence, USA.

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