AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).

Acute and Subchronic (28-Day) Oral Toxicity Study in Rats Fed with Novel
Surfactants
Submitted: July 8, 2003; Accepted: November 17, 2003; Published: April 7, 2004

Ranjit Madhukar Bidhe1 and Sangita Ghosh2
1

Indian Institute of Toxicology, KIM 2057, Sadashiv Peth, Vijaynagar Colony, Pune, India 411 030
BioZone Laboratories Inc, Pittsburg, CA 94565

2

dress the biologic stability problems associated with theconventional liposomes in vivo. To our knowledge, nosuch modified PEG-lipid has been reported for oral use.
The oral route of drug administration is generally preferred because of its versatility, safety, and relative patient comfort. Hence, there is an outstanding need forlipids that form liposomes, are stable for prolonged shelflife, and yet have no toxicity when administered orally.

ABSTRACTThe toxicity of 2 new synthetic lipids, 1,2-dioleoyl-racglycerol-3-dodecaethylene glycol, GDO-12 (lipid 1)
and 1,2-distearoyl-rac-glycerol-3-dodecaethylene glycol, GDS-12 (lipid 2) has been evaluated in acute andsubchronic toxicity studies. Acute oral toxicity studies
in male and female rats documented no deaths or treatment-related signs at high doses. The lipids wereindividually administered (by gavage) to male and female Sprague-Dawley rats at concentrations of 250,
500, and 1000 mg/Kg bodyweight for 28 days. All
animals survived the duration of the study, with no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or
histopathological findings. These studies establish thatboth GDO-12 (lipid 1) and GDS-12 (lipid 2) are nontoxic in rats following oral administration. The noobserved-adverse-effect level ranged between 250
mg/Kg and 1000 mg/Kg following oral administration.

We have developed 2 synthetic PEG-lipid conjugates foruse as a drug carrier and solubilizer. These lipids havethe general structure shown in Figure 1 and have a PEG12 head group and long hydrocarbon chains (oleoyl for 1
and stearoyl for 2). Liposomes formed spontaneously
upon hydration of 2 new lipids.

KEYWORDS: oral toxicity, rats, polyoxyethylene glycol (PEG), liposomes
INTRODUCTION
Liposomes have received much attention for their usefulness in reducing toxicity and improving therapeutic
effectiveness.1 Although liposomes are a very promising, broadly applicable, and highly researched novel
delivery system, they suffer from serious stability problems.2 Phenomena associated with the aqueous suspension of liposomes such as aggregation, fusion, and phospholipid hydrolysis limit their shelf life. Polyoxyethylene glycol-lipid (PEG-lipid) conjugates have been reported, although in injectable formulations3-7 that ad-

Figure 1. Chemical structure of 2 synthetic lipids: (A)PEG-12 glyceryl dioleate, GDO-12 (lipid 1), and (B)PEG-12 glyceryl distearate, GDS-12 (lipid 2).
However, no evaluation has been made so far regarding
the oral toxicity of any PEGylated lipid conjugate.
Safety evaluation would be more pertinent in view of thedifferences of the PEG head group of these types of lipids with respect to conventional phospholipids.

Corresponding Author: Sangita Ghosh, BioZone
Laboratories Inc, Pittsburg, CA 94565.
Tel: (925) 473-1000. Fax: (925) 473-1001.
Email: sghosh@biozonelabs.com.

1

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
The joint expert committee on food additive’s8 typicalcriteria for safety evaluation include physical appearance
and behavior, growth and body weight gain, food consumption, and absorption. In addition, the recommended
criteria include the evaluation of utilization of other nutrients such as nitrogen, fat, calcium, and phosphorus.
Digestibility and bioassay procedures for evaluation of
feed utilization, plasma hematology and blood chemistry, gross pathological examination on necroscopy, organ weight, and histopathology are also recommended.

with a bedding of husk, and 12-hour light/dark cycles
were provided. Feed and water were given ad lib. Environmental conditions were maintained at a temperatureof 22°C ± 2°C and a relative humidity of 60% ± 10%.

Preparation of Test Diets
GDO-12 (lipid 1) and GDS-12 (lipid 2) were diluted
with corn oil and administered to the rats at doses of 250
mg/kg, 500 mg/kg, and 1000 mg/kg, in the volume of 5
mL/kg. These test diets were freshly prepared everyday
for 28 days. The control animals were administered vehicle only.

A rigorous safety evaluation of these lipids, GDO-12(lipid 1) and GDS-12 (lipid 2) would enable their use as
liposomal carriers of drugs and nutritional supplements
in oral formulations. Therefore, we conducted a shortterm subchronic toxicity study in rats by feeding both
lipids individually at various levels for a period of 28days to assess the oral safety aspect of these PEGylated
lipids.

Experimental Design and Conduct
Forty healthy rats, 20 male and 20 female, were acclimatized to laboratory conditions for 7 days prior to initiation of dosing. They were randomly assigned to cages
and the individual animal was fur marked with picricacid. The females were nulliparous and nonpregnant.
Rats were assigned to treatment groups of 5 males and 5
females. The rats were deprived of feed for 16 hours
before and 3 hours after administration of the test substance. The test substance, diluted with corn oil, wasadministered by gavage to rats of both sexes using aball-tipped intubation needle fitted onto a syringe.

MATERIALS AND METHODS
Test Material
The lipids used in this work were 1,2-dioleoyl-racglycerol-3-dodecaethylene glycol (1) and 1,2-distearoylrac-glycerol-3-dodecaethylene glycol (2), synthesized
by BioZone Laboratories (Pittsburg, CA). They havebeen abbreviated as GDO-12 (lipid 1) and GDS-12(lipid 2) respectively. GDO-12 (lipid 1) was a liquid atroom temperature (RT) while GDS-12 (lipid 2) was a
waxy solid at RT. One mole of glycerol was reactedwith 2 moles of oleic acid at 150°C under nitrogen to
give glyceryl dioleate. Complete consumption of starting
material as followed by liquid chromatography-mass
spectrometry (LC-MS) indicated quantitative conversion. Glyceryl dioleate was then alkylated by passing
ethylene oxide gas for 8 hours using sodium methylate
as catalyst under nitrogen at 150°C until LC-MS analysis showed complete consumption of starting material.
Completion of reaction was also monitored by measuring acid value and saponification value to give final
product GDO-12 (lipid 1). For GDO-12 (lipid 1),
saponification value was 105.7 and acid value was
0.001. Synthesis of GDS-12 (lipid 2) was similar to thatof GDO-12 and stearic acid was used instead of oleic
acid. For GDS-12 (lipid 2), saponification value was104.7 and acid value was 1.0. The yields of the 2 lipids
were quantitative.

Observations of pharmacotoxic signs were made at 10,30, 60, and 120 minutes and at 4 and 6 hours after dosing during the first day and daily thereafter for 28 days.
The time of onset, intensity, and duration of these symptoms, if any, were recorded. All animals were observed
twice daily for mortality during the 28-day period ofstudy. The weight of each rat was recorded on day 0 and
at weekly intervals throughout the course of the study.
The group mean body weights were calculated. The
quantity of food consumed by groups consisting of 5 rats
each was recorded weekly, and the food consumption
per rat was calculated for control and dose groups. Theeyes of control and animals from different groups were
examined prior to the initiation of the dosing in week 4of the study period on day 28. Eye examination was carried out using a hand slit lamp after the induction of mydriasis with 0.5% solution of tropic amide sulfate.
All reagents used were of analytical grade. At the end of
the 28-day period the animals were fasted overnight.
The following morning, each animal was heparinizedand blood samples were collected from the orbital sinus.
The hematological parameters hemoglobin concentration (Hb),9 mean corpuscular volume (MCV), totalerythrocyte count (RBC), reticulocyte concentration(Rt), mean corpuscular hemoglobin (MCH), hematocrit

Animals
Sprague-Dawley rats (aged 6-8 weeks) were in polycarbonate cages. Each cage contained 5 rats of the same sex

2

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
(HCT), and total and differential leucocyte count were
determined by standard methods using Serono 9110
automated hematology analyzer (Serono-Baker Diagnostics Inc, Allentown, PA. Plasma concentrations of
glucose, total protein, albumin and blood urea nitrogen,sodium, and potassium were analyzed using a Boehringer Knoll autoanalyzer 4010 system (Boehringer
GmbH, Mannheim, Germany). The plasma activities ofserum alkaline phosphatase (SAP), serum glutamic pyruvic transaminase (SGPT), and serum glutamic oxaloacetic transaminase (SGOT) were also assayed using
Boehringer Knoll autoanalyzer 4010 system. Urine
samples were also collected at the end of the study period and analyzed for pH, glucose, proteins, ketones, andoccult blood. The rats were humanely killed on day 29
using CO2 asphyxiation technique. Necroscopy of all
animals was carried out and the weights of liver, kidneys, adrenals, and testes were recorded. The organ
weights were recorded as absolute values and their relative values (ie, percentage of the body weight) were calculated.

Figure 2. Growth curve of male rats fed GDO-12(lipid 1).

Feed conversion efficiency percentage was calculated asfollows:
Feed Conversion Efficiency (%) =Weekly Body Weight Gain ( g )× 100
Weekly Food Consumptio n ( g )(1)Histopathological observations were carried out in control and animals treated at the highest dose level of 1000
mg/kg. Tissue samples were preserved in 10% neutral
buffered formalin.
Statistical analysis was done using Bartlett’s homogeneity test, analysis of variance (ANOVA) and Dunnett test.
The Student t test was employed to compare the statistical significance between control and experimentalgroups.

Figure 3. Growth curve of female rats fed GDO-12(lipid 1).

Hematological parameters, hemoglobin concentration,
total and differential erythrocyte count, total and differential leucocyte count, hematocrit, and mean cell hemoglobin concentration, in both control and experimental
rats, were not significantly different (P > .05) for both
lipids 1 and 2 (Tables 3 and 4). All values were found to
be within the normal range for rats,10 and there were nodifferences between the groups.

RESULTS
It was observed that the animals fed with the novel lipids
GDO-12 (lipid 1) and GDS-12 (lipid 2) were healthy.
No unusual changes in behavior or in locomotor activity,
no ataxia, and no signs of intoxication were observed
during the 28-day period. No differences were found ingrowth between the control group and the animals fed
with different levels of the alkoxylated diesters GDO-12(lipid 1) and GDS-12 (lipid 2) (Figures 2, Figure 3,
Figure 4 & Figure 5). The food consumption (and therefore the feed conversion efficiency) of male and female
rats of control and experimental groups was similar, indicating that the feed intake and utilization was not affected (Tables 1 and 2).

The levels of plasma analytes, such as total protein,blood urea nitrogen (BUN), glucose, bilirubin, albumin,creatine, cholesterol, chloride, calcium, phosphorus, sodium, and potassium ions were not significantly different between the control and the experimental groups ofrats (P > .05) when fed with lipids 1 and 2 (Tables 5 and
6). Analysis of the urinary metabolite levels (glucose,
proteins, hemoglobin, and ketones) showed trace or no3

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
Table 1. Mean Food Consumption of the Animals During the Study Period (g/Animal)With GDO-12 (lipid 1)
Male
Control
250 mg/kg
500 mg/kg
1000 mg/kg
FemaleControl
250 mg/kg
500 mg/kg
1000 mg/kg

Day 0
10.42
10.74
10.04
10.58
Day 0
8.76
8.68
7.72
9.30

Day 7
12.60
13.12
11.92
12.16
Day 7
11.26
12.16
10.68
10.92

Day 14
14.80
15.16
14.22
14.54
Day 14
13.66
13.92
13.02
13.38

Day 21
16.14
15.92
15.08
15.48
Day 21
14.62
14.80
14.18
14.30

Day 28
17.76
18.08
17.00
17.58
Day 28
16.76
17.20
15.98
16.70

Table 2. Mean Food Consumption of the Animals During the Study Period (g/Animal)With GDS-12 (lipid 2)
Male
Control
250 mg/kg
500 mg/kg
1000 mg/kg
FemaleControl
250 mg/kg
500 mg/kg
1000 mg/kg

Day 0
10.42
10.48
9.72
10.20
Day 0
8.76
8.78
8.12
9.00

Day 7
12.60
12.90
12.28
12.44
Day 7
11.26
11.78
11.12
11.24

Day 14
14.80
15.02
14.36
14.64
Day 14
13.66
13.82
13.26
13.52

Day 21
16.14
15.96
15.36
15.60
Day 21
14.62
14.82
14.58
14.76

Day 28
17.76
17.84
17.04
17.56
Day 28
16.76
16.80
16.24
16.58

Figures 6 and 7 depict the organ-to-body mass ratios ofanimals at the end of 28 days’ feeding. No abnormalchanges were observed in organ mass with respect tobody mass of GDO-12 (lipid 1) and GDS-12 (lipid 2)
fed rats in comparison with controls. Observations ofgross pathology immediately after dissection, on rats ofall groups were found to be uniformly healthy, lacking
in any apparent pathological abnormalities.

presence of these in both control and experimental animals fed with lipids 1 and 2.

Figure 4. Growth curve of male rats fed GDS-12 (lipid2). Note: Possible changes (slight reduction) in locomotor activity were observed in 2 of the 5 male rats at dosesof 1000 mg/kg after 20 days. Hence, the growth curve atconcentration of 1000 mg/kg has been plotted to day 20.
Tables 7 and 8 detail the activities of enzymes analyzed
in plasma. No significant differences were observed inenzyme activities between the control and lipid-fed animals.

Figure 5. Growth curve of female rats fed GDS-12(lipid 2).
Histopathological examination of the liver and kidneys
in the control and the GDO-12 (lipid 1) and GDS-12

4

Dose
mg/kg

Hb
(g%)

RBC
(106/mm3)

Rt
(%)

HCT
(%)

MCV (µm3)

MCH
(pg)

7.55 ± 0.6
7.42 ± 0.4
7.5 ± 0.3
7.6 ± 0.3
7.4 ± 0.3
7.6 ± 0.4
7.5 ± 0.3
7.6 ± 0.3

14.5 ± 0.5
14.4 ± 0.3
14.46 ± 0.4
14.48 ± 0.2
14.4 ± 0.3
14.5 ± 0.3
14.5 ± 0.3
14.4 ± 0.3

Control
250
500
1000
Control
250
500
1000

Male

1.34 ± 0.4
1.4 ± 0.3
1.46 ± 0.3
1.44 ± 0.3
1.4 ± 0.4
1.4 ± 0.4
1.4 ± 0.3
1.5 ± 0.4

Rt(%)
44.0 ± 1.7
43.54 ± 0.8
43.82 ± 0.8
43.9 ± 0.8
43.7 ± 0.8
44.1 ± 1.0
43.9 ± 0.8
43.7 ± 0.9

HCT’
(%)
58.7 ± 2.4
58.7 ± 1.6
58.4 ± 1.3
57.8 ± 1.4
58.8 ± 1.6
58.3 ± 1.6
58.4 ± 1.1
58.6 ± 1.2

MCV
(µm3)
19.22 ± 0.85
19.38 ± 0.4
19.26 ± 0.3
19.04 ± 0.5
19.4 ± 0.4
19.2 ± 0.5
19.1 ± 0.5
19.4 ± 0.5

MCH
(pg)
32.9 ± 0.2
33.04 ± 0.3
33.02 ± 0.4
32.98 ± 0.2
32.9 ± 0.3
32.9 ± 0.2
32.9 ± 0.1
32.9 ± 0.2

MCHC
(%)

MCHC (%)

3.4 ± 0.2
3.4 ± 025
3.3 ± 0.2
3.48 ± 0.2
3.4 ± 0.3
3.4 ± 0.2
3.5 ± 0.3
3.4 ± 0.3

Platelets(105/mm3)

Platelets(105/mm3)

Total
Leucocytes(103/ mm3)
9.06 ± 1.2
9.32 ± 1.7
9.86 ± 2.2
9.08 ± 1.7
9.3 ± 1.8
9.4 ± 1.99.8 ± 1.99.4 ± 2.4

21.8
21.2
21.8
21.4
21
22
21
21

N%

74.8
75.8
75.0
75.4
75
76
75
76

L%

1
0.8
0.8
1
1
0.8
1
0.6

E%

2.4
2.2
2.4
2.2
2.4
2.2
2.0
2.2

M%

Control
250
500
1000
Control
250
500
1000

Male

Control
250
500
1000
Control
250
500
1000

Male

11.52 ± 0.9
11.17 ± 1.4
10.68 ± 1.3
11.55 ± 1.5
11.24 ± 1.2
11.1 ± 1.3
11.3 ± 1.3
11.15 ± 0.9
Chloride
mmol/L
102.66 ± 1.7
103.5 ± 1.5
103.08 ± 1.4
102.34 ± 2.9
103.28 ± 1.2
103.36 ± 1.3
103.98 ± 1.4
103.1 ± 2.3

Potassium
mmol/L
3.84 ± 0.1
3.64 ± 0.5
3.48 ± 0.4
3.7 ± 0.5
3.58 ± 0.3
3.57 ± 0.5
3.61 ± 0.4
3.71 ± 0.4

BUN (mg%)

Total Serum Protein(g%)
6.37 ± 0.2
6.35 ± 0.2
6.42 ± 0.3
6.44 ± 0.3
6.31 ± 0.2
6.48 ± 0.2
6.32 ± 0.2
6.58 ± 0.2*BUN indicates blood urea nitrogen.

Female

Dose mg/kg

Sex

Female

Dose mg/kg

Sex

Cholesterol(mg%)
62.68 ± 7.7
61.62 ± 3.2
60.0 ± 5.4
61.5 ± 5.6
61.08 ± 2.4
63.3 ± 4.7
61.12 ± 3.0
64.02 ± 7.7

Blood Sugar(mg%)
78.78 ± 3.8
80.8 ± 5.4
79.86 ± 1.9
78.78 ± 2.9
79.38 ± 2.5
79.58 ± 3.9
80.46 ± 3.9
79.72 ± 2.55

Calcium(mg%)
9.45 ± 0.2
9.4 ± 0.3
9.5 ± 0.3
9.47 ± 0.3
9.47 ± 0.2
9.34 ± 0.4
9.45 ± 0.3
9.46 ± 0.2

Phosphorus(mg%)
4.04 ± 0.3
4.0 ± 0.1
3.98 ± 0.3
3.92 ± 0.1
4.02 ± 0.3
3.98 ± 0.2
4.01 ± 0.2
3.95 ± 0.1

Bilirubin(mg%)
0.59 ± 0.09
0.63 ± 0.1
0.65 ± 0.1
0.62 ± 0.07
0.65 ± 0.07
0.58 ± 0.08
0.62 ± 0.12
0.66 ± 0.08

Albumin(mg%)
3.41 ± 0.2
3.42 ± 0.2
3.38 ± 0.2
3.31 ± 0.2
3.41 ± 0.2
3.43 ± 0.19
3.36 ± 0.29
3.37 ± 0.23

Creatine(mg%)
0.95 ± 0.04
0.95 ± 0.03
0.94 ± 0.02
0.95 ± 0.04
0.95 ± 0.02
0.95 ± 0.04
0.95 ± 0.03
0.96 ± 0.03

Sodiummmol/L139.38 ± 1.4
138.94 ± 2.2
138.16 ± 2.1
136.54 ± 5.3
139.2 ± 1.8
138.2 ± 1.8
138.68 ± 1.45
138.22 ± 3.9

Table 5. Plasma Biochemical, Na+, K+, Ca2+, and Cl− Profile of the Control and Experimental Groups Fed With GDO-12 (lipid 1) for 28 Days**Hb indicates hemoglobin; RBC, red blood cells; Rt, reticulocyte; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; N, neutrophils; L, lymphocytes; E, eosinophils; and M, monocytes. All values are expressed as mean ± SD.

FemaleRBC(106/mm3)

Hb
(g%)

Dose
mg/kg

Sex

Table 4. Hematological Data of Rats Fed With GDS-12 (lipid 2) for 28 Days*

Sex

Total
N%
L%
E%
M%
Leucocytes(103/mm3)
Male
Control
21.8
74.8
1
2.4
14.5 ± 0.5
7.55 ± 0.6
1.34 ± 0.4
44.06 ± 1.7
58.46 ± 2.4
19.22 ± .85
32.92 ± 0.2
3.4 ± 0.2
9.06 ± 1.2
250
21.2
75.6
1.2
2.4
14.46 ± 0.3
7.51 ± 0.3
1.5 ± 0.4
43.9 ± 0.9
58.52 ± 1.3
19.22 ± 0.4
32.94 ± 0.2
3.42 ± 0.5
9.2 ± 1.9
22.6
0.6
2.2
500
74.6
14.44 ± 0.4
7.52 ± 0.45
1.5 ± 0.3
43.62 ± 0.8
58.08 ± 2.4
19.18 ± .63
33.06 ± 0.4
3.5 ± 0.2
9.9 ± 2.1
1000
75.2
21.8
1.2
1.8
7.57 ± 0.3
1.52 ± 0.3
44.02 ± 1
58.18 ± 1.6
19.22± 0.37
33.02 ± 0.3
3.4 ± 0.3
10.0 ± 1.9
14.54 ± 0.3
Female21.2
2.4
Control
75.4
1
14.4 ± 0.3
7.44 ± 0.3
1.36 ± 0.4
43.68 ± 0.8
58.78 ± 1.6
19.38 ± 0.4
32.96 ± 0.3
3.35 ± 0.3
9.32 ± 1.8
250
22.4
75.8
0.8
2.2
14.5 ± 0.3
7.56 ± 0.4
1.42 ± 0.4
44.08 ± 1.0
58.34 ± 1.6
19.18 ± 0.5
32.86 ± 0.2
3.38 ± 0.2
9.36 ± 1.9
500
21.4
75.4
1
2.2
14.48 ± 0.3
7.52 ± 0.3
1.4 ± 0.3
43.92 ± 0.8
58.38 ± 1.1
19.14 ± 0.5
32.96 ± 0.2
3.5 ± 0.3
9.76 ± 1.9
21.0
0.6
2.4
1000
76.0
14.42 ± 0.3
7.58 ± 0.3
1.48 ± 0.3
43.78 ± 0.9
58.58 ± 1.2
19.38 ± 0.5
32.94 ± 0.2
3.37 ± 0.3
9.42 ± 2.4*Hb indicates hemoglobin; RBC, red blood cells; Rt, reticulocyte; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; N, neutrophils; L, lymphocytes; E, eosinophils; and M, monocytes. All values are expressed as mean ± SD.

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
Table 3. Hematological Data of Rats Fed with GDO-12 (lipid 1) for 28 Days*

Potassium
mmol/L
3.84 ± 0.1
3.87 ± 0.2
3.63 ± 0.3
3.63 ± 0.4
3.58 ± 0.3
3.75 ± 0.4
3.66 ± 0.4
3.52 ± 0.4

Dose
mg/kg
Control
250
500
1000
Control
250
500
1000

Chloride
mmol/L
102.66 ± 1.7
102.72 ± 1.3
102.66 ± 1.7
102.84 ± 1.9
103.28 ± 1.2
102.94 ± 2.0
102.6 ± 1.6
103.4 ± 2.0

BUN
(mg%)
11.52 ± 0.9
11.17 ± 1.8
11.35 ± 1.3
11.79 ± 1.7
11.24 ± 1.2
11.6 ± 1.2
11.48 ± 1.4
11.67 ± 1.2
Cholesterol(mg%)
62.68 ± 7.7
60.54 ± 5.5
61.76 ± 3.0
62.84 ± 2.0
61.08 ± 2.4
60.4 ± 7.9
61.34 ± 12.0
60.9 ± 5.8

Blood Sugar(mg%)
78.78 ± 3.8
81.12 ± 2.9
80.46 ± 5.5
79.77 ± 3.2
79.38 ± 2.5
79.8 ± 4.2
80.24 ± 3.6
79.08 ± 3.4

Calcium(mg%)
9.45 ± 0.2
9.3 ± 0.2
9.32 ± 0.3
9.36 ± 0.2
9.47 ± 0.2
9.43 ± 0.3
9.52 ± 0.3
9.34 ± 0.3

Phosphorus(mg%)
4.04 ± 0.3
4.07 ± 0.2
4.08 ± 0.2
3.91 ± 0.1
4.02 ± 0.3
4.0 ± 0.3
3.93 ± 0.1
3.98 ± 0.3

Bilirubin(mg%)
0.59 ± 0.09
0.64 ± 0.08
0.62 ± 0.1
0.65 ± 0.1
0.615 ± 0.07
0.61 ± 0.07
0.61 ± 0.09
0.61 ± 0.11

Albumin(mg%)
3.41 ± 0.2
3.39 ± 0.2
3.58 ± 0.1
3.31 ± 0.2
3.41 ± 0.2
3.55 ± 0.2
3.32 ± 0.3
3.36 ± 0.3

20.74 ± 2.05
20.42 ± 1.4
20.84 ± 1.3
20.44 ± 1.8
20.28 ± 2.4
20.84 ± 2.0
21.02 ± 2.00

19.98 ± 1.7
19.64 ± 1.5
19.86 ± 1.1
18.54 ± 1.3
19.32 ± 2.2
19.38 ± 1.5
19.92 ± 2.4

250
500
1000
Control
250
500
1000

24.32 ± 2.04

24.5 ± 2.2

24.2 ± 1.5

23.58 ± 2.4

24.22 ± 2.6

24.94 ± 1.6

23.66 ± 2.03

23.64 ± 1.6

SAP (IU/L)

14.6 ± 3.8

15.00 ± 3.54

13.4 ± 2.88

14.04 ± 3.05

15.4 ± 3.36

15.2 ± 4.09

14.8 ± 3.56

14.8 ± 3.9

ϒ GT (U/L)

6*SGPT indicates serum glutamic pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; SAP, serum alkaline phosphatase; and ϒ GT, gamma Glutamyl Transferase.

Female21.04 ± 1.5

19.10 ± 1.5

Control

Male

SGOT (IU/L)

SGPT (IU/L)

Dose mg/kg

Sex

Table 7. Enzyme Activities in Plasma of the Rats Fed With GDO-12 (lipid 1) for 28 Days*

Total Serum Protein(g%)
6.37 ± 0.2
6.44 ± 0.3
6.45 ± 0.3
6.43 ± 0.3
6.31 ± 0.2
6.32 ± 0.3
6.34 ± 0.2
6.37 ± 0.2

Dose
mg/kg
Control
250
500
1000
Control
250
500
1000

*BUN indicates blood urea nitrogen.

FemaleMale

Sex

FemaleMale

SexCreatine(mg%)
0.95 ± 0.04
0.94 ± 0.02
0.95 ± 0.03
0.95 ± 0.03
0.95 ± 0.02
0.95 ± 0.03
0.94 ± 0.03
0.94 ± 0.03

Sodiummmol/L139.38 ± 1.4
139.14 ± 3.5
138.3 ± 2.9
137.44 ± 3.3
139.2 ± 1.8
137.4 ± 4.7
138.74 ± 2.4
139.22 ± 3.9

Table 6. Plasma Biochemical, Na+, K+, Ca2+, and Cl− Profile of the Control and Experimental Groups Fed With GDS-12 (lipid 2) for 28 Days*

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).

AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
Table 8. Enzyme Activities in Plasma of the Rats Fed With GDS-12 (lipid 2) for 28 Days*
Sex

Dose mg/kg

SGPT (IU/L)

SGOT (IU/L)

SAP (IU/L)ϒ GT (U/L)

Control
19.10 ± 1.5
21.04 ± 1.5
23.64 ± 1.6
14.8 ± 3.9
25019.32 ± 1.5
20.7 ± 1.3
24.68 ± 2.4
13.4 ± 2.9
50019.92 ± 1.5
20.94 ± 1.75
24.08 ± 2.0
13.6 ± 3.21
1000
19.6 ± 2.1
21.14 ± 2.1
25.16 ± 1.9
13.2 ± 2.86
FemaleControl
18.54 ± 1.3
20.44 ± 1.8
20.44 ± 1.8
14.04 ± 3.05
250
19.92 ± 2.1
20.62 ± 1.96
20.62 ± 1.96
14.8 ± 3.7
50019.7 ± 2.4
20.8 ± 2.3
20.8 ± 2.3
14.8 ± 3.7
1000
19.54 ± 1.8
21.08 ± 1.5
21.08 ± 1.5
15.0 ± 3.5*SGPT indicates serum glutamic pyruvic transaminase; SGOT, serum glutamic oxaloacetic transaminase; SAP, serum alkaline phosphatase; and ϒ GT, gamma Glutamyl Transferase.
Male

Figure 6. (A) Percentage organ weight to body mass of male rats fed GDO-12 (lipid 1).
(B) Percentage organ weight to body mass of female rats fed GDO-12 (lipid 1).

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AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).

Figure 7. (A) Percentage organ weight to body mass of male rats fed GDS-12 (lipid 2).
(B) Percentage organ weight to body mass of female rats fed GDS-12 (lipid 2).

(lipid 2) fed groups showed no differences, indicatingthat feeding these synthetic lipids at these levels to therats did not result in any adverse toxicological effect onthese organs.

Liposome encapsulation of drug with GDO-12 (lipid 1)
and GDS-12 (lipid 2) does not require organic solventsand sonication methods. This characteristic increases thescope of using this drug delivery system for pharmaceutical use.

DISCUSSION

In structure, these lipids are similar to their phospholipid
analogs, which have been explored extensively as drugdelivery vehicles.11 However, for the first time, application of GDO-12 (lipid 1) and GDS-12 (lipid 2) as drugdelivery vehicles is being sought. (Formulations of drugs
incorporated in GDO-12 (lipid 1) or GDS-12 (lipid 2)
were made. A few examples follow. Example 1: Methotrexate (5% wt, Sigma, St Louis, MO) was added to
GDO-12 (lipid 1) (10% wt) and heated to 55°C and gen-The novel synthetic lipids used in this study are uniqueand different from conventional phospholipids. Unlike
phospholipids, GDO-12 (lipid 1) and GDS-12 (lipid 2)
lack a phosphate ester head group, and they are uncharged. In addition, these lipids have a polyoxyethylene
glycol head group. A unique feature of these lipids isthat they form vesicles spontaneously when hydrated.

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AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
glucose, protein, hemoglobin, bilirubin, or creatinine
indicating normal hepatocellular and nephrotic function.

tly mixed until dissolved. The resultant mixture was aclear solution. Deionized water (85% wt) was slowly
added to the solution and gently mixed for 5 minutes.
The resultant was an opaque yellow solution. The preparation was examined under a phase contrast microscopeand showed multilamellar liposomes. Example 2:
Weighed amounts of GDO-12 (lipid 1) (18 g), betamethasone dipropionate (50 mg), and cholesterol (100mg) were combined and heated to 50°C while mixing.
Uniphen-23 (LipoChemicals, Paterson, NJ) (1.5 mg) anddeionized water (81.85 g) were separately combined andthe mixture heated to 50°C. The 2 mixtures were commingled at 50°C while stirring gently. The final mixture
was cooled to room temperature. Examination by phase
contrast optical microscopy showed multilamellar liposomes.)

A review of the literature on the safety of PEGylated
lipids and other polymers suggests that they are safe foruse in parenterally administered pharmaceuticals.12
GDO-12 (lipid 1) and GDS-12 (lipid 2) are both PEGylated lipids that form spontaneous liposomes and havesuccessfully solubilized a variety of hydrophobic compounds like tetracaine, lidocaine, cholesterol, tretenoin,
bethamethasone propionate etc.13 In our efforts to determine the possible use of these lipids in oral formulations,
we have evaluated the acute and subchronic toxicity ofthese 2 lipids. Our findings indicate that these lipids arenontoxic and well tolerated for the 28-day study period
and therefore have potential for safe use in oral formulations. Further work toward developing formulations ofdifferent drugs using these 2 delivery vehicles andevaluating their efficacy as oral drug delivery systems isin progress and shall be reported elsewhere.

The purpose of this study was to look at the toxicity profile of the synthetic lipids. A 28-day study is considered
a subchronic study, which is well accepted for eliciting
any toxicity on long-term feeding. These lipids at various levels did not appear to retard growth or affect foodconsumption and utilization (Figures 2, Figure 3, Figure
4 & Figure 5). The feed conversion efficiency of thedifferent groups followed a similar pattern indicating anormal metabolism of the animals (Table 1).

ACKNOWLEDGEMENTS
We thank Dr. Sudhir Dagaonkar for his helpful discussions.

The mean food consumption of the animals in the control and experimental groups was similar (Tables 1 and
2). This finding indicates that the feed intake and utilization of protein and other nutrients were not affected by
the intake of the synthetic lipid. Moreover, there were nodifferences between the sexes with respect to feed conversion efficiency.

REFERENCES
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2. Hunt CA. Liposomes disposition in vivo. Liposome stability inplasma and implications for drug carrier function. Biochim Biophys
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4. Gabizon A. Liposome circulation time and tumor targeting: implications for cancer chemotherapy. Adv Drug Deliv Rev. 1995;16:285294.
5. Gabizon A, Catane R, Uziely B, et al. Prolonged circulation timeand enhanced accumulation in malignant exudates of doxorubicin
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7. Koning GA, Morselt HW, Velinova MJ, et al. Selective transfer ofa lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes
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8. Food and Agriculture Organization (FAO). Principles for the Safety
Assessment of Food Additives and Contaminant in Food. International Programme on Chemical Safety (IPCS) in co-operation withthe project FAO/WHO expert committee on food additives (JECFA).
Rome, Italy: Food and Agriculture Organization; 1987. Environmental Health Criteria 70.

There were no significant changes in the hematologicalparameters between the control and the experimentalgroups (Tables 3 and 4), suggesting that the lipids GDO12 (lipid 1) and GDS-12 (lipid 2) may not be toxic asthey do not affect the circulating red cells, nor the hematopoiesis and leucopoiesis that could otherwise havecaused a megaloblastic anemia, nor changes in packed
cell volume (PCV) and eosinophils. Plasma levels of
glucose, total proteins, albumin, blood urea nitrogen,sodium, and potassium ions were not affected by feeding
GDO-12 (lipid 1) and GDS-12 (lipid 2) (Tables 5 and 6).
This finding also indicates that the normal metabolismof the animals was not affected.
It is clear that the liver and kidneys play significant roles
in various metabolic processes. Therefore, emphasis wasplaced on the effect these lipids might have on the function of these organs. In addition, the liver plays an important role in xenobiotic function, while kidneys aresites for reabsorption. Feeding either GDO-12 (lipid 1)
or GDS-12 (lipid 2) did not alter the urinary levels of

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AAPS PharmSci 2004; 6 (2) Article 14 (http://www.aapspharmsci.org).
9. Dacie VJ, Lewis SM. Practical Haematology. International student
ed. Oxford, UK: Churchill Livingstone; 1984.
10. Brij MM, Rawnsley HM. Clinical, Biochemical and Hematological Reference Values in Normal Experimental Animals. Masson:
New York; 1977.
11. Harris JM, Zalipsky S. Poly(ethylene glycol) Chemistry and Biological Applications. Washington, DC: ACS Publications; 1997.

12. Lasic DD, Martin FJ. Stealth Liposomes. Boca Raton: CRC Press;
1995.
13. Keller BC, Lasic DD. Self-forming, thermodynamically stable
liposomes and their applications. US patent 6 610 322, August 26,
2003.

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