Halogenated pyrimidines (HP) (5-bromo-2′-deoxyuridine (BUdR) and 5-iodo-2′-deoxyuridine
(IUdR)) are effective radiosensitisers in cells that take them up (Fowler and Kinsella,
1996). They act by being incorporated into DNA in competition with thymidine during
S phase of the cell cycle and mediate radiosensitisation by increasing the susceptibility
of IUdR- or BUdR-substituted DNA to radiation-generated reactive free radicals, which
may also damage unsubstituted complementary-strand DNA (Fornace et al, 1990). In addition,
there is evidence that HP can inhibit the repair of radiation-induced DNA damage (Iliakis
et al, 1989). Exposure to clinically achievable steady-state concentrations of either
IUdR or BUdR significantly increases formation of single- and double-stranded DNA
breaks in vitro (Kinsella et al, 1987). HP-mediated radiosensitisation is directly
related to the extent of thymidine replacement in DNA (thymidine replacement index,
TRI) (Lawrence et al, 1990; Miller et al, 1992), which, in turn, is related to the
duration of drug exposure in vitro (Lawrence et al, 1990). Phase I clinical studies
using protracted intravenous infusions of IUdR in patients with hepatic metastases,
high-grade glioma, sarcomas and head and neck cancers resulted in TRI of 4–26% in
tumour tissue, which were significantly greater than the levels in adjacent normal
tissues (Speth et al, 1988; Cook et al, 1992). Haematological and mucosal toxicities
(including exacerbation of the local radiation response) were dose-limiting and directly
related to the duration of drug infusion (Urtasun et al, 1993; Epstein et al, 1994;
Sullivan et al, 1994). These findings may, in part, explain the failure of intravenous
infusional IUdR to yield a survival advantage when used as a radiosensitiser in the
treatment of gliomas, head and neck cancers and sarcomas (Goffman et al, 1992; Urtasun
et al, 1993; Epstein et al, 1994; Sullivan et al, 1994; Robertson et al, 1995; Urtasun
et al, 1996). In vitro data have suggested that repeated short duration drug exposures
may provide a means of achieving effective DNA incorporation and reducing dose-limiting
myelotoxicity (Lawrence et al, 1990), but relatively few cells will be targeted with
this approach since only cells in S phase at the time of drug administration will
incorporate the agent. Fowler and Kinsella (1996) have calculated that infusion durations
of 5–10 times greater than the population doubling time of clonogenic cells will be
required for clinically significant radiosensitisation. Therefore, although HP appear
to be a promising group of agents, their efficacy is limited by the amount of drug
incorporated into the tumour with relatively short durations of drug infusion and
the occurrence of dose-limiting systemic toxicity when more prolonged infusions are
administered.
One potential means of overcoming both of these obstacles is the development of liposome-targeted
HP. The inclusion of methoxypolyethylene glycol-derivatised (pegylated) lipids in
the bilayer membrane of liposomes effectively increases the longevity of liposomes
in the circulation (Papahadjopoulos et al, 1991) and increases their accumulation
in tumours (Huang et al, 1992). Preclinical studies have shown that cytotoxic drugs
entrapped in pegylated liposomes are active against a range of tumours (Harrington
et al, 2000a). Clinical studies of pegylated liposomal doxorubicin have confirmed
its activity against AIDS-related Kaposi's sarcoma (Northfelt et al, 1998; Stewart
et al, 1998) and breast and ovarian cancers (Muggia et al, 1997; Ranson et al, 1997)
with considerable attenuation of the adverse effects of the unencapsulated drug. Furthermore,
preclinical and Phase I/II trials of pegylated liposomal doxorubicin and radiotherapy
(RT) have supported the feasibility of this approach in head and neck and lung cancers
(Koukourakis et al, 1999; Harrington et al, 2000b, 2000c; Harrington et al, 2001a).
In the present study, we carried out experiments to examine the potential value of
a novel prodrug formulation of IUdR (3′, 5′-O-dipalmitoyl 5-iodo-2′-deoxyuridine,
dpIUdR) encapsulated in pegylated liposomes (PLIUdR) to act as a radiation sensitiser
in a human head and neck squamous cell cancer (HNSCC) model.
MATERIALS AND METHODS
In vitro radiosensitivity assay
Human HNSCC KB and HN5 cells were grown as monolayers in 75 cm2 tissue culture flasks
(Falcon, NJ, USA) in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum (FCS) (Gibco, Paisley, UK), nonessential amino acids, and antibiotics at
37°C in a humidified incubator with 5% CO2. Culture medium was supplied by the Media
Production Unit at the Imperial Cancer Research Fund, Clare Hall, Herts, UK. Under
these conditions, the doubling time of KB cells is approximately 17 h (data not shown).
The cells were plated at low density (5 × 104 cells flask−1) to ensure that they were
growing exponentially during the 48 h period in which they were exposed to 0, 10−6,
10−5 or 10−4 M unencapsulated IUdR (Nova Laboratories, Leicester, UK). For the assessment
of radiation survival after exposure to IUdR, the cells were trypsinised and plated
in appropriate dilutions in growth medium in six-well plates. At 4 h after plating,
the cells were irradiated (0, 4, 7 Gy) with a 111 TBq 137Cs source (CIS Bio International,
Gif-sur-Yvette, France) yielding a dose rate of about 2 Gy min−1. Following irradiation,
the cells were incubated for 6 days, changing the culture medium on alternate days
after day 3. After this time, surviving cells were trypsinised and plated onto 96-well
microtitre plates containing 100 μl of medium per well. After incubation overnight,
growth medium was removed and the wells were washed twice with phosphate-buffered
saline (PBS) and 60 μl of 3.75 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (NAG) in
0.05 M citrate buffer (0.1 M citric acid, 0.1 M trisodium citrate, 0.25% Triton X-100,
pH 5.0) was added to each well (Yagi et al, 1989). Cells were incubated at 37°C and
5% CO2 for 1 h, removed from the incubator and the colorimetric reaction was stopped
by the addition of 90 μl of glycine buffer (50 mM glycine, 5 mM EDTA, pH 10.4). The
optical density was read on a Titertek Multiskan® MCC/340 spectrophotometer with a
405 nm filter.
Thymidine replacement index
KB cells were grown as monolayers in 75 cm2 tissue culture flasks as detailed above.
The cells were plated at low density (5 × 104 cells flask−1) to ensure that they were
growing exponentially during the 48 h period in which they were exposed to 0, 10−6,
10−5 or 10−4 M unencapsulated IUdR. After this time, the cells were trypsinised, centrifuged
and the pellet was washed thrice with PBS and the DNA was extracted using a QIAamp
Tissue Kit (Qiagen Ltd, Sussex, UK) according to the manufacturer's instructions.
Digestion of DNA into deoxynucleotides was performed as follows: 100 μl of the DNA
preparation (containing 5–10 μg DNA) was incubated for 1 h at 37°C with 0.4–0.5 μl
DNase I (Boehringer Mannheim, Germany) and 5 μl 100 mM MgCl2; 0.4 μl of crotalus venom
phosphodiesterase I (Boehringer Mannheim) was added and incubated for 1 h at 37°C;
0.2 μl alkaline phosphatase (Boehringer Mannheim) was added and incubated for 1 h
at 37°C. The resulting nucleotide solution was adjusted to pH 5.5 before loading onto
the HPLC column. Standard nucleotides (2′-deoxycytidine [C], 2′-deoxyguanosine [G],
2′-deoxyadenosine [A] and thymidine [T]) were obtained from Sigma Chemical Co. (St
Louis, MO, USA). All HPLC experiments were performed using a C18 Phenomenex Columbus
column (2 × 250 mm, 5 μm particle size) with UV detection at 254 and 288 nm. The mobile
phase contained 10 mM ammonium acetate, pH 6.0, with 8.75% acetonitrile, pumped at
a flow rate of 1 ml min−1 at room temperature. The deoxynucleotides and IUdR were
detected at 254 and 288 nm. To calibrate the peaks on the HPLC, standard stock solutions
of IUdR and deoxynucleotides were diluted to yield several solutions with concentrations
of 2 × 10−4 to 2 × 10−6 M and a linear relationship was observed between the peak
heights and amounts loaded (data not shown).
Tumour xenograft model
All animal experiments were carried out with ethical committee approval and met the
standards laid out in the UKCCCR guidelines (Workman et al, 1998). Female nude mice
of mixed genetic background bred under specific pathogen-free conditions at the Imperial
Cancer Research Fund Animal Breeding Unit (South Mimms, Herts, UK) were used. Animals
were housed in sterile filter-top cages on sterile bedding and maintained on an irradiated
diet and autoclaved acidified water (pH 2.8) ad libitum. KB tumour cells were grown
to confluence in vitro in 175 cm2 tissue culture flasks as detailed above. Cells were
harvested and a single cell suspension was prepared. Tumour xenografts were set up
by subcutaneous injection of 5 × 106 tumour cells in 100 μl of culture medium into
the right flank of the mice. The animals were used for experiments approximately 15
days later, at which time the tumours were 6–8 mm in diameter. Starting 7 days after
inoculation the tumours were measured on alternate days on at least three occasions
before the commencement of the study. Three orthogonal tumour diameters were recorded
using Vernier calipers and the tumour diameter was calculated from the formula:
. Therapeutic irradiation (see below) was administered on day 15 after inoculation
and the measured tumour volume on this day was designated as the initial volume or
V
0. Subsequently, the tumour volume was assessed two or three times per week and the
absolute and relative (as compared to V
0) tumour volume was calculated. Mice were killed after the tumour had increased in
size to more than three times its original volume (3V
0). The time taken to reach 3V
0 was recorded and used as a surrogate measure of animal survival and was designed
to spare the animals from the physical distress of unnecessarily large tumour burdens
and to comply with the UKCCCR guidelines (Workman et al, 1998).
5-Iodo-2′-deoxyuridine for in vivo use
Unencapsulated 5-iodo-2′-deoxyuridine (Sigma-Aldrich Co. Ltd., Poole, UK) was dissolved
in a 20% solution of dimethyl sulphoxide in distilled water. Because of its water
solubility and low molecular weight, IUdR cannot be retained efficiently in liposomes.
Therefore, IUdR was derivatised by attaching two long-chain fatty acids (palmitic
acid) to the 3′ and 5′ positions of the IUdR sugar moiety to form 3′, 5′-O-dipalmitoyl-5-iodo-2′-deoxyuridine
(dpIUdR) to convert the agent into a hydrophobic, lipid soluble prodrug which was
stably incorporated into the bilayer of pegylated liposomes with the following lipid
composition (values expressed as a % molar ratio): HSPC (94.6%) and MPEG-DSPE (5.4%).
The agent was supplied as an isotonic, preservative-free solution in 20 ml vials at
a dpIUdR concentration of between 2.8 and 3.5 mg ml−1 (equivalent to IUdR concentrations
of 1.2–1.5 mg ml−1).
Administration of test agents
Unencapsulated IUdR was administered either as intravenous bolus injections or as
a continuous subcutaneous infusion. The intravenous bolus injections were administered
according to the following schedules: (1) single injections of 24 mg kg−1 given 16 h
before tumour irradiation; (2) as a series of four injections of 12 mg kg−1 on alternate
days to a total dose of 48 mg kg−1 over a 7-day period before irradiation. The continuous
infusions of unencapsulated IUdR were administered using subcutaneously implanted
Alzet® Model 2002 mini-osmotic pumps (Charles River UK, Ltd, Kent, UK) over a 7-day
period before irradiation. A dose of 48 mg kg−1 was delivered over the 7-day period
(6.9 mg kg day−1) by pumps with a mean fill volume of 226±9 μl and a mean pumping
rate of 0.46±0.03 μl h. PLIUdR was administered as intravenous bolus injections according
to the following protocols: (1) single injections of 24 mg kg−1 given 16 h before
tumour irradiation; (2) as a series of four injections of 12 mg kg−1 to a total dose
of 48 mg kg−1 over a 7-day period before irradiation. No attempt was made to deliver
subcutaneous infusions of PLIUdR.
Tumour irradiation
Tumour irradiation was performed using the 137Cs source with mice carefully positioned
within a specially constructed jig as described previously (Harrington et al, 2000c).
The system was calibrated as previously described (Harrington et al, 2000c) with lithium
fluoride thermoluminescent dosimeters (TLD) (Nuclear Enterprises, Reading, UK) that
were read in a Toledo 654 TLD reader (DA Pitman, Weybridge, UK) (data not shown).
Before therapeutic irradiation, the animals were anaesthetised with an intraperitoneal
injection of 100 μl of a 1 : 1 : 4 mixture of Hypnorm (fentanyl citrate 0.315 mg ml−1,
fluanisone 10 mg ml−1) (Janssen-Cilag Ltd, High Wycombe, UK), Hypnovel® (midazolam
5 mg ml−1) (Roche Products Ltd, Welwyn Garden City, UK) and water for injection BP
(Fresenius Health Care Group, Basingstoke, UK). Anaesthetised animals were positioned
such that the subcutaneous xenograft tumours were exposed to the radiation beam with
the rest of the animals’ bodies shielded by 4 cm thick lead. Great care was taken
to avoid direct pressure on the tumour mass in order to minimise the risk of creating
areas of pressure-induced hypoxia during irradiation. Animals were managed at all
times in accordance with UKCCCR standards (Workman et al, 1998).
Radiotherapy alone
The effect of single fractions of RT (SFRT) was initially assessed by irradiating
groups of mice bearing KB xenograft tumours with radiation doses of 4.5 Gy (n=17)
and 9 Gy (n=12) over a period of 385 and 770 s, respectively. The RT was delivered
at a dose rate of 0.7 Gy min−1 as determined by the dosimetric calibration outlined
above. Similarly, the effect of daily fractionated RT (FRT) to a dose of either 9 Gy
in three fractions over 3 days (n=11) or 15 Gy in five fractions over 5 days (n=10)
was determined in KB tumour-bearing mice. Again, all irradiations were carried out
at a dose rate of 0.7 Gy min−1 over a period of 257 s.
Radiotherapy and IUdR
The combination of RT and IUdR was tested according to a number of sequential protocols
with the aim of modelling the clinical use of IUdR as a radiation sensitiser in patients
with solid cancers. In the initial studies, tumour-bearing mice received intravenous
bolus doses of 24 mg kg−1 of IUdR, either in the form of unencapsulated IUdR or PLIUdR,
16 h before receiving SFRT at doses of either 4.5 or 9 Gy. In subsequent studies,
an attempt was made to model the clinical experience with IUdR in which prolonged
intravenous infusions of the drug have been administered as loading doses before RT.
Therefore, four separate intravenous bolus doses of 12 mg kg−1 of either unencapsulated
IUdR or PLIUdR were administered on alternate days over a 7-day period (−8 to −1 days)
before SFRT was delivered to a dose of either 4.5 or 9 Gy. Following on from the studies
using SFRT, a number of studies of FRT were performed in which IUdR was administered
according to the protracted loading schedule. Tumour-bearing mice received total doses
of 48 mg kg−1 of IUdR, either as four doses of 12 mg kg−1 of unencapsulated IUdR or
PLIUdR or as a subcutaneous infusion of unencapsulated IUdR administered over a 7-day
period as described above. FRT was administered to doses of either 9 Gy in three fractions
over 3 days or 15 Gy in five fractions over 5 days.
Immunohistochemistry
The monoclonal anti-BUdR antibody (clone BU-1), which recognises both IUdR and BUdR,
was used (Amersham International plc, Amersham, UK). KB tumour-bearing animals (three
for each treatment) received total doses of 48 mg kg−1 of IUdR, either as four doses
of 12 mg kg−1 of PLIUdR or as a subcutaneous infusion of unencapsulated IUdR administered
via Alzet® Model 2002 miniosmotic pumps over a 7-day period as described above. Control
animals received four bolus injections of PBS over the same time course as that used
for PLIUdR. The tumours were removed 24 h after completion of IUdR administration
and fixed in formalin at 4°C for 3 days. Thereafter, they were embedded in paraffin
and 5 μm sections were cut and mounted on slides coated with 0.1% poly-L-lysine (Sigma,
Poole, UK), dried overnight and stored at room temperature until use. The slides were
dewaxed and rehydrated through solutions of xylene, alcohol and water. They were then
incubated overnight at 4°C with 50 μl of BU-1 antibody that had previously been biotinylated
(3–4 molecules per molecule of antibody). After this incubation, streptavidin–horseradish
peroxidase (Dako Ltd, High Wycombe, UK) was added and subsequent colorimetric visualisation
was achieved by addition of 50 ml 3,3′-diaminobenzidine (BDH Laboratory Supplies,
Poole, UK) solution at a concentration of 0.3 mg ml−1.
Toxicity assessment
Serial measurements of animal weight were used as a surrogate measure of systemic
toxicity. The cutaneous radiation reaction in the treated area was observed in all
treatment groups. No attempt was made to measure haematological or biochemical parameters.
Statistical analysis
Relative tumour volumes were recorded for each animal in a treatment group. The times
taken to reach 3V
0 were recorded as an indication of the progression of the xenograft tumours as described
above. The Wilcoxon rank-sum test was used to test the difference between the times
taken to reach 3V
0 in the various test groups. Differences were considered to be significant at P<0.05.
RESULTS
In vitro radiation response of SCCHN cells
The effect of unencapsulated IUdR at concentrations between 10−4 and 10−6 M on the
survival of HN5 and KB cells after irradiation is presented in Figure 1A and B
Figure 1
Effect of IUdR treatment on the response of SCCHN cells to irradiation: (A) HN5 cells;
(B) KB cells. Cells were exposed to 0, 1, 10 or 100 μ
M unencapsulated IUdR for 48 h and then irradiated (0, 4 or 7 Gy) with a 137Cs source
at a dose rate of 2 Gy min−1. Following irradiation, cells were incubated for 6 days
and a p-nitrophenyl-N-acetyl-β-D-glucosaminide (NAG) cell survival assay was performed.
Significant radiosensitisation was seen at the 100 μ
M concentration for both the 4 and 7 Gy radiation doses in each cell line. The effect
of IUdR was most pronounced for the KB cells and this cell line was selected for subsequent
in vitro and in vivo analysis.
. Significant radiosensitisation was seen at the 10−4 M concentration for both the
4 and 7 Gy radiation doses in each cell line. This effect was more pronounced in the
KB cells (Figure 1B) and this cell line was used for subsequent experiments.
TRI after in vitro exposure of KB tumour cells
The retention times for C, G, T, A and IUdR, were 4.6±0.4, 5.0±0.4, 6.2±0.2, 7.3±0.2
and 10.8±0.2 min, respectively. IUdR was preferentially detected at 288 nm and the
deoxynucleotides at 254 nm. The compositions of all the deoxynucleotides showed good
agreement between control and treated cells, with the exception of thymidine that
was partially replaced by IUdR in treated cells. The percentage substitution by IUdR
was equal to the percentage by which thymidine was decreased from the control cells,
suggesting that thymidine was substituted by IUdR without any other effect on the
base content of the DNA (Table 1
Table 1
Thymidine replacement indices (TRI) for KB cells after 48 h period of exposure to
unencapulated IUdR at concentrations between 0 and 100 μ
M
IUdR concentration (μ
M)
TRI
Thymidine content in DNA (%)
0
0
29.8
1
2.2
27.6
10
2.8
27
100
7
22.8
).
SFRT and single bolus IUdR
The results of SFRT at doses of 4.5 and 9 Gy delivered 16 h after a single bolus injection
of 24 mg kg−1 of either unencapsulated IUdR or PLIUdR are presented in Table 2
Table 2
Effect of single dose unencapsulated IUdR or PLIUdR 24 mg kg−1 plus single fraction
RT against KB xenograft tumours in nude mice
Median time to 3V
0 (days)
Group
4.5 Gy
9 Gy
No drug
12.7
22.6
Unencapsulated IUdR plus RT
14.0
21.9
PLIUdR plus RT
12.4
22.7
P
Group
4.5 Gy
9 Gy
RT vs unencapsulated IUdR plus RT
>0.1
>0.1
RT vs PLIUdR plus RT
>0.1
>0.1
Unencapsulated IUdR plus RT vs PLIUdR plus RT
>0.1
>0.1
Median times to 3V
0 and statistical analysis.
. As compared to the untreated controls, neither unencapsulated IUdR nor PLIUdR alone
exerted a significant effect on tumour growth. In combination with SFRT doses of 4.5
and 9 Gy, there was no evidence of enhancement of the radiation response with either
agent (data not shown). These results prompted attempts to increase the area under
the curve of tumour exposure to IUdR by using more protracted dosing schedules.
SFRT and protracted administration of IUdR
The effect of repeated bolus administration of unencapsulated IUdR and PLIUdR (total
dose 48 mg kg−1 over 7 days) before SFRT at doses of either 4.5 and 9 Gy on the growth
of KB xenograft tumours is shown in Table 3
Table 3
Effect of multiple dose unencapsulated IUdR or PLIUdR (4 × 12 mg kg−1) plus single
fraction RT against KB xenograft tumours in nude mice
Median time to 3V
0 (days)
Group
4.5 Gy
9 Gy
No drug
12.7
22.6
Unencapsulated IUdR plus RT
13.7
24.3
PLIUdR plus RT
16.8
26.7
P
Group
4.5 Gy
9 Gy
RT vs unencapsulated IUdR plus RT
>0.1
>0.1
RT vs PLIUdR plus RT
<0.01
>0.1
Unencapsulated IUdR plus RT vs PLIUdR plus RT
>0.1
>0.1
Median times to 3V
0 and statistical analyses.
. Once again, neither of these agents exerted a significant independent effect on
tumour growth (data not shown). When administered according to this protracted schedule,
PLIUdR significantly increased the effect of SFRT at a dose of 4.5 Gy (P<0.01) but
not at a dose of 9 Gy (P>0.1). There was no evidence of enhancement of the effect
of SFRT at either dose level with unencapsulated IUdR (P>0.1). Furthermore, a direct
comparison between the effects of PLIUdR and unencapsulated IUdR revealed no significant
difference at 4.5 or 9 Gy (P>0.1 for both comparisons). There was no evidence of increased
local cutaneous radiation toxicity with unencapsulated IUdR or PLIUdR at either radiation
dose.
FRT and protracted administration of IUdR
The effect of FRT to a dose of either 9 Gy in three fractions in 3 days (9 Gy in 3F)
or 15 Gy in five fractions in 5 days (15 Gy in 5F) in combination with either a subcutaneous
infusion of 48 mg kg−1 of unencapsulated IUdR or four bolus injections of 12 mg kg−1
of PLIUdR over 7 days on the growth of KB tumour xenografts is shown in Table 4
Table 4
Effect of a subcutaneous infusion of unencapsulated IUdR (48 mg kg−1) or multiple
dose PLIUdR (4 × 12 mg kg−1) plus fractionated RT on KB xenograft tumours in nude
mice
Median time to 3V
0 (days)
Group
9 Gy in 3F
15 Gy in 5F
RT alone
13.2
18.8
sc unencapsulated IUdR 48 mg kg−1 plus RT
17.6
26.9
PLIUdR 4 × 12 mg kg−1 plus RT
21.6
34.2
P
Group
9 Gy in 3F
15 Gy in 5F
RT vs sc unencapsulated IUdR plus RT
>0.1
0.1>P>0.05
RT vs PLIUdR plus RT
<0.05
<0.01
sc unencapsulated IUdR plus RT vs PLIUdR plus RT
>0.1
>0.1
Median times to 3V
0 and statistical analyses.
and in Figure 2A and B
Figure 2
Effect of a subcutaneous infusion of unencapsulated IUdR (48 mg kg−1) or four intravenous
doses of 12 mg kg−1 PLIUdR (total dose 48 mg kg−1) on the response of KB xenograft
tumours to fractionated irradiation: (A) 9 Gy in three fractions over 3 days; (B)
15 Gy in five fractions over 5 days. Test agents were administered over a period of
7 days terminating 16 h before the delivery of the first dose of FRT. PLIUdR administered
according to this protracted schedule enhanced the effect of both FRT schedules (P<0.05
for 9 Gy in three fractions; P<0.01 for 15 Gy in five fractions). The effect of unencapsulated
IUdR did not reach statistical significance.
. When administered according to this schedule, PLIUdR significantly increased the
effect of FRT at doses of both 9 Gy in 3F (P<0.05) and 15 Gy in 5F Gy (P<0.01). However,
the subcutaneous infusion of unencapsulated IUdR did not significantly increase the
effect of either dose of FRT, although at the dose of 15 Gy in 5F it was of borderline
significance (0.1>P>0.05). A direct comparison between the effects of multiple dose
PLIUdR and subcutaneous unencapsulated IUdR revealed no significant difference at
either dose of FRT (P>0.1 for both comparisons). There was no evidence of increased
local radiation toxicity with subcutaneous unencapsulated IUdR or PLIUdR at either
FRT dose.
Immunohistochemistry
The results of staining tumour specimens from animals treated with PBS, unencapsulated
IUdR and PLIUdR with BU-1 monoclonal antibody are presented in Figure 3A, B and C
Figure 3
Immunohistochemistry of KB tumours from animals treated with (A) intravenous phosphate-buffered
saline (four doses of 100 μl over 7 days); (B) unencapsulated IUdR 48 mg kg−1 as a
continuous subcutaneous infusion over 7 days; (C) intravenous PLIUdR (four doses of
12 mg kg−1 over 7 days). Paraffin-embedded sections were stained with biotinylated
BU-1 monoclonal antibody followed by incubation with streptavidin–horseradish peroxidase
and addition of 3,3′-diaminobenzidine. No positive staining was seen in the phosphate-buffered
saline-treated group. The animals treated with unencapsulated IUdR showed low levels
of staining. There was very prominent nuclear staining in the animals treated with
PLIUdR.
, respectively. Significant positive staining of the nuclei of tumour cells was seen
in animals treated with PLIUdR and, to a lesser extent, unencapsulated IUdR. In contrast,
there was virtually no tumour staining seen in the animals treated with PBS.
Toxicity
There was no significant alteration in the weight of mice that received single intravenous
bolus doses of 24 mg kg−1 of either unencapsulated IUdR or PLIUdR, SFRT or combinations
of these two treatments (data not shown). However, multiple intravenous injections
of either unencapsulated IUdR or PLIUdR were associated with a temporary reduction
in the animals’ weights (<10%) over the week during which the injections were administered
(data not shown). No such alteration was detected for the animals that had the subcutaneous
infusion pumps inserted (data not shown). All groups that received FRT experienced
a reversible 6–15% reduction in mean weight during the first 10 days of the study
(data not shown). There was no evidence of enhanced cutaneous radiation reaction in
any of the treatment groups (data not shown).
DISCUSSION
In recent years, radiosensitising agents (cytotoxic drugs, hyperbaric oxygen, hypoxic
cell sensitisers and HP) have been shown to improve the response rate and outcome
of radical RT in a range of tumour types, including head and neck cancer (McGinn and
Kinsella 1992; Munro 1995; Saunders and Dische 1996; Overgaard et al, 1998; Pignon
et al, 2000). However, the combined use of RT and radiosensitising agents has a number
of potential drawbacks: increased local normal tissue radiation toxicity that may
necessitate radiation dose reductions and treatment delays (Tannock, 1996); increased
late local radiation morbidity (Henk, 1997); and dose-limiting systemic toxicities.
Liposome delivery offers the prospect of reducing or circumventing each of these problems
by targeting delivery of the radiosensitiser preferentially to the tumour tissue and
avoiding local and distant normal tissue deposition of the drug. We have recently
provided proof of principle for this strategy using RT and liposomal doxorubicin and
cisplatin in a KB tumour xenograft model (Harrington et al, 2000b). However, since
each of those agents has been shown to have a demonstrable antitumour effect in this
model (Harrington et al, 2000c), it was difficult to be clear that they were acting
as true radiosensitisers.
In this study, we have demonstrated that a novel prodrug formulation of IUdR encapsulated
within pegylated liposomes has no intrinsic antitumour efficacy but is able to enhance
the effect of both SFRT and FRT in a HNSCC xenograft model in nude mice. Significantly,
the liposomal agent was at least as effective as a sustained infusion of unencapsulated
IUdR. However, it must be borne in mind that unencapsulated IUdR was not administered
at the maximum tolerated dose in these experiments. Clearly, future studies will need
to compare these two agents at their maximal tolerated doses in order to provide a
more accurate picture of the radiosensitising effect of this liposomal prodrug formulation.
These data have considerable significance for the development of clinical strategies
using pegylated liposomal radiosensitisers. In clinical practice, the conventional
means of delivering IUdR has involved prolonged intravenous infusions over a period
of up to 14 days before commencing RT (Speth et al, 1988; Cook et al, 1992; Goffman
et al, 1992; Urtasun et al, 1993; Epstein et al, 1994; Sullivan et al, 1994; Robertson
et al, 1995; Urtasun et al, 1996). Such schedules have been associated with appreciable
systemic hematological toxicity and local catheter-related infusion site reactions
and have effectively prevented the development of this promising agent for clinical
use (Goffman et al, 1992; Urtasun et al, 1993; Sullivan et al, 1994; Epstein et al,
1994; Robertson et al, 1995; Urtasun et al, 1996). The data presented here suggest
that administration of a small number of short-duration intravenous infusions of dpIUdR
encapsulated in pegylated liposomes could achieve an equivalent radiosensitising effect
as more prolonged intravenous infusions. Further studies will be required to determine
if the liposomal agent is as active as protracted delivery of unencapsulated drug
at the maximum tolerated dose. Although there was no direct assessment of toxicity
in these studies, previous clinical experience with myelotoxic agents, such as doxorubicin,
entrapped in pegylated liposomes has revealed attenuation of haematological toxicity
(Muggia et al, 1997; Ranson et al, 1997).
The examination of the effect of various schedules of administration of unencapsulated
IUdR and PLIUdR provides some useful insights into the use of these agents. Enhancement
of the radiation response was not evident after single bolus injections of either
agent delivered 16 h (approximately one in vitro doubling time) before SFRT at doses
of 4.5 and 9 Gy. This lack of activity of PLIUdR may have been accentuated by relatively
slow kinetics of conversion of the lipophilic prodrug to its hydrophilic active component
and its subsequent diffusion into the interior of tumour cells to be incorporated
into their DNA. However, when the IUdR was delivered according to a protracted administration
schedule (approximately 10 in vitro doubling times), this resulted in enhanced efficacy
of the SFRT and FRT doses. These findings are in keeping with the previously published
preclinical and clinical data which suggest that for meaningful radiosensitisation
to occur IUdR should be present for a prolonged period to ensure maximal labelling
of tumour cells (Goffman et al, 1992; Urtasun et al, 1993; Epstein et al, 1994; Sullivan
et al, 1994; Robertson et al, 1995; Fowler and Kinsella, 1996; Urtasun et al, 1996).
The administration of unencapsulated IUdR by continuous subcutaneous infusion and
PLIUdR by multiple bolus injections appeared to be an effective means of achieving
this objective. Previous studies in nude mice bearing KB xenograft tumours have shown
that 111In-DTPA-labelled pegylated liposomes have a prolonged circulation half-life
of approximately 10 h and achieve maximal tumour uptake at 24 h after intravenous
injection (Harrington et al, 2000d). Biodistribution and pharmacokinetic studies in
patients with solid cancers have demonstrated similar data, with a circulation half-life
of approximately 75 h and maximal tumour uptake at 48–72 h (Harrington et al, 2001b).
Therefore, progressive accumulation of PLIUdR within tumour deposits may effectively
act as a local, prolonged, high-concentration infusion of the agent precisely in the
site where it is likely to exert maximum efficacy.
Therefore, in summary, this novel prodrug formulation of IUdR encapsulated in pegylated
liposomes appears to offer considerable promise for further development. Indeed, preliminary
pharmacokinetic and biodistribution studies have commenced in patients with head and
neck cancer.