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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
Received April 24, 2003; accepted June 5, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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; Ranson et al.,
1997; Gordon et al.,
2000). As reviewed by Allen et al., STEALTH liposomes have
dose-independent, log-linear pharmacokinetics
(Allen et al., 1995).
Encapsulating doxorubicin within these liposomes alters its pharmacokinetics
and biodistribution and results in a decrease in doxorubicin-associated
toxicities, including its dose-limiting cardiomyopathy and myelosuppression
(Berry et al., 1998;
Safra et al., 2000). The
dose-limiting toxicities of SL-DXR are mucocutaneous reactions such as
palmar-plantar erythrodysesthesia (PPE) and mucositis/stomatitis
(Gordon et al., 1995;
Uziely et al., 1995;
Lotem et al., 2000;
Hamilton et al., 2002).
Palmar-plantar erythrodysesthesia primarily affects the palms of the hands
and the soles of the feet. Patients who develop PPE experience erythema and
edema that can lead to blistering desquamation if the next dose is not delayed
or reduced. The current hypothesis for the development of PPE is that the
small size (100-nm diameter) and long circulation time
(t1/2 is approximately 48 h in humans) of SL-DXR allows
liposomes to accumulate in the skin. The basal layers of the skin are damaged
with prolonged exposure to doxorubicin as the liposomes slowly release their
contents. The accumulation of liposomes is thought to mimic the anatomical
distribution of lesions and to be greatest in regions of skin that are
subjected to pressure or irritation, such as the flexure creases of the hands,
soles of the feet, or belt lines (Gordon
et al., 1995; Lotem et al.,
2000).
This hypothesis is supported by current experimental and clinical data.
Liposomes with long circulation times accumulate in the skin of experimental
animals to a greater extent than liposomes with shorter circulation times
(Allen et al., 1991;
Papahadjopoulos et al., 1991).
In mice, this accumulation is dependent on liposome size; furthermore, mouse
paws (homologous to human hands and feet) accumulate more liposomes than skin,
supporting the idea of the pressure-dependent extravasation of liposomes into
cutaneous tissues (Charrois and Allen,
2003). This hypothesis is further supported by the observation
that Myocet, another liposome formulation of doxorubicin, does not produce PPE
and has myelosuppression as its dose-limiting toxicity. Myocet differs from
Doxil in having a larger mean diameter (160 versus 100 nm), a shorter plasma
t1/2 (6.7 versus 45.2 h), and a larger volume of
distribution (18.8 versus 4 liters)
(Cowens et al., 1993;
Gabizon et al., 1994a).
Clinical data suggest that PPE is more likely to develop after multiple
doses of SL-DXR. In addition, the likelihood of developing PPE is related to
the dose intensity of SL-DXR therapy, with patients receiving greater than 10
to 12 mg/m2/week more likely to develop symptoms
(Gabizon et al., 1994b;
Muggia et al., 1997;
Ranson et al., 1997). When PPE
develops, clinical interventions include lengthening the dose interval and/or
decreasing the dose intensity. Either of these interventions may compromise
the therapeutic outcome (Hensley et al.,
2001).
Despite the widespread clinical use of SL-DXR, few studies have looked at
the pharmacokinetics and biodistribution of repeat injections in experimental
models, and no studies have quantified the cutaneous localization of
doxorubicin from SL-DXR with repeat administration
(Amantea et al., 1999).
Therefore, a small-animal model for the pharmacokinetics and biodistribution
of doxorubicin (from SL-DXR) in plasma, tumor, and cutaneous tissues will be
beneficial in understanding the relationship between the dose schedule and
dose intensity of SL-DXR therapy and its therapeutic activity and toxicity. We
performed experiments studying the plasma pharmacokinetics and biodistribution
of doxorubicin to the skin and paws of mice as a function of time using either
the same dose of SL-DXR and different dose intervals (i.e., different dose
intensities) or different dose schedules with the same dose intensity. The
latter experiments included tumor tissue (4T1 murine mammary carcinoma) and
were performed to test the hypothesis that, for a given dose intensity, it is
therapeutically beneficial to administer larger infrequent doses than smaller
more frequent doses (Gabizon,
2001). Therapeutic experiments were also performed using the 4T1
murine mammary carcinoma model to determine whether altering either the dose
schedule or the dose intensity affects the therapeutic activity of SL-DXR.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Animals and Tumor Model. Female BALB/c mice (6-8 weeks) were purchased from the breeding colony at the Health Sciences Laboratory Animal Services (University of Alberta, Alberta, Canada). Mice were housed under standard conditions and had access to food and water ad libitum. All animal protocols were approved by the Health Sciences Animal Policy and Welfare Committee (University of Alberta) and are in accordance with the Guide to the Care and Use of Experimental Animals set forth by the Canadian Council on Animal Care.
Pharmacokinetics and Biodistribution. Pharmacokinetic and
biodistribution studies were carried out in either tumor-free mice or in mice
bearing murine mammary carcinoma (see below). The SLDXR was diluted in D5W,
and 200 µl was injected intravenously (i.v.) via the lateral tail vein. In
tumor-free mice, 4 doses of 9 mg/kg (27 mg/m2) SL-DXR were
administered either weekly (q1 week x 4), every 2 weeks (q2 weeks
x 4), or every 4 weeks (q4 weeks x 4) for a total dose of 36
mg/kg. The dose intensities for these schedules were 9 mg/kg/week (27
mg/m2/week), 4.5 mg/kg/week (13.5 mg/m2/week), and 2.25
mg/kg/week (6.75 mg/m2/week), respectively
(Freireich et al., 1966). In
experiments at the same dose intensity, tumor-bearing mice received 9
mg/kg/week (27 mg/m2/week) of SL-DXR using either 4 doses of 4.5
mg/kg (q3 day), 2 doses of 9 mg/kg (q1 week), or 1 dose of 18 mg/kg (q2
weeks). At various time points after each injection, mice were euthanized
(n = 4-5), blood was collected with a heparinized syringe, and plasma
was isolated by centrifugation (3,000g for 5 min). Organs were
removed and doxorubicin quantified as described below.
Pharmacokinetic parameters were calculated for total doxorubicin. The area under the concentration versus time curve (AUC) was calculated using the trapezoidal rule. Plasma half-lives (t1/2) were calculated using the formula: t1/2 = 0.693/kelm, where kelm is the elimination constant derived from the plasma concentration versus time curve. Tissue t1/2 was calculated in a similar manner, using the terminal slope of the tissue concentration versus time curve; t1/2 was not calculated for q3-day or q1-week dosing, as there were not sufficient time points in the terminal portion of the curves. For experiments using different dose intensities, the average steady state concentration (Css) was calculated by taking the fourth dose AUC (taken as steady state) as determined by the trapezoidal rule and dividing by the dose interval in hours.
Quantification of Doxorubicin. Total tissue doxorubicin was
quantified using a method similar to that of Mayer et al.
(1997). Briefly, tissue
homogenates of 10% w/v were prepared in water. Skin and paws were frozen in
liquid nitrogen and crushed with a mortar and pestle before homogenization
with a Polytron homogenizer (Brinkmann Instruments, Inc., Mississauga, ON,
Canada). Homogenates or 25% plasma (200 µl) was placed in a 2-ml
micro-centrifuge tube, and 100 µl of 10% (v/v) Triton X-100, 200 µl of
water, and 1,500 µl of acidified isopropanol (0.75 N HCl) were added. The
tubes were mixed thoroughly, and the doxorubicin and doxorubicin metabolites
(if any) were extracted overnight at -25°C. The next day, the tubes were
warmed to room temperature, vortexed for 5 min, centrifuged at
15,000g for 20 min, and stored at -80°C until analysis.
Doxorubicin was quantified fluorometrically (
excitation 470
nm and emission 590 nm). To correct for nonspecific
background fluorescence, the samples were analyzed using a standard curve
containing tissue extracts derived from drug-free mice. The data represent the
mean ± S.D. of triplicate aliquots from four to five mice and are
expressed as doxorubicin microequivalents per milliliter of plasma or per gram
of tissue, as this assay does not discriminate between doxorubicin and any
fluorescent metabolites that may have similar excitation and emission
profiles.
Tumor Implantation/Therapeutic Experiments. The 4T1 murine mammary
carcinoma was a generous gift from Dr. Fred Miller (Barbara Ann Karmanlos
Cancer Institute, Detroit, MI) and was maintained in minimal essential medium
supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml) at 37°C in a humidified incubator with a 5%
CO2 atmosphere (Aslakson and
Miller, 1992). Tumors were orthotopically implanted as previously
described (Moase et al.,
2001). Briefly, a small incision was made in the lower abdomen of
anesthetized mice, and 105 4T1 cells in 10 µl of supplemented
medium were implanted in the right no. 4 mammary fat pads. The incision was
closed with a surgical wound clip, which was removed 1 week later. For tissue
distribution studies in tumor-bearing mice, mice were injected with the chosen
dose of SL-DXR 10 days after tumor implantation when tumors were large enough
to excise. Studies were then performed as described above.
For therapeutic experiments, mice were treated 4 days after tumor implantation. Mice treated with different dose intensities received 9 mg/kg SL-DXR either q1 week x 2, q2 weeks x 2, or q4 weeks x 2. When the dose intensity was kept constant, mice received one dose of 18 mg/kg (54 mg/m2), 9 mg/kg (27 mg/m2) q1 week, or 4.5 mg/kg (13.5 mg/m2) q3 days for a total drug dose of 18 mg/kg. Tumor growth was monitored by measuring tumor diameters with calipers, and tumor volume was calculated using the formula: v = 0.4ab2, where a and b represent perpendicular diameters and a > b. The experiment was repeated once, and the data represent the mean ± S.D. from 5 to 10 mice except for the group receiving 18 mg/kg, where n = 4 to 5 (see Toxicity).
Statistical Analysis. Statistical comparisons were performed using a one-way ANOVA with a Tukey-Krammer post test or Student's t test (as appropriate) with Graph Pad InStat Version 3.01 for Windows 95/NT (GraphPad Software, Inc., San Diego CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Skin and paw drug concentrations for a dose schedule of q1 week x 4 are seen in Fig. 1, B and C, respectively. Similar to plasma, doxorubicin was not completely cleared from either tissue between doses. For the first three doses of SL-DXR, skin Cmax was reached 72 h postinjection (p < 0.001-0.05) and at 24 h after injection for the fourth dose. The nadir occurred at increasing drug levels with each subsequent dose. Skin AUCs increased 3-fold between the first and third doses and then appeared to reach steady state (Table 1).
The Cmax for total doxorubicin was reached in paws 72 h after the first dose but was earlier for subsequent doses (p < 0.01-0.001). Paws achieved higher drug concentrations than skin for the first two doses, as reflected in their higher AUC levels, but were similar to skin for the next two doses (Fig. 1, Table 1). The nadir drug levels for paws remained high throughout the study, and paw levels appeared to reach steady state after the first dose (the AUCs for paws did not change with subsequent doses). The higher drug levels in paws than in skin may be due to the pressure-dependent extravasation of liposomes as the mice walk around the cage, groom, feed, etc.
Doxorubicin levels in plasma, skin, and paws of mice receiving i.v. SL-DXR at a dose of 9 mg/kg q2 weeks are presented in Fig. 2. Extending the dose interval allowed plasma drug levels to fall to below detectable limits before the next dose of SL-DXR was given. As with the q1-week dosing schedule, plasma AUC values plateaued after the second dose (Table 1).
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Skin and paws reached Cmax for total doxorubicin for the q2 weeks x 3 schedule at approximately 72 h postinjection. Prolonging the dose interval allowed more drug to be cleared from the skin and paws, and the nadir drug levels were significantly lower than those reached for the q1-week dose schedule (p < 0.001 for skin and p < 0.01-0.001 for paws). Again, paw concentrations of doxorubicin were initially higher than those in skin. With subsequent doses, however, the skin Cmax increased (p < 0.05 for dose 1 versus doses 3 and 4), while unexpectedly the paw Cmax decreased significantly between the first and second doses (p < 0.05) and between the second and third doses (p < 0.01) (Table 1). These changes are also reflected in their respective AUC values (Table 1).
Figure 3 presents results for an i.v. dose schedule of 9 mg/kg SL-DXR q4 weeks x 4. Peak plasma levels were the same as for the previous two dosing schedules, and as was seen in mice receiving the q2 weeks x 4 dosing schedule, the longer dose interval resulted in plasma doxorubicin concentrations that were below detectable limits between doses. The t1/2 and AUC values were also similar to those for previous dosing schedules (Table 1).
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Skin and paw doxorubicin concentrations for this dose schedule are presented in Fig. 3, B and C, respectively. Again, the Cmax for total doxorubicin was achieved at approximately 72 h postinjection. For this dose schedule, the drug concentrations in both skin and paws fell to low levels before each successive injection. Skin Cmax and AUC values increased with each dose (Cmax, dose 1 versus dose 4, p < 0.05), whereas those for paws decreased, particularly between the first and subsequent doses (Cmax, p < 0.001 dose 1 versus dose 2) (Table 1).
The skin and paw clearance t1/2 values for doxorubicin are given in Table 1. Skin and paw t1/2 values could not be calculated for the q1-week dosing schedule. Modest increases in plasma t1/2 were observed for all dosing schedules from the first to fourth dose. Mice receiving 9 mg/kg q2 weeks or q4 weeks had greatly increased clearance t1/2 values for skin from the first to fourth dose. Mice receiving SL-DXR with a q4 weeks schedule had an increase in clearance t1/2 for paws, while skin t1/2 did not change appreciably. The average steady state drug concentration (Css) for each dose schedule was calculated by dividing the AUCss (fourth dose) by the dose interval in hours (Table 1). As expected, doubling the dose interval resulted in a halving of the Css values for all tissues.
Toxicity. PPE-like lesions were more frequent in mice receiving the
9 mg/kg q1 week x 4 dose schedule
(Table 2). The lesions included
hair loss on the mouse's muzzle (area exposed to pressure while the mouse
feeds) and red inflamed paws with mild swelling (the presence of lesions did
not, however, have an important effect on the weight of the paws; data not
shown). This is consistent with current clinical and laboratory data
demonstrating that PPE is more likely to occur with higher Doxil dose
intensities (Ranson et al.,
1997; Amantea et al.,
1999; Lotem et al.,
2000).
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During these experiments, some additional drug toxicity was observed, particularly for the weekly dose schedule. Four mice from the 9 mg/kg q1 week schedule were euthanized due to severe weight loss (three mice, no cause determined; 1 mouse, heart failure). Three mice from the 9 mg/kg q2 weeks schedule were euthanized (2 mice, no cause determined; 1 mouse, mild subacute cardiac and hepatic degeneration). In the 9 mg/kg q4 weeks group, 1 mouse was euthanized due to severe weight loss (no cause determined). The staff veterinary pathologist at the University of Alberta's Health Sciences Laboratory Animal Services performed all postmortem exams.
The total cumulative SL-DXR dose for these animals was high (36 mg/kg, 108 mg/m2). Since toxicity was encountered, mice in the therapeutic experiments received only two doses of SL-DXR (18 mg/kg, 54 mg/m2 total drug).
Pharmacokinetics for the Same Dose Intensity. To determine the effect of different dose schedules at the same dose intensity, we performed pharmacokinetic and biodistribution experiments in mice bearing the 4T1 murine mammary carcinoma and measured doxorubicin levels in plasma, tumor, skin, and paws. At 10 days postimplantation, when the tumors were well developed, mice were injected i.v. with a total dose of 18 mg/kg SL-DXR (54 mg/m2) given as either 4.5 mg/kg q3 days, 9 mg/kg q1 week x 2, or 18 mg/kg q2 weeks x 1. Tissue concentrations and pharmacokinetic parameters are given in Fig. 4 and Table 3.
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The results for plasma doxorubicin concentrations are presented in
Fig. 4A. For mice receiving 4.5
mg/kg q3 days, there was a significant increase in plasma
Cmax from the first dose to the second and subsequent
doses (p < 0.001). In addition, plasma levels appeared to reach
steady state after the second dose, as evidenced by the AUCs
(Table 3). As with naive mice,
in tumor-bearing mice receiving 9 mg/kg q1 week, there was detectable drug in
the plasma at 7 days after injection (Fig.
1A versus
4A). Interestingly, the plasma
t1/2 and AUC values were lower in tumor-bearing mice than
for naive mice receiving 9 mg/kg q1 week
(Table 3 versus
Fig. 1A). Distribution to the
tumor may account for the lower t1/2 and tissue AUC
values, which is consistent with results from studies using the C26 colon
carcinoma tumor model in BALB/c mice (Hong
et al., 1999).
A single dose of 18 mg/kg resulted in a plasma Cmax
approximately twice that of the first dose of the 9 mg/kg dose schedule and
approximately 4 times that of the first dose of the 4.5 mg/kg dose schedules
(Table 3). For each schedule,
there is also a linear relationship between the AUC of the first injection and
the dose (r2 = 0.9937). These observations are in line
with the dose independence of the plasma pharmacokinetics for single doses of
SL-DXR in this dose range (Allen and
Hansen, 1991; Gabizon et al.,
1994b).
Tumor levels of doxorubicin are given in
Fig. 4B. For mice receiving 18
mg/kg and for the first dose at the 4.5 or 9 mg/kg dose schedules, tumor
doxorubicin reached Cmax at 24 h, which was earlier than
skin and paw levels reached Cmax for the two higher doses.
The Cmax for the dose schedule was approximately double
that of the 9 mg/kg q1-week dose schedule and approximately 4-fold higher than
the Cmax for the 4.5 mg/kg q3-day dose schedule
(Fig. 4B), i.e., the
Cmax increased proportionately with dose. The tumor
AUC(0-
) values were similar for all
dose schedules (Table 3).
Skin drug levels from each of the dose schedules are seen in
Fig. 4C. Results for mice
receiving 9 mg/kg were similar to those for nontumor-bearing mice in that the
Cmax for skin doxorubicin in tumor-bearing mice peaked at
72 h postinjection. In addition, the Cmax and AUCs for the
second dose were higher than the first (p < 0.0008, t
test) (Table 1A versus
3; Fig.
1B versus
4C). As with tumor, the
doxorubicin Cmax in skin increased proportionately with
dose (Fig. 4C). The total
AUC(0-) values for the 18 mg/kg and 9
mg/kg dose schedules were similar, and higher than that seen for the 4.5 mg/kg
dose schedule (Table 3). These
results demonstrated that skin, like tumor, was exposed to sustained levels of
doxorubicin for all three dose schedules, although the 9 and 18 mg/kg
schedules resulted in exposure to higher drug concentrations.
As in naive mice, the paw concentrations of doxorubicin were higher than
skin concentrations in tumor-bearing mice (Fig.
4D versus
1C). For mice receiving 4.5
mg/kg (Fig. 4D), the
Cmax in paws continued to increase for 7 days after
initiation of therapy. The Cmax in paws also increased
proportionately with dose. The AUC(0-)
for the 18 mg/kg dose was higher than that for the 9 mg/kg x 2 dose
schedule, which in turn was higher than the AUC for the 4.5 mg/kg x 4
dose schedule (Table 3). The
increased paw AUC at higher doses may indicate a greater likelihood of
developing skin toxicities such as PPE at these doses. For mice receiving 18
mg/kg, the tumor, skin, and paw clearance t1/2 values were
117, 90, and 110 h, respectively. It is notable that the tissue
t1/2 values were considerably higher than those for plasma
t1/2 (Table
3). The values for skin and paws are consistent with results from
naïve mice receiving SL-DXR with different dose schedules.
Therapeutic Experiments. The results of therapeutic experiments in tumor-bearing mice receiving 9 mg/kg q1 week, q2 weeks, or q4 weeks by the i.v. route are presented in Fig. 5. Tumor volume can be difficult to measure when tumors exceed 400 mm3; however, tumor growth in control mice receiving sterile D5W was similar for all dose schedules. The therapeutic activities of SL-DXR were equivalent for mice receiving the drug for either a q1 week x 2 or a q2 weeks x 2 dose schedule. SL-DXR administered using a q4 weeks x 2 dose schedule appeared to have reduced therapeutic activity compared with the other two dose regimes. In other words, if the dose interval was too long, antitumor activity was affected adversely. This may have therapeutic implications, as clinical interventions for PPE include lengthening the dose interval or reducing the dose (i.e., reducing the dose intensity) to decrease the incidence and/or severity of PPE.
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The results for mice receiving the same dose intensity (9 mg/kg/week, 27
mg/m2/week) at different dosing schedules are presented in
Fig. 6. All three schedules
delayed tumor growth considerably. Nevertheless, the two dosing schedules with
larger doses given less frequently (9 mg/kg q1 week x 2 or18 mg/kg)
appeared to delay tumor growth to a greater extent than smaller doses given
more frequently. When this experiment was repeated, five mice had to be
euthanized due to toxicity in the group receiving 18 mg/kg. A gross postmortem
examination by the University of Alberta's Health Sciences Laboratory staff
veterinary pathologist found evidence of cardiac toxicity. This dose is well
below the reported LD50 of 38 mg/kg reported for a bolus injection
of SL-DXR in CD-1 mice (Working and Dayan,
1996). Whether this difference was because of strain-specific
differences in sensitivity to doxorubicin or was tumor-related was not
examined further. No further experiments were carried out with this dose.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Ranson et al., 1997). If our
murine results can be extrapolated to humans, then dose delay appears to be
useful in controlling PPE because it allows time for drug to be cleared from
the skin and for existing lesions to heal. As shown here, however, the
advantages of dose delay may be offset by reduced therapeutic activity.
A recent review of toxicities associated with Doxil in patients with
metastatic breast cancer provides support for our model
(Lyass et al., 2000). The
recommended dose intensity for these patients was 
12
mg/m2/week (e.g., 40-50 mg/m2 q4 weeks), and the average
plasma t1/2 was 79.4 h, corresponding to 8.5 times the
plasma t1/2 for SL-DXR administered every 4 weeks. Our
data mimic these clinical data in that a dose interval of q2 weeks (13.5
mg/m2/week) corresponds to 8.5 plasma t1/2 (the
average plasma t1/2 in naive mice was 39.4 h).
Interestingly, our experiments show that a dose of 13.5 mg/m2/week
resulted in good therapeutic efficacy combined with low levels of PPE-like
symptoms.
It is also important to note that the plasma t1/2 did
not change substantially for multiple doses of SL-DXR, although there was a
modest increase in t1/2 after the first dose for each
schedule. This is significant because the development of PPE has been
correlated to the plasma half-life of SL-DXR
(Lyass et al., 2000). If
SL-DXR is cytotoxic to cells of the mononuclear phagocyte system (MPS), which
is responsible for clearing liposomes, then multiple dose regimes could result
in extended t1/2 as a result of impaired clearance
mechanisms (Daemen et al.,
1995). We conclude that the dose schedules used in this study did
not impair MPS function to a degree that affected the pharmacokinetics of
SL-DXR. This lack of substantial MPS toxicity with SL-DXR is consistent with
studies from other laboratories (Storm et
al., 1998).
The observation that skin and paw pharmacokinetic parameters were different
from those for plasma is interesting. Plasma drug levels fell to low values
between doses for even a q1-week dose schedule, while skin and paws drug
levels remained elevated for several days. Plasma levels in mice have been
important for determining the dosing schedule for liposomal drugs in efficacy
studies, and a q1 week schedule is often chosen. This schedule is based on
clearance of inert liposomal markers such as 125I-tyraminylinulin
in naive mice (t1/2 of 18-24 h in liposomes of similar
composition to those used in these studies)
(Allen et al., 1993). Hence,
within 1 week (>8 half-lives), this marker would be cleared almost
completely from the plasma of mice. Nevertheless, the clearance rate of
doxorubicin is approximately 2-fold longer than the clearance rate of
125I-tyraminylinulin (an average 39 h in naive mice), and 8
half-lives, in this case, corresponds to one dose every 2 weeks. The
difference between the t1/2 of doxorubicin and
125I-tyraminylinulin liposomes reflects differences in release
rates and volumes of distribution of the two compounds. Furthermore, loading
doxorubicin into liposomes has been shown to increase their circulation times
in other models (Bally et al.,
1989). Regardless of the model, these data demonstrate that
pharmacokinetic studies that do not follow the pharmacologically active agent
should be interpreted with caution.
The half-life of SL-DXR was shorter in tumor-bearing mice than in naive
mice. This is consistent with work by Hong et al.
(1999), who found that the
t1/2 for SL-DXR was shorter in mice bearing subcutaneous
implants of the C26 colon carcinoma (19.1 h) than in naive mice (25.1 h). This
can partially be explained by significant distribution of drug-loaded
liposomes to tumors.
Our results show that half-lives for elimination of drug from skin, paws, and tumors were longer than that for plasma. A longer t1/2 will lead to retention of drug in tumors and, arguably, improved antitumor effects, but longer t1/2 values in cutaneous tissues will lead to unwanted side effects such as PPE. The challenge is to find the proper balance between minimizing PPE and maintaining therapeutic activity. Our studies show that increasing the dose interval to q2 weeks did not significantly affect the therapeutic outcome in our tumor model; however, extending the dose interval to q4 weeks compromised the therapeutic activity.
Skin concentrations of doxorubicin and their respective AUCs continued to
increase with each successive dose (Table
1). This may be a consequence of skin cytotoxicity accompanied by
inflammation. It is well known that inflamed tissue, like tumor tissue, has
increased capillary permeability and can accumulate liposomes via the enhanced
permeability and retention effect
(Matsumura and Maeda, 1986;
Maeda et al., 2000). This will
increase localization of liposomes into skin with subsequent injections in a
vicious cycle. Alternatively, since our dorsal skin samples were not subject
to pressure or irritation, the increased localization of liposomes into skin
may reflect an increase to steady-state levels, which normally occurs within
three to five doses. For drug clearance, an interval of five half-lives
results in approximately 3% of the total dose remaining in tissues. For skin,
five half-lives would be approximately 23 days, roughly corresponding to the
q4-week dosing interval that produced the lowest incidence of PPE-like
lesions.
One unexpected observation was the decrease in the Cmax for paws using the q2- and q4-week dose schedules. This decrease was not due to the alterations in the plasma pharmacokinetics (i.e., t1/2 values did not decrease). Therefore, fewer liposomes localized in paws. This may be a result of doxorubicin-associated tissue damage causing tissue remodeling or scarring, which would reduce the ability of subsequent doses to accumulate. Alternatively, it could be due to a reduction in the pressure-dependent extravasation of liposomes if mice developed "sore paws" (PPE-like lesions) and moved around their cages less, although this was not specifically measured.
The cytotoxicity of doxorubicin is not cell cycle-dependent; therefore, one can speculate that the antitumor activity of doxorubicin might be dependent upon tumor Cmax. Our therapeutic studies demonstrated that SL-DXR doses of 9 mg/kg q1 week or 18 mg/kg doxorubicin, which result in higher peak concentrations of total drug, had better therapeutic activity than smaller doses (4.5 mg/kg) given more frequently.
As previously observed (Charrois and
Allen, 2003) and as verified in these experiments, the 4T1 tumor
accumulates liposomes at a faster rate than either skin or paws. Therefore, it
may be possible to reduce the incidence of SL-DXR-associated cutaneous
toxicities by engineering a liposomal drug delivery system, e.g., a triggered
release system, that accumulates in tumors and releases its contents before
maximal liposome accumulation in skin or paws. This hypothesis is supported by
work by Needham et al. (2000),
who demonstrated improved therapeutic outcomes in tumor-bearing mice when
doxorubicin release was triggered by hyperthermia in single tumors (i.e., not
metastatic disease).
Our study measured total doxorubicin, which includes both
liposome-encapsulated and released drug. An important consideration in
pharmacokinetic, biodistribution, and therapeutic studies with liposomes is
the bioavailability of the drug. As long as the drug, e.g., doxorubicin,
remains encapsulated within the liposomes, it is not bioavailable and will
have no biological activity, including no antitumor effect. It is possible to
have high tissue AUCs for liposomal drugs, but low levels of efficacy, if the
drug is released very slowly since minimal therapeutic levels of drug in
tissues may not be reached. At the opposite end of the spectrum, if the drug
is released too rapidly, before the liposomes localize in target tissues, the
therapeutic effects may not be different from the administration of
nonencapsulated drug. To determine optimum drug release rates, it will be
necessary to develop methods for measuring bioavailable drug in the target
tissues and in the tissues that are subject to toxic side effects; this has
been a relatively neglected area of liposome research
(Krishna et al., 2001).
Nevertheless, several laboratories are developing methods to trigger the
release of liposomal contents once the liposomes have accumulated in target
tissues such as tumors (Adlakha-Hutcheon et
al., 1999; Kong et al.,
2000; Goldberg et al.,
2002).
In summary, these studies using a murine model reinforce the importance of dose schedule and dose intensity on the therapeutic activity and cutaneous toxicity of SL-DXR and provide the first experimental data on the pharmacokinetics and biodistribution of liposomal doxorubicin in tumor and cutaneous tissue for multiple dosing schedules. They also provide experimental evidence supporting the utility of a mouse model for predicting side effects and therapeutic activity in the clinic.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SL-DXR, pegylated (STEALTH) liposomal doxorubicin (Doxil/Caelyx); PPE, palmar-plantar erythrodysesthesia; D5W, dextrose 5% in sterile water; q, every; AUC, area under the plasma/tissue concentration versus time curve; MPS, mononuclear phagocyte system.
Address correspondence to: Theresa M. Allen, Department of Pharmacology, University of Alberta, -31 Medical Sciences Building, Edmonton, AB Canada T6G 2H7. E-mail: terry.allen{at}ualberta.ca
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,
18 mg/kg q2 weeks (1 dose); 
, 9 mg/kg q1 week (2 doses); 
, 4.5
mg/kg q3 days (4 doses).
