with clearance (P = 0.006). Pre-treatment monocyte counts increased over cycles (P < 0.001) and were associated with an increase in clearance at cycle 3 (P = 0.015). The hand–foot–skin syndrome was significantly more severe in patients of advanced age or longer PLD half-life.Conclusions This study showed (1) increased systemic drug exposure over subsequent cycles; (2) association of age with increased drug exposure, reduced DNA repair capability and worse skin toxicity; (3) a relation between monocyte count and drug clearance.
Keywords Liposomes · Doxorubicin · Pharmacokinetics · DNA damage · Monocytes · Age · Drug toxicity
Introduction
Encapsulation of doxorubicin in pegylated liposomes has enhanced drug delivery to tumor tissues, resulting in good efficacy and reduced toxicity profiles: these reasons pro-vide a strong rationale for considering pegylated liposo-mal doxorubicin (PLD) safe treatment for elderly patients [1–3]. However, recent studies have shown good response rates, but a remarkable incidence of side effects (grade 3–4 toxicities, unplanned hospital admissions and toxic deaths) in patients over 70 treated with PLD-adapted doses (−20 % compared with standard doses), suggesting overall poor feasibility in unselected elderly patients [4, 5].
Studies of PLD pharmacokinetics (PK) [6] show pro-tracted and elevated drug plasma levels, reduced exit from the blood compartment and low leakage from liposomes (<5–10 %). Plasma clearance (CL) was highly variable in patients, independently of body surface area (BSA) and inversely related to patients’ age, patients younger than 60 having PLD CL more than double that of older patients [7].
Abstract Purpose Pegylated liposomal doxorubicin (PLD) is often used in elderly people, due to its improved tolerability. However, clinical and pharmacological data in the subset of patients over 70 are scanty.Methods PLD safety was evaluated in 35 patients (aged ≥70 years) who were treated with PLD as a single agent for 165 cycles. Doxorubicin plasma levels, leukocyte DNA breaks and monocyte count variations were measured as markers of drug exposure, DNA repair capability and retic-uloendothelial system activation, respectively. A correlation between these markers and age was sought.Results Treatment was generally well tolerated. Skin erythrodysesthesia was the most frequent side effect, and no severe (G4) toxicity occurred. PLD plasma half-life generally correlated with age (P < 0.001) and was particu-larly prolonged in octogenarians (P = 0.005). Doxorubicin clearance significantly declined up to 70 % at cycle 7. DNA breaks increased over the first two cycles (P = 0.007) and were inversely correlated with age (P = 0.007) and directly
M. Gusella (*) · A. Bononi · Y. Modena · D. Menon · C. Barile · G. Crepaldi · A. Inno · F. Pasini Department of Oncology, ULSS 18-Rovigo, S Maria della Misericordia Hospital, Rovigo General Hospital, Viale Tre Martiri 140, 45100 Rovigo, Italye-mail: [email protected]
M. Gusella · L. Bertolaso · P. Franceschetti · E. Pezzolo · C. Bolzonella Laboratory of Pharmacology and Molecular Biology, Department of Oncology, ULSS 18-Rovigo, San Luca Hospital, Viale Grisetti 265, 45027 Trecenta, Rovigo, Italy
R. Padrini Clinical Pharmacology Section, Department of Medicine DIMED, University of Padova, Via Giustiniani 2, 35128 Padua, Italy
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To date, knowledge of PLD PK in the setting of patients over 70 and octogenarians is limited, but improving it may be of clinical importance, because PK parameters have been associated with treatment toxicities [8].
Despite low leakage from liposomes, as the persistent presence of doxorubicin in circulating blood cells has been shown [9], we studied DNA damage and repair capacity in peripheral leukocytes as surrogates of normal cells under PLD exposure.
Pre-treatment monocyte count has been associated with liposome CL [7]. Monocytes share a common ori-gin with macrophages and dendritic cells constituting the reticuloendothelial system (RES) and represent the main clearance pathway of PLD. RES engulfs drug-containing nanoparticles, but repeated administration of PLD pro-gressively reduces the elimination rate over the first three cycles, probably because of doxorubicin-induced damage to phagocyte cells [10].
Whether the reduction in RES activity continues beyond the third cycle and/or is influenced by age is unclear. Elderly patients have reduced DNA integrity and repair efficiency [11] and consequently may be more sensitive to doxorubicin injure.
Our study enrolled only patients over 70 and investi-gated the PK profiles of PLD administered as a single agent for more than three cycles and its relation with age and tox-icity. PLD effects on leukocyte DNA damage repair activity and monocyte count were also evaluated.
Methods
Patients were eligible if they were ≥70 years, had ECOG performance status (PS) ≤2 and clinical indication for monotherapy with PLD as adjuvant treatment or for locally advanced/metastatic tumors. Prior to treatment, patients were evaluated by comprehensive geriatric assessment (CGA) screening tests (aCGA, G8, VES13) [12].
Patients received PLD (40 mg/m2) intravenously over 1 h, every 4 weeks. This reduced dose, compared with standard schedules (50 mg/m2), was chosen on account of previous reports about its diffuse use in routine practice [13] and better tolerability not associated with efficacy loss [14].
Unless otherwise clinically indicated, response was eval-uated after 4 cycles and treatment was further continued until progression, toxicity or best clinical response.
A PK study was planned for at least the first 3 cycles. Blood samples were taken before infusion and at 1 h, 7 days, 14 and 21 days after the end of administration.
DNA repair activity in leukocytes (Comet assay; see below) was measured during the first two cycles.
Physical examination, hemogram, routine blood chemis-try and toxicity were assessed before treatment and weekly during the first four cycles, afterward monthly. Echocar-diography was performed before treatment and every 3–4 cycles. Toxic manifestations were graded according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE in www.ctep.cancer.gov/). The study was approved by the local Ethic Committee for clinical trials, and all patients provided their written informed consent.
Pharmacokinetic analysis
Plasma doxorubicin was measured by HPLC and fluori-metric detection, as previously described [15]. Total plasma concentrations were determined for two reasons: (1) All previous reports demonstrating a relationship between plasma PLD levels and clinical outcome did not distinguish intact PLD from PLD-released free doxorubicin [9, 16]; (2) free doxorubicin plasma levels, leaked out of liposomes, were demonstrated to be very low (<0.1–0.2 mg/L) [6], and thus unlikely to exert any clinically important effect.
Each time-course of PLD plasma concentrations was analyzed with the 1-compartment PK model with Graph-Pad Prism software. The following parameters were cal-culated: elimination rate constant (K), plasma half-life (T1/2 = 0.693/K) and peak plasma level (Cmax) as the drug concentration measured 1 h after infusion. The area under the drug concentration–time curve (AUC) was cal-culated as Cmax/K, plasma clearance normalized by body surface area (CL/BSA) was calculated as dose/AUC/m2, and the volume of distribution normalized by body surface area (VD/BSA) was calculated as dose/Cmax/m2.
Comet assay
The comet assay is a sensitive and reproducible method for detecting the presence of DNA single- or double-strand breaks produced by repair system activation under cyto-toxic drug exposure in clinical samples [17].
Single- and double-strand DNA breaks in peripheral leukocytes were determined by the alkaline version of the comet assay, following the procedure of Collins et al. [18]. Briefly, suspensions of 5 × 104 blood cells were embed-ded in 0.7 % agarose and allowed to gel on microscopic slides. A lysis solution was applied for 1 h, and electropho-resis for 20 min at 25 V and 300 mA with alkaline buffer (pH > 13) was performed. Slides were stained with ethid-ium bromide immediately before analysis through a fluo-rescence microscope. Image analysis was performed with dedicated IAS 2000 software, version 7.0 (Delta Sistemi, Rome). Healthy cells appeared as intact nuclei (undam-aged DNA was retained in the head region), whereas cells
with DNA breaks appeared as smaller nuclei with smears (broken DNA fragments migrating to the tail region after electrophoresis).
A value of >0.02 in the tail/head fluorescence ratio was used to define cells with significant DNA breaks. The num-ber of such cells was determined by examining at least 100 cells for each sample and expressed as a percentage.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism software (version 3.00 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com).
P values of <0.05 were considered significant. The paired t test, ANOVA for repeated measures, linear regres-sion and the chi-square test were used to compare PK parameters and to evaluate association between PKs and clinical observations. Data were expressed as median val-ues and ranges. The coefficient of variation of the means (CV) was used as a normalized measure of the dispersion of continuous parameters.
Monocyte counts were expressed as number of cells ×103/μl; comparisons between white blood cell counts were performed by transforming pre-treatment cell counts at cycle 3 in percentage of cycle 1, for each patient.
Results
Thirty-five patients received PLD for locally advanced/metastatic (n = 31) or adjuvant treatment (n = 4) of solid cancers. The characteristics of the study population are listed in Table 1. No patient presented clinical evidence of ascites or peritoneal exudate. Most patients had a PS score of 1 (63 %). According to the results of CGA screening, patients were classified as frail (40 %) or not frail (51 %); 30 and 48 % had 2 or 3 comorbidities, respectively, con-sisting mainly of arterial hypertension and diabetes. Eleven patients were chemotherapy-naïve, 4 had received only hormonal treatment, and 18 had been pre-treated with chemotherapy (4 cases including anthracyclines). For two patients, previous treatment was unknown.
Clinical data
Overall, 165 cycles (median 4, range 1–10) were adminis-tered; in the first 4 cycles, 123 cycles out of the initially planned 140 (88 %) were administered, at a relative dose intensity (RDI) of 88.4 %. The RDI of cycles 5–7 was 74.5 %. Three patients received 1 cycle and 2 patients 2 cycles and then stopped therapy due to deteriorating PS (n = 4) or allergic reaction (n = 1). Four patients stopped chemotherapy after 3 cycles, due to progressive disease
(n = 2) or toxicity (n = 2; hand–foot syndrome G3 and thrombocytopenia G3); 13 patients interrupted after 4 cycles, because of completion of planned therapy (n = 4) or progression (n = 9); 13 patients continued treatment for more than 4 cycles (1 patient 5 cycles, 4 patients 6 cycles, 3 patients 7 cycles, 2 patients 8 cycles, 2 patients 9 cycles, 1 patient 10 cycles) up to progression (n = 3), completion of therapy (n = 4) or toxicity (n = 6); this last appeared mainly between cycles 6 and 8 and consisted of hand–foot syndrome (HFS) G2–3 (n = 3), fever of unknown origin (n = 1), mucositis G3 (n = 1) or fatigue G3 (n = 1). There was no evidence of reduction in the left ventricular ejection fraction with echocardiographic monitoring.
PK results
PK was analyzed in 96 cycles (median 3, range 1–7) (Fig. 1). Following the first administration, median plasma concentrations progressively dropped from a post-infusion value of 23.2 mg/L (range 15.2–33.6) to 0.05 mg/L (range 0.0–0.56) at day 28. First-cycle pharmacokinetic param-eters showed wide inter-patient variability. Median val-ues and ranges (in brackets) were as follows: (1) plasma T1/2 = 82.9 h (15.1–132.5); (2) AUC = 2,282 mg h/L (332–4,712); (3) CL = 14.5 mL/h/m2 (7.7–117.3); (4) VD = 1.7 L/m2 (1.2–2.6).
Twenty-eight patients received 3 cycles without dose reduction; of these, 16 completed PK analysis (Fig. 1). In
these patients, in the first 3 cycles, Cmax concentrations and VD remained stable, while median T1/2 increased by 13.5 % (95.5 vs 86.4 h, P = 0.03; range –13 to +52 %) and AUC by 21 % (3,462 vs 2,855 mg h/L, P = 0.007; range −10 to +59 %); accordingly, CL/m2 decreased by 16 % (12.4 vs 14.8 mL/h/m2, P = 0.002; range −37 to +11 %).
Six patients were studied beyond the third cycle for a total of 13 cycles; the data showed a progressive and
significant decrease in the CL (−30 %) and increase in T1/2 (+30 %) and AUC (+48 %) at cycle 7, compared with basal values.
PK association with age
Age was associated with a reduced elimination rate: over-all, a linear correlation was found between plasma T1/2 and age (r = 0.44, P < 0.001). The mean T1/2 of the first 3 cycles was 30 % longer in patients aged >77 years than in those aged ≤77 (93.7 vs 72 h, P = 0.02); at any sampling time considered, drug plasma concentrations were at least twofold higher in patients over 77 than in those under 77 (Table 2).
In the subset of octogenarians, these findings were even more evident: T1/2 was +42 % (106 vs 74.9 h, P = 0.005), AUC +43 % (3,840 vs 2,664 mg h/L, P = 0.037) and CL/m2 −48 % (11.3 vs 21.7 mL/h/m2, P = 0.045) than that of younger patients (Table 2).
DNA breaks and monocyte counts
The comet assay was performed in 15 patients weekly over cycles 1 and 2. There was a considerable inter-individual difference in the percentage of circulating leukocytes with DNA breaks between pre- and inter-cycle samples. At cycle 1, the median basal level of DNA breaks was 22 (range 14–52), 42 (range 20–58, P = 0.007) after 1 week, 33 after 2 weeks and 30 after 3 weeks from drug infusion and returned to pre-therapy values after 4 weeks. Similar findings were observed at cycle 2 (Fig. 2a). There was an inverse association between age and the global mean num-ber of post-infusion breaks (r2 = 0.54; P = 0.007) (Fig. 2b). This finding was particularly evident in patients >80, who had fewer DNA breaks than those ≤80 (31 ± 5 vs 43 ± 10, respectively, P = 0.04). DNA breaks were also associated with PLD CL: patients with CL below the median value had fewer circulating damaged cells than patients with higher CL values (31.8 ± 5.1 vs 42.0 ± 10.5 mL/h/m2, respectively, P = 0.006) (Fig. 2c).
0 1 2 3 4 5 6 70
2500
5000
7500
p=0.0007
Cycles
PL
D A
UC
(m
g x
h/L
)
0 1 2 3 4 5 6 70
25
50
Cycles
PL
D C
L (
ml/h
/m2 )
p=0.006
a
b
Fig. 1 PLD AUC (a) and CL (b): gradual changes with regular cycles
Table 2 PLD plasma levels according to median age (77 years) and 80-year cutoff
a Peak plasma levelb Plasma levels reported as percentage (%) of Cmax value
Pre-treatment monocyte counts (×103/μL) significantly increased over cycles: at cycles 1, 2 and 3, pre-treatment counts were 0.41 ± 0.13, 0.51 ± 0.22 and 0.53 ± 0.20, respectively (ANOVA P = 0.0001; post-test for linear trend P < 0.0004), with a mean increase from baseline to
cycle 3 of +24 ± 28 %. Instead, granulocyte and lym-phocyte counts did not significantly change from baseline (−3.0 ± 47, +1.0 ± 25 %, respectively). Accordingly, sig-nificant differences were found between monocytes ver-sus granulocytes (124 ± 28 vs 97 ± 47 %, respectively,
0 1 2 3 4 5 6 7 80
10
20
30
40
Weeks
**
***
*
*
Blo
od
cel
l DN
A b
reak
s **
60 70 80 90 1000
25
50
75
p=0.007
Age (years)
Inte
rcyc
le D
NA
bre
aks
< median >median 0
25
50
75
p=0.006
CL/m2
Inte
rcyc
le D
NA
bre
aks
a
b
c
Fig. 2 Comet assay results as function of time of chemotherapy (a), age (b) and PLD clearance (c). Arrow PLD administration. *P < 0.05; **P < 0.01
Fig. 3 Monocyte count association with PLD CL at cycle 3
G0 G1 G2-30
50
100
150
PPE grade
PL
D t
1/2
(h)
ANOVA p=0.021Trend p=0.007
G0 G1 G2-360
70
80
90
PPE grade
Ag
e (y
ears
)
ANOVA p=0.03Trend p <0.001
a
b
Fig. 4 HFS (also called PPE) association with PLD T1/2 (a) and age (b)
522 Cancer Chemother Pharmacol (2014) 73:517–524
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P = 0.0006) and monocytes versus lymphocytes (124 ± 28 vs 101 ± 25 %, respectively, P = 0.0009).
A significant positive association between pre-treatment monocyte counts and PLD CL was found only at the third course of treatment (r2 = 0.35, P = 0.015) (Fig. 3).
Association between skin toxicity, PK and age
HFS was a frequent side effect. Grades of HFS toxic-ity were as follows: no toxicity: n = 13, G1: n = 8, G2: n = 6, G3: n = 3, not evaluable due to early termination of treatment: n = 5. Patients with G2 and G3 toxicities were grouped, because the clinical difference is sometimes not easy to classify.
Increased T1/2 (up to 38 %) was associated with worse HFS (Fig. 4a): T1/2 was 104.1 ± 22.7 h in patients with G2–3 HFS, 90.7 ± 14.3 in G1 and 75.5 ± 22.2 in the absence of toxicity (ANOVA P = 0.02; linear trend P = 0.007).
Similarly, increased AUC was associated with worse HFS: Higher AUC (4,098 ± 1,268 mg/h/L) was associ-ated with G2–3 HFS; lower AUC values (3,462 ± 906 and 2,585 ± 1,014 mg h/L) were associated with G1 and no toxicity, respectively (ANOVA P = 0.006; linear trend P = 0.02).
CL was 10.4 ± 3.7 mL/h/m2 in G2–3, 11.6 ± 2.5 in G1 and 19.6 ± 15.8 in the absence of toxicity (ANOVA P = 0.03).
HFS was also associated with age (Fig. 4b): Patients without skin manifestations had a mean age of 75.1 ± 3.2 compared with patients suffering from G1 or G2–3 toxicity, who were 77.7 ± 5.7 and 80.4 ± 3.4 years old, respectively (ANOVA P = 0.003; linear trend P < 0.001). It should be noted that all the octogenarians complained of some degree of skin toxicity.
Discussion
This study evaluated for the first time the PK of PLD given as a single agent, over repeated administrations, in patients >70 years old and its correlation with age and toxicity. We also assessed nuclear DNA breaks in peripheral blood leu-kocytes as markers of individual drug-induced DNA dam-age repair activity and monocyte count variations as surro-gate markers of drug-induced RES reactivity.
In previous reports, with doses in the range 35–70 mg/m2, mean PLD half-life was 36 h in pediatric patients [19] and about 60–90 h in middle-aged patients (range 50–65 years) [6].
In our study, PLD plasma half-life in cycle 1 was within this range (median = 79.6 h). However, T1/2 was
significantly longer in patients over 77 (+30 %) and over 80 (+42 %), compared with younger patients.
PLD plasma concentrations were influenced by age, as well: although peak values at the end of drug infusion were similar, the following weekly levels were at least double in the subset of patients over 77.
This phenomenon was more evident in the subset of octogenarians, who presented significantly longer T1/2, higher AUC and lower CL (Table 2).
La-Beck et al. [7] reported CL values of 54.6 ± 28.5 mL/h/m2 in patients <60 years at cycle 1, while in the older group (aged between 60 and 77), mean CL was 20.8 ± 9.4 mL/h/m2 (CV % 45.2) in women and 27.4 ± 12.5 (CV % 45.6) in men. In our series, the mean PLD CL of the 29 women was 18.9 ± 20.6 mL/h/m2 (CV % 109.2) and 27.3 ± 27.4 mL/h/m2 (CV % 100.3) in 6 men (data not shown). Mean CL values were therefore sim-ilar to those of La-Beck et al. [7], whereas the CVs were at least double, revealing wider inter-individual variability.
Recently, Gabizon et al. [10] reported a 33 % reduction in PDL CL (from 24 mL/h to 16 mL/h) from cycle 1 to cycle 3 in 12 patients with a median age of 61 (range 33–78). We confirmed the reduction in CL over the first three cycles (−16 %) and demonstrated a further CL decline continuing up to cycle 7 (−30 %). The CL decrease was slower in our study than previously reported [10]: as the PLD doses were comparable, the inconsistency was prob-ably due to age differences in patient selection. As a con-sequence of CL reduction over subsequent cycles, our PK data also showed prolonged T1/2 and increased AUC, up to +30 and +48 %, respectively (Fig. 1).
From a clinical point of view, age was associated with skin toxicity. In general, older patients suffered from a sig-nificantly greater degree of HFS, all octogenarians com-plaining of HFS, probably in relation to their more pro-longed T1/2 and lower CL (Fig. 4) .
On the whole, treatment was tolerated without G4 tox-icities. During the first 4 cycles, the RDI was 88.4 %, and only 8.5 % of patients interrupted treatment due to toxic-ity. In cycles 5–7, the RDI fell to 74.5 %, partly because of delays in re-staging procedures and partly late toxicity; nevertheless, a not negligible proportion of patients (8/13) continued therapy beyond cycle 6, showing that prolonged treatment is feasible in one subset of patients.
Chemotherapy was stopped, mostly for prudential rea-sons, considering the high risk of side effects in the elderly. The finding of a progressive increase in AUC (up to 48 % in this study) supports this approach, because more intense drug exposure may predispose to a higher risk of toxicity [8]. For reasons which are unclear, this study did not detect any difference in PK parameters in frail compared with fit patients (data not shown).
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Toxicities observed in our population were generally consistent with those of other reports (3, 14), supporting the tolerability of this PLD schedule.
Instead, other studies [4, 5] reported higher G 3–4 hema-tological and non-hematological toxicities (neutropenia 8.4 %, HFS 13.6 % and infection 18.6 %) and even pos-sible treatment-related deaths. Falandry et al. [5] concluded that PLD feasibility was poor in elderly unselected patients. Cross-comparisons among these studies are problematic, since our research aimed mainly at PK analysis, while the others were phase II clinical trials. In a clinical setting, elderly patients may present different tolerance and com-pliance, and treating physicians, outside trials, may have variable attitudes about continuing treatment. Another con-founding factor is due to different patient selection, particu-larly critical in elderly patients because of previous treat-ments, comorbidities and fragility.
To our knowledge, this is the first study to document in vivo DNA breaks in leukocytes during PLD exposure and their correlation with age and drug CL (Fig. 2).
The mechanisms by means of which doxorubicin pro-duces DNA damage are not yet fully understood. How-ever, at clinically important drug concentrations, they are most likely represented by nucleotide oxidation (recog-nized by the base excision repair system), intercalation/interstrand cross-links/adduct formation (recognized by the nucleotide excision repair system) and inhibition of topoi-somerase II (by stabilization of an intermediate reaction in which DNA strands have been already cut and DNA reseal-ing is impeded) [20]. In all these cases, doxorubicin does not directly induce fragmentation of DNA strands, but, as breaks depend on DNA repair and supercoiling regulation system activities, the total number of breaks detected by the comet assay during doxorubicin exposure may be seen as a marker of activity and of the integrity of the DNA repair and maintenance machinery [17].
We observed that, during inter-cycle periods, DNA alter-ations increased significantly over the basal level, peaked in the first week post-infusion (1.5-fold), decreased in paral-lel with PLD plasma decay in the following weeks and had returned to base level by day 28, which is the day sched-uled in the clinical setting for recycling.
In our study, comet test scores were inversely associated with age (P = 0.007) (Fig. 2b) and the subset of octoge-narians in particular presented a significantly reduced num-ber of break-positive peripheral blood leukocytes. These data match the reduced DNA repair capability demon-strated in the elderly [11, 22, 23], although apoptosis and/or removal of damaged leukocytes from circulation can-not be excluded. In addition, lower PLD CL values were associated with fewer DNA breaks (Fig. 2c). This finding may have two explanations: lower CL values are inherently associated with age, or the reduced DNA repair capacity
observed in leukocytes may be similar in the healthy tis-sues involved in drug catabolism (SRE), thus inducing higher susceptibility to drug injury and negative effects on drug elimination rate.
The reduction in DNA breaks observed during inter-cycle periods may be interpreted as completed repair activ-ity or inhibition of repair. One in vitro study demonstrated down-regulation of DNA repair enzyme expression under continuous doxorubicin exposure [21]. If this is so, DNA damage accumulation may be responsible for functional impairment of the cells involved in PLD elimination, favor-ing reduced drug CL in subsequent cycles. In any case, further studies are needed to clarify whether DNA repair activity plays a role in PLD CL inter- and intra-subject variability.
It has recently been reported that there is a borderline correlation (P = 0.09) between changes in pre-treatment monocyte counts and PLD CL, measured before cycles 1 and 3 [7]. With a larger number of hemogram determina-tions, we found a significant association between pre-treat-ment monocyte count and CL (P = 0.015) only at cycle 3 (Fig. 3): at that time, most of our patients had increased monocytes (corresponding to a median of +30 % compared with baseline). This increase did not appear to be related to hematological recovery after chemotherapy, since it was independent of granulocyte and lymphocyte counts, which never significantly increased over basal values. Because monocytes belong to the family of macrophagic cells (the principal effectors of the systemic elimination of circulat-ing liposomes), their increase suggests RES activation as a reaction to PLD administration. Fitting this hypothesis, it has been demonstrated in an animal models that empty pegylated liposomes undergo accelerated blood clearance after two injections at several days’ interval, because the second dose accumulates rapidly in Kupffer cells, as if the protective effect of pegylated envelope against macrophage uptake was reduced [24]. Our observation that RES activa-tion was not accompanied by an increase but, on the con-trary, by a reduction in CL, may be due to the toxic content of liposomes. Accelerated uptake induced by repeated PLD administration may generate doxorubicin overload in RES and give rise to a poisoning effect, resulting in reduced plasma CL. The association between the number of circu-lating monocytes and PLD elimination, observable at cycle 3, may be related to the inter-individual variable capacity of RES to cope with toxic insults. Whether monocyte count is a surrogate marker predicting PLD clearance requires fur-ther investigations and is now a field of ongoing research in our institution.
In conclusion, this study involving only patients over 70 showed: (1) increased systemic exposure to drug over subsequent cycles (increased T1/2 and AUC and reduced CL); (2) an association of age with increased drug systemic
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exposure, reduced DNA repair and more severe hand–foot syndrome; (3) the possible relation of monocyte count with drug-induced RES reactivity.
Acknowledgments The authors would like to thank Dr. Eros Fer-razzi for critical review of the paper and the nurses at the Oncology Unit of Rovigo General Hospital for caring for the patients in this trial. This research was partly funded by CARIPARO Foundation, Italy.
Conflict of interest None.
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