Alberta Locatelli

Human pharmacokinetic characterization and in vitro study of the interaction between doxorubicin and paclitaxel in patients with breast cancer.

PURPOSE We performed a pharmacologic investigation of paclitaxel (PTX) infused over 3 hours and bolus doxorubicin (DOX) to assess the role of sequence, interval between drugs, and duration of doxorubicin infusion on paclitaxel and anthracycline plasma disposition. We also explored possible mechanisms of pharmacokinetic interference involving the physiologic role of the multidrug resistance phenotype in anthracycline and taxane biliary excretion. PATIENTS AND METHODS Pharmacokinetics was performed in 80 cycles and 36 women with previously untreated metastatic breast cancer. PTX, DOX, and their metabolites 6 alpha-hydroxyl-PTX (6 alpha OH-PTX) and doxorubicinol (DOL) were measured by high-pressure liquid chromatography (HPLC). Human breast cancer MCF-7 wild-type (WT) and resistant (TH) cell lines were cultured in whole human plasma to study anthracycline retention after treatment with different combinations of PTX, Cremophor EL (CEL) (PEG35 castor oil; BASF, Parsippany, NJ), and DOX. RESULTS Pharmacokinetic interference between PTX and DOX was responsible for nonlinearity of DOX plasma disposition and increased concentrations of DOX and DOL. These effects were PTX dose-dependent, DOX concentration-dependent, and likely a result of interference at the level of liver elimination. In view of the physiologic role of P-glycoproteins (P-gp) in xenobiotic biliary excretion, retention of DOX was assessed in MCF-7 WT and MCF-7 TH cells. Intracellular was significantly higher in MCF-7 WT than MCF-7 TH (P < .05). However, concomitant exposure to DOX, PTX, and CEL caused similar DOX retention in both MCF-7 WT and TH cells. CONCLUSION PTX, as clinically formulated in CEL, is responsible for a nonlinear disposition of DOX and DOL. Nonlinearity is PTX- and DOX-dependent, and possibly caused by competition for biliary excretion of taxanes and anthracyclines mediated by P-gp. Nonlinearity indicates that even minor modifications of dose and infusion duration of DOX and PTX may lead to unpredictable pharmacodynamic consequences. The postulated role of P-gp suggests that CEL is clinically active, and advises caution in designing combinations of PTX with other drugs that are substrate for P-gp.

Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel

Purpose: To retrospectively evaluate the effects of six known allelic variants in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes on the pharmacokinetics of the anticancer agent paclitaxel (Taxol). Experimental Design: A cohort of 97 Caucasian patients with cancer (median age, 57 years) received paclitaxel as an i.v. infusion (dose range, 80-225 mg/m2). Genomic DNA was analyzed using PCR RFLP or using Pyrosequencing. Pharmacokinetic variables for unbound paclitaxel were estimated using nonlinear mixed effect modeling. The effects of genotypes on typical value of clearance were evaluated with the likelihood ratio test within NONMEM. In addition, relations between genotype and individual pharmacokinetic variable estimates were evaluated with one-way ANOVA. Results: The allele frequencies for the CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP3A4*3, CYP3A5*3C, and ABCB1 3435C>T variants were 0.7%, 9.2%, 2.1%, 0.5%, 93.2%, and 47.1%, respectively, and all were in Hardy-Weinberg equilibrium. The population typical value of clearance of unbound paclitaxel was 301 L/h (individual clearance range, 83.7-1055 L/h). The CYP2C8 or CYP3A4/5 genotypes were not statistically significantly associated with unbound clearance of paclitaxel. Likewise, no statistically significant association was observed between the ABCB1 3435C>T variant and any of the studied pharmacokinetic variables. Conclusions: This study indicates that the presently evaluated variant alleles in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes do not explain the substantial interindividual variability in paclitaxel pharmacokinetics.

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (‘Iressa’) is independent of EGFR expression level†

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR‐TKI) ZD1839 (‘Iressa’) has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]‐thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity. J. Cell. Physiol. 198: 259–268, 2004© 2003 Wiley‐Liss, Inc.

Targeting TRAIL Agonistic Receptors for Cancer Therapy

Based on preclinical studies demonstrating that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) exerts a potent and cancer cell–specific proapoptotic activity, recombinant TRAIL as well as agonistic anti–TRAIL-R1 and anti–TRAIL-R2 antibodies recently entered clinical trials. Additionally, gene therapy approaches using TRAIL-encoding adenovirus (Ad-TRAIL) are currently being developed to overcome the limitations inherent to TRAIL receptor targeting, i.e., pharmacokinetic of soluble TRAIL, pattern of receptor expression, and tumor cell resistance. To optimize gene therapy approaches, CD34+ cells transduced with Ad-TRAIL (CD34-TRAIL+) have been investigated as cellular vehicles for TRAIL delivery. Transduced cells exhibit a potent tumor killing activity on a variety of tumor cell types both in vitro and in vivo and are also cytotoxic against tumor cells resistant to soluble TRAIL. Studies in tumor-bearing nonobese diabetic/severe combined immunodeficient mice suggest that the antitumor effect of CD34-TRAIL+ cells is mediated by both direct tumor cell killing due to apoptosis and indirect tumor cell killing due to vascular-disrupting mechanisms. The clinical translation of cell and gene therapy approaches represent a challenging strategy that might achieve systemic tumor targeting and increased intratumor delivery of the therapeutic agent.

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

Background Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. Methods and Principal Findings Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. Conclusions These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) with weekly paclitaxel

BACKGROUND The additive cytotoxicity in vitro prompted a clinical study evaluating the non-prodrug rapamycin analogue ridaforolimus (AP23573; MK-8669; formerly deforolimus) administered i.v. combined with paclitaxel (PTX; Taxol). MATERIALS AND METHODS Patients with taxane-sensitive solid tumors were eligible. The main dose escalation foresaw 50% ridaforolimus increments from 25 mg with a fixed PTX dose of 80 mg/m(2), both given weekly 3 weeks in a 4-week cycle. Collateral levels with a lower dose of either drug were planned upon achievement of the maximum tolerated dose in the main escalation. Pharmacodynamic studies in plasma, peripheral blood mononuclear cells (PBMCs) and skin biopsies and pharmacokinetic (PK) interaction studies at cycles 1 and 2 were carried out. RESULTS Two recommended doses were determined: 37.5 mg ridaforolimus/60 mg/m(2) PTX and 12.5 mg/80 mg/m(2). Most frequent toxic effects were mouth sores (79%), anemia (79%), fatigue (59%), neutropenia (55%) and dermatitis (48%). Two partial responses were observed in pharyngeal squamous cell and pancreatic carcinoma. Eight patients achieved stable disease > or =4 months. No drug interaction emerged from PK studies. Decrease of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) phosphorylation was shown in PBMCs. Similar inhibition of phosphorylation of 4E-BP1 and mitogen-activated protein kinase was present in reparative epidermis and vascular tissues, respectively. CONCLUSION Potential antiangiogenic effects and encouraging antitumor activity justify further development of the combination.

Clinical and pharmacological phase I evaluation of Exherin™ (ADH-1), a selective anti-N-cadherin peptide in patients with N-cadherin-expressing solid tumours

BACKGROUND Upregulation of N-cadherin promotes dysregulated cell growth, motility, invasiveness, plus maintenance of vascular stability and is associated with cancer progression in several human tumour types. N-cadherin is expressed also on tumour cells and the anti-N-cadherin cyclic pentapeptide ADH-1, tested in the present study, can exert a direct antitumour effect. PATIENTS AND METHODS Adult patients with advanced solid malignancies expressing N-cadherin on tumour biopsies carried out in the previous 12 months received escalating i.v. doses of ADH-1 given weekly (initially for 3 of 4 weeks, then every week). Plasma pharmacokinetics (PK) was studied at cycle 1. Blood flow changes were assessed after first dosing in all patients treated in the initial regimen. RESULTS In all, 129 patients were screened, 65 (50%) were N-cadherin positive, and 30 were enrolled. The doses ranged from 150 to 2400 mg/m(2); no maximum tolerated dose was reached. Treatment was well tolerated with asthenia as the most frequent adverse event. Two patients with ovarian cancer showed prolonged disease stabilisation while one patient with fallopian tube carcinoma achieved a mixed response. PK was linear in the range of doses tested. CONCLUSION ADH-1 is the first anti-N-cadherin compound tested in humans. In N-cadherin-positive patients, ADH-1 showed an acceptable toxicity profile, linear PK and hints of antitumour activity in gynaecological cancers.

Phase IB Study of the mTOR Inhibitor Ridaforolimus With Capecitabine

PURPOSE Synergistic/additive cytotoxicity in tumor models and widespread applicability of fluoropyrimidines in solid tumors prompted the study of the combination of the mammalian target of rapamycin (mTOR) inhibitor, non-prodrug rapamycin analog ridaforolimus, with capecitabine. PATIENTS AND METHODS Thirty-two adult patients were treated. Intravenous ridaforolimus was given once weekly for 3 weeks and capecitabine was given from days 1 to 14 every 4 weeks. Ridaforolimus was given at 25, 37.5, 50, or 75 mg with capecitabine at 1,650 mg/m(2) or 1,800 mg/m(2) divided into two daily doses. Pharmacokinetics of both drugs were determined during cycles 1 and 2. Pharmacodynamic studies in peripheral blood mononuclear cells (PBMCs) and wound tissue of the skin characterized pathways associated with the metabolism or disposition of fluoropyrimidines and mTOR and ERK signaling. RESULTS Two recommended doses (RDs) were defined: 75 mg ridaforolimus/1,650 mg/m(2) capecitabine and 50 mg ridaforolimus/1,800 mg/m(2) capecitabine. Dose-limiting toxicities were stomatitis and skin rash. One patient achieved a partial response lasting 10 months and 10 of 29 evaluable patients had stable disease for ≥ 6 months. The only pharmacokinetic interaction was a ridaforolimus-induced increase in plasma exposure to fluorouracil. PBMC data suggested that prolonged exposure to capecitabine reduced the ridaforolimus inhibition of mTOR. Ridaforolimus influenced the metabolism of fluoropyrimidines and inhibited dihydropyrimidine dehydrogenase, behavior similar to that of rapamycin. Inhibition of the target thymidylate synthase by capecitabine was unaffected. mTOR and ERK signaling was inhibited in proliferating endothelial cells and was more pronounced at the RD with the larger amount of ridaforolimus. CONCLUSION Good tolerability, feasibility of prolonged treatment, antitumor activity, and favorable pharmacologic profile support further investigation of this combination.

Clinical and Pharmacologic Study of the Epirubicin and Paclitaxel Combination in Women With Metastatic Breast Cancer

PURPOSE A pharmacokinetic interaction may cause increased cardiotoxicity of paclitaxel (PTX) and high cumulative dose of doxorubicin. We tested antitumor activity, tolerability, and pharmacokinetics of the lesser cardiotoxic epirubicin (EPI) and PTX (ET combination). PATIENTS AND METHODS Twenty-seven women with untreated metastatic breast cancer, median age of 56 years, and prominent visceral involvement (74%) were studied. Three-weekly EPI (90 mg/m(2)) and PTX (200 mg/m(2) over 3 hours) were given for a maximum nine cycles. EPI was administered 24 hours before PTX (E --> T) in cycle 1, and 15 minutes before PTX (ET) thereafter. EPI, epirubicinol (EOL), EPI-glucuronide (EPI-glu), EOL-glucuronide (EOL-glu), PTX, and 6alpha-OH-PTX were measured in plasma and urine in 14 women. RESULTS Patients received 205 cycles of ET and a median EPI dose of 720 mg/m(2). Grade 4 neutropenia (49% of cycles) was the most frequent toxicity. Cardiac contractility was decreased in five patients. Mild congestive heart failure occurred in two (7.4%). Response rate was 76% (28% complete). Median overall survival was 29 months. On the basis of intrapatient comparison in the first 24 hours of E --> T and ET cycles, PTX did not affect EPI disposition, but significantly increased plasma exposure to EOL (by 137%), EPI-glu (threefold) and EOL-glu (twofold). Urinary excretion of EPI dose went from 8.2% in E --> T to 11.8% in ET cycles. Clearance of PTX was 30% slower in ET than E --> T. ET cycles caused lower neutrophil nadir than E --> T (644 +/- 327 v 195 +/- 91, P <.05) CONCLUSION ET is feasible, devoid of excessive cardiac toxicity, and active. A reciprocal pharmacokinetic interference between the two drugs has pharmacodynamic consequences, and suggests a direct effect of PTX on EPI metabolism requiring ad hoc investigation.

Clinical pharmacokinetics of the new oral camptothecin gimatecan: The inter-patient variability is related to α1-acid glycoprotein plasma levels

AIM OF THE STUDY To determine the pharmacokinetics of gimatecan, a camptothecin with a lipophilic substitution in position 7, given orally to patients participating in the phase I study. METHODS Pharmacokinetics was evaluated in 78 patients after oral daily dose for 5 days a week for 1, 2 or 3 weeks by HPLC with a fluorescence detector. RESULTS Gimatecan was mainly present in plasma as lactone (>85%), the active form as DNA-topoisomerase I poison. The AUC(0-24) on the first day of treatment normalised per daily dose (mg/m(2)), ranged from 194 to 2909 ng h/mL/mg/m(2). The half-life was 77.1+/-29.6h, consequently C(max) and AUC rose 3-6-fold after multiple dosing. Multivariate analysis indicated the daily dose (p<0.0001) and the alpha(1)-acid glycoprotein (AGP) plasma levels (p<0.0001) as main predictors of gimatecan AUC(0-24). In the overall analysis, daily dose and AGP plasma levels explained 85% of the deviance. The hydroxy metabolite ST1698 was present in plasma at low levels with AUC values of 5-15% of gimatecan. In mice, orally treated with gimatecan, plasma and tissue levels were 2-fold higher after treatment with a pro-inflammatory agent causing AGP induction. CONCLUSIONS Gimatecan is orally absorbed and its variable plasma levels seem to be related to AGP plasma concentrations. Data obtained in mice, together with the fact that AGP levels largely exceeded gimatecan plasma concentrations, suggest that the increased gimatecan levels in patients with high AGP levels are not related to the binding of the drug to AGP with consequent reduced tissue drug distribution, but possibly to other mechanism associated with inflammation being AGP simply a marker of the inflammation process.