Cellular Uptake and Efflux of Palbociclib In Vitro in Single Cell and Spheroid Models.

Adequate drug distribution through tumors is essential for treatment to be effective. Palbociclib is a cyclin-dependent kinase 4/6 inhibitor approved for use in patients with hormone receptor positive, human epidermal growth factor receptor 2 negative metastatic breast cancer. It has unusual physicochemical properties, which may significantly influence its distribution in tumor tissue. We studied the penetration and distribution of palbociclib in vitro, including the use of multicellular three-dimensional models and mathematical modeling. MCF-7 and DLD-1 cell lines were grown as single cell suspensions (SCS) and spheroids; palbociclib uptake and efflux were studied using liquid chromatography-tandem mass spectrometry. Intracellular concentrations of palbociclib for MCF-7 SCS (C max 3.22 µM) and spheroids (C max 2.91 µM) were 32- and 29-fold higher and in DLD-1, 13- and 7-fold higher, respectively, than the media concentration (0.1 µM). Total palbociclib uptake was lower in DLD-1 cells than MCF-7 cells in both SCS and spheroids. Both uptake and efflux of palbociclib were slower in spheroids than SCS. These data were used to develop a mathematical model of palbociclib transport that quantifies key parameters determining drug penetration and distribution. The model reproduced qualitatively most features of the experimental data and distinguished between SCS and spheroids, providing additional support for hypotheses derived from the experimental data. Mathematical modeling has the potential for translating in vitro data into clinically relevant estimates of tumor drug concentrations. SIGNIFICANCE STATEMENT: This study explores palbociclib uptake and efflux in single cell suspension and spheroid models of cancer. Large intracellular concentrations of palbociclib are found after drug exposure. The data from this study may aid understanding of the intratumoural pharmacokinetics of palbociclib, which is useful in understanding how drug distributes within tumor tissue and optimizing drug efficacy. Biomathematical modelling has the potential to derive intratumoural drug concentrations from plasma pharmacokinetics in patients.

Impact of age and medical comorbidity on adjuvant treatment outcomes for stage III colon cancer: a pooled analysis of individual patient data from four randomized, controlled trials.

BACKGROUND: Adjuvant oxaliplatin plus capecitabine or leucovorin/5-fluorouracil (LV/5-FU) (XELOX/FOLFOX) is the standard of care for stage III colon cancer (CC); however, there is disagreement regarding oxaliplatin benefit in patients aged >70. In most analyses, the impact of medical comorbidity (MC) has not been assessed. Efficacy and safety of adjuvant XELOX/FOLFOX versus LV/5-FU were compared with respect to age and MC using pooled data from four randomized, controlled trials, selected for access to patient-level MC data and including commonly endorsed and utilized regimens. PATIENTS AND METHODS: Individual data from patients with stage III CC in NSABP C-08, XELOXA, X-ACT, and AVANT were pooled, excluding bevacizumab-treated patients. Patients were grouped by treatment, MC (low versus high), or age (<70 versus ≥70), and compared for disease-free survival (DFS), overall survival (OS), and adverse events (AEs). Multivariable Cox proportional hazards regression controlled for gender, T stage, and N stage. RESULTS: DFS benefits were shown for XELOX/FOLFOX versus LV/5-FU regardless of age or MC, although benefits were modestly attenuated for patients aged ≥70. Hazard ratios were 0.68 (P < 0.0001) and 0.77 (P < 0.014) for <70 and ≥70 age groups; 0.69 (P < 0.0001) and 0.59 (P < 0.0001) for Charlson Comorbidity Index ≤1 and >1 groups; and 0.70 (P < 0.0001) and 0.58 (P < 0.0001) for National Cancer Institute Combined Index ≤1 and >1 groups. OS was also significantly improved in all groups. Grade 3/4 serious AE rates were comparable across cohorts and MC scores and higher in patients aged ≥70. Oxaliplatin-relevant grade 3/4 AEs, including neuropathy, were comparable across ages and MC scores. CONCLUSIONS: Results further support consideration of XELOX or FOLFOX as standard treatment options for the adjuvant management of stage III CC in all age groups and in patients with comorbidities, consistent with those who were eligible for these clinical trials.

'Being there' for women with metastatic breast cancer: a pan-European patient survey.

BACKGROUND: Understanding their experiences of diagnosis is integral to improving the quality of care for women living with advanced/metastatic breast cancer. METHODS: A survey, initiated in March 2011, was conducted in two stages. First, the views of 47 breast cancer-related patient groups in eight European countries were sought on standards of breast cancer care and unmet needs of patients. Findings were used to develop a patient-centric survey to capture personal experiences of advanced breast cancer to determine insights into the 'trade-off' between extending overall survival and side effects associated with its treatment. The second online survey was open to women with locally advanced or metastatic breast cancer, or their carers, and responders were recruited through local patient groups. Data were collected via anonymous local language questionnaires. RESULTS: The online stage II survey received a total of 230 responses from 17 European countries: 94% of respondents had locally advanced or metastatic breast cancer and 6% were adult carers. Although the overall experience of care was generally good/excellent (77%), gaps were still perceived in terms of treatment choice and information provision. Treatment choice for patients was felt to be lacking by 32% of responders. In addition, 68% of those who responded would have liked more information about future medical treatments and research, with 57% wishing to receive this information from their oncologist. Two-thirds (66%) of women with advanced breast cancer, or their carers, believed life-extending treatment to be important so that they can spend more time with family and friends, and 67% said that the treatment was worthwhile, despite potential associated side effects. CONCLUSION: These findings show a continuing need to provide women with advanced breast cancer with better information and emphasise the importance that these patients often place on prolonging survival.

Adjuvant epirubicin followed by cyclophosphamide, methotrexate and fluorouracil (CMF) vs CMF in early breast cancer: results with over 7 years median follow-up from the randomised phase III NEAT/BR9601 trials.

BACKGROUND: The National Epirubicin Adjuvant Trial (NEAT) and BR9601 trials tested the benefit of epirubicin when added to cyclophosphamide, methotrexate and 5-fluorouracil (E-CMF) compared with standard CMF in adjuvant chemotherapy for women with early breast cancer. This report details longer follow-up with interesting additional time-dependent analyses. METHODS: National Epirubicin Adjuvant Trial used epirubicin (E) 3-weekly for four cycles followed by classical (c) CMF for four cycles (E-CMF) compared with cCMF for six cycles. BR9601 used E 3-weekly for four cycles followed by CMF 3-weekly for four cycles, compared with CMF 3-weekly for eight cycles. RESULTS: In all, 2391 eligible patients were randomised and with a median 7.4-year follow-up, E-CMF confirmed a significant benefit over CMF in both relapse-free survival (RFS) (78% vs 71% 5 years RFS, respectively, hazard ratio (HR)=0.75 (95% CI: 0.65-0.86), P<0.0001) and overall survival (OS) (84% vs 78% 5 years OS, respectively, HR=0.76 (95% CI: 0.65-0.89), P=0.0007). Interaction of treatment effect and prognostic factors was demonstrated for duplication of chromosome 17 centromeric enumeration (Ch17CEP) as previously reported. Poor prognostic factors at diagnosis (ER and PR negative and HER2 positive) showed time-dependent annual hazard rates for RFS and OS. In univariate analysis, these factors demonstrated more favourable HRs for RFS after 5 years. Treatment effects also suggested a differential benefit for E-CMF within the first 5 years for poor prognosis tumours. CONCLUSION: Longer follow-up has confirmed E-CMF as significantly superior to CMF for all patients. Ch17CEP duplication was the only biomarker that demonstrated significant treatment interaction. Standard poor prognostic factors at diagnosis were time-dependent, and after 5 years disease-free, poor prognosis patients demonstrated favourable HRs for survival.

Designing phase II trials in cancer: a systematic review and guidance.

BACKGROUND: Literature reviews of cancer trials have highlighted the need for better understanding of phase II statistical designs. Understanding the key elements associated with phase II design and knowledge of available statistical designs is necessary to enable appropriate phase II trial design. METHODS: A systematic literature review was performed to identify phase II trial designs applicable to oncology trials. The results of the review were used to create a library of currently available designs, and to develop a structured approach to phase II trial design outlining key points for consideration. RESULTS: A total of 122 papers describing new or adapted phase II trial designs were obtained. These were categorised into nine levels, reflecting the practicalities of implementation, and form a library of phase II designs. Key design elements were identified by data extraction. Along with detailed description of the key elements and the library of designs, a structured thought process was developed to form a guidance document for choice of phase II oncology trial design. CONCLUSION: The guidance offers researchers a structured and systematic approach to identifying phase II trial designs, highlighting key issues to be considered by both clinicians and statisticians and encouraging an interactive approach to more informed trial design.

A mathematical model of doxorubicin penetration through multicellular layers.

Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 microM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive "flip-flop" mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.

Drug delivery in a tumour cord model: a computational simulation.

The tumour vasculature and microenvironment is complex and heterogeneous, contributing to reduced delivery of cancer drugs to the tumour. We have developed an in silico model of drug transport in a tumour cord to explore the effect of different drug regimes over a 72 h period and how changes in pharmacokinetic parameters affect tumour exposure to the cytotoxic drug doxorubicin. We used the model to describe the radial and axial distribution of drug in the tumour cord as a function of changes in the transport rate across the cell membrane, blood vessel and intercellular permeability, flow rate, and the binding and unbinding ratio of drug within the cancer cells. We explored how changes in these parameters may affect cellular exposure to drug. The model demonstrates the extent to which distance from the supplying vessel influences drug levels and the effect of dosing schedule in relation to saturation of drug-binding sites. It also shows the likely impact on drug distribution of the aberrant vasculature seen within tumours. The model can be adapted for other drugs and extended to include other parameters. The analysis confirms that computational models can play a role in understanding novel cancer therapies to optimize drug administration and delivery.

A phase I and pharmacokinetic study of LY231514, the multitargeted antifolate.

LY231514 is a novel antifolate that principally inhibits thymidylate synthase, but with additional folate-dependent enzyme targets. A Phase I study of single-agent LY231514 administered as a daily i.v. infusion over 10 minutes for 5 days, repeated every 3 weeks, was conducted to evaluate the maximum tolerated dose, pharmacokinetic profile, and antitumor activity of the drug using this schedule. Thirty-eight patients with advanced malignancies that were refractory or not amenable to standard therapy were treated with a total of 116 courses of LY231514, escalating treatment doses through 10 dose levels, from 0.2-5.2 mg/m2/day. No objective clinical responses were observed, although minor antitumor activity not fulfilling the response criteria was seen in three patients. A maximum tolerated dose of 4.0 mg/m2/day was determined, with neutropenia as the predominant dose-limiting toxicity. Reversible disturbances of liver biochemistry, fulfilling the protocol definitions of dose-limiting toxicity, were also observed. Other toxicities included diarrhea, mucositis, skin rash, and fatigue. Pharmacokinetic studies were performed at all treatment levels. Analysis showed a linear relation between administered dose and both maximum plasma concentration (Cmax) and area under the plasma concentration/time curve. The drug was cleared with a day 1 total body clearance of 108.9 +/- 38.8 ml/min/m2, with plasma concentrations declining with a mean harmonic terminal half-life of 1.4 +/- 0.98 h. When given by this schedule, LY231514 is tolerable, and Phase II studies are in progress.

Protein kinase C: a target for anticancer drugs?

Protein kinase C (PKC) is a family of serine/threonine kinases that is involved in the transduction of signals for cell proliferation and differentiation. The important role of PKC in processes relevant to neoplastic transformation, carcinogenesis and tumour cell invasion renders it a potentially suitable target for anticancer therapy. Furthermore, there is accumulating evidence that selective targeting of PKC may improve the therapeutic efficacy of established neoplastic agents and sensitise cells to ionising radiation. This article reviews the rationale for targeting PKC, focuses on its role in breast cancer and reviews the preclinical and clinical data available for the efficacy of PKC inhibition.

Entry into clinical trials in breast cancer: the importance of specialist teams. Scottish Breast Cancer Focus Group and Scottish Cancer Therapy Network.

The aim of this study was to identify the factors influencing entry of women with invasive breast cancer into clinical trials in Scotland. Women diagnosed during 1987 and 1993 were identified from cancer registry data records and their case notes reviewed. Entry into clinical trials was recorded, along with clinical and demographic data for 4688 patients. In 1987, the proportion of women entering clinical trials was 12.3% and, allowing for shorter follow-up, this appeared unchanged in 1993. Patients seen by surgeons with a high case load and those referred to an oncologist were approximately seven times and three times, respectively, more likely to enter a clinical trial (P < 0.0001). The area of Scotland (Health Board) where the woman was first treated also influenced study entry (P < 0.0001), whereas social deprivation had no effect (P = 0.93). Older women, especially those over 80 years of age, were less likely to enter studies (P = 0.05). Extending the management of patients by specialist multidisciplinary teams should increase recruitment into clinical trials and help to identify better treatments for women with breast cancer.

Improvements in survival for women with breast cancer in Scotland between 1987 and 1993: impact of earlier diagnosis and changes in treatment.

We investigated changes in survival, and their causes, in women with early breast cancer diagnosed in Scotland. The Scottish Cancer Registry identified 1617 and 2077 such women, without metastases at diagnosis who underwent surgery as part of their primary treatment, diagnosed in 1987 and 1993, respectively. There was a statistically significant 11% improvement in 8-year survival between 1987 and 1993. Survival improved across almost all clinical/pathological, treatment and health care delivery/deprivation categories; improvement was not limited to those women diagnosed through the screening programme. In a multivariate model, improved survival appeared to be explained largely by screening and clinical/pathological prognostic factors. Deprivation also had an adverse effect on survival; however, the geographical variation in survival observed for women diagnosed in 1987 was not apparent by 1993. We did not demonstrate a significant independent effect of surgical caseload on survival. We conclude that survival has increased partly as a consequence of screening and earlier diagnosis, but also due to improvements in the organisation and delivery of care.

Epirubicin in patients with liver dysfunction: development and evaluation of a novel dose modification scheme.

This study aimed to develop an epirubicin dose modification scheme in women with breast cancer and liver dysfunction. We first identified target areas under the concentration-time curve (AUCs) of 2400 and 1600 ng/ml.h from pharmacokinetic studies in 15 women with normal liver tests. In a second group of 16 women with abnormal liver biochemistry, the relationship between raised asparate aminotransferase (AST) and epirubicin clearance was: dose=AUC (97.5-34.2xlog AST). Adaptive dosing was evaluated prospectively in a third group of 41 women with serum AST > or =2xnormal+/-raised bilirubin. The median AUCs were 2444 and 1608 ng/ml.h, close to the high and low target AUCs, respectively. Variability in AUC was lower with adaptive dosing than in a fourth group given an unadjusted dose of epirubicin (coefficient of variation=25.8, 30.0 and 46.5%, respectively; P=0.06). Epirubicin dosing based on AST is safe and may reduce pharmacokinetic variability.

Patient acceptability and practical implications of pharmacokinetic studies in patients with advanced cancer.

We have studied the practical implications and acceptability to patients of pharmacokinetic studies in 34 women receiving anthracyclines for advanced breast cancer. The following parameters were recorded: age, ECOG performance status, psychological state (Rotterdam Symptom Checklist), cytotoxic drug and dose, number of venepunctures for treatment and sampling, and time when the sampling cannula was removed. Immediately after finishing pharmacokinetic sampling, patients completed a questionnaire which revealed that (i) all patients understood sampling was for research, (ii) 35% of patients experienced problems with sampling, (iii) benefits from participation were perceived by 56% of patients. Of 20 patients later questioned after completion of their treatment course, 40% recalled difficulties with blood sampling. Factors identifying in advance those patients who tolerate pharmacokinetic studies poorly were not identified but the number of venepunctures should be minimised. Patients may also perceive benefits from 'non-therapeutic' research.

Doxorubicin in advanced breast cancer: influence of schedule on response, survival and quality of life.

The influence of scheduling of doxorubicin on response, survival and quality of life was assessed in a randomised trial in patients with advanced breast cancer, none of whom had previously received cytotoxic chemotherapy for advanced disease. 28 patients received 75 mg/m2 doxorubicin every 3 weeks for four courses (arm 1) and 31 patients received 25 mg/m2 weekly for 12 courses (arm 2). Response rates and median time to progression were similar in the two arms and median survival was 8 months in both arms. However, amongst patients receiving treatment every 3 weeks, psychological distress measured using the Rotterdam symptom checklist fell significantly over the course; no such change was observed in those treated weekly. Physical symptoms related to cancer improved during treatment similarly for both groups.

DNA: still a target worth aiming at? A review of new DNA-interactive agents.

DNA acts as the final target for most clinically effective cytotoxic agents, but the lack of selectivity for tumor cells has raised questions about the value of developing new DNA-interactive agents. Three new classes of cytotoxic agents are reviewed; each interacts directly with DNA but cytotoxicity appears to be mediated through novel mechanisms, including the interaction with specific proteins by DNA-bound drug molecules. Irofulven is the lead compound of the illudin family of molecules. It causes a novel type of DNA damage whose repair is dependent on functioning DNA helicases. Pre-clinical and clinical synergy between irofulven and agents which inhibit topoisomerases has been observed. Clinical trials with irofulven have shown significant activity and phase II studies in pancreatic, ovarian and prostatic cancer are ongoing. Toxicity in the form of myelosuppression and fatigue have been shown to be schedule dependent, with intermittent administration appearing to significantly reduce toxicity. DNA-interacting agents which alkylate bases exposed in the minor groove have been derived from a number of natural sources. The minor groove alkylation appears to be sequence specific; although the significance of this specificity for cytotoxicity is unclear, one proposed mechanism is through inhibition of expression of particular genes. Three cyclopropylpyrroloinole analogues which cause sequence specific minor groove alkylation are currently under clinical assessment. Myelosuppression is the dose limiting toxicity and is biphasic in its time course. Moderate activity in phase I trials has been observed. Ecteinascidins represent one of the increasing number of groups of drugs isolated from marine organisms. Ecteinascidin-743 (ET-743) is the most advanced in its clinical development. Binding to the minor groove of DNA occurs, although with a different base specificity from other compounds. The cytotoxic effects of ET-743 may occur by inhibition of the inducible transcription of a number of genes by sequestration of specific transcription factors. Clinical trials of ET-743 have shown significant activity, and phase II trials are underway in soft tissue sarcoma and breast cancer. Hepatic toxicity and myelosuppression are predictable and appear associated with peak plasma concentrations, whereas efficacy seems to be improved with prolonged infusion.

Toxicity of intra-arterial doxorubicin in locally advanced breast cancer.

Four patients with inoperable, locally advanced breast cancer were treated i.a. with 25-35 mg/m2 doxorubicin given as a 6-h infusion on 2 successive days. In each patient, the catheter was introduced percutaneously via the femoral or brachial artery using local anaesthetic and positioned in the internal mammary artery without complications. However, within 48 h of starting treatment all four patients developed extensive erythema over the chest wall, which progressed to superficial ulceration in one case. Two patients also developed a raised hemidiaphragm and phrenic nerve paralysis that was associated with a pleural effusion in one case. This study closed prematurely because of unacceptable local toxicity; thus, we cannot assess the activity of doxorubicin given in this way. If this approach to local control is to be tested further in locally advanced breast cancer, lower doxorubicin doses should be used, or different drugs selected.

Activity and toxicity of carboplatin and iproplatin in relapsed high-grade glioma.

A total of 15 patients with relapsed high-grade glioma were treated with carboplatin (400 mg/m2) or iproplatin (300 mg/m2). All had received previous radiotherapy, and 12 had previously undergone chemotherapy. One of the ten patients treated with carboplatin and one of the five treated with iproplatin achieved a partial remission as determined by repeat computerised tomographic (CT) scan. Myelosuppression was considerable, as three patients developed grade IV neurotoxicity, which was fatal in one case. Although carboplatin and iproplatin showed activity against malignant glioma, the study was closed because of unacceptable toxicity.

Sequential chemotherapy in good-prognosis patients with small-cell lung cancer.

A sequential combination chemotherapy regimen was evaluated in 23 patients with small-cell lung cancer (16, limited disease; 7, extensive disease). All patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, normal serum sodium and albumin levels and alkaline phosphatase values of less than 1.5 times the upper limit of normal. Treatment comprised ifosfamide and either vindesine or vincristine given on weeks 0, 2 and 4; cisplatin and etoposide given on weeks 6, 9 and 12; and doxorubicin and methotrexate given on weeks 15 and 17. The overall response rate at the end of chemotherapy was 91% and the complete response rate was 43%. Treatment was generally well tolerated and the delivered dose intensity was 83% of that projected. Median survival was 54 weeks, with 4 patients (17%) being alive 2 years after the completion of therapy.

Epirubicin in hepatocellular carcinoma: pharmacokinetics and clinical activity.

The pharmacokinetics and clinical activity of epirubicin were investigated in 16 patients with hepatocellular carcinoma (HCC) who received epirubicin at 75 mg/m2; the drug was given intravenously to 7 patients and via the hepatic artery to 9 patients (7 of whom also underwent embolisation). Lignocaine (1 mg/kg) was also given intravenously to 15 patients, and the metabolite monoethylglycinexylidide (MEGX) was measured as an indicator of liver function. Epirubicin clearance correlated with serum aspartate aminotransferase (AST), albumin and bilirubin values in patients treated intravenously or intraarterially. Although the route of administration did not affect the median total plasma clearance of epirubicin, early- and intermediate-phase clearance was higher following intraarterial administration. MEGX levels correlated with serum bilirubin levels but there was no correlation with albumin or AST values or epirubicin clearance. The rate of response to epirubicin was 3/13 (23%; 95% confidence interval, 8%-50%). Intravenous epirubicin was tolerated well, but intraarterial treatment was associated with significant morbidity. These data confirm that although current recommended dose adjustments are based primarily on serum bilirubin levels, altered epirubicin pharmacokinetics correlate more strongly with AST and albumin values than with serum bilirubin concentrations. However, at this dose and schedule, epirubicin has only modest activity against HCC.

Comparative pharmacokinetics of doxorubicin given by three different schedules with equal dose intensity in patients with breast cancer.

The pharmacokinetics of doxorubicin given according to three different schedules with a similar dose-time intensity have been studied and compared in 16 women with metastatic breast cancer. Six patients were treated with doxorubicin 75 mg/m2 by i.v. bolus repeated every 3 weeks; 5 patients received doxorubicin by 4-day continuous infusion every 3 weeks (4 at 75 mg/m2 and 1 at 60 mg/m2); 5 patients received 25 mg/m2 by i.v. bolus given weekly. Timed blood samples were collected and plasma levels of doxorubicin and its metabolite doxorubicinol were measured by high-performance liquid chromatography with fluorescence detection. Peak plasma concentrations were measured, and areas under the concentration-time curves calculated. Peak plasma levels of doxorubicin were significantly lower with the 4-day infusion than with either of the bolus injections. The 4-day infusion, however, gave significantly greater total exposure to doxorubicin and doxorubicinol, as indicated by area under the concentration-time curve, than weekly or 3-weekly bolus treatment. A single bolus injection of doxorubicin 25 mg/m2 yielded a total exposure to doxorubicin approximately half that achieved with a 75 mg/m2 bolus injection. Over a 3-week period, therefore, total exposure to doxorubicin would be greater with the weekly low-dose schedule than with the 3-weekly administration. We conclude that drug scheduling has significant effects on doxorubicin pharmacokinetics.