Guido Poggi

Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation

A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.

Validation of an LC–MS/MS method for the determination of epirubicin in human serum of patients undergoing Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE)

Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE) is a new delivery system to administrate drugs in a controlled manner useful for application in the chemoembolization of colorectal cancer metastases to the liver. DEM-TACE is focused to obtain higher concentrations of the drug to the tumor with lower systemic concentrations than traditional cancer chemotherapy. Therefore a specific, precise and sensitive LC-ESI-MS/MS assay procedure was properly designed to detect and quantify epirubicin at the concentrations expected from a transarterial chemoembolization with microspheres. Serum samples were kept acidic (pH approximately of 3.5) and sample preparation consisted of a solid phase extraction (SPE) procedure with HLB OASIS cartridges using a methylene chloride/2-propanol/methanol mixture solution to recover epirubicin. The analyses consisted of reversed-phase high-performance liquid chromatography (rp-HPLC) coupled with tandem mass spectrometry (MS/MS). Accuracy, precision and matrix effect of this procedure were carried out by analyzing four quality control samples (QCs) on five separate days. The validation parameters were assessed by recovery studies of spiked serum samples. Recoveries were found to vary between 92 and 98% at the QC levels (5, 40, 80 and 150 microg/L) with relative standard deviation (RSD) always less than 3.7%. The limit of detection (LOD) was set at 1 microg/L. The developed procedure has been also applied to investigate the different capability of two types of commercially available microspheres to release epirubicin into the human circulatory system.

Simultaneous measurement of doxorubicin and reduced metabolite doxorubicinol by UHPLC–MS/MS in human plasma of HCC patients treated with TACE

A sensitive, selective, accurate and precise method for simultaneous quantification of doxorubicin (DOX) and doxorubicinol (DOXol) in human plasma of patients diagnosed as having intermediate stage unresectable hepatocellular carcinoma (HCC) was developed. The method was based on electrospray tandem mass spectrometry in selected reaction monitoring mode. DOX, DOXol and trofosfamide, an internal standard, were extracted from plasma by using a simple solid phase extraction (SPE) procedure after the addition of 0.1 M hydrochloric acid. A 200-μL aliquot of the extracted sample reconstituted in mobile phase was analyzed on a Zorbax SB-C18 UHPLC column (50 mm × 2.1 mm, 1.8 μm particle size) in 8 min. The mobile phase consisted of acetonitrile and 0.1% formic acid pH 4.5 (95:05 v/v). Good accuracy and precision of this method were demonstrated by determination of spiked plasma QC samples in four consecutive days. The SPE extraction recoveries ranged from 72.3 to 77.3% and 75.5 to 98.4% for doxorubicin and doxorubicinol, respectively. The intra-day and inter-day precisions were less than 11.4%. The limit of quantitation was 1.0 ng/mL for both compounds. The calibration curves of DOX and DOXol were analyzed by weighted linear regression with 1/x as a weighting factor. They were linear over the concentration range of 1.0-100.0 ng/mL with R(2) greater than 0.99. This developed method was successfully applied to study plasma pharmacokinetics in patients affected by HCC and treated with transarterial chemoemolization practices (TACEs) using HepaSphere™ pre-loaded with DOX in a standardized procedure.

Massive hepatic infarction complicating ultrasound-guided percutaneous radiofrequency thermal ablation

Radiofrequency ablation (RFA) is an effective treatment option for patients with unresectable liver tumors (1). Although RFA has been considered a safe technique, investigations of its complications are limited. We describe here a well-compensated cirrhotic patient who had massive hepatic infarction after RFA of a small hepatocellular carcinoma (HCC). A 72-year-old woman with a diagnosis of hepatitis C virus-related cirrhosis presented with a 3.6 cm mass in the fourth segment of the liver, seen on abdominal ultrasound (US). The lesion was located between the right portal vein and the inferior vena cava. Color-Doppler US examination showed that both veins were patent. The patient’s a fetoprotein was 356 ng/ml. Enhanced computed tomography (CT) scanning of the abdomen failed to show the typical contrast pattern of HCC with the absence of early enhancement during the arterial phase within the tumor, but cytological examination of a specimen of the lesion confirmed that the nodule was a HCC. The patient’s past medical history included high-grade non-Hodgkin’s lymphoma, diagnosed 20 years earlier and successfully treated with six cycles of chemotherapy (CHOP regimen) and para-aortic radiotherapy. Seven years before admission, a single nodule of HCC had been discovered in the first hepatic segment and surgically removed. We proposed surgical resection of this newly diagnosed HCC, but the patient refused. So we decided to treat it with thermal ablation. The radiofrequency (RF) system that we used consists of an insulated 18-gauge needle electrode with a 3 cm tip exposure attached to a 500 kHz RF generator (Radionics, Burlington, MA). The treatment was performed percutaneously with two insertions of the needle into the tumor under US guidance. The RF current was emitted over 24min. The patient was treated under sedation with both 7mg of midazolam maleate and 4ml of fentanyl citrate. At the end of the procedure, US examination did not reveal any complications. The next morning the patient complained of profound asthenia and severe tenderness in the right upper quadrant. On examination, she appeared ill and uncomfortable; her temperature was 38.1 1C, blood pressure 105/60, heart rate 92 bpm; there were no signs of jaundice. The rest of the clinical examination was normal. Laboratory values were: 3100white blood cells/mm, with 65% polymorphonuclear cells; hemoglobin 12.5 g/dl, platelet count 153.000/mm, aspartate aminotrasferase (AST) 4000UI/l, alanine aminotrasferase (ALT) 2650UI/l, alkaline phosphatase 222UI/l, total bilirubin 1.1mg/dl and international normalized ratio 0.96. The levels of AST and ALT rose to 6490 and 4770UI/ml, respectively, on the second day and then slowly decreased. Two days after the procedure, colorDoppler US revealed right portal vein thrombosis and right hepatic artery occlusion. An abdominal enhanced CT scan demonstrated a large ischemic wedge-shaped hypo-echoic area within the right lobe and two contiguous fluid areas diagnosed as bilomas (Fig. 1a, b). At discharge, 11 days after the RFA procedure, the patient was afebrile but complained of mild asthenia; the levels of AST and ALT had dropped to 58 and 159UI/l, respectively. Three months after RFA the patient gained weight and an abdominal US showed ascites. Twelve months after RFA the patient died of hepatic failure. RFA-related complications can be distinguished into two categories: major complications are those that, if left untreated, threaten the patient’s life, leading to substantial morbidity and disability, or a prolonged stay in hospital and all other complications are considered minor (2). The most common major complications are infection and bleeding; the others include biloma, pneumothorax, haemothorax, ground pad burns and bowel injuries. Liver infarcts are very uncommon given the liver’s double blood supply from both the portal vein and the hepatic artery. Common causes of non-neoplastic portal vein thrombosis are portal hypertension, an abdominal wound, sepsis and splenectomy. Although heat injury to the vessels Liver International 2004: 24: 704–705 Printed in Denmark. All rights reserved Copyright r Blackwell Munksgaard 2004

Intravenous or Oral Vinorelbine Plus Capecitabine As First-Line Treatment in HER2– Metastatic Breast Cancer: Joint Analysis of 2 Consecutive Prospective Phase II Trials

BACKGROUND The purpose of this study was to assess the activity and safety of the combination of vinorelbine (VNR) and capecitabine (CAP) as first-line treatment in HER2-negative (HER(-)) metastatic breast cancer (MBC). PATIENTS AND METHODS Patients (42) enrolled in trial A received intravenous (i.v.) VNR 25 mg/m2 on days 1 and 8 of a 21-day cycle combined with CAP 1000 mg/m2 twice daily for 14 consecutive days followed by 1 week of rest. Trial B (46 patients) followed trial A when the oral formulation of VNR became available at our institution. Patients received oral VNR (60 mg/m(2) on days 1-8) combined with the same CAP schedule as in trial A. RESULTS The response rate (RR) in trial A was 73.2% (95% confidence interval [CI], 56.4-82.8), including 12.2% complete responses (CRs). Clinical benefit was achieved in 78% of patients (95% CI, 63.2-87.9). In trial B, overall RR was 76% (95% CI, 62.0-86.0), with 13% CRs and clinical benefit of 80.4% (95% CI, 66.8-89.3). In trial A, median progression-free survival (PFS) was 8.2 months (range, 6-14+ months) and median overall survival (OS) was 32.4 months (range, 17-36+ months). In trial B, median PFS and OS were 8.8 months (range, 8-21+ months) and 34.3 months (14-39+ months), respectively. Treatment-related toxicity was manageable. Quality of life assessment showed a statistically significant difference regarding body image (p = .001), sexual functioning (p = .02), and future perspectives (p = .03) in women receiving chemotherapy fully by the oral route. CONCLUSION This joint analysis shows that both tested schedules can produce high objective RRs with encouraging PFS, manageable toxicity profile, and suggested benefit on some aspects of quality of life for the fully oral combination.

Multimodality treatment of unresectable hepatic metastases from pancreatic glucagonoma.

Glucagonomas are pancreatic islet cell tumors arising from the alpha cells which belong to neuroendocrine tumors. They frequently metastasize to the liver. We report the case of a 52- year old man with a pancreatic glucagonoma with synchronous multiple liver metastases treated by surgery, transarterial chemoembolization, percutaneous radiofrequency thermal ablation and long-acting octreotide. Our report confirms that a multimodal approach is very effective in patients with unresectable liver metastases from pancreatic endocrine tumors providing long-lasting palliation and probably prolonging survival.

Acute Thrombocytopenia: An Unusual Complication Occurring After Drug-Eluting Microspheres Transcatheter Hepatic Chemoembolization

Image-guided transcatheter hepatic chemoembolization (TACE) is accepted worldwide as an effective treatment for patients with unresectable hepatocellular carcinoma and liver metastases from neuroendocrine tumors, colorectal carcinomas, and uveal melanomas. Although the technique is relatively safe, it has been associated with several complications. We report the cases of two patients with colorectal liver metastases who developed acute thrombocytopenia a few hours after TACE. To our knowledge, acute thrombocytopenia occurring after TACE with drug-eluting microspheres has not yet been reported. Here we discuss the hypothetical etiopathogenetic mechanisms.

Treatment of Chronic Hepatitis C in a Patient Affected by Systemic Sclerosis

The currently recommended treatment for patients infected with hepatitis C virus (HCV) is pegilated interferon α (IFN α) plus ribavirin. Despite the numerous benefits of this therapy, there is an increasing concern regarding his tolerance. Among the most common side effects, interferon may trigger the onset or exacerbation of autoimmune diseases. When chronic hepatitis C coexists with an autoimmune disorder, it is not clear whether using interferon is better than avoiding it. We evaluated the disease state of a 55-year old female affected by sistemic sclerosis (SSc), during and after therapy with IFNα pegilated plus ribavirin for chronic HCV infection. We were worried about the potential worsening of the autoimmune disease during the therapy, but we were confident that we would give our patient a short course of peginterferon and ribavirin. A mild, asymptomatic worsening of lung SSc was observed during IFN administration, without life threatening symptoms. After 24 months follow up we observed the maintenance of the virological response and a good control of the rheumatological disease. Thus, in liver disease at high risk of progression and concomitant SSc, the antiviral therapy with IFNα is a feasible approach.

Trattamento loco regionale dell’epatocarcinoma non resecabile mediante chemioembolizzazione con microparticelle caricate con epirubicina: valutazione di efficacia, tollerabilità e analisi farmacocinetica in uno studio clinico monocentrico

Image-guided transcatheter arterial chemoembolization (TACE) is the first-line treatment for patients with intermediate stage HCC not candidate for curable therapies. Recently, microembolizing particles have been introduced in clinical practise; they are able to be loaded with chemotherapic agents, in particular doxorubicin, cisplatin and epirubicin. By now, two types of microparticles are mostly used in clinical practise: DC Bead® and HepaSphere Microsphere®. Drug Eluting Beads (DEBs)-TACE reduces side effects and improve local efficacy because the microparticles have a double effect: they embolize the arteries feeding the neoplastic lesion and they gradually realise the chemotherapic agent in the tumoral bed, to obtain high intratumoral drug levels and low systemic drug concentrations. The purpose of this study is to determine the prevalence of complication in patients affected by intermediate stage HCC treated with DEBs-TACE. Between January 2007 and December 2009, 80 patients have been treated with DEBs-TACE for a total of 125 treatments. Everyone was affected by intermediate stage HCC, mostly BCLC B; they received Drug Eluting Beads (DEBs)-TACE with DC Bead or HepaSphere MicroSphere pre-loaded with epirubicin (50 mg/vial). Biochemical blood analysis were performed before, 4 and 24 hours after the procedure to monitor some hematologic parameters. Side effects were reported following the Common Toxicity Criteria in order to evaluate post-TACE tossicity. Tumor response was assessed after 40 days from the procedure with CT scans according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST). This study shows that the use of drug-eluting microspheres did not increase the risk of post-procedural complication and the prevalence of major adverse events are similar to conventional TACE, according to data reported in literature. We, also, noticed no differences in the prevalence of adverse events between patients treated with DC Bead-TACE and HepaSphereTACE. Moreover, in a selected group of 20 patients, 12 treated with DC Bead and 8 with HepaSphere, we performed peripheral blood samples analysis at the end of the embolic solution delivery and at 5, 10, 20, 40, 60, 120, 180, 360, 1,440 minutes after the procedure to assess epirubicin serum pharmacokinetic. The pharmacokinetic study showed low peak serum epirubicin concentrations as median peak level was 73.5+/-24.5 ng/mL for the DC Bead group and 33.9+/-11.0 ng/mL for the HepaSphere cohort. The highest drug concentration was observed after microspheres injection at 5 minutes in all 20 patients. The PK profile never dropped to zero up until the end of the experiment. In the time-interval included between 1 and 24 hours, persisting levels of epirubicin were detected in patients’peripheral blood samples, ranging from 2.3 to 24.2 ng/mL for both embolics. This study suggests that, after an earlier release effect, occurring during the very first few minutes, both microspheres are capable of a sustained kinetic release.

Analisi retrospettiva della prevalenza di complicanze correlate alle procedure di chemioembolizzazione intra-arteriosa epatica (TACE) nelle differenti classi di rischio

Transcatheter hepatic chemoembolization (TACE) is widely used in the treatment of unresectable hepatic tumors. Although considered relatively safe, TACE has been associated with several complications. The aim of this study was to determine the prevalence of complications and correlate them with some know risk factors. Between 2004 and 2009 we treated 155 patients (106 men and 49 women) with 297 sessions of TACE. 193 patients had primitive liver tumor and 104 had metastases from different primitivities. The patients were aged 49–86 years. TACE procedures were performed either with drug loaded microspheres (136 sessions with DCBead®; 124 with Hepasphere®) and with iodized oil (33 with Lipiodol®). The chemoterapeutic agent used was Epidoxorubicin in 217 sessions, Irinotecan in 30 and Oxaliplatin in 50. Our data showed that major complications occurred in 16.5% of patients. Specifically we found acute pancreatitis (2.7%), liver abscess (3%), cholecystistis (3.7%) and autoimmune thrombocytopenia (3.4%). Around 80 % of patients had postembolization syndrome that is not considered a complication but rather an expected outcome of embolotherapy. Complications occurred more frequently in diabetic than in non diabetic patients (26.7% vs 13.1%; p=0.006). Conversely we didn’t find any statistically significant differences according to the embolization agents used (Lipiodol® vs microspheres), the chemotherapic agent (Epidoxorubin vs Oxalplatin vs Irinotecan ), the age of the patients and the histology (primary vs metastatic tumors). All patients received antibiotic therapies before and after TACE; no statistically significant differences were found among the different classes of antibiotics used. Also no more complications were found with combined therapies (TACE+RFTA) than with TACE alone. Among the know risk factors only diabetes increases the prevalence of complications of TACE. TACE with Lipiodol® is more painful than drug loaded microspheres.