Osteoporosis medication dispensing for older Australian women from 2002 to 2010: Influences of publications, guidelines, marketing activities and policy

Purpose Developments in anti-osteoporosis medications (AOMs) have led to changes in guidelines and policy, which, along with media and marketing strategies, have had an impact upon the prescribing of AOM. The aim was to examine patterns of AOM dispensing in older women (aged 76–81 years at baseline) from 2002 to 2010. Methods Administrative claims data were used to describe AOM dispensing in 4649 participants (born in 1921–1926 and still alive in 2011) in the Australian Longitudinal Study on Women's Health. The patterns were interpreted in the context of changes in guidelines, indications for subsidy, publications (scholarly and general media), and marketing activities. Results Total use of AOM increased from 134 DDD/1000/day in 2002 to 216 DDD/1000/day in 2007 but then decreased to 184 DDD/1000/day in 2010. Alendronate was the most commonly dispensed AOM but decreased from 2007, while use of risedronate (2002 onward), strontium ranelate (2007 onward) and zoledronic acid (2008 onward) increased. Etidronate and hormone replacement therapy (HRT) prescriptions gradually decreased over time. The decline in alendronate dispensing coincided with increases of other bisphosphonates and publicity about potential adverse effects of bisphosphonates, despite relaxing indications for bone density testing and subsidy for AOM. Conclusions Overall dispense of AOM from 2002 reached a peak in 2007 and thereafter declined despite increases in therapeutic options and improved subsidised access. The recent decline in overall AOM dispensing seems to be explained largely by negative publicity rather than specific changes in guidelines and policy.

Pre-eclampsia : The role of soluble VEGF receptor-1 and related anti-angiogenic factors beyond

Pre-eclampsia is a pregnancy complication that affects about 5% of all pregnancies. It is known to be associated with alterations in angiogenesis -related factors, such as vascular endothelial growth factor (VEGF). An excess of antiangiogenic substances, especially the soluble receptor-1 of VEGF (sVEGFR-1), has been observed in maternal circulation after the onset of the disease, probably reflecting their increased placental production. Smoking reduces circulating concentrations of sVEGFR-1 in non-pregnant women, and in pregnant women it reduces the risk of pre-eclampsia. Soluble VEGFR-1 acts as a natural antagonist of VEGF and placental growth factor (PlGF) in human circulation, holding a promise for potential therapeutic use. In fact, it has been used as a model to generate a fusion protein, VEGF Trap , which has been found effective in anti-angiogenic treatment of certain tumors and ocular diseases. In the present study, we evaluated the potential use of maternal serum sVEGFR-1, Angiopoietin-2 (Ang-2) and endostatin, three central anti-angiogenic markers, in early prediction of subsequent pre-eclampsia. We also studied whether smoking affects circulating sVEGFR-1 concentrations in pregnant women or their first trimester placental secretion and expression in vitro. Last, in order to allow future discussion on the potential therapy based on sVEGFR-1, we determined the biological half-life of endogenous sVEGFR-1 in human circulation, and measured the concomitant changes in free VEGF concentrations. Blood or placental samples were collected from a total of 268 pregnant women between the years 2001 2007 in Helsinki University Central Hospital for the purposes above. The biomarkers were measured using commercially available enzyme-linked immunosorbent assays (ELISA). For the analyses of sVEGFR-1, Ang-2 and endostatin, a total of 3 240 pregnant women in the Helsinki area were admitted to blood sample collection during two routine ultrasoundscreening visits at 13.7 ± 0.5 (mean ± SD) and 19.2 ± 0.6 weeks of gestation. Of them, 49 women later developing pre-eclampsia were included in the study. Their disease was further classified as mild in 29 and severe in 20 patients. Isolated early-onset intrauterine growth retardation (IUGR) was diagnosed in 16 women with otherwise normal medical histories and uncomplicated pregnancies. Fifty-nine women remaining normotensive, non-proteinuric and finally giving birth to normal-weight infants were picked to serve as the control population of the study. Maternal serum concentrations of Ang-2, endostatin and sVEGFR-1, were increased already at 16 20 weeks of pregnancy, about 13 weeks before the clinical manifestation of preeclampsia. In addition, these biomarkers could be used to identify women at risk with a moderate precision. However, larger patient series are needed to determine whether these markers could be applied for clinical use to predict preeclampsia. Intrauterine growth retardation (IUGR), especially if noted at early stages of pregnancy and not secondary to any other pregnancy complication, has been suggested to be a form of preeclampsia compromising only the placental sufficiency and the fetus, but not affecting the maternal endothelium. In fact, IUGR and preeclampsia have been proposed to share a common vascular etiology in which factors regulating early placental angiogenesis are likely to play a central role. Thus, these factors have been suggested to be involved in the pathogenesis of IUGR. However, circulating sVEGFR-1, Ang-2 and endostatin concentrations were unaffected by subsequent IUGR at early second trimester. Furthermore, smoking was not associated with alterations in maternal circulating sVEGFR-1 or its placental production. The elimination of endogenous sVEGFR-1 after pregnancy was calculated from serial samples of eight pregnant women undergoing elective Caesarean section. As typical for proteins in human compartments, the elimination of sVEGFR-1 was biphasic, containing a rapid halflife of 3.4 h and a slow one of 29 h. The decline in sVEGFR-1 concentrations after mid-trimester legal termination of pregnancy was accompanied with a simultaneous increase in the serum levels of free VEGF so that within a few days after pregnancy VEGF dominated in the maternal circulation. Our study provides novel information on the kinetics of endogenous sVEGFR-1, which serves as a potential tool in the development of new strategies against diseases associated with angiogenic imbalance and alterations in VEGF signaling.

Tonically Active Kainate Receptors (tKARs) : A Novel Mechanism Regulating Neuronal Function in the Brain

Fast excitatory transmission between neurons in the central nervous system is mainly mediated by L-glutamate acting on ligand gated (ionotropic) receptors. These are further categorized according to their pharmacological properties to AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), NMDA (N-Methyl-D-aspartic acid) and kainate (KAR) subclasses. In the rat and the mouse hippocampus, development of glutamatergic transmission is most dynamic during the first postnatal weeks. This coincides with the declining developmental expression of the GluK1 subunit-containing KARs. However, the function of KARs during early development of the brain is poorly understood. The present study reveals novel types of tonically active KARs (hereafter referred to as tKARs) which play a central role in functional development of the hippocampal CA3-CA1 network. The study shows for the first time how concomitant pre- and postsynaptic KAR function contributes to development of CA3-CA1 circuitry by regulating transmitter release and interneuron excitability. Moreover, the tKAR-dependent regulation of transmitter release provides a novel mechanism for silencing and unsilencing early synapses and thus shaping the early synaptic connectivity. The role of GluK1-containing KARs was studied in area CA3 of the neonatal hippocampus. The data demonstrate that presynaptic KARs in excitatory synapses to both pyramidal cells and interneurons are tonically activated by ambient glutamate and that they regulate glutamate release differentially, depending on target cell type. At synapses to pyramidal cells these tKARs inhibit glutamate release in a G-protein dependent manner but in contrast, at synapses to interneurons, tKARs facilitate glutamate release. On the network level these mechanisms act together upregulating activity of GABAergic microcircuits and promoting endogenous hippocampal network oscillations. By virtue of this, tKARs are likely to have an instrumental role in the functional development of the hippocampal circuitry. The next step was to investigate the role of GluK1 -containing receptors in the regulation of interneuron excitability. The spontaneous firing of interneurons in the CA3 stratum lucidum is markedly decreased during development. The shift involves tKARs that inhibit medium-duration afterhyperpolarization (mAHP) in these neurons during the first postnatal week. This promotes burst spiking of interneurons and thereby increases GABAergic activity in the network synergistically with the tKAR-mediated facilitation of their excitatory drive. During development the amplitude of evoked medium afterhyperpolarizing current (ImAHP) is dramatically increased due to decoupling tKAR activation and ImAHP modulation. These changes take place at the same time when the endogeneous network oscillations disappear. These tKAR-driven mechanisms in the CA3 area regulate both GABAergic and glutamatergic transmission and thus gate the feedforward excitatory drive to the area CA1. Here presynaptic tKARs to CA1 pyramidal cells suppress glutamate release and enable strong facilitation in response to high-frequency input. Therefore, CA1 synapses are finely tuned to high-frequency transmission; an activity pattern that is common in neonatal CA3-CA1 circuitry both in vivo and in vitro. The tKAR-regulated release probability acts as a novel presynaptic silencing mechanism that can be unsilenced in response to Hebbian activity. The present results shed new light on the mechanisms modulating the early network activity that paves the way for oscillations lying behind cognitive tasks such as learning and memory. Kainate receptor antagonists are already being developed for therapeutic use for instance against pain and migraine. Because of these modulatory actions, tKARs also represent an attractive candidate for therapeutic treatment of developmentally related complications such as learning disabilities.

Estudo das alterações cardiovasculares e metabólicas em modelo experimental de programação metabólica: efeito de extrato da casca de uva Vitis vinífera

Estudos epidemiológicos e experimentais têm sugerido que fatores de risco cardiovasculares podem ser parcialmente atribuídos às influências do ambiente em que vive o indivíduo, e que a nutrição materna influencia na programação de alterações metabólicas e cardiovasculares no indivíduo adulto e que caracterizam a síndrome metabólica (SM). Em contrapartida, estudos prévios de nosso laboratório demonstram que o extrato da casca de uva Vitis labrusca (GSE) possui efeito vasodilatador, antihipertensivo e antioxidante. Desta forma, o objetivo deste estudo foi avaliar o efeito do tratamento oral com GSE (200mg/kg/dia), sobre as alterações cardiovasculares e metabólicas e estresse oxidativo observados na prole adulta (fêmea e machos) com 3 e 6 meses, cujas mães foram submetidas a uma dieta rica em gordura (hiperlipídica) durante a lactação. Quatro grupos de ratas foram alimentados com dietas experimentais: controle (7% de gordura); controle + GSE (7% de gordura + GSE), hiperlipídica (24% de gordura); hiperlipídica + GSE (24% de gordura + GSE) durante a lactação. Após o desmame, todos os filhotes passaram a ser alimentados com uma dieta controle e foram sacrificados aos 3 ou 6 meses de idade. A pressão arterial sistólica (PAS) foi medida por pletismografia de cauda e o efeito vasodilatador da acetilcolina (ACh) foi avaliado em leito arterial mesentérico (LAM) perfundido. Foram avaliados o peso corporal, adiposidade (intra-abdominal e gonadal), níveis plasmáticos de colesterol total, triglicerídeos, glicose e insulina, e a resistência à insulina (RI) foi calculada pelo índice de HOMA IR. As expressões do IRS-1, Akt e GLUT-4 foram determinadas em músculo soleus. O dano oxidativo, níveis de nitritos e a atividade das enzimas antioxidantes: superóxido dismutase, catalase e glutationa peroxidase foram dosados no plasma e homogenato de LAM. A PAS e tecido adiposo foram aumentados nas proles adultas de ambos os sexos e idades do grupo hiperlipídico e revertidos pelo tratamento com o GSE. A resposta vasodilatadora à ACh em LAM não foi diferente entre os grupos de ambos os sexos, mas foram reduzidas com o envelhecimento. Nas proles fêmeas e machos do grupo hiperlipídico também foram observados o aumento dos níveis de triglicerídeos, de glicose e RI em ambas as idades e foram reduzidos pelo GSE. No grupo hiperlipídico houve redução nas expressões de IRS-1, Akt e GLUT-4 e o GSE reverteu estas expressões. Os níveis plasmáticos de malondialdeído estavam aumentados e os níveis de nitrito diminuídos no grupo hiperlipídico, de ambos os sexos e idades e foram revertidos pelo GSE. As atividades das enzimas antioxidantes no plasma e no mesentério foram reduzidas no grupo hiperlipídico e restauradas pelo GSE. Em conclusão, O GSE parece proteger as proles fêmeas e machos, cujas mães foram expostas a uma dieta hiperlipídica durante a lactação, dos fatores de riscos cardiovasculares, proporcionando uma fonte alternativa nutricional para a prevenção da SM.

Application of matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in determining arginine esterase from snake venom

Using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The homogeneities and molecular weights of three arginine esterases from snake venom, which possessing therapeutic use in myocardial infarction, were determined and compared, MALDI-TOF-MS is possessed of high accuracy, high sensitivity and rapidity. MALDI-TOF-MS and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can provide complementary and confirmatory results information. MALDI-TOF-MS can be directly used as an important method for the purification of snake venom complexes successfully.

Evolução da resistência aos antibióticos β-lactâmicos em Staphylococcus aureus

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Considerações sobre o papel da química bioinorgânica na saúde

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Design, synthesis and development of novel indolocarbazole derivatives as potential anti-cancer agents

This thesis describes work carried out on the design of new routes to a range of bisindolylmaleimide and indolo[2,3-a]carbazole analogs, and investigation of their potential as successful anti-cancer agents. Following initial investigation of classical routes to indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycons, a new strategy employing base-mediated condensation of thiourea and guanidine with a bisindolyl β-ketoester intermediate afforded novel 5,6-bisindolylpyrimidin-4(3H)-ones in moderate yields. Chemical diversity within this H-bonding scaffold was then studied by substitution with a panel of biologically relevant electrophiles, and by reductive desulfurisation. Optimisation of difficult heterogeneous literature conditions for oxidative desulfurisation of thiouracils was also accomplished, enabling a mild route to a novel 5,6-bisindolyluracil pharmacophore to be developed within this work. The oxidative cyclisation of selected acyclic bisindolyl systems to form a new planar class of indolo[2,3-a]pyrimido[5,4-c]carbazoles was also investigated. Successful conditions for this transformation, as well as the limitations currently prevailing for this approach are discussed. Synthesis of 3,4-bisindolyl-5-aminopyrazole as a potential isostere of bisindolylmaleimide agents was encountered, along with a comprehensive derivatisation study, in order to probe the chemical space for potential protein backbone H-bonding interactions. Synthesis of a related 3,4-arylindolyl-5-aminopyrazole series was also undertaken, based on identification of potent kinase inhibition within a closely related heterocyclic template. Following synthesis of approximately 50 novel compounds with a diversity of H-bonding enzyme-interacting potential within these classes, biological studies confirmed that significant topo II inhibition was present for 9 lead compounds, in previously unseen pyrazolo[1,5-a]pyrimidine, indolo[2,3-c]carbazole and branched S,N-disubstituted thiouracil derivative series. NCI-60 cancer cell line growth inhibition data for 6 representative compounds also revealed interesting selectivity differences between each compound class, while a new pyrimido[5,4-c]carbazole agent strongly inhibited cancer cell division at 10 µM, with appreciable cytotoxic activity observed across several tumour types.

Synthesis and evaluation of novel azaindolocarbazole derivatives as cancer chemotherapeutics

This thesis details the design and implementation of novel chemical routes towards a series of highly propitious 7-azaindolyl derivatives of the indolocarbazole (ICZ) and bisindolylmaleimide (BIM) families, with subsequent evaluation for use as cancer chemotherapeutic agents. A robust synthetic strategy was devised to allow the introduction of a 7-azaindolyl moiety into our molecular template. This approach allowed access to a wide range of β-keto ester and β-keto nitrile intermediates. Critical analysis identified F-ring modulation as a major theme towards the advancement of ICZ and BIM derivatives in drug therapy. Thus, the employment of cyclocondensation methodology furnished a number of novel aminopyrazole, isoxazolone, pyrazolone and pyrimidinone analogues, considerably widening the scope of the prevalent maleimide functionality. Photochemical cyclisation provided for the first reported aza ICZ containing a six-membered F-ring. Another method towards achieving the aza ICZ core involved use of a Perkin-type condensation approach, with chemical elaboration of the headgroup instigated post-aromatisation. Subsequent use of a modified Lossen rearrangement allowed access to further analogues containing a six-membered F-ring. Extensive screening of the novel aza ICZ and BIM derivatives was carried out against the NCI-60 cancer cell array, with nine prospective candidates selected for continued biological evaluation. From these assays, a number of compounds were shown to inhibit cancer cell growth at concentrations of below 10 nM. Indeed, the most active aza ICZ tested is currently under assessment by the Biological Evaluation Committee of the NCI due to excellent antiproliferative activity demonstrated across the panel of cell lines, with a mean GI50 of 34 nM, a mean total growth inhibition (TGI) of 4.6 μM and a mean cytotoxicity (LC50) of 63.1 μM. Correlation to known topoisomerase I (topo I) inhibitors was revealed by COMPARE analysis, and subsequent topo I-mediated DNA cleavage assays showed inhibitory activity below 1 μM for several derivatives.

Synthesis of iminophosphine and phosphinoiminol cyclometallated Pt (II) and Pt (IV) chloro complexes and studies into their biological and photophysical properties

This thesis focuses on the synthesis and analysis of novel chloride based platinum complexes derived from iminophosphine and phosphinoamide ligands, along with studies on their reactivity towards substitution and oxidation reactions. Also explored here are the potential applications of these complexes for biological and luminescent purposes. Chapter one provides an extensive overview of platinum coordination chemistry with examples of various mixed donor ligands along with the history of platinum anticancer therapy. It also looks at metals in medicine, both for biological functions as well as for therapeutic purposes and gives a background to some other applications for platinum complexes. Chapter two outlines the design and synthetic strategies employed for the development of novel platinum (II) chloride complexes from iminophosphine and phosphinoamide ligands. Also reported is the cyclometallation of these complexes to form stable tridentate mixed donor platinum (II) compounds. In Chapter three the development of a direct method for displacing a chloride from a platinum metal centre with a desired phosphine is reported. Numerous methods for successful oxidation of the platinum (II) complexes will also be explored, leading to novel platinum (IV) complexes being reported here also. The importance of stabilisation of the displaced anion, chloride, by the solvent system will also be discussed in this chapter. Chapter four investigates the reactivity of the platinum (II) complexes towards two different biomolecules to form novel platinum bio-adducts. The potential application of the platinum (II) cyclometallates as chemotherapeutics will also be explored here using in-vitro cancer cell testing. Finally, luminescence studies are also reported here for the ligands and platinum complexes reported in chapter two and three to investigate potential applications in this field also. Chapter five provides a final conclusion and an overall summary of the entire project as well as identifying key areas for future work.

Usual interstitial pneumonia

We report the case of a 49-year old woman with an idiopathic pulmonary fibrosis (IPF) initially diagnosed as a systemic lupus erythematosus. The IPF is an uncommon clinical entity with an estimated prevalence from 3 to 6 cases per 100,000 in the general population of the United States. This disease is characterised by an insidious onset, a pejorative course and poor survival prognosis (median survival: 2.8 years). The diagnosis is often difficult and depends on the exclusion of other diseases associated with interstitial lung injury. It is generally established only after collegial coordination between the clinician, the radiologist and the pathologist. New consensuses are now published to establish a clear and explicit classification of the IPF. Moreover, because of the poor results obtained with conventional immunosuppressive drugs, new treatments are proposed.

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease.

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

Paclitaxel versus doxorubicin as first-line single-agent chemotherapy for metastatic breast cancer: a European Organization for Research and Treatment of Cancer Randomized Study with cross-over.

PURPOSE: To compare the efficacy of paclitaxel versus doxorubicin given as single agents in first-line therapy of advanced breast cancer (primary end point, progression-free survival ¿PFS) and to explore the degree of cross-resistance between the two agents. PATIENTS AND METHODS: Three hundred thirty-one patients were randomized to receive either paclitaxel 200 mg/m(2), 3-hour infusion every 3 weeks, or doxorubicin 75 mg/m(2), intravenous bolus every 3 weeks. Seven courses were planned unless progression or unacceptable toxicity occurred before the seven courses were finished. Patients who progressed within the seven courses underwent early cross-over to the alternative drug, while a delayed cross-over was optional for the remainder of patients at the time of disease progression. RESULTS: Objective response in first-line therapy was significantly better (P =.003) for doxorubicin (response rate ¿RR, 41%) than for paclitaxel (RR, 25%), with doxorubicin achieving a longer median PFS (7.5 months for doxorubicin v 3.9 months for paclitaxel, P <.001). In second-line therapy, cross-over to doxorubicin (91 patients) and to paclitaxel (77 patients) gave response rates of 30% and 16%, respectively. The median survival durations of 18.3 months for doxorubicin and 15.6 months for paclitaxel were not significantly different (P =.38). The doxorubicin arm had greater toxicity, but this was counterbalanced by better symptom control. CONCLUSION: At the dosages and schedules used in the present study, doxorubicin achieves better disease and symptom control than paclitaxel in first-line treatment. Doxorubicin and paclitaxel are not totally cross-resistant, which supports further investigation of these drugs in combination or in sequence, both in advanced disease and in the adjuvant setting.

An EORTC-IDBBC phase I study of gemcitabine and continuous infusion 5-fluorouracil in patients with metastatic breast cancer resistant to anthracyclines or pre-treated with both anthracyclines and taxanes.

The aim of this study was to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and potential activity of combined gemcitabine and continuous infusion 5-fluorouracil (5-FU) in metastatic breast cancer (MBC) patients that are resistant to anthracyclines or have been pretreated with both anthracyclines and taxanes. 15 patients with MBC were studied at three European Organization for Research and Treatment of Cancer centres. 13 patients had received both anthracylines and taxanes. Gemcitabine was given intravenously (i.v.) on days 1 and 8, and 5-FU as a continuous i.v. infusion on days 1 through to 14, both drugs given in a 21-day schedule at four different dose levels. Both were given at doses commonly used for the single agents for the last dose level (dose level 4). One of 6 patients at level 4 (gemcitabine 1200 mg/m2 and 5-FU 250 mg/m2/day) had a DLT, a grade 3 stomatitis and skin toxicity. One DLT, a grade 3 transaminase rise and thrombosis, occurred in a patient at level 2 (gemcitabine 1000 mg/m2 and 5-FU 200 mg/m2/day). Thus, the MTD was not reached. One partial response and four disease stabilisations were observed. Only 1 patient withdrew from the treatment due to toxicity. The MTD was not reached in the phase I study. The combination of gemcitabine and 5-FU is well tolerated at doses up to 1200 mg/m2 given on days 1 and 8 and 250 mg/m2/day given on days 1 through to 14, respectively, every 21 days. The clinical benefit rate (responses plus no change of at least 6 months) was 33% with one partial response, suggesting that MBC patients with prior anthracycline and taxane therapy may derive significant benefit from this combination with minimal toxicity.