Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Application of Inductive Logic Programming to Structure-Based Drug Design

Enot, D. and King, R. D. (2003) Application of Inductive Logic Programming to Structure-Based Drug Design. 7th European Conference on Principles and Practice of Knowledge Discovery in Databases (PKDD '03). Springer LNAI 2838 p156-167

Structure based drug design with inductive logic programming

David P. Enot and Ross D. King (2003). Structure based drug design with inductive logic programming. The ACS National Meeting Spring 2003, New Orleans

The use of Inductive Logic Programming in drug design

David P. Enot and Ross D. King (2002) The use of Inductive Logic Programming in drug design. Proceedings of the 14th EuroQSAR Symposium (EuroQSAR 2002). Blackwell Publishing, p247-250

Avaliação do perfil de prescrição num Serviço Hospitalar de Psiquiatria

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

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Strategies to prevent potentially inappropriate prescribing and adverse drug reactions in older patients

INTRODUCTION: Potentially inappropriate prescribing (PIP) is a major contributor to adverse drug reactions and overall increased healthcare costs. The aim of this thesis was to identify, develop and implement strategies with the potential to prevent PIP and ADRs in older patients. METHODS: A systematic review of the qualitative literature (Meta-synthesis); A qualitative study in Irish hospitals; A randomised controlled trial (RCT) to assess the impact of an online educational module on doctors’ prescribing knowledge and confidence; Exploration of the potential for a frailty index score to enable doctors identify patients at increased risk of PIP and ADRs; Exploration of the potential for the SHiM tool to enable doctors to optimise prescribing for older patients. RESULTS: The meta-synthesis identified four key concepts: (i) Desire to please the patient; (ii) Feeling of being forced to prescribe; (iii) Tension between experience and guidelines; (iv) Prescriber fear. Similar themes also emerged from the qualitative study. In the RCT, the online educational module resulted in a highly significant 22% difference in test scores between intervention and control groups. The studies exploring the frailty index score showed a significant positive relationship between a patient’s frailty status and their likelihood of experiencing PIP/ADRs. Patients above a frailty threshold of 0.16 were at least twice as likely to experience PIP/ADRs. SHiM was found to be a useful tool in terms of reconciling patients’ medications. However, the evidence for it being capable of preventing clinically relevant adverse events was poor. CONCLUSION:Qualitative research in this thesis has proposed novel theories relating to the causative factors of PIP in older patients. In doing so, it has identified several areas for intervention and laid down a road map for future research.

Randomized, controlled trial investigating short-term health-related quality of life with doxorubicin and paclitaxel versus doxorubicin and cyclophosphamide as first-line chemotherapy in patients with metastatic breast cancer: European Organization for Research and Treatment of Cancer Breast Cancer Group, Investigational Drug Branch for Breast Cancer and the New Drug Development Group Study.

PURPOSE: To compare health-related quality of life (HRQOL) in patients with metastatic breast cancer receiving the combination of doxorubicin and paclitaxel (AT) or doxorubicin and cyclophosphamide (AC) as first-line chemotherapy treatment. PATIENTS AND METHODS: Eligible patients (n = 275) with anthracycline-naive measurable metastatic breast cancer were randomly assigned to AT (doxorubicin 60 mg/m(2) as an intravenous bolus plus paclitaxel 175 mg/m(2) as a 3-hour infusion) or AC (doxorubicin 60 mg/m(2) plus cyclophosphamide 600 mg/m(2)) every 3 weeks for a maximum of six cycles. Dose escalation of paclitaxel (200 mg/m(2)) and cyclophosphamide (750 mg/m(2)) was planned at cycle 2 to reach equivalent myelosuppression in the two groups. HRQOL was assessed with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire C30 and the EORTC Breast Module at baseline and the start of cycles 2, 4, and 6, and 3 months after the last cycle. RESULTS: Seventy-nine percent of the patients (n = 219) completed a baseline measure. However, there were no statistically significant differences in HRQOL between the two treatment groups. In both groups, selected aspects of HRQOL were impaired over time, with increased fatigue, although some clinically significant improvements in emotional functioning were seen, as well as a reduction in pain over time. Overall, global quality of life was maintained in both treatment groups. CONCLUSION: This information is important when advising women patients of the expected HRQOL consequences of treatment regimens and should help clinicians and their patients make informed treatment decisions.

Evaluating intra-articular drug delivery for the treatment of osteoarthritis in a rat model.

Osteoarthritis (OA) is a degenerative joint disease that can result in joint pain, loss of joint function, and deleterious effects on activity levels and lifestyle habits. Current therapies for OA are largely aimed at symptomatic relief and may have limited effects on the underlying cascade of joint degradation. Local drug delivery strategies may provide for the development of more successful OA treatment outcomes that have potential to reduce local joint inflammation, reduce joint destruction, offer pain relief, and restore patient activity levels and joint function. As increasing interest turns toward intra-articular drug delivery routes, parallel interest has emerged in evaluating drug biodistribution, safety, and efficacy in preclinical models. Rodent models provide major advantages for the development of drug delivery strategies, chiefly because of lower cost, successful replication of human OA-like characteristics, rapid disease development, and small joint volumes that enable use of lower total drug amounts during protocol development. These models, however, also offer the potential to investigate the therapeutic effects of local drug therapy on animal behavior, including pain sensitivity thresholds and locomotion characteristics. Herein, we describe a translational paradigm for the evaluation of an intra-articular drug delivery strategy in a rat OA model. This model, a rat interleukin-1beta overexpression model, offers the ability to evaluate anti-interleukin-1 therapeutics for drug biodistribution, activity, and safety as well as the therapeutic relief of disease symptoms. Once the action against interleukin-1 is confirmed in vivo, the newly developed anti-inflammatory drug can be evaluated for evidence of disease-modifying effects in more complex preclinical models.

Study protocol: the Adherence and Intensification of Medications (AIM) study--a cluster randomized controlled effectiveness study.

BACKGROUND: Many patients with diabetes have poor blood pressure (BP) control. Pharmacological therapy is the cornerstone of effective BP treatment, yet there are high rates both of poor medication adherence and failure to intensify medications. Successful medication management requires an effective partnership between providers who initiate and increase doses of effective medications and patients who adhere to the regimen. METHODS: In this cluster-randomized controlled effectiveness study, primary care teams within sites were randomized to a program led by a clinical pharmacist trained in motivational interviewing-based behavioral counseling approaches and authorized to make BP medication changes or to usual care. This study involved the collection of data during a 14-month intervention period in three Department of Veterans Affairs facilities and two Kaiser Permanente Northern California facilities. The clinical pharmacist was supported by clinical information systems that enabled proactive identification of, and outreach to, eligible patients identified on the basis of poor BP control and either medication refill gaps or lack of recent medication intensification. The primary outcome is the relative change in systolic blood pressure (SBP) measurements over time. Secondary outcomes are changes in Hemoglobin A1c, low-density lipoprotein cholesterol (LDL), medication adherence determined from pharmacy refill data, and medication intensification rates. DISCUSSION: Integration of the three intervention elements--proactive identification, adherence counseling and medication intensification--is essential to achieve optimal levels of control for high-risk patients. Testing the effectiveness of this intervention at the team level allows us to study the program as it would typically be implemented within a clinic setting, including how it integrates with other elements of care. TRIAL REGISTRATION: The ClinicalTrials.gov registration number is NCT00495794.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Spending on postapproval drug safety.

Withdrawals of high-profile pharmaceuticals have focused attention on post-approval safety surveillance. There have been no systematic assessments of spending on postapproval safety. We surveyed drug manufacturers regarding safety efforts. Mean spending on postapproval safety per company in 2003 was 56 million dollars (0.3 percent of sales). Assuming a constant safety-to-sales ratio, we estimated that total spending on postapproval safety by the top twenty drug manufacturers was 800 million dollars in 2003. We also examined, using regression analysis, the relationship between the number of safety personnel and the number of initial adverse-event reports. This study offers information for the debate on proposed changes to safety surveillance.

Economics of new oncology drug development.

PURPOSE: Review existing studies and provide new results on the development, regulatory, and market aspects of new oncology drug development. METHODS: We utilized data from the US Food and Drug Administration (FDA), company surveys, and publicly available commercial business intelligence databases on new oncology drugs approved in the United States and on investigational oncology drugs to estimate average development and regulatory approval times, clinical approval success rates, first-in-class status, and global market diffusion. RESULTS: We found that approved new oncology drugs to have a disproportionately high share of FDA priority review ratings, of orphan drug designations at approval, and of drugs that were granted inclusion in at least one of the FDA's expedited access programs. US regulatory approval times were shorter, on average, for oncology drugs (0.5 years), but US clinical development times were longer on average (1.5 years). Clinical approval success rates were similar for oncology and other drugs, but proportionately more of the oncology failures reached expensive late-stage clinical testing before being abandoned. In relation to other drugs, new oncology drug approvals were more often first-in-class and diffused more widely across important international markets. CONCLUSION: The market success of oncology drugs has induced a substantial amount of investment in oncology drug development in the last decade or so. However, given the great need for further progress, the extent to which efforts to develop new oncology drugs will grow depends on future public-sector investment in basic research, developments in translational medicine, and regulatory reforms that advance drug-development science.

Assessing teen risk behavior and later drug use

Gemstone Team Risky Business

Returns to R&D on new drug introductions in the 1980s.

This study finds that the mean IRR for 1980-84 U.S. new drug introductions is 11.1%, and the mean NPV is 22 million (1990 dollars). The distribution of returns is highly skewed. The results are robust to plausible changes in the baseline assumptions. Our work is also compared with a 1993 study by the OTA. Despite some important differences in assumptions, both studies imply that returns for the average NCE are within one percentage point of the industry's cost of capital. This is much less than what is typically observed in analyses based on accounting data.