Circular Sponge against miR-21 Enhances the Antitumor Activity of Doxorubicin against Breast Cancer Cells.

Breast cancer is the most common type of cancer in women, with chemotherapy being the main strategy. However, its effectiveness is reduced by drug resistance mechanisms. miR-21 is upregulated in breast cancer that has been linked to drug resistance and carcinogenic processes. Our aim was to capture miR-21 with a circular sponge (Circ-21) and thus inhibit the carcinogenic processes and drug resistance mechanisms in which it participates. Proliferation, migration, colony formation, cell cycle, and poly [ADP-ribose] polymerase 1 (PARP-1) and vascular endothelial growth factor (VEGF) detection assays were performed with MCF7 breast cancer cells and MCF10A non-tumor cells. In addition, doxorubicin resistance tests and detection of drug resistance gene expression were performed in MCF7 cells. Reduction in proliferation, as well as migration and colony formation, increased PARP-1 expression, inhibition of VEGF expression and cell cycle arrest in G2/M phase were displayed in the Circ-21 MCF7, which were not observed in the MCF10A cells. Furthermore, in the MCF7 cells, the Circ-21 enhanced the antitumor activity of doxorubicin and decreased the expression of resistance genes: ABCA1, ABCC4, and ABCC5. Based on these results, the use of Circ-21 can be considered a first step for the establishment of an effective gene therapy in the treatment of breast cancer.

Antitumor Effect of Traditional Drugs for Neurological Disorders: Preliminary Studies in Neural Tumor Cell Lines.

Glioblastoma multiforme is the most common malignant primary brain tumor in adults. Despite new treatments developed including immunomodulation using vaccines and cell therapies, mortality remains high due to the resistance mechanisms presented by these tumor cells and the function of the blood-brain barrier that prevents the entry of most drugs. In this context of searching for new glioblastoma therapies, the study of the existing drugs to treat neurological disorder is gaining great relevance. The aim of this study was to determine, through a preliminary in vitro study on human glioblastoma (A172, LN229), anaplastic glioma (SF268) and neuroblastoma (SK-N-SH) cell lines, the possible antitumor activity of the active principles of several drugs (levomepromazine, haloperidol, lacosamide, valproic acid, levetiracetam, glatiramer acetate, fingolimod, biperiden and dextromethorphan) with the ability to cross the blood-brain barrier and that are commonly used in neurological disorders. Results showed that levetiracetam, valproic acid, and haloperidol were able to induce a relevant synergistic antitumor effect when associated with the chemotherapy currently used in clinic (temozolomide). Regarding the mechanism of action, haloperidol, valproic acid and levomepromazine caused cell death by apoptosis, while biperiden and dextromethorphan induced autophagy. Fingolimod appeared to have anoikis-related cell death. Thus, the assayed drugs which are able to cross the blood-brain barrier could represent a possibility to improve the treatment of neural tumors, though future in vivo studies and clinical trials will be necessary to validate it.

Synthetic Circular miR-21 Sponge as Tool for Lung Cancer Treatment.

Lung cancer is the most common cancer in the world and several miRNAs are associated with it. MiRNA sponges are presented as tools to inhibit miRNAs. We designed a system to capture miRNAs based on circular RNAs (circRNA). To demonstrate its usefulness, we chose miR-21, which is upregulated and implicated in lung cancer. We constructed a miR-21 sponge and inserted it into a vector that facilitates circular RNA production (Circ-21) to study its effect on growth, colony formation, and migration in lung cancer cell lines and multicellular tumor spheroids (MTS). Circ-21 induced a significant and time-dependent decrease in the growth of A549 and LL2 cells, but not in L132 cells. Furthermore, A549 and LL2 cells transfected with Circ-21 showed a lower number of colonies and migration than L132. Similar findings were seen in A549 and LL2 Circ-21 MTS, which showed a significant decrease in volume growth, but not in L132 Circ-21 MTS. Based on this, the miR-21 circular sponge may suppress the processes of tumorigenesis and progression. Therefore, our system based on circular sponges seems to be effective, as a tool for the capture of other miRNAs.

Nanoparticles in Colorectal Cancer Therapy: Latest In Vivo Assays, Clinical Trials, and Patents.

Colorectal cancer (CRC) is the third most common cancer worldwide. Its poor response to current treatment options in advanced stages and the need for efficient diagnosis in early stages call for the development of new therapeutic and diagnostic strategies. Some of them are based on the use of nanometric materials as carriers and releasers of therapeutic agents and fluorescent molecules, or even on the utilization of magnetic materials that provide very interesting properties. These nanoformulations present several advantages compared with the free molecular cargo, including increased drug solubility, bioavailability, stability, and tumor specificity. Moreover, tumor multidrug resistance has been decreased in some cases, leading to improved treatment effectiveness by reducing drug dose and potential side effects. Here, we present an updated overview of the latest advances in clinical research, in vivo studies, and patents regarding the application of nanoformulations in the treatment of CRC. Based on the information gathered, a wide variety of nanomaterials are being investigated in clinical research, even in advanced phases, i.e., close to reaching the market. In sum, these novel materials can offer remarkable advantages with respect to current therapies, which could be complemented or even replaced by these nanosystems in the near future.

Novel MicroRNA Sponges to Specifically Modulate Gene Expression in Colon Cancer Cells.

MicroRNA (miRNA) sponges allow the selective blockade of a complete family of associated miRNAs, which induce post-transcriptional gene silencing in their target through binding to 3'UTR mRNA. miRNA-365 and miRNA-145 are downregulated in colorectal cancer (CRC) but not in healthy tissues. Based on this, we constructed two vectors by inserting miRNA sponges (one for miRNA-365 and other for miRNA-145), and used enhanced green fluorescent protein (EGFP) as a 3'UTR reporter gene to analyze the ability of each sponge to catch its respective miRNA. Quantitative polymerase chain reaction (qPCR) results corroborated that the expression levels of both miRNAs were lower in CRC cell lines than in normal colon cell lines. Flow cytometry analysis revealed a decrease of the EGFP expression levels in the cell lines transfected with both sponges, being higher on the normal cell line while CRC cell lines presented a minimal decline. Also, this decrease was inversely proportional to the levels of expression of both miRNAs obtained by qPCR. These results were corroborated by fluorescence microscopy, showing a similar decrease in fluorescence. We propose a new vector system to carry in a specific way the expression of genes to CRC cells without affecting healthy cells, preventing damage to healthy tissues.

Adverse reproductive health outcomes in a cohort of young women with breast cancer exposed to systemic treatments.

BACKGROUND: Breast cancer is the most common cancer in young women. Fortunately current survival rates of BC are significant which makes future fertility very important for quality of life of BC survivors. Chemotherapy carries a significant risk of infertility in BC patients so it is important to support fertility preservation decisions in premenopausal women. Amenorrhea has long been used as a surrogate marker of infertility in cancer patients but more reliable ovarian reserve (OR) markers are available. This study aimed to prospectively measure levels of OR in a cohort of young women with breast cancer exposed to chemotherapy, to identify adverse reproductive health outcomes in this population and to assess the influence of patient and treatment-related factors in those outcomes. METHODS: This prospective observational study included premenopausal women with breast cancer aged 18-40 years at diagnosis and proposed for (neo) adjuvant chemotherapy. Patients were evaluated before, during and a minimum of 9 months after the end of chemotherapy. Reproductive health outcomes: menses, hormonal and ultrasound OR markers, recovery of ovarian function and Premature Ovarian Insufficiency (POI). RESULTS: A total of 38 patients were included (mean age 32.9 ± 3.5 years). Levels of OR significantly decreased during the study. At the last follow up, 35 patients had AMH below the expected values for age; eight presented postmenopausal FSH; ten had not recovered their ovarian function and five met the defined criteria for POI. Age and baseline AMH were positively correlated with AMH at the last follow-up. AMH levels were higher in the group of patients treated with trastuzumab and lower in those under hormonal therapy, at the last follow-up. CONCLUSIONS: Significant effects of systemic treatments on several reproductive outcomes and a strong relation of those outcomes with patient's age and baseline level of AMH were observed. Our results point to a possible lower gonadotoxicity when treatment includes targeted therapy with trastuzumab. Also, this investigation highlights the lack of reliable OR markers in women under hormonal therapy.

Double origin of the extensor hallucis longus muscle: a case report.

Most of the anatomic variations of the extensor hallucis longus (EHL) muscle are related to the tendon of insertion. We show a double origin of the EHL from the medial aspect of the fibula and the lateral aspect of the tibia. A 27-year-old male with a double closed fracture of tibia and fibula showed an involuntary extension of the big toe during foot plantar flexion after surgery. A tendon fibrosis by the fixation plates could be the cause of the foot functional alteration. Interestingly, the anatomic variation described could be related to the postsurgical foot dysfunction, since when the fibrotic tissue was removed the normal extension of big toe recovered. As illustrated in this case report, knowledge of anatomic variations is very useful, particularly in the context of foot surgery.

The challenge of drug resistance in pancreatic ductal adenocarcinoma: a current overview.

Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates among all cancer types. Its delayed diagnosis precludes curative resection, thus most of the current therapies against PDAC are based on chemo- and radiotherapy. Unfortunately, these strategies are insufficient to improve its poor prognosis. Despite the advances made in chemotherapy (e.g. nab-paclitaxel and gemcitabine), many patients with PDAC are unable to benefit from them due to the rapid development of drug resistance. Currently, more than 165 genes have been found to be implicated in drug resistance of pancreatic tumors, including different integrins, mucins, NF-κB, RAS and CXCR4. Moreover, drug resistance in PDAC is thought to be mediated by the modulation of miRNAs (e.g. miRNA-21, miRNA-145 and miRNA-155), which regulate genes that participate in cell proliferation, invasion and metastasis. Finally, cancer stem cells are intimately related to drug resistance in PDAC due to their ability to overexpress ABC genes -involved in drug transport-, and enzymes such as aldehyde dehydrogenases -implicated in cellular drug metabolism- and poly (ADP-ribose) polymerases -involved in drug-induced DNA damage repair. Understanding the mechanisms involved in drug resistance will contribute to the development of efficient therapeutic strategies and to improve the prognosis of patients with PDAC.

Downregulated microRNAs in the colorectal cancer: diagnostic and therapeutic perspectives.

Colorectal cancer (CRC), the third most common cancer in the world, has no specific biomarkers that facilitate its diagnosis and subsequent treatment. The miRNAs, small single-stranded RNAs that repress the mRNA translation and trigger the mRNA degradation, show aberrant levels in the CRC, by which these molecules have been related with the initiation, progression, and drug-resistance of this cancer type. Numerous studies show the microRNAs influence the cellular mechanisms related to the cell cycle, differentiation, apoptosis, and migration of the cancer cells through the post-transcriptionally regulated gene expression. Specific patterns of the upregulated and down-regulated miRNA have been associated with the CRC diagnosis, prognosis, and therapeutic response. Concretely, the downregulated miRNAs represent attractive candidates, not only for the CRC diagnosis, but for the targeted therapies via the tumor-suppressing microRNA replacement. This review shows a general overview of the potential uses of the miRNAs in the CRC diagnosis, prognosis, and treatment with a special focus on the downregulated ones. [BMB Reports 2018; 51(11): 563-571].

DNA methylation patterns as molecular biomarkers: an overview in colorectal cancer

DNA methylation patterns may be used as innovative biomarkers for some pathologies including cancer. They show a great accessibility due to their stability and presence in body fluids. In addition, these epigenetic modifications may be used as prognosis markers or therapeutic targets. Concretely, in colorectal cancer (CRC), the third most common cancer in the world in both men and women, a continuous genetic and epigenetic alteration occurs during neoplastic transformation in colonic epithelial cells. This accumulation of alterations leads to the transformation of normal colonic epithelial cells to adenocarcinomas, and these genetic alterations are promoted by aberrant methylation of promoter regions and subsequent gene silencing. Many of these genes have been reported to be methylated in the tissue, plasma and stool of CRC patients, suggesting that they may have great potential to be used as biomarkers for the early detection of CRC. The aim of this study is to review changes in the methylation pattern of the genes that can be used as novel diagnostic and prognostic biomarkers of CRC

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Antineoplastic Agents and (In)fertility: Informing Patients to Improve Decisions.

PURPOSE: Infertility is a potential adverse effect of cancer treatment, and future fertility is an important issue for cancer patients. In Portugal, the Centre for Fertility Preservation of CHUC, EPE, conducted a project to develop and disseminate oncofertility information resources. In this study, we report the results of the specific component of this program, which intended to produce information resources that promote patients' awareness of the subject and to support decisions concerning fertility preservation. METHODS: Guidance for writing health information for patients and criteria for developing decision aids were gathered. Information needs were assessed (literature review and locally applied questionnaire). Resources were pre-tested with a sample of patients and professionals. Their readability, presentation quality, and ability to support decisions were evaluated. RESULTS: General information handouts on infertility risk and decision aids about fertility preservation options were developed and positively evaluated. The resources are currently being distributed in collaboration with several national organizations. CONCLUSIONS: Through our multidisciplinary information program, reproductive-age cancer patients now have access to relevant information resources that will support timely, shared decision-making concerning fertility preservation.

Decision on Fertility Preservation in Cancer Patients: Development of Information Materials for Healthcare Professionals.

Infertility is a potential side effect of cancer chemotherapy. As the number of adolescent and young adult (AYA)-aged survivors increases, future fertility becomes an important issue. However, many patients are not adequately informed and oncologists point the lack of information as a barrier to discussion. Our aim was to produce information materials tailored to oncologists' needs to promote and support discussion on infertility risk and fertility preservation (FP) with AYA-aged patients. After literature review, information materials were successfully developed and are currently being distributed to healthcare professionals in Portugal, with the collaboration of several national organizations. These information materials will contribute to shared informed decisions regarding FP in AYA-aged patients.

Factors associated with ovarian function recovery after chemotherapy for breast cancer: a systematic review and meta-analysis.

STUDY QUESTION: Which factors related to patient, treatment or disease are associated with ovarian function recovery after chemotherapy in premenopausal women with breast cancer? SUMMARY ANSWER: Younger age and GnRH agonist (GnRHa) administration during chemotherapy were significantly associated with menses recovery, but this recovery was less likely in patients exposed to taxanes. WHAT IS ALREADY KNOWN: To date, published meta-analyses have only assessed GnRHa administration as a possible factor for ovarian function recovery, and their results were conflicting. Current guidelines present distinct recommendations regarding the use of GnRHa for fertility preservation (FP) in women with breast cancer. STUDY DESIGN, SIZE, DURATION: A systematic review and meta-analysis of published studies in the English, Portuguese, French or Spanish languages (1990-2015), ongoing trials or completed trials (1990-2015) and conference proceedings (2000-2015) were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: We searched the MEDLINE, Embase, LILACS, Scielo, Toxline and DART databases, online trial registries and conference proceedings. Studies were eligible if they included premenopausal women with early breast cancer treated with chemotherapy, reported ovarian function recovery data and identified factor(s) associated with recovery. Two authors independently screened the studies, extracted data and assessed the risk of bias. An odds ratio (OR) was estimated from the number of recovery events. A meta-analysis was conducted using a random-effects model. MAIN RESULTS AND THE ROLE OF CHANCE: Fifteen articles were included. Five different factors were analysed: younger age and baseline levels of anti-Müllerian hormone (patient-related factors), co-administration of GnRHa, addition of taxanes to anthracycline-based chemotherapy and addition of endocrine therapy to chemotherapy (treatment-related factors). Menses recovery was the most used marker. Younger age (≤40 years) and exposure to GnRHa were positively associated with menses recovery (OR 6.07 and 2.03, respectively) but exposure to taxanes adversely affected recovery (OR 0.49). Significant heterogeneity among studies was found. LIMITATIONS, REASONS FOR CAUTION: A general limitation of the included studies is the use of menses as the main recovery marker. Regarding GnRHa, the substantial heterogeneity and conflicting results limit the interpretation of our results. Studies that use additional markers and have a longer follow-up are needed. WIDER IMPLICATIONS OF THE FINDINGS: The decision for using chemotherapy regimens with taxanes must take into account their potential adverse effects on female fertility. Considering the conflicting results regarding GnRHa agonist use, other fertility preservation strategies should also be considered. STUDY FUNDING/COMPETING INTERESTS: No external funding was received. There are no conflicts of interest to declare. PROSPERO REGISTRATION NUMBER: This review was registered at PROSPERO (CRD42015013494).

Last Advances in Nanocarriers-Based Drug Delivery Systems for Colorectal Cancer.

Colorectal cancer is the third most common type of cancer in both, men and women. The development of metastasis is very frequent, especially in patients with advanced stage, who require intensive chemotherapy that often results in poor response and significant morbidity. The undesirable effects of intensive chemotherapy on normal cells and the development of multidrug resistance are two of the main causes of treatment failure. Recent advances in nanotechnology allow to target cancer cells using cytotoxic drugs without affecting normal cells. Nanocarriers such as liposomes, polymeric nanoparticles and carbon nanotubes, among others, are able to improve drug distribution and bioavailability, cytotoxic concentration in the tumor mass and drug delivery to tumor tissue and, at the same time, reduce side effects. Current research studies are being conducted to develop new biomaterials that improve the characteristics of these nanomolecules. Several preclinical assays have disclosed the efficacy of nanotherapy in colon cancer, although further clinical trials will be necessary to demonstrate its efficacy. This review discusses the current status and the potential advantages of using nanocarrier-based drug delivery systems for colorectal cancer.

Enhanced antitumoral activity of doxorubicin against lung cancer cells using biodegradable poly(butylcyanoacrylate) nanoparticles.

Doxorubicin (Dox) is widely used for the combined chemotherapy of solid tumors. However, the use of these drug associations in lung cancer has low antitumor efficacy. To improve its efficacious delivery and activity in lung adenocarcinoma cells, we developed a biodegradable and noncytotoxic nanoplatform based on biodegradable poly(butylcyanoacrylate) (PBCA). The reproducible formulation method was based on an anionic polymerization process of the PBCA monomer, with the antitumor drug being entrapped within the nanoparticle (NP) matrix during its formation. Improved drug-entrapment efficiencies and sustained (biphasic) drug-release properties were made possible by taking advantage of the synthesis conditions (drug, monomer, and surfactant-agent concentrations). Dox-loaded NPs significantly enhanced cellular uptake of the drug in the A549 and LL/2 lung cancer cell lines, leading to a significant improvement of the drug's antitumoral activity. In vivo studies demonstrated that Dox-loaded NPs clearly reduced tumor volumes and increased mouse-survival rates compared to the free drug. These results demonstrated that PBCA NPs may be used to optimize the antitumor activity of Dox, thus exhibiting a potential application in chemotherapy against lung adenocarcinoma.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells.

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell-microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 µM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.

Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter.

Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA) to direct E gene expression (pCEA-E) towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells.

ABC transporters as differentiation markers in glioblastoma cells.

UNLABELLED: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumour, characterized by a high aggressivity, a huge heterogeneity attending a hierarchical model and resistance to therapy. Drug resistance has been correlated with the presence of the ABC efflux transporters which are able to exclude drugs for the cellular cytoplasm. In the nucleus of the GBM, initiating cells (ICs) can self-renew and give rise to cancer stem cells, which differ to the side population cells and the different cellular subtypes that form the mass around them. The ICs do not express or express ATP binding cassette (ABC) at very low levels, but this expression may increase with the differentiation process. We suggest that the differentiation process may be responsible of chemoresistance of the GBM cells. We compared three ABC transporters expression: ABCA1, MRP4 and MRP5, in the ICs obtained from 9 patients with GBM and their respective differentiated GBM cells. We show an overexpression of the three ABC transporters in the differentiated GBM cells in comparison to ICs. IMPLICATIONS OF THE HYPOTHESIS: The blockade of these ABC transporters could help to improve the drug effectivity and thus reduce the tumour growth and prevent the tumour recurrence.

Cancer stem cells and their implication in breast cancer.

BACKGROUND: The cancer stem cell (CSC) hypothesis on the origin of cancer has recently gained considerable support. CSCs are tumour cells with the capacity for self-renewal and differentiation that direct the origin and progression of the disease and may be responsible for relapse, metastasis and treatment failures. DESIGN: This article reviews breast CSCs (BCSCs) phenotyping, clinical implications and clinical trials focused on BCSCs in breast cancer. Relevant studies were found through PubMed and Clinicaltrials.gov databases. RESULTS: Cancer stem cells are identified and isolated using membrane and cell activity markers; in the case of BCSCs, these are CD44(+) /CD24(low/-) and show aldehyde dehydrogenase activity, alongside their capacity to grow and form mammospheres. The presence of stem cell properties is associated with a worse outcome. Hence, these cells have important clinical implications, and elucidation of the mechanisms underlying their activity will allow the development of novel effective therapies and diagnostic instruments, improving the prognosis of these patients. CONCLUSIONS: Standard treatments are directed against the tumour mass and do not eliminate CSCs. There is therefore a need for specific anti-CSC therapies, and numerous authors are investigating new targets to this end, as reported in this review. It is also necessary for clinical trials to be undertaken to allow this new knowledge to be applied in the clinical setting. However, there have been few trials on anti-BCSCs therapies to date.

Assessing the impact of multi-compartment compliance aids on clinical outcomes in the elderly: a pilot study.

BACKGROUND: Medication non-adherence is a major problem for elderly people. Multicompartment compliance aids (MCAs) have been advocated as a solution for this problem. OBJECTIVE: To assess the impact of using MCAs in self-reported adherence and clinical biomarkers of elderly patients followed in a community pharmacy. SETTING: One community pharmacy at Sabugal (Portugal). METHODS: A four-month prospective, non-randomised, controlled study was performed. Autonomous patients aged 65 or more using 3 or more medicines and under follow-up in the pharmacy were invited to participate. All patients were offered to receive their medication in MCAs prepared in the pharmacy. Patients refusing the MCA were used as control. The intervention consisted of providing 4 weekly MCAs during the monthly visit. All patients received regular pharmacy counselling. Blood pressure (BP), lipid profile and glycaemia were assessed at baseline and monthly for all the patients. Morisky self-reported scale was applied at baseline and at the end of the study. Bivariate analysis and generalized estimation equations (GEE) were used. MAIN OUTCOME MEASURE: Self-reported medication adherence, clinical biomarkers: BP, lipid profile, glycaemia. RESULTS: 54 patients between 65 and 90 years were under follow-up. 44 patients accepted the MCA, constituting the intervention group. No difference in the baseline biomarkers between both groups was found. The bivariate pre-post analysis yielded significant improvements in the intervention groups, but not in the control, for glycaemia (p < 0.001), HDL-c (p = 0.018), and systolic (p < 0.001) and diastolic (p = 0.012) BP. However, when introducing the 'time in follow-up' in the GEE model, all the differences became non-significant, except systolic BP, but the time remained significant for all the biomarkers. CONCLUSION: MCAs apparently improve several clinical biomarkers in a cohort of patients under pharmacist's follow-up. When including the time in pharmacist's followup in a GEE, the effect of the MCA disappeared, remaining only the time as a significant variable. Not considering the time in follow-up may be overestimating the effect of MCAs.