Were medicine quality and pharmaceutical management contributing factors in diminishing artemisinin efficacy in Guyana and Suriname?

BACKGROUND: Recent studies in Guyana and Suriname unveiled diminished efficacy of artemisinin derivatives based on day-3 parasitaemia. The migrant characteristics of the population at risk and the potential development of resistance pose a serious health threat in the region. Assessment of factors that may have contributed to this situation is warranted, and analysis of the data generated in those countries on quality and pharmaceutical managements of anti-malarials may contribute to a better understanding of this occurrence. METHODS: Data on malaria medicine quality and pharmaceutical management, generated in the context of the Amazon Malaria Initiative (AMI), was reviewed and discussed. RESULTS: Numerous substandard artemisinin-containing malaria medicines were identified in both countries, particularly in Guyana, where a larger number and variety of anti-malarials were sampled. Poor quality was more frequent in the private and informal sector than in the public sector, posing a greater threat to the populations at risk, which are mostly located in hard to reach areas with scarce public facilities. Stock-outs identified in the public sector in Guyana could enhance the need to access those alternative sectors, exacerbating the risk of utilizing poor quality medicines. The availability of monotherapies and other non-recommended therapies for Plasmodium falciparum malaria, could also have contributed to the diminished efficacy. The type of quality deficiencies identified -reduced content of active pharmaceutical ingredient (API) and/or poor dissolution- and the irrational use of non-recommended treatments could result in non-sustained or lower levels of API in blood, favouring survival of more resistant mutants by exposing parasites to sub-lethal doses of the active ingredient. CONCLUSIONS: The quality of malaria medicines and the availability and use of non-recommended treatments could have played a role in the diminished efficacy of artemisinin derivatives described in Guyana and Suriname. However, also other factors need to be considered and a more comprehensive and extensive assessment on quality and pharmaceutical management is necessary to establish a tighter cause-effect correlation. Nevertheless, relevant authorities in these and neighbouring countries should take into consideration the reviewed data to properly address the problem when implementing corrective actions.

Quality of anti-malarials collected in the private and informal sectors in Guyana and Suriname.

BACKGROUND: Despite a significant reduction in the number of malaria cases in Guyana and Suriname, this disease remains a major problem in the interior of both countries, especially in areas with gold mining and logging operations, where malaria is endemic. National malaria control programmes in these countries provide treatment to patients with medicines that are procured and distributed through regulated processes in the public sector. However, availability to medicines in licensed facilities (private sector) and unlicensed facilities (informal sector) is common, posing the risk of access to and use of non-recommended treatments and/or poor quality products. METHODS: To assess the quality of circulating anti-malarial medicines, samples were purchased in the private and informal sectors of Guyana and Suriname in 2009. The sampling sites were selected based on epidemiological data and/or distance from health facilities. Samples were analysed for identity, content, dissolution or disintegration, impurities, and uniformity of dosage units or weight variation according to manufacturer, pharmacopeial, or other validated method. RESULTS: Quality issues were observed in 45 of 77 (58%) anti-malarial medicines sampled in Guyana of which 30 failed visual & physical inspection and 18 failed quality control tests. The proportion of monotherapy and ACT medicines failing quality control tests was 43% (13/30) and 11% (5/47) respectively. A higher proportion of medicines sampled from the private sector 34% (11/32) failed quality control tests versus 16% (7/45) in the informal sector. In Suriname, 58 medicines were sampled, of which 50 (86%) were Artecom®, the fixed-dose combination of piperaquine-dihydroartemisinin-trimethoprim co-blistered with a primaquine phosphate tablet. All Artecom samples were found to lack a label claim for primaquine, thus failing visual and physical inspection. CONCLUSIONS: The findings of the studies in both countries point to significant problems with the quality of anti-malarial medicines available in private and informal sector facilities as well as the availability of therapy not compliant with national treatment guidelines. They also stress the need to strengthen regulatory control efforts on the availability of anti-malarial medicines in these sectors and in endemic areas.

Implementation of basic quality control tests for malaria medicines in Amazon Basin countries: results for the 2005-2010 period.

BACKGROUND: Ensuring the quality of malaria medicines is crucial in working toward malaria control and eventual elimination. Unlike other validated tests that can assess all critical quality attributes, which is the standard for determining the quality of medicines, basic tests are significantly less expensive, faster, and require less skilled labour; yet, these tests provide reproducible data and information on several critical quality attributes, such as identity, purity, content, and disintegration. Visual and physical inspection also provides valuable information about the manufacturing and the labelling of medicines, and in many cases this inspection is sufficient to detect counterfeit medicines. The Promoting the Quality of Medicines (PQM) programme has provided technical assistance to Amazon Malaria Initiative (AMI) countries to implement the use of basic tests as a key screening mechanism to assess the quality of malaria medicines available to patients in decentralized regions. METHODS: Trained personnel from the National Malaria Control Programmes (NMCPs), often in collaboration with country's Official Medicine Control Laboratory (OMCL), developed country- specific protocols that encompassed sampling methods, sample analysis, and data reporting. Sampling sites were selected based on malaria burden, accessibility, and geographical location. Convenience sampling was performed and countries were recommended to store the sampled medicines under conditions that did not compromise their quality. Basic analytical tests, such as disintegration and thin layer chromatography (TLC), were performed utilizing a portable mini-laboratory. RESULTS: Results were originally presented at regional meetings in a non-standardized format that lacked relevant medicines information. However, since 2008 information has been submitted utilizing a template specifically developed by PQM for that purpose. From 2005 to 2010, the quality of 1,663 malaria medicines from seven AMI countries was evaluated, mostly collected from the public sector, 1,445/1,663 (86.9%). Results indicate that 193/1,663 (11.6%) were found not to meet quality specifications. Most failures were reported during visual and physical inspection, 142/1663 (8.5%), and most of these were due to expired medicines, 118/142 (83.1%). Samples failing TLC accounted for 27/1,663 (1.6%) and those failing disintegration accounted for 24/1,663 (1.4%). Medicines quality failures decreased significantly during the last two years. CONCLUSIONS: Basic tests revealed that the quality of medicines in the public sector improved over the years, since the implementation of this type of quality monitoring programme in 2005. However, the lack of consistent confirmatory tests in the quality control (QC) laboratory, utilizing methods that can also evaluate additional quality attributes, could still mask quality issues. In the future, AMI countries should improve coordination with their health authorities and their QC lab consistently, to provide a more complete picture of malaria medicines quality and support the implementation of corrective actions. Facilities in the private and informal sectors also should be included when these sectors constitute an important source of medicines used by malaria patients.

2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.

Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.

Importance of medicine quality in achieving universal health coverage.

OBJECTIVE: To assess the importance of ensuring medicine quality in order to achieve universal health coverage (UHC). METHODS: We developed a systems map connecting medicines quality assurance systems with UHC goals to illustrate the ensuing impact of quality-assured medicines in the implementation of UHC. The association between UHC and medicine quality was further examined in the context of essential medicines in low- and middle-income countries (LMICs) by analyzing data on reported prevalence of substandard and falsified essential medicines and established indicators for UHC. Finally, we examined the health and economic savings of improving antimalarial quality in four countries in sub-Saharan Africa: the Democratic Republic of the Congo (DRC), Nigeria, Uganda, and Zambia. FINDINGS: A systems perspective demonstrates how quality assurance of medicines supports dimensions of UHC. Across 63 LMICs, the reported prevalence of substandard and falsified essential medicines was found to be negatively associated with both an indicator for coverage of essential services (p = 0.05) and with an indicator for government effectiveness (p = 0.04). We estimated that investing in improving the quality of antimalarials by 10% would result in annual savings of $8.3 million in Zambia, $14 million in Uganda, $79 million in two DRC regions, and $598 million in Nigeria, and was more impactful compared to other potential investments we examined. Costs of substandard and falsified antimalarials per malaria case ranged from $7 to $86, while costs per death due to poor-quality antimalarials ranged from $14,000 to $72,000. CONCLUSION: Medicines quality assurance systems play a critical role in reaching UHC goals. By ensuring the quality of essential medicines, they help deliver effective treatments that lead to less illness and result in health care savings that can be reinvested towards UHC.

Synthesis, antiproliferative, and pharmacokinetic properties of 3- and 17-double-modified analogs of 2-methoxyestradiol.

The syntheses of 21 analogs of 2-methoxyestradiol are presented, including ENMD-1198 which was selected for advancement into Phase 1 clinical trials in oncology. These analogs were evaluated for antiproliferative activity using breast tumor MDA-MB-231 cells, for antiangiogenic activity in HUVEC proliferation assays, and for estrogenic activity in MCF-7 cell proliferation. The most active analogs were evaluated for iv and oral pharmacokinetic properties via cassette dosing in rat and in mice pharmacokinetic models.

Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure-activity relationships compared to 2-methoxyestradiol.

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.

Synthesis and structure-activity relationships of 16-modified analogs of 2-methoxyestradiol.

A series of 16-modified 2-methoxyestradiol analogs were synthesized and evaluated for antiproliferative activity toward HUVEC and MDA-MB-231 cells, and for susceptibility to conjugation. In addition, the estrogenicity of these analogs was accessed by measuring cell proliferation of the estrogen-dependent cell line MCF7 in response to compound treatment. It was observed that antiproliferative activity dropped as the size of the 16 substituent increased. Selected analogs tested in glucuronidation assays had similar rates of clearance to 2-methoxyestradiol, but had enhanced clearance in sulfonate conjugation assays.

2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors alpha and beta.

2-Methoxyestradiol (2ME(2)) is an endogenous metabolite of 17beta-estradiol (E(2)) that arises from the hydroxylation and subsequent methylation at the 2-position. In vitro 2ME(2) inhibits a large variety of tumor and nontumor cell lines from diverse origins, as well as several stages of the angiogenic cascade. In vivo it has been shown to be a very effective inhibitor of tumor growth and angiogenesis in numerous models. Although various molecular targets have been proposed for this compound, the mechanism of action is still uncertain. As this molecule emerges as a drug candidate it is important to assess the estrogen receptors (ERs) as molecular targets for 2ME(2). The purpose of this study was to investigate whether 2ME(2) is able to engage ERs as an agonist and whether its antiproliferative activities are mediated through ERs. We confirm that 2ME(2) has a lower binding affinity for ERalpha as compared with E(2) and other E(2) metabolites and antagonists, and we demonstrate that the affinity of 2ME(2) for ERbeta is even lower. When assessed in the presence of galangin, a cytochrome P450 enzyme inhibitor, at concentrations at which 2ME(2) interacts with ERalpha in an in vitro binding assay, it does not stimulate the proliferation of an estrogen-dependent breast carcinoma cell line. Similar IC(50) values for inhibition of proliferation and induction of apoptosis are obtained in estrogen-dependent and estrogen-independent human breast cancer cell lines, irrespective of the expression of ERalpha and ERbeta. Moreover, the estrogen antagonist ICI 182,780 does not inhibit the antiproliferative activity of 2ME(2). In E(2)-responsive cells such as MCF-7 and human umbilical vascular endothelial cells, high levels of E(2) inhibit the antiproliferative activity of ICI 182,780 but not of 2ME(2). Collectively, these results suggest that 2ME(2) is distinct among estradiol metabolites because of its inability to engage ERs as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of ERalpha and ERbeta.

2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway.

2-Methoxyestradiol (2ME2), a natural metabolite of estradiol, is a potent antitumor and antiangiogenic agent. In vitro, 2ME2 inhibits the proliferation of a wide variety of cell lines and primary cultures, and in numerous models in vivo, it has been shown to be an effective inhibitor of tumor growth and angiogenesis. 2ME2 is currently in several Phase I and Phase II clinical trials under the name Panzem. Although various molecular targets have been proposed for this compound, the mechanism by which 2ME2 exerts its effects is still uncertain. This study shows that 2ME2 uses the extrinsic pathway for induction of apoptosis. 2ME2 treatment results in up-regulation of death receptor 5 (DR5) protein expression in vitro and in vivo and renders cells more sensitive to the cytotoxic activities of the DR5 ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). 2ME2-induced apoptosis requires caspase activation and kinetic studies show the sequential activation of caspase-8, caspase-9, and caspase-3. Blockage of death receptor signaling by expression of dominant-negative Fas-associated death domain severely attenuates the ability of 2ME2 to induce apoptosis. Because 2ME2 administration has not manifested dose-limiting toxicity in the clinic, DR5 expression may serve as a surrogate marker for biological response.

Tissue factor/factor VIIa inhibitors block angiogenesis and tumor growth through a nonhemostatic mechanism.

An association between cancer and thrombosis has been recognized for more than a century. However, the manner by which tumor growth is regulated by coagulation in vivo remains unclear. To assess the role of coagulation on tumor growth, in vivo, we tested coagulation inhibitors specific for either tissue factor (TF)/factor VIIa (fVIIa) complexes or factor Xa (fXa) for antitumor activity. Here, we show that two inhibitors of TF/fVIIa, TF pathway inhibitor (TFPI) and the nematode anticoagulant protein rNAPc2, inhibit both primary and metastatic tumor growth in mice. In addition, we show that rNAPc2 is also a potent inhibitor of angiogenesis. In contrast, rNAP5, a second nematode anticoagulant protein that specifically inhibits fXa, does not exhibit antitumor activity. Because the hemostatic activity of TF/fVIIa is mediated through activation of fXa, these data suggest that proteolytic activity of TF/fVIIa promotes tumor growth and angiogenesis through a novel proangiogenic mechanism and independently of hemostasis.

2-methoxyestradiol inhibits hypoxia-inducible factor 1alpha, tumor growth, and angiogenesis and augments paclitaxel efficacy in head and neck squamous cell carcinoma.

PURPOSE: Head and neck squamous cell carcinomas have been reported to overexpress hypoxia-inducible factor (HIF)-1alpha, a transcription factor that promotes expression of angiogenesis factors and resistance to programmed and therapy-induced cell death. 2-Methoxyestradiol (2ME2) is a natural compound with HIF-1alpha inhibitory activity that is currently being evaluated in phase 1 and 2 clinical trials for advanced solid tumors and multiple myeloma. To our knowledge, this is the first study to evaluate the effects of 2ME2 in head and neck squamous cell carcinoma. EXPERIMENTAL DESIGN: In the present study, we investigated the effects of 2ME2 alone and in combination with paclitaxel, an active agent in recurrent or advanced head and neck squamous cell carcinoma. RESULTS: 2ME2 exhibited antiproliferative and cytotoxic effects in a panel of five head and neck squamous cell carcinoma cell lines in the 0.5 to 10 micromol/L range, including induction of G2-M blockade, caspase-3/7 activation, and apoptosis at 48 hours. 2ME2 resulted in decreased nuclear HIF-1alpha-binding activity and affected the expression of downstream genes, such as bid, a proapoptotic bcl-2 family member, and vascular endothelial growth factor, a proangiogenic cytokine. The up-regulation of Bid (57.5% at 12 hours, P < 0.0006) and inhibition of vascular endothelial growth factor secretion (57.7% at 24 hours, P < 0.015; and 50.3% at 48 hours, P < 0.0006) could be partially attributed to the effects on HIF-1alpha, because HIF-1alpha small interfering RNAs produced similar effects. Finally, in vivo, in a xenograft model of head and neck squamous cell carcinoma using UM-SCC-11A cells, 2ME2 exhibited antitumor and antiangiogenic activity, as measured by CD31 immunostaining. CONCLUSIONS: These results provide support for the use of 2ME2 in combination with paclitaxel for the treatment of recurrent or advanced head and neck squamous cell carcinoma.

Monitoring the quality of medicines: results from Africa, Asia, and South America.

Monitoring the quality of medicines plays a crucial role in an integrated medicines quality assurance system. In a publicly available medicines quality database (MQDB), the U.S. Pharmacopeial Convention (USP) reports results of data collected from medicines quality monitoring (MQM) activities spanning the period of 2003-2013 in 17 countries of Africa, Asia, and South America. The MQDB contains information on 15,063 samples collected and tested using Minilab® screening methods and/or pharmacopeial methods. Approximately 71% of the samples reported came from Asia, 23% from Africa, and 6% from South America. The samples collected and tested include mainly antibiotic, antimalarial, and antituberculosis medicines. A total of 848 samples, representing 5.6% of total samples, failed the quality test. The failure proportion per region was 11.5%, 10.4%, and 2.9% for South America, Africa, and Asia, respectively. Eighty-one counterfeit medicines were reported, 86.4% of which were found in Asia and 13.6% in Africa. Additional analysis of the data shows the distribution of poor-quality medicines per region and by therapeutic indication as well as possible trends of counterfeit medicines.

Identification and characterization of a very low density lipoprotein receptor-binding peptide from tissue factor pathway inhibitor that has antitumor and antiangiogenic activity.

Tissue factor pathway inhibitor (TFPI) is the major physiologic inhibitor of the extrinsic coagulation pathway. We have previously shown that TFPI is also a potent inhibitor of endothelial proliferation in vitro and of primary and metastatic tumor growth in vivo. Surprisingly, the antitumor activity of TFPI was demonstrated to be independent of its anticoagulant activity, suggesting a possible nonhemostatic mechanism of action for TFPI in these models. This antitumor mechanism may involve the very low density lipoprotein (VLDL) receptor because the in vitro antiproliferative activity of TFPI is mediated through interaction with the VLDL receptor. In the current study, we identify a 23-amino acid fragment of TFPI (TFPIc23) localized to the C-terminus, which mediates binding to the VLDL receptor. The TFPIc23 peptide inhibits endothelial cell proliferation through an apoptotic mechanism and blocks vessel outgrowth in the in vitro assays, and this activity is mediated through interaction with the VLDL receptor. In vivo, this peptide potently inhibits angiogenesis in Matrigel and chick chorioallantoic membrane models and also inhibits metastatic tumor growth. Our data demonstrate that this VLDL receptor-binding fragment of the TFPI molecule has apoptotic, antiangiogenic, and antitumor activity and suggests a possible mechanism whereby TFPI can regulate angiogenesis and tumor growth independently of its anticoagulant activity.

Withaferin A is a potent inhibitor of angiogenesis.

The medicinal plant Withania somnifera is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda , the major Traditional Indian medicine system, extracts from W. somnifera are distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somnifera root extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50 =12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.