What makes joint assessment procedures attractive to the innovative industry: successes, challenges, and proposed improvements

Regulatory harmonization and convergence have been identified as the key driver in promoting efficient evaluation of medicines, reducing workload, and supporting earlier access to medicines on the African continent. There has been great progress to date in enhancing regulatory harmonization and convergence on the African continent via the Regional Economic Communities (RECs) and with the establishment of the Africa Medicines Agency (AMA). In this article, the International Federation of Pharmaceutical Manufacturers and Associations (IFPMA) Africa Regulatory Network (ARN) presents its perspective based on the available literature review and results from a survey conducted with innovative biopharmaceutical companies to gather experiences using regional joint assessment procedures (JAPs) in Africa, such as the East African Community Medicines Regulatory Harmonization (EAC-MRH), the West African Medicines Regulatory Harmonization (WA-MRH), and the Southern African Development Community Medicines Regulatory Harmonization (SADC-MRH) initiative through the ZAZIBONA Collaborative Procedure for Medicines Registration (ZaZiBoNa), and provides best practices in this evolving landscape. The article also assesses other collaborative registration pathways available to facilitating registration of pharmaceutical products in African countries, such as WHO Collaborative Registration Procedures (CRP), Swissmedic’s Marketing Authorisation for Global Health Products (MAGHP) and EU Medicines for All (EU-M4ALL). Benefits and challenges of each of the existing pathways are discussed in this article. Main benefits include building more expert capacity and improved collaboration amongst experts, as well as shorter review timelines in some cases. Key challenges include the lack of predictability in the adherence to procedural timelines as defined per guidelines, lengthy timeline to achieve national marketing authorization following joint assessment, the lack of dedicated personnel, administrative issues during the submission process as well as additional country-specific requirements on top of JAP-specific requirements. Our recommendations for improvements include harmonization of requirements across countries and regions and with international standards, appropriate resource allocation for JAP activities to ensure adherence to timelines, use of JAPs throughout the entire product lifecycle and all product categories, adequate use of digital technologies, and improved communication and transparency with applicants. These improvements will allow industry to better plan their filing strategies for the region which will lead to overall improved usability of the JAPs in Africa and enable faster patient access

Heme Mediated STAT3 Activation in Severe Malaria

The mortality of severe malaria [cerebral malaria (CM), severe malaria anemia (SMA), acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)] remains high despite the availability associated with adequate treatments. Recent studies in our laboratory and others have revealed a hitherto unknown correlation between chemokine CXCL10/CXCR3, Heme/HO-1 and STAT3 and cerebral malaria severity and mortality. Although Heme/HO-1 and CXCL10/CXCR3 interactions are directly involved in the pathogenesis of CM and fatal disease, the mechanism dictating how Heme/HO-1 and CXCL10/CXCR3 are expressed and regulated under these conditions is still unknown. We therefore tested the hypothesis that these factors share common signaling pathways and may be mutually regulated.We first clarified the roles of Heme/HO-1, CXCL10/CXCR3 and STAT3 in CM pathogenesis utilizing a well established experimental cerebral malaria mouse (ECM, P. berghei ANKA) model. Then, we further determined the mechanisms how STAT3 regulates HO-1 and CXCL10 as well as mutual regulation among them in CRL-2581, a murine endothelial cell line.The results demonstrate that (1) STAT3 is activated by P. berghei ANKA (PBA) infection in vivo and Heme in vitro. (2) Heme up-regulates HO-1 and CXCL10 production through STAT3 pathway, and regulates CXCL10 at the transcriptional level in vitro. (3) HO-1 transcription is positively regulated by CXCL10. (4) HO-1 regulates STAT3 signaling.Our data indicate that Heme/HO-1, CXCL10/CXCR3 and STAT3 molecules as well as related signaling pathways play very important roles in the pathogenesis of severe malaria. We conclude that these factors are mutually regulated and provide new opportunities to develop potential novel therapeutic targets that could be used to supplement traditional prophylactics and treatments for malaria and improve clinical outcomes while reducing malaria mortality. Our ultimate goal is to develop novel therapies targeting Heme or CXCL10-related biological signaling molecules associated with development of fatal malaria

Consumer perceptions of plant based beverages: The Ghanaian consumer's perspective

This study identified factors influencing consumer desirability for a concept beverage from tiger nut milk and cocoa pulp using qualitative focus groups with four demographic groups: mothers, young adults, adults, and middle-aged adults. Specifically, Ghanaian consumers’ knowledge of plant-based beverages (PBB), sensory attributes driving preferences and selection, and willingness to purchase were investigated. Focus group discussions were thematically analyzed by Attride-Stirling's method using ATLAS.ti.7 software. Most consumers were knowledgeable of PBB. Health and nutrition were main drivers of consumption while taste, cost, availability, and culture were barriers to consumption. Drivers of consumer preference of PBB differed across demographic groups. Environmentally conscious consumers (mothers, adults, and middle-aged) preferred paper packaging while young adults preferred plastic packaging for convenience. Middle-aged adults preferred unsweetened versions for health reasons and mothers preferred unflavoured PBB for their children. Consumers expressed willingness to purchase the concept beverage, citing innovation, taste, and health benefits as main drivers. Industrial Relevance: Increased consumer awareness of the relationship between diet and disease and the importance of consuming healthful foods has informed a switch to more plant-based diets. Our results provide valuable insights to facilitate the development of an acceptable plant-based beverage for Ghanaian consumers

HO-1 and CXCL10 are induced by Heme and its synthetic inducer in mouse endothelial CRL2581 cell line.

<p>We exposed mouse brain endothelial cell line CRL-2581 in vitro with Heme at different concentration. The results show that expression of HO-1 mRNA (A) and protein (B) are significantly up-regulated by Heme and HO-1 enzymatic inducer-CoPP treatment. HO-1 expression peaked at 5–10 µM for Heme and 5–15 µM for CoPP. HO-1 mRNA (C) and protein (D) were down-regulated by HO-1 enzymatic activity inhibitor-ZnPP. The expression of CXCL10 mRNA (E) were also significantly up-regulated by Heme and CoPP treatment. The CXCL10 promoter activity is proportional to amounts of CXCL10 luc (F) within the range between 100 ng to 1000 ng when treated with Heme. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g007" target="_blank">Figure 7G</a> showed that Heme enhanced the CXCL10 promoter activity in a dose-dependent matter within the range from 1 µM to 5 µM. Data are shown as mean ± SD. *<i>P</i><0.05; ** P<0.01.</p

STAT3 activation is induced by Heme in CRL-2581 cells.

<p>Decreased activation of STAT3 by siRNA-STAT3 (siSTAT3) and AG490 down regulates HO-1 and CXCL10 protein. pSTAT3 was increased by Heme from 1 µM and peaked at 5 µM and then decreased (A). When pSTAT3 was reduced by siSTAT3 (B) or pharmacological inhibitor of Jak, AG490 (C), HO-1 protein expression was inhibited correspondingly. These data indicated that Heme induced production of HO-1 by way of STAT3 signaling pathway. Reduced activation of STAT3 caused by siSTAT3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g008" target="_blank">Figure 8D</a>) and AG490 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g008" target="_blank">Figure 8E</a>) also caused reduced expression of CXCL10. We futher found when CXCL10 was blocked by anti-CXCL10 antibody, HO-1 induction by Heme was decreased to one half, indicating that CXCL10 directly induce HO-1 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g008" target="_blank">Figure 8F</a>). Reduced HO-1 expression by siHO-1 increased CXCL10 expression which implies that HO-1 also modulates CXCL10 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g008" target="_blank">Figure 8G</a>). Interestingly, pSTAT3 level was increased by CoPP and decreased by ZnPP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g008" target="_blank">Figure 8H</a>), which indicates that HO-1 also regulates STAT3 signaling.</p

Plasma levels of Heme and HO-1 are increased in ECM mice infected with <i>P. berghei</i> ANKA.

<p>In left panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g002" target="_blank">Figure 2</a>, plasma levels of Heme were significantly increased in PBA-infected wild type C57 BL/6 mice (n = 6) than those of non-infected controls (n = 6), indicating that plasma Heme was associated with PBA infection in mice. HO-1 plasma levels were significantly higher in the PBA-infected wild type C57 BL/6 mice compared to controls, suggesting HO-1 may be a protective factor against Heme (right panel). CXCL10−/− infected mice showed the same pattern as CXCL10−/− non-infected mice regarding plasma concentration of Heme and HO-1. When infected with PBA, CXCL10−/− mice do not present the same increase in the levels of Heme or HO-1 as WT mice do during infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g002" target="_blank">Figure 2</a>). Data are shown as mean ± SD (n = 6 per group).</p

High expression level of HO-1 protein and phosphorylated STAT3 (pSTAT3) occur in kidney, brain and lung tissues of mice infected with <i>P. berghei</i> ANKA (n = 6 per group).

<p>HO-1 and the active STAT3 molecules-pSTAT3^Tyr705 protein were examined in kidney (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g004" target="_blank">Fig. 4A</a>), brain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g004" target="_blank">Fig. 4B</a>) and lung (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g004" target="_blank">Fig. 4C</a>) of some mice by Western blot. <i>P. berghei</i> infection up-regulates HO-1, pSTAT3 protein in various tissues of C57BL/6 mice. pSTAT3 level peaked on day 2 in kidney and lung and on day 4 in brain, STAT3 level appeared earlier than those of HO-1, which peaking on day 4 in kidney and lung, on day 8 in brain respectively. However, <i>P. berghei</i> infection failed to up-regulate HO-1 protein in <i>CXCL10−/−</i> mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g004" target="_blank">Figure 4D–F</a>). Corresponding densitometric analysis of the bands performed with the ImageQuant program were shown below the Western blot. Data are shown as mean ± SD (n = 6 per group). *<i>P</i><0.05; ** P<0.01.</p

Primers for real-time polymerase chain reaction.

<p>Primers for real-time polymerase chain reaction.</p

<i>Hmox1</i>(HO-1) messenger RNA (mRNA) expression in kidney, brain, and lung as determined by quantitative RT-PCR.

<p>Data are shown as mean ± SD (n = 6 per group). *<i>P</i><0.05; ** P<0.01. mRNA expression of HO-1 in kidney, brain and lung tissues of mice infected with <i>P. berghei</i> ANKA are increased significantly. The data showed the mean ratio of HO-1 mRNA expression to GAPDH expression (n = 6) in mice on day 2, 4 and 8 respectively. Infection of C57BL/6 WT or <i>CXCL10</i>−/− mice with <i>P. berghei</i> (C = non-infected control In = PBA infectetion) up-regulated HO-1 mRNA in kidney (A), brain (B) and lung (C), suggesting HO-1 expression may be protective against <i>P. berghei</i> induced damage. Mice deficient in CXCL10 down regulated HO-1 in both uninfected and infected C57BL/6 mice (white vs. red; black vs. yellow), suggesting that transcription of mouse HO-1 gene is positively regulated by CXCL10.</p

ECM is associated with high levels of <i>CXCL10</i> expression in vital organs in C57BL/6 WT mice.

<p>Left panel: CXCL10 messenger RNA (mRNA) expression in kidney, brain, and lung as determined by quantitative RT-PCR. Data are shown as mean ± SD (n = 6 per group). *<i>P</i><0.05; ** P<0.01. ECM onset in C57BL/6 mice infected with <i>P. berghei</i> ANKA reveals that levels of CXCL10 mRNA were much higher in kidney, brain and lung tissues in infected than uninfected mice on day 4 and day 8 respectively (left panel). Right panel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g006" target="_blank">Figure 6A–6F</a>): representative examples of HO-1 immunohistochemistry staining in kidney, brain and lung of C57BL/6 wild type mice. Membrane and cytoplasma staining of CXCL10 protein were much stronger in the kidney, brain and lung of PBA infected WT C57 BL/6 mice compared to corresponding controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034280#pone-0034280-g006" target="_blank">Figure 6B vs. 6A, 6D vs. 6C, 6F vs.6E</a>).</p