The appointment-based model in community pharmacies: Patient demographics and reimbursable clinical services uptake in Ontario.

Background: Community pharmacies typically require patients to request medication refills. The appointment-based model (ABM) is a proactive approach that synchronizes refills and schedules patient-pharmacist appointments. These appointments provide opportunities for medication reviews, medication optimization and health promotion services. The primary aim of this study was to describe the types of patients who received an ABM service in a community pharmacy in Ontario in 2017. The secondary aim was to describe reimbursable clinical service uptake. Methods: In September 2017, the ABM was implemented across 3 Ontario community pharmacies within a Canadian pharmacy banner. Patients who filled at least 1 chronic oral medication and consented to enrolment were eligible. In December 2018, data were extracted from pharmacies using pharmacy management software. Descriptive statistics and frequencies were generated. Results: Analysis of 131 patients (51.1% female; mean ± SD age 70.8 ± 10.5 years) revealed patients were dispensed a mean ± SD of 5.1 ± 2.7 medications, and 73 (55.7%) experienced polypharmacy. Hypertension (87.8%) and dyslipidemia (68.7%) were the most common medical conditions. There were 74 (56.5%) patients who received ≥1 medication review service (MedsCheck). Of 79 unique drug therapy problems (DTPs) identified, the most common categories related to patients needing additional drug therapy and adverse drug reactions. Discussion and conclusion: Patients enrolled in the ABM were generally older adults experiencing polypharmacy. The ABM presented opportunities for DTP identification and delivery of reimbursed services. Findings support continued exploration of the ABM to support integration of clinical services within community practice.

Glutathione­iron hybrid nanozyme-based colorimetric sensor for specific and stable detection of thiram pesticide on fruit juices.

Given the potential dangers of thiram to food safety, constructing a facile sensor is significantly critical. Herein, we presented a colorimetric sensor based on glutathione­iron hybrid (GSH-Fe) nanozyme for specific and stable detection of thiram. The GSH-Fe nanozyme exhibits good peroxidase-mimicking activity with comparable Michaelis constant (Km = 0.551 mM) to the natural enzyme. Thiram pesticides can specifically limit the catalytic activity of GSH-Fe nanozyme via surface passivation, causing the change of colorimetric signal. It is worth mentioning that the platform was used to prepare a portable hydrogel kit for rapid qualitative monitoring of thiram. Coupling with an image-processing algorithm, the colorimetric image of the hydrogel reactor is converted into the data information for accurate quantification of thiram with a detection limit of 0.3 µg mL-1. The sensing system has good selectivity and high stability, with recovery rates in fruit juice samples ranging from 92.4% to 106.9%.

Narciclasine, a novel topoisomerase I inhibitor, exhibited potent anti-cancer activity against cancer cells.

DNA topoisomerases are essential nuclear enzymes in correcting topological DNA errors and maintaining DNA integrity. Topoisomerase inhibitors are a significant class of cancer chemotherapeutics with a definite curative effect. Natural products are a rich source of lead compounds for drug discovery, including anti-tumor drugs. In this study, we found that narciclasine (NCS), an amaryllidaceae alkaloid, is a novel inhibitor of topoisomerase I (topo I). Our data demonstrated that NCS inhibited topo I activity and reversed its unwinding effect on p-HOT DNA substrate. However, it had no obvious effect on topo II activity. The molecular mechanism of NCS inhibited topo I showed that NCS did not stabilize topo-DNA covalent complexes in cells, indicating that NCS is not a topo I poison. A blind docking result showed that NCS could bind to topo I, suggesting that NCS might be a topo I suppressor. Additionally, NCS exhibited a potent anti-proliferation effect in various cancer cells. NCS arrested the cell cycle at G2/M phase and induced cell apoptosis. Our study reveals the antitumor mechanisms of NCS and provides a good foundation for the development of anti-cancer drugs based on topo I inhibition.

LepR+ niche cell-derived AREG compromises hematopoietic stem cell maintenance under conditions of DNA repair deficiency and aging.

The cross talk between extrinsic niche-derived and intrinsic hematopoietic stem cell (HSC) factors controlling HSC maintenance remains elusive. Here, we demonstrated that amphiregulin (AREG) from bone marrow (BM) leptin receptor (LepR+) niche cells is an important factor that mediates the cross talk between the BM niche and HSCs in stem cell maintenance. Mice deficient of the DNA repair gene Brca2, specifically in LepR+ cells (LepR-Cre;Brca2fl/fl), exhibited increased frequencies of total and myeloid-biased HSCs. Furthermore, HSCs from LepR-Cre;Brca2fl/fl mice showed compromised repopulation, increased expansion of donor-derived, myeloid-biased HSCs, and increased myeloid output. Brca2-deficient BM LepR+ cells exhibited persistent DNA damage-inducible overproduction of AREG. Ex vivo treatment of wild-type HSCs or systemic treatment of C57BL/6 mice with recombinant AREG impaired repopulation, leading to HSC exhaustion. Conversely, inhibition of AREG by an anti-AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2fl/fl mice rescued HSC defects caused by AREG. Mechanistically, AREG activated the phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, promoted HSC cycling, and compromised HSC quiescence. Finally, we demonstrated that BM LepR+ niche cells from other DNA repair-deficient and aged mice also showed persistent DNA damage-associated overexpression of AREG, which exerts similar negative effects on HSC maintenance. Therefore, we identified an important factor that regulates HSCs function under conditions of DNA repair deficiency and aging.

Discovery of novel thymidylate synthase (TS) inhibitors that influence cancer angiogenesis and metabolic reprogramming in NSCLC cells.

Based on previous work, further search for more effective and less damaging thymidylate synthase (TS) inhibitors was the focus of this study. After further optimization of the structure, in this study, a series of (E)-N-(2-benzyl hydrazine-1-carbonyl) phenyl-2,4-deoxy-1,2,3,4-tetrahydro pyrimidine-5-sulfonamide derivatives were synthesized and reported for the first time. All target compounds were screened by enzyme activity assay and cell viability inhibition assay. On the one hand, the hit compound DG1 could bind directly to TS proteins intracellularly and promote apoptosis in A549 and H1975 cells. Simultaneously, DG1 could inhibit cancer tissue proliferation more effectively than Pemetrexed (PTX) in the A549 xenograft mouse model. On the other hand, the inhibitory effect of DG1 on NSCLC angiogenesis was verified both in vivo and in vitro. In parallel, DG1 was further uncovered to inhibit the expression of CD26, ET-1, FGF-1, and EGF by angiogenic factor antibody microarray. Moreover, RNA-seq and PCR-array assays revealed that DG1 could inhibit NSCLC proliferation by affecting metabolic reprogramming. Collectively, these data demonstrated that DG1as a TS inhibitor could be promising in treating NSCLC angiogenesis, deserving further investigation.

Implementation of the appointment-based model in community pharmacies: An analysis of refills and adherence.

BACKGROUND: Traditionally, much of community pharmacy practice relies on patients to request their own medication refills. These refills are often not aligned, which has been shown to decrease adherence and workflow efficiencies. The appointment-based model (ABM) is designed to proactively synchronize refills and schedule patient-pharmacist appointments. OBJECTIVES: To describe the characteristics of patients enrolled in the ABM; and to compare the number of distinct refill dates, number of refills, and adherence for antihypertensives, oral antihyperglycemics, and statins 6-months and 12-months pre-post ABM implementation. METHODS: In September 2017, the ABM was implemented across independent community pharmacies within a pharmacy banner in Ontario, Canada. In December 2018, a convenience sample of three pharmacies was extracted. Demographic and clinical characteristics were collected on program enrollment (index) date for individual patients and their medication fill histories were used to investigate adherence measures including distinct number of refill dates, number of refills, and proportion of days covered. Descriptive statistics were analyzed using StataCorp. RESULTS: Analysis of 131 patients (48.9% male; mean age 70.8 years ± 10.5 SD) filled on average 5.1 ± 2.7 medications with 73 (55.7%) experiencing polypharmacy. Patients had a significant reduction in mean number of refill dates (6.8 ± 3.8 SD six-months pre-enrollment, 4.9 ± 3.1 SD six-months post-enrollment, p < 0.0001). Adherence to chronic medications remained high (PDC ≥95%). CONCLUSION: The ABM was implemented for a cohort of established users, already highly adherent to their chronic medications. Results demonstrate reduced filling complexity and fewer refill dates while also sustaining the high baseline adherence across all chronic medications studied. Future studies should investigate patient perspectives and potential clinical benefits of the ABM.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited ß-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Synthesis and evaluation of novel HER-2 inhibitors to exert anti-breast cancer ability through epithelial-mesenchymal transition (EMT) pathway.

Human epidermal growth factor receptor 2 (HER-2) is an essential member of the receptor tyrosine kinase (RTK) superfamily and has been reported as a critical method for treating HER-2 positive breast cancer. Here, we retained (E)-4-methyl-2-(4-(trifluoromethyl)styryl)oxazole, a fragment of HER-2 inhibitor Mubritinib, and synthesized 32 novel compounds from it. We screened out the most potential compound Q7j with HER-2 positive breast cancer cells through MTT assays, which possessed low toxicity on normal cells (MCF7-10A). Subsequently, wound healing, transwell, western blotting, and immunofluorescence experiments were performed, and it was found that compound Q7j could suppress cell migration by inhibiting the phosphorylation of HER-2 and affecting the expression of EMT-related proteins. Moreover, the SKBR3 orthotopic xenograft model confirmed that compound Q7j was more effective than Mubritinib in inhibiting the proliferation of cancer cells. In general, compound Q7j was a potential HER-2 inhibitor in treating breast cancer, which may be of great significance for developing and improving HER-2 small molecule inhibitors.

Discovery of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure as novel EGFR/HER-2 dual-target inhibitors against cancer growth and angiogenesis.

Targeting EGFR and HER-2 is an essential direction for cancer treatment. Here, a series of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure was designed and synthesized to serve as EGFR/HER-2 dual-target inhibitors. The kinase assays verified that target compounds could inhibit the kinase activity of EGFR and HER-2 selectively. The results of CCK-8 and 3D cell viability assays confirmed that target compounds had excellent anti-proliferation ability against breast cancer cells (MCF-7 and SK-BR-3) and lung cancer cells (A549 and H1975), particularly against SK-BR-3 cells, while the inhibitory effect on healthy breast cells (MCF-10A) and lung cells (Beas-2B) was weak. Among them, the hit compound YH-9 binded to EGFR and HER-2 stably in molecular dynamics studies. Further studies found thatYH-9could induce the release of cytochrome c and inhibit proliferation by promoting ROS expression in SK-BR-3 cells. Moreover,YH-9could diminish the secretion of VEGF and bFGF factors in SK-BR-3 cells, then inhibited tube formation and angiogenesis. Notably,YH-9could effectively inhibit breast cancer growth and angiogenesis with little toxicity in the SK-BR-3 cell xenograft model. Taken together,in vitroandin vivoresults revealed that YH-9 had high drug potential as a dual-target inhibitor of EGFR/HER-2 to inhibit breast cancer growth and angiogenesis.

Quinacrine-CASIN combination overcomes chemoresistance in human acute lymphoid leukemia.

Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidence that prolonged and intensive chemotherapy often fails to eradicate leukemic stem cells, which are protected by the bone marrow niche and can induce relapse. Thus, new therapeutic approaches to overcome chemoresistance are urgently needed. By conducting an ex vivo small molecule screen, here we have identified Quinacrine (QC) as a sensitizer for Cytarabine (AraC) in treating acute lymphoblastic leukemia (ALL). We show that QC enhances AraC-mediated killing of ALL cells, and subsequently abrogates AraC resistance both in vitro and in an ALL-xenograft model. However, while combo AraC+QC treatment prolongs the survival of primary transplanted recipients, the combination exhibits limited efficacy in secondary transplanted recipients, consistent with the survival of niche-protected leukemia stem cells. Introduction of Cdc42 Activity Specific Inhibitor, CASIN, enhances the eradication of ALL leukemia stem cells by AraC+QC and prolongs the survival of both primary and secondary transplanted recipients without affecting normal long-term human hematopoiesis. Together, our findings identify a small-molecule regimen that sensitizes AraC-mediated leukemia eradication and provide a potential therapeutic approach for better ALL treatment.

Novel Allosteric Inhibitors of Deoxyhypusine Synthase against Malignant Melanoma: Design, Synthesis, and Biological Evaluation.

Based on the novel allosteric site of deoxyhypusine synthase (DHPS), two series of 30 novel 5-(2-methoxyphenoxy)-2-phenylpyrimidin-4-amine derivatives as DHPS inhibitors were designed and synthesized. Among them, compound 8m, with the best DHPS inhibitory potency (IC50 = 0.014 µM), exhibited excellent inhibition against melanoma cells, which was superior to that of GC7. Besides, molecular docking and molecular dynamics (MD) simulations further proved that compound 8m was tightly bound to the allosteric site of DHPS. Flow cytometric analysis and enzyme-linked immunosorbent assay (ELISA) showed that compound 8m could inhibit the intracellular reactive oxygen species (ROS) level. Furthermore, by western blot analysis, compound 8m effectively activated caspase 3 and decreased the expressions of GP-100, tyrosinase, eIF5A2, MMP2, and MMP9. Moreover, both Transwell analysis and wound healing analysis showed that compound 8m could inhibit the invasion and migration of melanoma cells. In the in vivo study, the tumor xenograft model showed that compound 8m effectively inhibited melanoma development with low toxicity.

A newly identified polyunsaturated macamide alleviates dextran sulfate sodium-induced colitis in mice.

Macamides are a class of bioactive amide alkaloids found only in maca (Lepidium meyenii). Recent studies have shown that macamide-rich extracts possess various biological activities, such as antioxidative, immune-enhancing, and reproductive health-improving activities. In the present study, N-benzyl docosahexaenamide (NB-DHA), a newly identified macamide with the highest degree of unsaturation among all identified macamides, was identified from the maca extract. Microalgae oil, a docosahexaenoic acid-rich substance, was used as the starting material for the synthesis of NB-DHA. The effects of NB-DHA in colitis-induced mice were evaluated. NB-DHA significantly alleviated weight loss, shortening of colon length, and occult blood occurrence in mice with dextran sulfate sodium-induced colitis. Histological analysis revealed that following the administration of NB-DHA in mice with colitis, the infiltration of inflammatory cells and levels of proinflammatory factors, such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and myeloperoxidase, decreased, whereas the level of the anti-inflammatory factor IL-10 increased. Furthermore, the decreased expression of intestinal tight junction proteins caused by colitis was upregulated by the administration of NB-DHA. These results indicate that NB-DHA could be developed as a therapeutic agent for ulcerative colitis.

FANCD2 and HES1 suppress inflammation-induced PPARÉ£ to prevent haematopoietic stem cell exhaustion.

The Fanconi anaemia protein FANCD2 suppresses PPARƔ to maintain haematopoietic stem cell's (HSC) function; however, the underlying mechanism is not known. Here we show that FANCD2 acts in concert with the Notch target HES1 to suppress inflammation-induced PPARƔ in HSC maintenance. Loss of HES1 exacerbates FANCD2-KO HSC defects. However, deletion of HES1 does not cause more severe inflammation-mediated HSC defects in FANCD2-KO mice, indicating that both FANCD2 and HES1 are required for limiting detrimental effects of inflammation on HSCs. Further analysis shows that both FANCD2 and HES1 are required for transcriptional repression of inflammation-activated PPARg promoter. Inflammation orchestrates an overlapping transcriptional programme in HSPCs deficient for FANCD2 and HES1, featuring upregulation of genes in fatty acid oxidation (FAO) and oxidative phosphorylation. Loss of FANCD2 or HES1 augments both basal and inflammation-primed FAO. Targeted inhibition of PPARƔ or the mitochondrial carnitine palmitoyltransferase-1 (CPT1) reduces FAO and ameliorates HSC defects in inflammation-primed HSPCs deleted for FANCD2 or HES1 or both. Finally, depletion of PPARg or CPT1 restores quiescence in these mutant HSCs under inflammatory stress. Our results suggest that this novel FANCD2/HES1/PPARƔ axis may constitute a key component of immunometabolic regulation, connecting inflammation, cellular metabolism and HSC function.

Mesenchymal PGD2 activates an ILC2-Treg axis to promote proliferation of normal and malignant HSPCs.

Cyclooxygenase (COX)-dependent production of prostaglandins (PGs) is known to play important roles in tumorigenesis. PGD2 has recently emerged as a key regulator of tumor- and inflammation-associated functions. Here we show that mesenchymal stromal cells (MSCs) from patients with acute myeloid leukemia (AML) or normal MSCs overexpressing COX2 promote proliferation of co-cultured hematopoietic stem and progenitor cells (HSPCs), which can be prevented by treatment with COX2 knockdown or TM30089, a specific antagonist of the PGD2 receptor CRTH2. Mechanistically, we demonstrate that PGD2-CRTH2 signaling acts directly on type 2 innate lymphoid cells (ILC2s), potentiating their expansion and driving them to produce Interleukin-5 (IL-5) and IL-13. Furthermore, IL-5 but not IL-13 expands CD4+CD25+IL5Rα+ T regulatory cells (Tregs) and promotes HSPC proliferation. Disruption of the PGD2-activated ILC2-Treg axis by specifically blocking the PGD2 receptor CRTH2 or IL-5 impedes proliferation of normal and malignant HSPCs. Conversely, co-transfer of CD4+CD25+IL5Rα+ Tregs promotes malignant HSPC proliferation and accelerates leukemia development in xenotransplanted mice. Collectively, these results indicate that the mesenchymal source of PGD2 promotes proliferation of normal and malignant HSPCs through activation of the ILC2-Treg axis. These findings also suggest that this novel PGD2-activated ILC2-Treg axis may be a valuable therapeutic target for cancer and inflammation-associated diseases.

Persistent DNA damage-induced NLRP12 improves hematopoietic stem cell function.

NOD-like receptor 12 (NLRP12) is a member of the nucleotide-binding domain and leucine-rich repeat containing receptor inflammasome family that plays a central role in innate immunity. We previously showed that DNA damage upregulated NLRP12 in hematopoietic stem cells (HSCs) of mice deficient in the DNA repair gene Fanca. However, the role of NLRP12 in HSC maintenance is not known. Here, we show that persistent DNA damage-induced NLRP12 improves HSC function in both mouse and human models of DNA repair deficiency and aging. Specifically, treatment of Fanca-/- mice with the DNA cross-linker mitomycin C or ionizing radiation induces NLRP12 upregulation in phenotypic HSCs. NLRP12 expression is specifically induced by persistent DNA damage. Functionally, knockdown of NLRP12 exacerbates the repopulation defect of Fanca-/- HSCs. Persistent DNA damage-induced NLRP12 was also observed in the HSCs from aged mice, and depletion of NLRP12 in these aged HSCs compromised their self-renewal and hematopoietic recovery. Consistently, overexpression of NLRP12 substantially improved the long-term repopulating function of Fanca-/- and aged HSCs. Finally, persistent DNA damage-induced NLRP12 maintains the function of HSCs from patients with FA or aged donors. These results reveal a potentially novel role of NLRP12 in HSC maintenance and suggest that NLRP12 targeting has therapeutic potential in DNA repair disorders and aging.

Hes1 deficiency causes hematopoietic stem cell exhaustion.

The transcriptional repressor Hairy Enhancer of Split 1 (HES1) plays an essential role in the development of many organs by promoting the maintenance of stem/progenitor cells, controlling the reversibility of cellular quiescence, and regulating both cell fate decisions. Deletion of Hes1 in mice results in severe defects in multiple organs and is lethal in late embryogenesis. Here we have investigated the role of HES1 in hematopoiesis using a hematopoietic lineage-specific Hes1 knockout mouse model. We found that while Hes1 is dispensable for steady-state hematopoiesis, Hes1-deficient hematopoietic stem cells (HSCs) undergo exhaustion under replicative stress. Loss of Hes1 upregulates the expression of genes involved in PPARγ signaling and fatty acid metabolism pathways, and augments fatty acid oxidation (FAO) in Hes1 f/f Vav1Cre HSCs and progenitors. Functionally, PPARγ targeting or FAO inhibition ameliorates the repopulating defects of Hes1 f/f Vav1Cre HSCs through improving quiescence in HSCs. Lastly, transcriptome analysis reveals that disruption of Hes1 in hematopoietic lineage alters expression of genes critical to HSC function, PPARγ signaling, and fatty acid metabolism. Together, our findings identify a novel role of HES1 in regulating stress hematopoiesis and provide mechanistic insight into the function of HES1 in HSC maintenance.