A series of seven 2-amino-4-oxo-6-substituted thieno [2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno [2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC 50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno [2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.

Abstract2-Amino-4-oxo-6-substituted pyrrolo [2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo [2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR-and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.

BackgroundPhysical therapy can prevent functional impairments and improve the quality of life of patients after hospital discharge. However, the effect of early mobilization on patients with a critical illness remains unclear. This study was performed to assess the evidence available regarding the effect of early mobilization on critically ill patients in the intensive care unit (ICU).MethodsElectronic databases were searched from their inception to March 21, 2019. Randomized controlled trials (RCTs) comprising critically ill patients who received early mobilization were included. The methodological quality and risk of bias of each eligible trial were assessed using the Cochrane Collaboration tool. Data were extracted using a standard collection form each included study, and processed using the Mantel-Haenszel (M-H) or inverse-variance (I-V) test in the STATA v12.0 statistical software.ResultsA total of 1,898 records were screened. Twenty-three RCTs comprising 2,308 critically ill patients were ultimately included. Early mobilization decreased the incidence of ICU-acquired weakness (ICU-AW) at hospital discharge (three studies, 190 patients, relative risk (RR): 0.60, 95% confidence interval (CI) [0.40, 0.90]; p = 0.013, I2 = 0.0%), increased the number of patients who were able to stand (one study, 50 patients, 90% vs. 62%, p = 0.02), increased the number of ventilator-free days (six studies, 745 patients, standardized mean difference (SMD): 0.17, 95% CI [0.02, 0.31]; p = 0.023, I2 = 35.5%) during hospitalization, increased the distance the patient was able to walk unassisted (one study, 104 patients, 33.4 (0–91.4) meters vs. 0 (0–30.4) meters, p = 0.004) at hospital discharge, and increased the discharged-to-home rate (seven studies, 793 patients, RR: 1.16, 95% CI [1.00, 1.34]; p = 0.046). The mortality (28-day, ICU and hospital) and adverse event rates were moderately increased by early mobilization, but the differences were statistically non-significant. However, due to the substantial heterogeneity among the included studies, and the low quality of the evidence, the results of this study should be interpreted with caution. Publication bias was not identified.ConclusionsEarly mobilization appears to decrease the incidence of ICU-AW, improve the functional capacity, and increase the number of ventilator-free days and the discharged-to-home rate for patients with a critical illness in the ICU setting.

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo [2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-͕4-[3-2-amino-4-oxo-4,7-, respectively) were inhibitory, with 2 Ͼ Ͼ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [ 3 H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/ hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ϳ50 and 75%, respectively. By an in situ GARFTase assay, 2 was ϳ20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.

Highly pathogenic avian influenza (HPAI), subtype H5N1, was first officially reported in Indonesia in 2004. Since then the disease has spread and is now endemic in large parts of the country. This study investigated the statistical relationship between a set of risk factors and the presence or absence of HPAI in Indonesia during 2006 and 2007. HPAI was evaluated through participatory disease surveillance (PDS) in backyard village chickens (the study population), and risk factors included descriptors of people and poultry distribution (separating chickens, ducks and production sectors), poultry movement patterns and agro-ecological conditions. The study showed that the risk factors “elevation”, “human population density” and “rice cropping” were significant in accounting for the spatial variation of the PDS-defined HPAI cases. These findings were consistent with earlier studies in Thailand and Vietnam. In addition “commercial poultry population”, and two indicators of market locations and transport; “human settlements” and “road length”, were identified as significant risk factors in the models. In contrast to several previous studies carried out in Southeast Asia, domestic backyard ducks were not found to be a significant risk factor in Indonesia. The study used surrogate estimates of market locations and marketing chains and further work should focus on the actual location of the live bird markets, and on the flow of live poultry and poultry products between them, so that patterns of possible transmission, and regions of particular risk could be better inferred.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.

Mesenchymal stem cells (MSCs), the non-hematopoietic progenitor cells, are multi-potent stem cells from a variety of tissues with the capability of self-renewal, proliferation, differentiation into multi-lineage cell types, as well as anti-inflammatory and immunomodulatory. These properties make MSCs an ideal source of cell therapy in bone and joint diseases. This review describes the advances of animal study and preliminary clinical application in the past few years, related to MSC-based cell therapy in the common bone and joint diseases, including osteoarthritis, rheumatoid arthritis, osteoporosis, osteonecrosis of the femoral head and osteogenesis imperfecta. It highlights the promising prospect of MSC in clinical application of bone and joint diseases.

Purpose AZD1775, a first-in-class, small-molecule inhibitor of the Wee1 tyrosine kinase, is under evaluation as a potential chemo- and radiosensitizer for treating glioblastoma. This study was to prospectively, quantitatively, and mechanistically investigate the penetration of AZD1775 across the human blood–brain barrier (BBB). Experimental Design AZD1775 plasma and tumor pharmacokinetics were evaluated in 20 patients with glioblastoma. The drug metabolism, transcellular passive permeability, and interactions with efflux and uptake transporters were determined using human derived in vitro systems. A whole-body physiologically based pharmacokinetic (PBPK) model integrated with a four-compartment permeability-limited brain model was developed for predicting the kinetics of AZD1775 BBB penetration and assessing the factors modulating this process. Results AZD1775 exhibited good tumor penetration in patients with glioblastoma, with the unbound tumor-to-plasma concentration ratio ranging from 1.3 to 24.4 (median, 3.2). It was a substrate for ABCB1, ABCG2, and OATP1A2, but not for OATP2B1 or OAT3. AZD1775 transcellular passive permeability and active efflux clearance across MDCKII–ABCB1 or MDCKII–ABCG2 cell monolayers were dependent on the basolateral pH. The PBPK model well predicted observed drug plasma and tumor concentrations in patients. The extent and rate of drug BBB penetration were influenced by BBB integrity, efflux and uptake active transporter activity, and drug binding to brain tissue. Conclusions In the relatively acidic tumor microenvironment where ABCB1/ABCG2 transporter-mediated efflux clearance is reduced, OATP1A2-mediated active uptake becomes dominant, driving AZD1775 penetration into brain tumor. Variations in the brain tumor regional pH, transporter expression/activity, and BBB integrity collectively contribute to the heterogeneity of AZD1775 penetration into brain tumors.