Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

At present, the methods and enzymology of the UDP-glucuronosyltransferases (UGTs) lag behind that of the cytochromes P450 (CYPs). About 15 human UGTs have been identified, and knowledge about their regulation, substrate selectivity, and tissue distribution has progressed recently. Alamethicin has been characterized as a treatment to remove the latency of microsomal glucuronidations. Most UGT isoforms appear to have a distinct hepatic and/or extrahepatic expression, resulting in significant expression in kidney, intestine, and steroid target tissues. The gastrointestinal tract possesses a complex expression pattern largely containing members of the UGT1A subfamily. Thus, these forms are poised to participate in the first pass metabolism of oral drugs. The authors and others have identified a significant expression of UGT1A1 in human small intestine, an enzyme possessing considerable allelic variability and a polymorphic expression pattern in intestine. Intestinal glucuronidation therefore plays a major role not only in first pass metabolism, but also in the degree of interindividual variation in overall oral bioavailability. Due to issues such as significant genetic variability and tissue localization in first-pass organs, clearance due to UGT1A1 should be minimized for new drugs.

International monitoring of drinking water and sanitation shapes awareness of countries’ needs and informs policy, implementation and research efforts to extend and improve services. The Millennium Development Goals established global targets for drinking water and sanitation access; progress towards these targets, facilitated by international monitoring, has contributed to reducing the global disease burden and increasing quality of life. The experiences of the MDG period generated important lessons about the strengths and limitations of current approaches to defining and monitoring access to drinking water and sanitation. The methods by which the Joint Monitoring Programme (JMP) of WHO and UNICEF tracks access and progress are based on analysis of data from household surveys and linear regression modelling of these results over time. These methods provide nationally-representative and internationally-comparable insights into the drinking water and sanitation facilities used by populations worldwide, but also have substantial limitations: current methods do not address water quality, equity of access, or extra-household services. Improved statistical methods are needed to better model temporal trends. This article describes and critically reviews JMP methods in detail for the first time. It also explores the impact of, and future directions for, international monitoring of drinking water and sanitation.

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BackgroundThe Sustainable Development Goals include commitments to end poverty, and promote education for all, gender equality, the availability of water and decent work for all. An important constraint is the fact that each day, many millions of women and children, and much less frequently men, carry their household’s water home from off-plot sources. The burden of fetching water exacerbates gender inequality by keeping women out of education and paid employment. Despite speculation about the potential health impacts of fetching water, there is very little empirical evidence. We report the first large study of the health impacts of carrying water on women and children.MethodsA cross-sectional survey was conducted in South Africa, Ghana and Vietnam during 2012. It investigated water carrying methods and health status. Because areas of self-reported pain were correlated we undertook factor analysis of sites of reported pain, to interpret patterns of pain reporting. Regression analysis using Generalised Estimating Equations (GEE) investigated water carrying as a risk factor for general health and self-reported pain.ResultsPeople who previously carried water had increased relative risk of reporting pain in the hands (risk ratio RR 3.62, 95% confidence interval CI 1.34 to 9.75) and upper back (RR 2.27, 95% CI 1.17 to 4.40), as did people who currently carry water (RR hand pain 3.11, 95% CI 1.34 to 7.23; RR upper back pain 2.16, 95% CI 1.25 to 3.73). The factor analysis results indicate that factor 1, ‘axial compression’, which is correlated with pain in the head and upper back, chest/ribs, hands, feet and abdomen/stomach, is associated with currently (0.30, 95% CI 0.17 to 0.43) or previously (0.21, 95% CI 0.01 to 0.42) carrying water. Factor 2, ‘soft tissue strain’, which is correlated with pain in the neck, shoulders/arms, lower back and hips/pelvis or legs, is marginally negatively associated with currently (-0.18, 95% CI -0.32 to -0.04) carrying water. The factor ‘axial compression’ was more strongly associated with carrying water containers on the head.ConclusionsParticipants who reported a history of current or past water carrying more frequently reported pain in locations most likely to be associated with sustained spinal axial compression in the cervical region. Given the fact that cervical spinal conditions are globally one of the more common causes of disability, our findings suggest that water carrying, especially by head loading is a major contributing factor in musculoskeletal disease burden in low income countries. Our findings support the proposed indicator for monitoring SDG6.1: “Percentage of population using safely managed drinking water services at home.”

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.