Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Design, Synthesis, and In Vitro and In Vivo Evaluation of Cereblon Binding Bruton's Tyrosine Kinase (BTK) Degrader CD79b Targeted Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an established modality that allow for targeted delivery of a potent molecule, or payload, to a desired site of action. ADCs, wherein the payload is a targeted protein degrader, are an emerging area in the field. Herein we describe our efforts of delivering a Bruton's tyrosine kinase (BTK) bifunctional degrader 1 via a CD79b mAb (monoclonal antibody) where the degrader is linked at the ligase binding portion of the payload via a cleavable linker to the mAb. The resulting CD79b ADCs, 3 and 4, exhibit in vitro degradation and cytotoxicity comparable with that of 1, and ADC 3 can achieve more sustained in vivo degradation than intravenously administered 1 with markedly reduced systemic exposure of the payload.

Continuous low-dose infusion of patupilone increases the therapeutic index in mouse and rat tumour models.

Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C0/Cmin ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity.

Continuous low plasma concentrations of everolimus provides equivalent efficacy to oral daily dosing in mouse xenograft models of human cancer.

PURPOSE: Everolimus is a drug used successfully in a number of different oncology indications, but significant on-target toxicities exist. We explored the possibility of improving the therapeutic index (TI) by studying alternative means of administering the drug based upon low continuous dosing. METHODS: All studies were performed using naïve nude mice or nude mice bearing s.c. human renal 786-O tumours or human breast MDA-MB-468 tumours. Everolimus was administered via a standard emulsion, either i.v., p.o., i.p., s.c., or via s.c. osmotic mini-pumps (MP) or via poly-lactic-co-glycolic (PLGA)-microparticles (PLGA-µP) prepared from everolimus powder injected s.c. Total-drug levels in blood, plasma or tissues were quantified ex vivo by LC-MS/MS. Efficacy studies were performed over 2-3 weeks and toxicity assessed by changes in body weight, glucose and white blood cell count. Effects on tumour activity biomarkers were quantified using reverse-phase protein array. RESULTS: Everolimus administration s.c. in an emulsion decreased the absorption rate but increased the C max and bio-availability of everolimus compared to standard approaches of administration p.o. or i.p. Everolimus administration s.c. via MP or PLGA-µP reduced the C max and provided continuous low concentrations of everolimus in the plasma, which inhibited tumour pS6/S6 to a similar degree to oral administration. Toxicities such as changes in body weight or white blood cell count were unaffected. Provided the everolimus concentration was above the free unbound IC50 for proliferation of the tumour cell line, efficacy could be achieved equivalent to that provided by standard oral administration. However, an overall improvement in the TI could not be demonstrated. CONCLUSIONS: Continuous low plasma concentrations of everolimus can provide strong efficacy in preclinical models, which if translatable to the clinic may reduce on-target toxicities and so increase the TI.

Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 µmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

Determinants of 6-month maternal satisfaction with breastfeeding experience in a multicenter prospective cohort study.

Although a personally defined experience, successful breastfeeding is usually measured with regard to duration. This study investigated the determinants of maternal satisfaction with breastfeeding experience for 907 mothers enrolled in a prospective cohort study. Despite a median breastfeeding duration (18 weeks) that fell short of recommendations, 822 mothers (90.6%) rated their breastfeeding experience as very or fairly satisfactory. Anticipated breastfeeding duration was a determinant of satisfaction only for women who actually breastfeed < 2 months; in this subgroup of mothers, satisfaction rates ranged from 84.6% for those who anticipated breastfeeding < 2 months to 69.8% for those who anticipated breastfeeding > 4 months (P = .01). Smoking during pregnancy and experiencing breastfeeding difficulties after discharge were independently associated with decreased satisfaction. Eliciting the mother's expectations regarding breastfeeding duration may help the lactation consultant in providing appropriate guidance. Future studies should assess maternal satisfaction using validated instruments.

CD-ROM-based program for breastfeeding mothers.

The vast majority of breastfeeding mothers in Western countries have routine access to multimedia and Internet resources at home. The aim of this study was to assess the effectiveness of a CD-ROM-based intervention in increasing the rates of breastfeeding. We conducted a pre- and post-intervention study involving four control and four intervention maternity units in France. All breastfeeding mothers in intervention units were given a CD-ROM-based program addressing various breastfeeding topics. The primary outcome was any breastfeeding at 4 weeks assessed by follow-up telephone interview. The secondary outcomes included breastfeeding duration, breastfeeding difficulties after discharge and satisfaction with the breastfeeding experience. The rates of any breastfeeding at 4 weeks varied from 88.6% (209/236) to 87.9% (211/240) and from 86.0% (222/258) to 88.0% (228/259) for mothers enrolled in intervention and control maternity units, respectively (P for interaction=0.54). The hazard of breastfeeding discontinuation for mothers enrolled in intervention units did not vary significantly across study periods after adjusting for education level, epidural anaesthesia, breastfeeding assessment score and return to work (P for interaction=0.18). The rates of breastfeeding at 4 weeks remained unchanged when restricting the analysis to the mothers who actually received (87.8% [173/197]) or used [88.2% (105/119)] the CD-ROM during the post-intervention period. No significant differences were found in secondary outcomes between the two study groups. A CD-ROM-based intervention for breastfeeding mothers provides no additional benefit to usual post-natal care. Further study is needed to assess the effectiveness of multimedia packages as part of more intensive multifaceted interventions.

Requesting physicians' experiences regarding infectious disease consultations.

BACKGROUND: Solicited consultations constitute a substantial workload for infectious disease (ID) specialists in the hospital setting. The objectives of this survey were to describe requesting physicians' experiences regarding ID consultations. METHODS: A cross-sectional survey was conducted in a university-affiliated hospital in France in 2009. All physicians were eligible (n = 530) and received a self-administered questionnaire. The main outcomes were reasons for request and opinion. Secondary outcomes were frequency of request and declared adherence to recommendations. RESULTS: The participation rate was 44.7% (237/530). Among the responders, 187 (79%) had solicited the ID consultation service within the previous year. Ninety-three percent of the responders (173/187) were satisfied with the ID consultation. The main reasons for requesting consultations were the need for therapeutic advice (93%), quality of care improvement (73%) and the rapidity of access (61%). ID consultations were requested several times a month by 52% (72/138) of senior physicians and by 73% (36/49) of residents (p = 0.01). Self-reported adherence to diagnostic and therapeutic recommendations was 83% and 79%, respectively. CONCLUSION: The respondent requesting physicians expressed great satisfaction regarding ID consultations that they requested principally to improve patient care and to assist in medical decision making.

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.).

BACKGROUND: In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies 1 showed that the use of the beta-D-glucosyl Yariv reagent (beta-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that beta-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation 2. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used beta-GlcY to block SE in order to identify genes potentially involved in this process. RESULTS: Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. beta-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by beta-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by beta-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. CONCLUSION: The use of two different chicory genotypes differing in their responsiveness to SE induction, together with beta-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

[Developing patient information sheets in general practice. Proposal for a methodology]. / Elaboration de fiches d'information pour les patients en médecine générale. Proposition d'une méthodologie.

BACKGROUND: Health information is patients' wish and right. For general practitioners, it is a duty, a legal obligation and a pre-requisite in any preventive approach. Written information must complete oral information since it improves health care quality. However, in general practice, there are no patient documents which are scientifically valid, understandable and efficient in terms of communication. OBJECTIVE: To develop a method for creating patient information sheets and to experiment its feasibility through the development of 125 sheets focused on the most common clinical conditions in general practice. METHOD: Research and literature review pour the development of specifications, and creation of 125 sheets following these specifications. RESULTS: The specifications developed consist of the 10 following steps: selection of the topic and the objectives, literature review, selection of the sections, drafting, validation of the scientific contents, assessment among patients, validation of the layout, selection of the media, delivery to patients and update. Following these specifications, we developed 125 information sheets. Each of these was reviewed by several physicians and assessed with R. Flesh readability test (the established acceptable threshold value was 40). The 30 sheets associated with the lowest scores were selected and reviewed to improve their overall readability. CONCLUSION: Even though some difficulties cannot be avoided when developing patient information sheets, each physician or physician association can create its own documents following the proposed specifications and thus deliver a customized message.

Breastfeeding outcomes for mothers with and without home access to e-technologies.

AIM: To estimate the percentage of breastfeeding mothers with home access to e-technologies and to compare breastfeeding outcomes for mothers with and without access to e-technologies. METHODS: We conducted a prospective observational study of 550 breastfeeding mothers discharged from nine maternity units in France. RESULTS: Overall, 435 mothers (79%; 95% confidence interval [95% CI], 75-82) had home access to e-technologies. Mothers with access to e-technologies were less likely to be unemployed (6% vs. 15%, p = 0.004), to smoke during pregnancy (8% vs. 16%, p = 0.03), to have a breastfeeding assessment score <8 (39% vs. 59%, p < 0.001) and to use a pacifier (23% vs. 41%, p < 0.001). Although mothers with access to e-technologies had a longer median breastfeeding duration than those without home access to e-technologies (19 vs. 16 weeks, p = 0.02), adjusted hazard ratios for breastfeeding discontinuation (0.85; 95% CI, 0.60-1.21), overall satisfaction rates (73% vs. 67%, p = 0.19) and breastfeeding difficulties after discharge (58% vs. 61%, p = 0.60) were not different for the two groups. CONCLUSION: A vast majority of breastfeeding mothers have home access to e-technologies in France. However, access to e-technologies was not independently associated with better breastfeeding outcomes in this study.